LIST OF FIGURES

ABSTRACT

Low biohydrogen (H2) yields and limited use of process by-products from dark fermentation (DF) of waste biomass is limiting its scaled-up application. This study aims to investigate the effects of culture pH, substrate concentration, pre-treatment of substrate and inoculum adaptation on H2 yields during the DF of three organic wastes biomass (i.e.

food waste, rice straw and olive mill wastewater). The results showed that the biodegradability of the substrates is important for the selection and application of optimal operational parameters aimed at enhancing H2 production.

Moreover, long-term operational feasibility and stability of dark fermentative H2 production was demonstrated using food waste and cheese whey in two semi-continuous thermophilic DF reactors. The effect of Organic Loading Rates (OLRs), Hydraulic

Retention Times (HRTs) and co-substrates (buffalo manure) addition, as a source of alkalinity, on culture pH and H2 production stability was discussed. The results showed that combination of OLR, HRT and co-substrate addition could play a vital role in the culture pH and stability of H2 production.

The by-products of DF process were utilized for H2 production via photo fermentation (PF), while the waste stream generated from coupling of DF and PF processes was converted to methane in anaerobic digestion. The three-step conversion of food waste in a biorefinery concept increased the total energy yields. Moreover, PF also showed a good potential for concomitant production of H2 and polyhydroxybutyrate (biopolymer).

Likewise, dry fermentation of waste biomass could be promising for the production of bioenergy and biochemicals (organic acids and alcohols) in a biorefinery concept.

xii SINTESI La produzione di Idrogeno mediante Dark-Fermentation (DF) rappresenta ad oggi uno dei processi biologici più promettenti nel campo della valorizzazione energetica delle biomasse di scarto. Sebbene, tale bio-tecnologia presenti un potenziale notevole, le basse rese in termini di produzione di idrogeno e l'assenza di metodologie che prevedano il riutilizzo dei sottoprodotti di pregio, rendono l'applicazione di tale processo non sostenibile a scala reale.

Il lavoro risulta articolato in quattro fasi distinte. Nella prima fase sono stati investigati gli effetti sulla produzione biologica di H2 di specifici parametri operativi. In particolare, si è proceduto ad analizzare: i) l'effetto del pH, ii) l'effetto combinato del pH e della concentrazione iniziale di substrato, iii) l'effetto di pretrattamenti del substrato, iv) l'utilizzo di biomasse microbiche adattate. In fase di sperimentazione sono state utilizzate tre differenti tipologie di substrato. I risultati hanno mostrato che la biodegradabilità dei differenti substrati risulta fondamentale nella corretta definizione dei parametri di processo al fine di massimizzare la produzione di Bio-idrogeno.

La seconda parte dell'attività sperimentale è stata dedicata alla messa a punto e alla conduzione di due reattori di DF operanti in regime di termofilia e alimentati con frazione organica di rifiuto solido urbano e reflui caseari, mediante i quali viene dimostrata la fattibilità e la stabilità del processo di DF nel lungo periodo. Vengono, altresì, discussi gli effetti dovuti all'applicazione di differenti Carichi Organici (Organic Loading Rates -OLRs), differenti Tempi di Ritenzione Idraulica (Hydraulic Retention Times -HRTs) e dell'aggiunta di substrati ad elevato tenore di alcalinità. I risultati hanno dimostrato che la combinazione di OLR, HRT e l'aggiunta di co-substrato (refluo bufalino) possono giocare un ruolo fondamentale nella stabilità del processo di DF.

In un contesto di bio-raffineria, nella terza parte del lavoro viene proposto un sistema integrato costituito dall'abbinamento del processo di Dark Fermentation ai processi di Photo Fermentation (PF) e di digestione anaerobica (AD). Attraverso tale sistema a triplo stadio, è stato possibile non solo incrementare la resa energetica totale, ma, aspetto non trascurabile, ottenere mediante l'applicazione del processo di PF, la sintesi del biopolimero Poly-Hydroxy-Butyrate (PHB) che può essere utilizzato per la produzione di bioplastiche.

Infine, nella quarta ed ultima parte dello studio, è stato valutato il processo di DF in condizioni dry. Tale applicazione ha consentito la contestuale produzione di bio-energia xiii e di ulteriori bio-prodotti (e.g. alcoli, acidi organici etc.), amplificando ulteriormente il concetto di bio-raffineria.

xiv

RÉSUMÉ

La fermentation sombre est un procédé utilisant des déchets organiques dont le passage à l'échelle pilote est limité par les faibles rendements de production d'hydrogène ainsi que par l'utilisation des sous-produits métaboliques. Cette étude a pour premier objectif d'étudier des paramètres opératoires, par exemple, l'effet du pH, de la concentration en substrat, du prétraitement du substrat et de l'adaptation de l'inoculum microbien sur la fermentation sombre de trois types de déchets différents (i.e. déchets alimentaires, paille de riz et les eaux usées de pressoirs à d'olives). Il a été montré que la biodégradabilité des substrats jouait un rôle majeur dans le choix des paramètres opérationnels utilisés pour optimiser la production d'hydrogène.

De plus, la faisabilité et la stabilité à long terme de la production d'hydrogène par le procédé de fermentation sombre ont été observées en utilisant des déchets agroalimentaires et du petit lait dans deux réacteurs thermophiles fonctionnant en mode semi-continu. En particulier, il a été discuté de l'influence de la charge organique (OLR), du temps de rétention hydraulique (HRT) et de l'addition de co-substrats (fumier de buffle) comme source d'alcalinité. Ainsi, cette étude a permis de montrer que la combinaison de ces trois paramètres pouvait jouer un rôle important sur le pH et la stabilité de la production d'hydrogène.

Les sous-produits métaboliques de la fermentation sombre ont également été utilisés pour produire de l'hydrogène via la photo-fermentation, alors que les déchets générés par le couplage de la fermentation sombre et de la photo-fermentation ont été valorisés pour la production de méthane par digestion anaérobie. Ce concept de bioraffinerie basé sur la conversion en trois étapes des déchets agroalimentaires augmente le rendement énergétique global du procédé. Par ailleurs, le potentiel important du procédé de photofermentation pour la production concomitante de polyhydroxybutyrate (polymère) et de l'hydrogène a ainsi été démontré.

En conclusion, la fermentation par voie sèche de déchets organiques pour la production de bioénergie et de produits biochimiques (i.e. acides organiques et alcools) paraît prometteuse dans un contexte d'optimisation de la production d'énergies et de biomolécules au sein d'une bioraffinerie environnementale.

SAMENVATTING

Lage biowaterstof (H2) produktierendementen en beperkt gebruik van bijproducten van in vergisting (dark fermentaiton, DF) van biomassa beperken de opschaling van dit process. Deze studie onderzocht het effect van pH, combinatie van substraat concentratie en cultuur pH, voorbehandeling van het substraat en entmateriaal op de H2 opbrengst via DF van drie verschillende types afvalbiomassa, met name (i.e. keukenafval, rijststro en afvalwater van olijfolieproductie. Uit het onderzoek bleek dat de biologische afbreekbaarheid van de substraten een belangrijke rol speelde bij de selectie en toepassing van de optimale operationele parameters ter verbetering van de H2-productie.

De operationele haalbaarheid en stabiliteit op lange termijn van H2-productie via DF werd gedemostreerd met keukenafval en wei in twee semi-continue thermofiele DF reactoren.

Het effect van de organische belasting OLRs), hydraulische retentietijd (HRT) en toevoeging van co-substraten (buffel mest als bron van alkaliteit) op de cultuur pH en H2 productiestabiliteit zijn bestudeerd. Uit deze studie bleek dat een combinatie van OLR, HRT en co-substraat toediening een belangrijke rol kunnen spelen in de pH van de fermentor en de stabiliteit van de H2-productie.

Bovendien werden de bijprodukten van het DF-proces gebruikt voor H2 productie via photofermentatie (PF), terwijl de afvalstroom gegenereerd uit de koppeling van DF en PF processen omgezet werd naar methaan via anaërobe vergisting (AD). De drie-staps conversie van keukenafval in een bioraffinage concept verhoogde de totale energie opbrengst. Bovendien toonde PF een goede potentie voor de gelijktijdige productie van H2 en polyhydroxybutyraat (biopolymeer). Ook droge fermentatie kan veel belovend zijn voor voor de productie van bio-energie en biochemicaliën (VFAs en alcoholen) in een bioraffinage concept op basis van afvalbiomassa.

xvi ACKNOWLEDGEMENT Persuing a doctoral degree was one of the aspirations of my life. I am grateful to all my friends, colleagues, mentors, supervisors and my family who have continuously inspired and supported during this journey. I would like to dedicate this Doctoral Thesis to all these people who did not gave up on me during the difficult times and shared joyful

Background and problem statement

The global reserves of fossil fuels are depleting due to their increasing consumption in energy and chemical sectors. In addition, environment is facing severe pollution problems due to the gaseous emissions (NOx, SOx, CO, CO2, etc.) and waste generated from the production and use of fossil fuels. The scientific community has widely accepted the fact that the increasing CO2 levels has impacted global warming phenomena, which is threatening the entire earth's ecosystem [START_REF] Ciais | Carbon and Other Biogeochemical Cycles[END_REF]. Therefore, a current need is to explore and invest in alternative ways to harness the energy and chemicals from the clean renewable sources that are carbon neutral and can reduce the global CO2 emissions at the same time.

In this context, hydrogen gas (H2) could represent a promising alternative energy carrier due to its social, economic and environmental credentials (Kotay & Das, 2008). The net energy content of the H2 per unit mass is higher than other conventional fuels. The lower heating value (LHV) of hydrogen varies between 2.4-2.8 and is 4 times higher than that of methane, gasoline and coal respectively [START_REF] Marbán | Towards the hydrogen economy?[END_REF]. H2 is a carbon-free clean fuel as the ultimate by-products of combustion is only water. Thus, H2 carries a long term potential to reduce consumption of fossil fuels that can be helpful in combating global warming and pollution problems.

A preliminary major challenge in the use of this promising source of energy carrier lies in the sustainable production of H2. In commercial applications, H2 have been produced from natural gas by steam reforming process, coal gasification and water electrolysis (Kotay & Das, 2008;[START_REF] Manish | Comparison of biohydrogen production processes[END_REF]. At present, steam reforming of methane is the cheapest H2 production method. However, for the equivalent amount of energy, it is four times more costly than gasoline [START_REF] Crabtree | The Hydrogen Economy[END_REF]. [START_REF] Bartels | An economic survey of hydrogen production from conventional and alternative energy sources[END_REF] reported an estimated cost of 0.36-1.83 $/kg and 2.48-3.17 $/kg for H2 production from coal and natural gas, respectively. H2 from conventional sources are economically convenient compared to biological routes for H2 production (i.e. thermophilic dark fermentation process), which costs about $28.35/kg H2 (€21/kg H2 with €1=$1.35 in 2011) (HYVOLUTION, 2011). In a study, [START_REF] Das | Advances in biohydrogen production processes: An approach towards commercialization[END_REF] reported a low production cost of energy as H2 from dark fermentation of sewage sludge ($1.3/Million British Thermal unit, MBTU) compared to natural gas ($2-$7/MBTU, in 2007) and gasoline ($23.5/MBTU in 2008).

However, most of the physical and chemical H2 production processes are highly energy intensive and/or dependent on fossil fuels. Moreover, the physical and chemical technologies do not reduce the consumption of fossil fuels or CO2 emissions. This only shifts the point of CO2 emissions to H2 producing industries from vehicular emissions or emission from stationary hydrogen power stations, which makes them less attractive from environmental point of view. On the other hand, H2 produced from biological processes, also known as biohydrogen is expected to be less energy intensive and can be produced from renewable sources (Das & Veziroglu, 2001;Hallenbeck & Ghosh, 2009).

H2 can be produced biologically by autotrophic as well as heterotrophic microorganisms (Hallenbeck & Ghosh, 2009) (detailed in Chapter 2). Autotrophic conversions are mediated by microalgae utilizing inorganic carbondioxide as a carbon source whereas heterotrophs convert the organic carbon sources into simpler compounds producing molecular H2. There are two types of heterotrophic conversions; one driven by light energy (photofermentation) and other that occurs in absence of light (dark fermentation).

Dark fermentation (DF) represents one of the most promising and cost-effective technologies for biohydrogen production due its faster conversion efficiencies. Moreover, DF process can utilize wide range of renewable complex waste biomass as feedstock and production of other valuable platform biochemicals of economic interest (Ghimire et al., 2015a). Currently, the major barriers in application of DF in scaled-up systems for H2 production are: low H2 yields and the high cost of production mainly due to the high cost of feedstock (Ren et al., 2011). Moreover, an inherent challenge of DF systems is to maximize the process conversion efficiencies, utilization and valorization of the byproducts and minimize the ecological footprint of the process by reducing the water and energy input to the process.

In order to achieve a scaled-up development of dark fermentative processes, an immediate attention is required to improve H2 yields utilizing the low cost materials like waste biomass such as agricultural residues, organic waste generated from municipalities and industries, that could also give competitive economic advantage (Chong et al., 2009;Kapdan & Kargi, 2006). The H2 yields and production rates can be enhanced by optimizing the operational parameters such as culture pH and temperature and substrate concentration as well as by inoculum enrichment and substrate pre-treatment (Guo et al., 2010;Urbaniec & Bakker, 2015). Moreover, knowledge gaps in the long-term operational feasibility of the DF process for continuous H2 production needs to be filled for its development.

The by-products of DF process, which mostly includes volatile fatty acids (VFAs), lactic acid, alcohols and un-hydrolyzed residues, can be utilized in other biological systems for their valorization by energy recovery or can be used as a feedstock in production of platform chemicals of economic interests [START_REF] Agler | Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform[END_REF]Bastidas-Oyanedel et al., 2015;Ghimire et al., 2015a). The dark fermentation effluent (DFE) can be converted to H2 photo fermentation (PF) process, which is mediated by purple non sulfur bacteria (PNSB). In addition to H2 production, PNSB are known to synthesize polyhydroxybutyrate (PHB), a precursor for biopolymers (Hustede et al., 1993).

Likewise, un-utilized biomass residues as well as the waste streams generated from coupling of DF and PF process can be further converted to methane in anaerobic digestion process [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF]. In this way, utilization of DF by-products can lead to realization of a biorefinery concept that could help in industrial development of DF technology. Moreover, Solid State Dark Fermentation (SSDF) process has been recently

proposed for biorefinery concept due to its inherent characteristics such as higher process yields and less energy and water requirements (Motte et al., 2015;Elsamadony and Tawfik, 2015). Therefore, a study of major limitations in SSDF is necessary to exploit this technology. In this context, a general scheme of the present doctoral research is reported in Figure 1.1.

Scope of the PhD thesis

The main objectives of this research were to study the process influencing parameters in the DF of complex organic waste and the valorization of the by-products in a biorefinery concept. The specific objectives were: i. To assess the effect of different operational parameters on dark fermentative H2 production from different complex waste biomass.

ii. To study long term continuous H2 production from food and cheese whey waste with an emphasis on pH control.

iii. To investigate the integration of DF in a biorefinery concept coupling with photo fermentation and anaerobic digestion to maximize energy yields and valorize the by-products. iv. To assess the limitations in the application of SSDF for H2 and organic acids productions.

To achieve these four major aims, the research activities that were carried out are outlined as follows:

Chapter 1 explains the motivation, hypothesis and scheme of the doctoral research.

Chapter 2 provides the comprehensive state-of-art in parameters influencing the DF of complex waste biomass and use of by-products. Moreover, it also discusses the potential application of photofermentation processes to valorize the dark fermentation by-products by H2 and biopolymer production.

Chapter 3 presents the influence of different operational parameters in DF of complex waste biomass. Various operational parameters such as inoculum sources and enrichment methods, pH, temperature and substrate concentration were studied.

Chapter 4 demonstrates the effects of different reactor operating conditions, such as organic loading rates (OLRs) and hydraulic retention times (HRTs), on long-term operational feasibility of H2 production. This chapter also discusses the use of low OLRs and co-substrate addition as pH controlling strategies using food waste and cheese whey waste, respectively, in two separate studies.

Chapter 5 discusses the potential for the integration of DF process in a biorefinery concept. The coupling with photofermentation and anaerobic digestion by using DFE was studied to explore the potential for futher energy recovery. Similarly, this section presents the prospective of photofermentation process for maximizing the valorization of DFE via concomitant H2 and biopolymer production. Moreover, possible limitations during the conversion of waste biomass in SSDF were studied.

Chapter 6 highlights the major findings and the implications of the research and provides future recommendations. [START_REF] Marbán | Towards the hydrogen economy?[END_REF] Towards the hydrogen economy? Int. J. Hydrogen Energy 32, 1625-1637.

Motte, J.-C., Sambusiti, C., Dumas, C., & Barakat, A. (2015). Combination of dry dark fermentation and mechanical pretreatment for lignocellulosic deconstruction: An innovative strategy for biofuels and volatile fatty acids recovery. Applied Energy, 147, 67-73.

Ren, N., Guo, W., Liu, B., Cao, G., Ding, J., 2011. Biological hydrogen production by dark fermentation: challenges and prospects towards scaled-up production. Curr. Opin. Biotechnol. 22, 365-70.

Urbaniec, K., Bakker, R.R., 2015. Biomass residues as raw material for dark hydrogen fermentation -A review. Int. J. Hydrogen Energy 40, 3648-3658.

CHAPTER 2 STATE OF THE ART IN DARK FERMENTATION OF COMPLEX WASTE

BIOMASS BY MIXED CULTURE AND UTILIZATION OF DARK FERMENTATION EFFLUENTS IN PHOTO FERMENTATION

The section 2.1 of this chapter has been published as [START_REF] Ghimire | A review on dark fermentative biohydrogen production from organic biomass: Process parameters and use of by-products[END_REF]. A review on dark fermentative biohydrogen production from organic biomass: Process parameters and use of by-products. Applied Energy 144, 73-95

The section 2.2 of this chapter has been submitted as Ghimire, A., Luongo, V., Frunzo, L., Pirozzi, F., Lens, P.N.L., Esposito, G., 2016. Engineering strategies for enhancing photofermentative biohydrogen production by purple non-sulfur bacteria using dark fermentation effluents. In Microbial Fuels:

Technologies and Applications. Taylor and Francis Group, CRC Press. Boca Raton, FL, USA.

Dark fermentative biohydrogen production from organic biomass

This section summarises the state of the art in the dark fermentative biohydrogen production from organic biomass such as agricultural residues, agro-industrial wastes and organic municipal waste. In spite of its potential, this technology needs further research and development to improve the biohydrogen yield by optimizing substrate utilization, microbial community enrichment and bioreactor operational parameters such as pH, temperature and H2 partial pressure. On the other hand, the technical and economic viability of the processes need to be enhanced by the use of valuable by-products from dark fermentation, which mostly includes volatile fatty acids. This paper reviews a range of different organic biomasses and their biohydrogen potential from laboratory to pilotscale systems. A review of the advances in H2 yield and production rates through different seed inocula enrichment methods, bioreactor design modifications and operational conditions optimization inside the dark fermentation bioreactor is presented. The prospects of valorizing the co-produced volatile fatty acids in photofermentation and bioelectrochemical systems for further H2 production, methane generation and other useful applications have been highlighted. A brief review on the simulation and modeling of the dark fermentation processes and their energy balance has been provided. Future prospects of solid state dark fermentation are discussed.

Introduction

Environmental friendly energy carriers and sources are the most highlighted topic in the energy and environmental sector. The current global energy demand is mostly dependent on reserves of fossil fuels, which are depleting, and the world is facing severe pollution problems from the by-products of fossil fuels uses [START_REF] Marbán | Towards the hydrogen economy?[END_REF]. The scientific community has widely accepted the fact that the increasing CO2 level due to the use of fossil resources is impacting the greenhouse gas effect and global warming.

Therefore, different ways to harness the energy from clean renewable sources are being developed, but the search for reliable energy sources is still on.

In the past years, the research and development interests have been directed towards renewable energy technologies like the anaerobic digestion (AD) of organic biomass and waste. For alternative energy carriers, hydrogen could be the fuel of the future because of its high energy content, environmental friendliness of production, and also because it can give substantial social, economic and environmental credentials (Kotay and Das, 2008).

Hydrogen is a carbon-free clean fuel, as the only final by-product of its combustion is water (Kotay and Das, 2008). Hydrogen can also be helpful in addressing global warming and increasing pollution problems. Furthermore, it is preferred over methane owing to its wider industrial applications, i.e. H2 is used in the synthesis of ammonia and hydrogenation of edible oil, petroleum, coal and shale oil [START_REF] Kothari | Fermentative hydrogen production -An alternative clean energy source[END_REF]. Hydrogen can be directly used either in combustion engines because of its highest energy per unit weight, i.e. 143 GJ per ton (Kotay and Das, 2008) among known gaseous biofuels or to produce electricity via fuel cell technologies [START_REF] Alves | Overview of hydrogen production technologies from biogas and the applications in fuel cells[END_REF]. Thus, the creation of a hydrogen economy which incorporates the production and use of hydrogen as an energy carrier could in the future lead to sustainable energy systems [START_REF] Ekins | The prospects for a hydrogen economy (1): hydrogen futures[END_REF][START_REF] Marbán | Towards the hydrogen economy?[END_REF].

The major challenge in the use of this promising energy carrier lies in its sustainable production and storage. In commercial applications, hydrogen has been produced from natural gas (48%) and oil (30%) by steam reforming processes, and also by other industrial methods such as coal gasification (18%) and water electrolysis (4%) [START_REF] Balat | Potential importance of hydrogen as a future solution to environmental and transportation problems[END_REF]. However, these processes are highly energy intensive and use non-renewable sources of energy, which makes them less attractive from an environmental point of view.

In order to produce a cleaner and more sustainable fuel, the hydrogen should come from processes that avoid or minimize CO2 emissions.

Hydrogen can be produced from biological processes that are less energy intensive and more environmental friendly in terms of global reduction of CO2. These renewable biohydrogen producing technologies have potential to become cost competitive as they can use low value waste biomass as feedstock (Kotay and Das, 2008), e.g. municipal, agricultural and industrial organic waste and wastewater. Biohydrogen can be produced by both autotrophic and heterotrophic microorganisms (Figure 2.1) (Das and Veziroglu, 2008;Kotay and Das, 2008). In autotrophic conversions (also known as direct or indirect biophotolysis), solar energy is directly converted to hydrogen via photosynthetic reactions mediated by photosynthetic microorganisms, i.e. microalgae, protists and photosynthetic bacteria. Under heterotrophic conditions, the organic substrates are transformed into simpler organic compounds with simultaneous production of molecular hydrogen (Das and Veziroglu, 2008;Li and Fang, 2007a). There are two types of heterotrophic conversions, photo-fermentation carried out by photosynthetic bacteria and dark fermentation (DF) carried out by anaerobic bacteria that convert carbohydrates into biohydrogen. DF is the most studied and promising technology for biohydrogen production owing to its higher production rates and treatment capacity for organic wastes. Several substrates rich in carbohydrates are also usable, such as first generation fuel crops (e.g. sugar cane, wheat, corn, and sugar beets) as well as second generation biomass like agricultural residues as well as industrial waste and wastewater (Das and Veziroglu, 2008). In recent years, there are increasing research activities in this domain, as shown by the increasing number of peer-reviewed articles with "dark fermentation" in the title (Figure 2.2). [START_REF] Scholar | Google Scholar Keywords searches and abstracts[END_REF]Scopus, 2014) At present, DF process development at industrial scale is limited by its lower hydrogen yield compared to its theoretical maximum yield of 4 moles of H2 per moles of hexose, as well as the estimated costs associated with the H2 production. There are areas for improvement to achieve higher H2 yields and production rates by optimizing the design and operation of DF bioreactors [START_REF] Show | Bioreactor and process design for biohydrogen production[END_REF]. The H2 production cost in scaled-up systems can be minimized by using low cost renewable materials such as waste biomass as feedstock (Kapdan and Kargi, 2006;Ren et al., 2011). Inoculum enrichment methods (De Gioannis et al., 2013;Li and Fang, 2007a;Ntaikou et al., 2010;Show et al., 2012;Wong et al., 2014) can improve the H2 yield, and pre-treatment of substrates can also enhance the biohydrogen production by improving the biodegradability of substrates (Ariunbaatar et al., 2014;Monlau et al., 2013b;Motte et al., 2014). Recently, there has been growing interest on coupled processes to obtain a higher H2 yield by integrating DF with processes like photofermentation (PF) (Rai et al., 2014;[START_REF] Redwood | Integrating dark and light bio-hydrogen production strategies: towards the hydrogen economy[END_REF] or bioelectrochemical systems (Chookaew et al., 2014;[START_REF] Guwy | Fermentative biohydrogen production systems integration[END_REF]Moreno et al., 2015). Because of the profitable production of biomethane, a coupled DF-methanogenic stage has also been a popular choice which increases the sustainability of the coupled-process by improving the energy recovery from the DF residues (Elbeshbishy and Studies, 2011;Gómez et al., 2011;Gottardo et al., 2013).

The aim of this paper is to provide an updated overview of advancements in biohydrogen production via DF of organic biomass. Regardless of the increasing number of research articles and reviews published, there is a need to provide an extended overview of dark fermentative biohydrogen production with the utilization of by-products and the future challenges and prospects for its up-scaled development. This review provides an insight on the factors that influence the biochemical pathways in dark fermentative biohydrogen production to increase the H2 yield and post-utilization of DF residues to realize its future sustainability. To summarize, this review provides an extended insight on a) possible feedstock or substrate sources and their biohydrogen potential (BHP), b) factors that influence the fermentative H2 yield: (i) inoculum sources and enrichment methods, (ii) pre-treatment of substrates and (iii) bioreactor operation and design (culture pH, temperature and OLR, HRT, H2 partial pressure, nutrients and elements addition), c) utilization of DF residues, d) pilot scale systems and e) challenges and future prospects:

(i) modeling and simulation of DF process, (ii) energy balance and conversion of organic carbon, (iii) natural pH control and (iv) future prospects of solid state dark fermentation.

Microbiology and biochemical pathways of DF

In DF processes, carbohydrate-rich substrates are broken down anaerobically by hydrogen-producing microorganisms, such as facultative anaerobes and obligate anaerobes. Molecular hydrogen (H2) is produced in the process of disposing the excess electrons through the activity of the hydrogenase enzyme (Das and Veziroglu, 2001;Li and Fang, 2007a). Under anaerobic environments, protons (H + ) can act as electron acceptors to neutralize the electrons generated by oxidation of organic substrates, consequently producing H2. In contrast with aerobic respiration, where oxygen is reduced and water is the final product (Das and Veziroglu, 2008;Wang and Wan, 2009).

In the DF of glucose as the model substrate, H2 -producing bacteria initially convert glucose to pyruvate through glycolytic pathways producing adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and the reduced form of nicotinamide adenine dinucleotide (NADH) (Li and Fang, 2007a). Pyruvate is further oxidized to acetyl coenzyme A (acetyl-CoA), carbon dioxide (CO2) and H2 by pyruvate ferredoxin oxidoreductase and hydrogenase. Depending on the type of microorganism and environmental conditions, pyruvate may also be converted to acetyl-CoA and formate which may be further converted into H2 and CO2. Also, acetyl-CoA might be converted to acetate, butyrate, and ethanol (Li and Fang, 2007a). DF of complex carbohydrates by mixed anaerobic microbiota can result in a wide range of intermediates and by-products depending on the operational parameters, such as substrate type, substrate loading rate, pH, temperature and other operating and environmental conditions, as they also influence the microbial community structure in bioreactors. These biochemical pathways (Figure 2.3) can be mediated by strict anaerobes (Clostridia, methylotrophs, rumen bacteria, methanogenic bacteria, archea, etc.), facultative anaerobes (Escherichia coli, Enterobacter, Citrobacter), and even aerobes (Alcaligenes, Bacillus) (Li and Fang, 2007a). Acetate and butyrate are the most common products of DF (Hawkes et al., 2007). When the metabolic pathway is such that it favors the production of acetic acid, the stoichiometric yield of H2 is 4 moles for each mole of glucose (i.e. 544 mL H2/g hexose at 25 °C) as in equation 2.1, whereas the yield of H2 is 2 moles for a mole of glucose (i.e.

272 mL H2/g hexose at 25 °C) when the final product is butyric acid (equation 2.2) (Li and Fang, 2007a). However, the actual hydrogen yield is lower than the theoretical yield as part of the substrate is utilized for biomass production and the degradation of the substrates might follow other biochemical pathways without hydrogen production [START_REF] Hallenbeck | Biological hydrogen production; fundamentals and limiting processes[END_REF][START_REF] Nath | Improvement of fermentative hydrogen production: various approaches[END_REF]. Under some conditions, the metabolic pathways lead to ethanol and acetate production, lowering the stoichiometric hydrogen yield to 2 moles of H2 for a mole of glucose (i.e. 272 mL H2/g hexose at 25 °C) as represented in equation 2.3 (Li and Fang, 2007a):

C6H12O6 + 2H2O → CH3CH2OH + CH3COOH + 2CO2 + 2H2 (2.3)
A widely studied clostridia species, Clostridium butyricum, is responsible for the production of butyric acid as the major product of fermentation together with acetate and hydrogen (Hawkes et al., 2007). Another fermentation pathway is the production of propionate by Clostridium articum which is a hydrogen consuming pathway (equation 2.4). Similarly, metabolic pathways leading to only ethanol and lactic acid production by Clostridium barkeri yield no hydrogen (equations 2.5 and 6) (Khanal et al., 2003): Hawkes et al. (2007) and Kim et al. (2006) proposed the molar ratio of butyric to acetic acid (B/A ratio) as a quantitative indicator of the biohydrogen yield associated with microbial metabolic pathways. Kim et al., 2006) found that B/A ratios were directly proportional to H2 yields (mol H2/mol hexose) during DF of sucrose in CSTR reactors operated at an organic loading rate (OLR) of 10 -60 g Chemical Oxygen Demand (COD)/L, pH 5.5 and 12 h hydraulic retention time (HRT). They also reported that a B/A ratio higher than 2.6 indicated an efficient H2 production by anaerobic microbiota. In DF with mixed cultures, when a B/A ratio of 3:2 is generally observed, results in a H2 yield of 2.5 moles H2 per mole of hexose fermented as given in equation 2.7 (Hawkes et al., 2007):

C6H12O6 + 2H2 → 2CH3CH2COOH + 2H2O (2.4) C6H12O6 → 2CH3CH2OH + 2CO2 (2.5) C6H12O6 → CH3CHOHCOOH + 2CO2 (2.6)
4C6H12O6 + 2H2O → 3CH3CH2CH2COOH + 2CH3COOH + 8CO2 + 10H2 (2.7)
In contrast, Guo et al. (2013) showed in their study performed with lignocellulosic substrates that this ratio might not give a good indication, particularly in batch tests where homoacetogenic activity prevails. Therefore, higher acetate concentrations cannot always give an indication of a higher H2 yield. Some homoacetogens belonging to the genus Clostridium (e.g. C. aceticum) can lower the H2 yield by converting H2 and CO2 to acetate or can convert hexose directly to acetate (Hawkes et al., 2007;Kim et al., 2006). However, analysis of soluble metabolites can give an indication of the fermentation pathways and thus the H2 production performance.

Clostridia have been identified as the dominant hydrogen producing microorganisms in DF operated with mesophilic mixed cultures at a pH of 5.5 (Fang et al., 2002). Fang and

Zhang (Fang et al., 2002) identified that 64.6% of all the microorganisms were affiliated with three Clostridium species (Clostridiaceae), 18.8% with Enterobacteriaceae, and 3.1% with Streptococcus bovis (Streptococcaceae) based on the phylogenetic analysis of the rDNA sequences. Interestingly, Rafrafi et al. [START_REF] Rafrafi | Sub-dominant bacteria as keystone species in microbial communities producing bio-hydrogen[END_REF] reported recently that sub-dominant species, in spite of their low abundance, can also have substantial impact on the hydrogen production performance. The presence of some species like E. coli can aid in increasing the H2 yield by diverting the metabolic pathways to the acetate and butyrate hydrogen producing pathways (equation 2.7), while other species communities such as Bacillus spp. and Lactobacillus spp. can lower the H2 yield by diverting the pathway to lactate accumulation (equation 2.6).

Other results of the identification of the microbial diversity by community fingerprinting techniques in the thermophilic DF of rice straw showed that hydrolytic and fermentative bacteria such as Clostridium pasteurianum, Clostridium stercorarium and Thermoanaerobacterium saccharolyticum dominated in the sludge of a repeated fedbatch reactor (Chen et al., 2012). Shin et al. (Shin et al., 2004) detected the hydrogen producing microorganisms Thermoanaerobacterium thermosaccharolytium and Desulfotomaculum geothermicum in a thermophilic acidogenic culture, while

Thermotogales strains and Bacillus species were detected in a mesophilic acidogenic culture by Polymerase Chain Reaction (PCR)-Denaturing Gradient Gel Electrophoresis (DDGE) analysis during DF of food waste. In another study, Quemeneur et al. [START_REF] Quéméneur | Functional versus phylogenetic fingerprint analyses for monitoring hydrogen-producing bacterial populations in dark fermentation cultures[END_REF] investigated the potential of a molecular capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) fingerprinting method based on the hydA functional genes to better describe the bacterial community dynamics in a mixed dark fermentative culture at different pH conditions.

Some undesirable microorganisms which lower the total H2 yield might be present in mixed cultures of fermentative microorganisms, either by consuming the H2 produced or by altering the biochemical pathways of the H2 synthesis (Li and Fang, 2007a). The main H2 consumers include methanogens, homoacetogenic bacteria and sulfate reducing bacteria (SRB). The activity of these hydrogen consumers can be controlled by inoculum pre-treatment methods or bioreactor operating conditions (Guo et al., 2010;Wang and Wan, 2009). The activity of methanogens and SRB can be significantly reduced by operating at a pH below 6 along with the control of the HRT and OLR. Therefore, hydrogen production via a mixed dark fermentative culture is a complex microbial system, influenced by a number of parameters such as substrate types, substrate pretreatment, inoculum type, inoculum enrichment method, bioreactor design and operation.

Potential sources of organic biomass for fermentative biohydrogen production

The substrate plays an important role in the H2 yield, H2 production rate and the overall economy of the process. These are mainly dependent on the substrate´s carbohydrate content, bioavailability and biodegradation rate (Chong et al., 2009;Guo et al., 2010;Kapdan and Kargi, 2006;Ntaikou et al., 2010;Ren et al., 2011). Carbohydrate rich substrates have been extensively used in DF studies, in particular pure glucose, sucrose and starch mixtures (Wang and Wan, 2009). But renewable biohydrogen production requires the substrate or feedstock to come from renewable resources (Hawkes et al., 2007;Ren et al., 2011). Second generation biomass sources, such as waste biomass, are abundant and can thus support the supply of renewable substrates for DF (Guo et al., 2010;Kapdan and Kargi, 2006;Show et al., 2012). Besides biohydrogen and volatile fatty acids as valuable by-products, DF also offers biological treatment of the organic waste.

In more recent dark fermentative studies, complex substrates have been considered, such as the organic fraction of municipal solid waste (OFMSW) (Chen et al., 2012;[START_REF] Nissilä | Thermophilic hydrogen production from cellulose with rumen fluid enrichment cultures: Effects of different heat treatments[END_REF][START_REF] Tawfik | Continuous hydrogen production from co-digestion of municipal food waste and kitchen wastewater in mesophilic anaerobic baffled reactor[END_REF]Valdez-vazquez et al., 2005;[START_REF] Zhang | Enhanced biohydrogen production from cornstalk wastes with acidification pretreatment by mixed anaerobic cultures[END_REF], agricultural residues like lignocellulosic biomasses (e.g. rice straw, wheat straw and corn stalks), agro-industrial wastes like those from food processing industries (e.g. olive mill wastewater and cheese whey), effluents from livestock farms and aquatic plants (Kapdan and Kargi, 2006;Show et al., 2012). With the integration of DF within a biorefinery concept, the waste generated from biofuel production such as crude glycerol (Chookaew et al., 2014;[START_REF] Varrone | Enrichment of activated sludge for enhanced hydrogen production from crude glycerol[END_REF], de-oiled algal cake (Venkata Subhash and Venkata Mohan, 2014) or cotton seed cake [START_REF] Panagiotopoulos | Biodiesel and biohydrogen production from cottonseed cake in a biorefinery concept[END_REF] can be utilized as a substrate, while dark fermentative metabolites can be utilized in the production of microalgal biomass (Liu et al., 2013;[START_REF] Lo | Sequential dark-photo fermentation and autotrophic microalgal growth for high-yield and CO2-free biohydrogen production[END_REF]Turon et al., 2015) and biodiesel [START_REF] Fei | The effect of volatile fatty acids as a sole carbon source on lipid accumulation by Cryptococcus albidus for biodiesel production[END_REF], which in turn can serve as feedstock for DF processes. a mL H2/g COD, b mL H2/g VSremoved, c mL/g TS, d mL H2/g carbohydrate, e mL H2/g total sugar, f mL H2/g algal biomass

Based on their availability, novel low-cost substrate sources need to be explored and assessed for their biohydrogen potential (BHP). Table 2.1 presents the biohydrogen production potential of different organic biomasses by dark fermentative process. The fermentation pathways depend on the substrates and the microbial metabolism (Li and Fang, 2007a). It has been well established that the type of substrate influences the biohydrogen yields (Choi and Ahn, 2013;Guo et al., 2013). Monlau et al. (2012) and Guo et al. (2013) in their studies reported that the soluble and readily accessible sugars represent the main fraction of biomass that can be converted into hydrogen. However, the biohydrogen production also depends on a number of parameters such as inoculum type and enrichment methods, bioreactor design and operation conditions. The latter are covered in the sections below.

Agricultural residues

Agricultural residues, which mainly include lignocellulosic wastes, are an economically viable and renewable source of second generation carbon neutral biofuels [START_REF] Mtui | Recent advances in pretreatment of lignocellulosic wastes and production of value added products[END_REF].

These include plant biomass waste, which generally contains cellulose, hemicellulose and lignin formed by of photosynthesis. Agricultural residues are produced when economically valuable products of the crops are harvested and the residues such as straw, stover, peelings, cobs stalks, bagasse and others are left over [START_REF] Mtui | Recent advances in pretreatment of lignocellulosic wastes and production of value added products[END_REF]. The 2010 global annual production of agricultural residues was around 5.1 billion dry tonnes [START_REF] Eisentraut | Sustainable production of second-generation biofuels: potential and perspectives in major economies and developing countries[END_REF]. The waste generated by the agricultural, forestry and aquaculture sectors is increasing with the increasing population and thus the waste from this sector will be increasing further in the future. Guo et al. (Guo et al., 2010) have reported the potentials and challenges of agricultural wastes as substrates for biohydrogen production.

Examples of agricultural residues as a potential feedstock sources for DF processes and recent advancements in their application are discussed below.

Lignocellulosic waste

Rice straw is an example of a typical agricultural residue. It is the world's third largest agricultural residue, after maize and wheat, with a reported global yearly production of approximately 916 million tons in 2009 [START_REF] Mussoline | The Anaerobic Digestion of Rice Straw-A Review[END_REF]. Thus, the use of this abundant biomass as a feedstock in dark fermentative hydrogen production might hold future potential for feedstock supply. Similarly, wheat straw, barley straw, corn stalk, corncobs and others could be potential DF feedstock. The cellulose and hemicellulose part of these wastes can be hydrolyzed into carbohydrates which are further biologically converted to organic acids and biohydrogen in DF processes (Table 2.2). The composition of typical lignocellulosic crop residues in terms of cellulose, hemicellulose and lignin content is presented in Table 2.2 The main limitation in the utilization of these valuable resources lies in the complex structure of lignocellulosic materials: a cross-linking between polysaccharides (cellulose and hemicellulose) and lignin via ester and ether linkages, which decreases their biodegradability [START_REF] Hendriks | Pretreatments to enhance the digestibility of lignocellulosic biomass[END_REF][START_REF] Mtui | Recent advances in pretreatment of lignocellulosic wastes and production of value added products[END_REF][START_REF] Quéméneur | Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures[END_REF][START_REF] Zheng | Pretreatment of lignocellulosic biomass for enhanced biogas production[END_REF]. Therefore, prior to DF, these biomasses are often subjected to physical, chemical and biological pre-treatment to increase their digestibility [START_REF] Brodeur | Chemical and physicochemical pretreatment of lignocellulosic biomass: a review[END_REF][START_REF] Harmsen | Literature Review of Physical and Chemical Pretreatment Processes for Lignocellulosic Biomass[END_REF][START_REF] Hendriks | Pretreatments to enhance the digestibility of lignocellulosic biomass[END_REF][START_REF] Mtui | Recent advances in pretreatment of lignocellulosic wastes and production of value added products[END_REF][START_REF] Quéméneur | Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures[END_REF][START_REF] Saritha | Biological pretreatment of lignocellulosic substrates for enhanced delignification and enzymatic digestibility[END_REF]Taherzadeh and Karimi, 2008;[START_REF] Zheng | Pretreatment of lignocellulosic biomass for enhanced biogas production[END_REF].

Livestock waste (manure)

Livestock wastes include solid animal manure waste, fodder waste (which generally contains a lignocellulosic fraction) and wastewater, which include urine and feces. A large quantity of livestock manure comes from cattle feedlots, poultry and swine buildings, identified as pollution sources, which pose threats to the atmospheric and water environment (Cantrell et al., 2008). The current practices of management of livestock waste include its application in agricultural fields as well as biological stabilization or treatment such as composting and AD. The former management practice contributes in uncontrolled greenhouse gas emissions (mainly CH4) from land applications. Manure management practices can reduce direct and indirect greenhouse gas emissions by generating energy in the form of biogas from the manure prior to its land application (Cantrell et al., 2008;Wu et al., 2009;[START_REF] Xing | Biohydrogen production from dairy manures with acidification pretreatment by anaerobic fermentation[END_REF].

However, manure substrates need physical and chemical treatment to inhibit the methanogenic activity that consumes H2 (Cheong and Hansen, 2006;Wu et al., 2009).

Another, problem that might occur during the use of this feedstock type is the inhibition of the biohydrogen production by ammonia as its high nitrogen content might cause failure of the bioreactor: swine, poultry and dairy manure have a low C/N ratio (C/N ratio of swine manure: 12.8) (Yin et al., 2014) and high levels of ammoniacal nitrogen (cattle slurry: 1.04 -1.9 g/L and chicken manure 7.0 -12.8g/L) (Callaghan et al., 2002). [START_REF] Salerno | Inhibition of biohydrogen production by ammonia[END_REF] reported that hydrogen production is possible at high concentrations up to 7.8 g N/L in continuous flow systems if the microbial culture is initially acclimated to a lower ammonia concentration of 0.8 g N/L. total ammonia) However, the biohydrogen production decreases when the total ammonia concentration increases to above 2 g N/L (Cavinato et al., 2012). Also, high sulfate concentrations in swine manure can inhibit the biohydrogen production due to the presence of hydrogen consuming sulfate reducers (Guo et al., 2010).

Because of the high nitrogen content of animal manure, it can be used as a co-digestion substrate for nitrogen supplementation of other agricultural residues to maintain a suitable carbon to nitrogen ratio. Wu et al. (2009) reported a H2 yield between 1.18 and 1.63 mol H2/mol glucose in a fermentation of swine manure supplemented with glucose. [START_REF] Xing | Biohydrogen production from dairy manures with acidification pretreatment by anaerobic fermentation[END_REF] achieved an enhanced H2 yield of 31.5 mL/g Volatile Solids (VS) with acidification pretreated dairy manures while treating 70 g VS/L of substrate at operating pH 5.0.

Industrial waste

Agro-industries waste such as palm oil mill wastewater [START_REF] Mohammadi | Effects of different pretreatment methods on anaerobic mixed microflora for hydrogen production and COD reduction from palm oil mill effluent[END_REF][START_REF] O-Thong | Optimization of simultaneous thermophilic fermentative hydrogen production and COD reduction from palm oil mill effluent by Thermoanaerobacterium-rich sludge[END_REF][START_REF] O-Thong | Improvement of biohydrogen production and treatment efficiency on palm oil mill effluent with nutrient supplementation at thermophilic condition using an anaerobic sequencing batch reactor[END_REF]Tabatabaei et al., 2009) and olive mill wastewater (OMWW) [START_REF] Eroglu | Biological hydrogen production from olive mill wastewater with two-stage processes[END_REF]Ntaikou et al., 2009), tapioca industries and food industries such as brewery and dairy industries [START_REF] Castelló | Feasibility of biohydrogen production from cheese whey using a UASB reactor: Links between microbial community and reactor performance[END_REF][START_REF] Gadhe | Optimization of conditions for hydrogen production from complex dairy wastewater by anaerobic sludge using desirability function approach[END_REF]Kargi et al., 2012b;S Venkata Mohan et al., 2008) produce large quantities of carbohydrate rich non-toxic waste in the form of solid waste and wastewater. It can be potential substrates for dark fermentative biohydrogen production. [START_REF] Ren | Biohydrogen production from molasses by anaerobic fermentation with a pilot-scale bioreactor system[END_REF] demonstrated that waste molasses are an excellent substrate in a pilot scale system operated under mesophilic conditions (35 °C) where very good results were obtained in terms of H2 production rate (232 mL H2/L/h) and yield (26.13 mol/kgCODremoved). The production of large quantities of this type of waste biomass supports its utilization in up-scaled DF systems for continuous biohydrogen production. Similarly, cheese whey, a waste by-product generated by cheese manufacturing industries and characterized by high organic loads, comprising mainly carbohydrates (lactose), protein and lipids, is a very good potential substrate for biohydrogen production (Moreno et al., 2015;Teli et al., 2014;Venetsaneas et al., 2009).

Organic fraction of municipal waste

Organic fraction of municipal waste (OFMSW) generally constitutes food waste which contains a high biodegradable carbohydrates fraction with 85-95% volatile solids and 75-85% moisture content making it a good substrate for DF (Guo et al., 2010). Food waste present in municipal waste is mainly responsible for methane emissions and leachate production from landfills [START_REF] Jiang | Volatile fatty acids production from food waste: effects of pH, temperature, and organic loading rate[END_REF]. AD has been proposed as the most suitable treatment option for OFMSW or food waste with energy recovery and other environmental credentials (Esposito et al., 2012). Thus, food waste has been used extensively in DF experiments (Cavinato et al., 2012;Kim et al., 2008;[START_REF] Lee | Bioproduction of hydrogen from food waste by pilotscale combined hydrogen/methane fermentation[END_REF]Pan et al., 2008;Shin et al., 2004). Gioannis et al. (De Gioannis et al., 2013) have reviewed the studies of DF processes utilizing OFMW or food waste for dark fermentative biohydrogen production.

Large quantities of waste biosolids or sludge are generated from municipal wastewater treatment plants which generally contain carbohydrates or polysaccharides and proteins [START_REF] Wang | Producing hydrogen from wastewater sludge by Clostridium bifermentans[END_REF]. Several researchers have used the available carbohydrates present in these biosolids in fermentative hydrogen production [START_REF] Cai | Enhanced biohydrogen production from sewage sludge with alkaline pretreatment[END_REF]Kim et al., 2004).

However, the sludge needs pre-treatment, such as ultrasonication, acidification, sterilization, freezing-thawing or alkaline pre-treatment, to facilitate the fermentative process [START_REF] Cai | Enhanced biohydrogen production from sewage sludge with alkaline pretreatment[END_REF][START_REF] Wang | Producing hydrogen from wastewater sludge by Clostridium bifermentans[END_REF]. Besides, Kim et al. (2004) demonstrated the usefulness of sewage sludge as co-substrate in the DF of food waste.

Factors affecting DF pathways and H2 yield

DF via mixed cultures is a complex system where environmental factors and bioreactor operation conditions such as temperature, pH and H2 partial pressure regulate metabolic pathways of hydrogen producing microorganisms (Guo et al., 2010;Li and Fang, 2007a;[START_REF] Liu | Hydrogen and methane production from household solid waste in the two-stage fermentation process[END_REF]Wang and Wan, 2009). In addition, substrate types and their pretreatment methods, bioreactors configurations, inoculum sources and enrichments also influence the biohydrogen production. Three categories of parameters that influence the DF pathways, and thus the yield of biohydrogen, can be distinguished (Figure 2.4). These parameters are reviewed below and compared in relation to H2 yield and production rate.

Inoculum and enrichment methods

The hydrogen producing seed inoculum or culture is very important for the startup of the hydrogen production process. Several studies using pure cultures have been done using a range of substrates (Table 2.3). Various species of Clostridia and Enterobacter are widely used in pure cultures (Table 2.3). Lee et al. (2011) and Elsharnouby et al. [START_REF] Elsharnouby | A critical literature review on biohydrogen production by pure cultures[END_REF] have reviewed the studies of DF performed with pure cultures. H2 synthesizing bacteria exist commonly in environments such as soil, wastewater sludge and compost. All these materials can thus be used as an inoculum for fermentative H2 production (Li and Fang, 2007a). Indeed, cow dung, anaerobic sludge, municipal solid waste, soil and compost are some of the common sources of mixed cultures. A mixed culture of hydrogen producers is generally preferred over a pure culture due to its practicability for environmental engineering applications, economic benefits in operation (as it can economize asepsis costs), easiness in control based on differential kinetics of microbial subgroups and broader feedstock choice (Li and Fang, 2007a;Valdez-vazquez et al., 2005;Wang and Wan, 2009). However, enrichment of mixed cultures becomes necessary to enhance the biohydrogen production on the one hand and inhibit hydrogen consumers such as methanogens and homoacetogens, often present in these mixed inocula (Wang and Wan, 2009), on the other hand. Table 2.4 summarizes the common pretreatment measures adopted for enrichment of hydrogen producers.

Pre-treatment of the inoculum to obtain an enrichment of hydrogen producers often relies on the spore forming characteristics of H2 producers such as Clostridium, which are ubiquitous in anaerobic sludge and sediments (Faloye et al., 2013;Li and Fang, 2007a;S Venkata Mohan et al., 2008;Wang and Wan, 2008). These organisms have a better chance to survive the harsh conditions during the pre-treatment of the inoculum than the non-spore forming bacteria such as methanogens, as the spores can germinate again under favorable conditions (Li and Fang, 2007a;Wong et al., 2014). Heat treatment of mixed cultures for the enrichment of H2 producers is a simple, inexpensive and effective method (Li and Fang, 2007a;Wang and Wan, 2009). However, the effect of heat treatment might be different depending on the inoculum source such as activated sludge or anaerobic sludge (Wang and Wan, 2009). Some studies (O-Thong et al., 2009;Zhu and Beland, 2006) reported a lower hydrogen yield by a heat shock treated seed inoculum than obtained by other pre-treatment methods. This could be due to the inhibition of other nonspore forming hydrogen producing bacteria which might destabilize the main hydrogen production pathways. Similarly, acid or base treatment is based on the notion that the activity of methanogens drops sharply at a pH below 6.3 or above 7.8 (Li and Fang, 2007a), while the activity of Clostridia sp. and other hydrogen producers is not affected by an acidic pH (below pH 6).

Other pre-treatment methods such as chemical pretreatment and aeration are directed towards the selective inhibition of methanogens present in anaerobic sludge, which are very sensitive to changes in environmental conditions. Besides being strict anaerobes, methanogens are sensitive to many chemicals (Li and Fang, 2007a). Thus, oxygen can inhibit their activity during aeration (Wang and Wan, 2008;Zhu and Beland, 2006). Wang and Wan (2008) aerated the inoculum sludge with air for 24 hours to inhibit the activity of methanogens. Likewise, chemical inhibitors like sodium 2-bromoethasulfonic acid (BESA), iodopropane, chloroform and acetylene are used to inhibit methanogens (Li and Fang, 2007a;O-Thong et al., 2009;Venkata Mohan et al., 2008;Wang et al., 2011;Zhu and Beland, 2006). Thus, selective inhibitors like chloroform or BESA selectively inhibit the activity of H2 consumer methanogens. In methanogens, BESA functions by inhibiting the activity of co-enzyme M reductase complex, which is a key co-enzyme of methanogenesis (Venkata Mohan et al., 2008;Zhu and Beland, 2006). The effect of inoculum enrichment methods on H2 production is different based on the source of inoculum (Table 2.5). However, in order to select an inoculum pre-treatment method for scaled-up systems, several parameters needs to be considered, such as operational costs, feasibility or complexity of the methods, time for the enrichment of the hydrogen producing seed, use of the DF residues in the post treatment processes. Table 2.6 gives a simple assessment of the commonly applied inoculum pre-treatment methods based on the authors' information from the literature. The selection of a chemical treatment method such as using BESA inhibits the methanogens, which will give problems when the DF residues are to be used in AD. In addition, BESA is not environmental friendly and expensive to use a large industrial scale (Li and Fang, 2007a).

Likewise, heat shock treatment requires large energy inputs, which makes it less attractive for large-scale applications. Acid and shock load pre-treatment can be applied at large scale to select the hydrogen producing inocula without net energy concerns.

There have been some dark fermentative studies done without the addition of seed inoculum, utilizing the microorganisms present in the waste itself [START_REF] Favaro | Effects of inoculum and indigenous microflora on hydrogen production from the organic fraction of municipal solid waste[END_REF]Kim et al., 2009). The fermentative hydrogen production took longer than in the tests with inoculum supply. Nonetheless, inoculum pre-treatment is important in batch tests or at process start-up. A high rate hydrogen producing microbial community can be develop in the fermentative bioreactors when applying appropriate reactor operating conditions [START_REF] Castelló | Feasibility of biohydrogen production from cheese whey using a UASB reactor: Links between microbial community and reactor performance[END_REF]Fang and Liu, 2002;Lee et al., 2008;[START_REF] Zahedi | Dark fermentation from real solid waste. Evolution of microbial community[END_REF].

Design and operation of bioreactors

The process design for dark fermentation depends mostly on substrates which limits the operational conditions of bioreactors such as culture temperature (mesophilic or thermophilic), reactor configurations (reactor types, wet, semi-dry or dry conditions) and feeding modes (mono substrate or co-substrates) (Motte et al., 2013). Weiland [START_REF] Weiland | Biomass Digestion in Agriculture: A Successful Pathway for the Energy Production and Waste Treatment in Germany[END_REF] reported the several types of bioreactors used for the conversion of agricultural biomass to energy through upscaled AD systems. Although these bioreactors are designed for biomethanation by AD, these can be used for biohydrogen production after modification of some operational parameters (Guo et al., 2010).

Bioreactor configuration

Different DF bioreactor configurations have been used in laboratory studies for a wide range of substrates (see Tables 2.2 (Motte et al., 2014(Motte et al., , 2013;;Robledo-Narváez et al., 2013;Valdez-Vazquez and Poggi-Varaldo, 2009) has focused on the application of high solids processes such as semi-dry (10-20 % TS) and dry (>20 % TS) DF processes for biohydrogen production, as the interests in the conversion of second generation lignocellulosic biomass (mostly agro-industrial residues) is growing.

Studies have correlated the biohydrogen production with the size of the microbial population and therefore different cell retention strategies have been investigated [START_REF] Show | Bioreactor and process design for biohydrogen production[END_REF][START_REF] Show | Critical assessment of anaerobic processes for continuous biohydrogen production from organic wastewater[END_REF][START_REF] Zhang | Biohydrogen production with anaerobic fluidized bed reactors-A comparison of biofilm-based and granule-based systems[END_REF]. The latter include sludge granulation and biofilm systems to increase the bacterial concentration in the reactor. The results of these studies showed that the volumetric hydrogen production rate of a bioreactor depends on the ability to maintain a high microbial density. [START_REF] Gavala | Biological hydrogen production in suspended and attached growth anaerobic reactor systems[END_REF] showed higher hydrogen production rates in a UASB (which has a granular biomass retention) than in a CSTR at low retention times (19.05 and 8.42 mmol H2/h/l, respectively at 2 h HRT), while the CSTR reactor gave higher hydrogen yields (mmol H2/mol glucose) at all HRTs tested.

This suggests a compromise should be sought between technical efficiency (based on H2 yields) and economic efficiency (based on H2 production rate), when one of these two systems is selected. [START_REF] Show | Critical assessment of anaerobic processes for continuous biohydrogen production from organic wastewater[END_REF] compared the performance of a CSTR and an AFBR for biohydrogen production using different biomass growth strategies with glucose as the substrate. The different bioreactor configurations used in their research were: suspended sludge CSTR system, granular sludge CSTR system, granular sludge AFBR system and biofilm AFBR system. The maximum H2 yield of their suspended sludge CSTR system, granule reactor and biofilm amounted to, respectively, 1.92 mol H2/mol glucose at a HRT of 6-12 hours, 1.83±0.09 mol H2/mol glucose at a HRT of 0.5 hours and 1.81 ± 0.08 mol H2/mol glucose at a HRT of 0.5 hours.

Besides the specific advantages of these different bioreactor systems, the major drawbacks are the washout of hydrogen-producing bacteria at short HRT in CSTR systems, low conversion rates in granular reactor systems and rapid biofilm development leading to fragmentation and separation from the supporting media in biofilm systems [START_REF] Show | Critical assessment of anaerobic processes for continuous biohydrogen production from organic wastewater[END_REF]. In another study by [START_REF] Zhang | Biohydrogen production with anaerobic fluidized bed reactors-A comparison of biofilm-based and granule-based systems[END_REF], their biohydrogen production potential of biofilm based and granule based reactors were compared. They concluded that the granule based system was advantageous as it gave better results in terms of biomass retention without being subjected to washout of the biomass support carriers.

The incompatibility of the use of high organic loading rates and rapid microbial growth in biofilm systems makes them thus less attractive than granular systems. Show et al.

(2010) recommended the column-shaped granular reactor for fermentative biohydrogen production from wastewater though the system is not suitable for digestion of substrates with a high solids content or for a longer retention time in which anaerobic granules may disaggregate. High rate bioreactors are necessary to convert complex organic biomass like OFMSW and agricultural waste.

Hydraulic retention time (HRT)

The HRT can affect substrate hydrolysis and thus the production of intermediates and products, thus affecting fermentative H2 production. Besides hydrolysis, the HRT can also be used as control parameter of the methanogenic activity. Some studies have demonstrated the effect of the HRT on the biohydrogen production in DF processes [START_REF] Dos Reis | Effect of upflow velocity and hydraulic retention time in anaerobic fluidized-bed reactors used for hydrogen production[END_REF]Kim et al., 2006;Liu et al., 2008;[START_REF] Pakarinen | The effect of organic loading rate and retention time on hydrogen production from a methanogenic CSTR[END_REF]. The different growth rates of hydrogen producers and consumers make it possible to use the HRT as a controlling parameter to inhibit the activity of H2 consumers in the DF. It has been reported that low HRTs favor hydrogen production as the methanogens are washed out, and hydrogen production increases as the HRT decreases (Kim et al., 2006;Liu et al., 2008;Oh et al., 2004;[START_REF] Pakarinen | The effect of organic loading rate and retention time on hydrogen production from a methanogenic CSTR[END_REF]. However, the optimum HRT for biohydrogen production in DF depends on the type of substrates used as the hydrolysis rate depends on the biodegradability of the substrates (Tables 2.2, 2.6 and 2.7).

However, the HRT alone is not sufficient to fully suppress the methanogenic activity ( Liu et al., 2008). Liu et al. (2008) investigated the effects of pH and HRT on hydrogen production using household solid waste as a substrate in a hyperthermophilic (70 °C)

CSTR. The effect of the HRT (1, 2, 3, 4 and 6 days) at a constant pH of 7 and the effect of pH (5, 5.5, 6, 6.5 and 7) at a constant HRT of 3 days was investigated. The results of the experiments at different HRTs and constant pH 7 showed unstable H2 production with subsequent methanogenic activities at the end. However, a combination of pH 5.5 and HRT of 3 days gave the optimum biohydrogen production conditions.

pH and temperature

The operational pH and temperature are the most crucial parameters that determine the optimum metabolic pathways of hydrogen synthesis as well as the inhibition of the hydrogen consuming processes which may occur simultaneously [START_REF] Hu | Influence of particle size and pH on anaerobic degradation of cellulose by ruminal microbes[END_REF]Khanal et al., 2003). An acidic operational pH (below 6) mainly inhibits the methanogenic activity under both mesophilic and thermophilic conditions, but the inhibition of hydrogen consuming homoacetogenic activity can only be achieved under thermophilic conditions at the initial pH of 5.5 [START_REF] Luo | Long-term effect of inoculum pretreatment on fermentative hydrogen production by repeated batch cultivations: homoacetogenesis and methanogenesis as competitors to hydrogen production[END_REF]. Thus, the control of the process pH and temperature plays an important role in achieving high biohydrogen conversion rates by minimizing the activity of the hydrogen consumers.

The pH is one of the key parameters that can influence the metabolic pathways as it may directly affect the hydrogenase activity, an iron containing enzyme which plays a major role in DF [START_REF] Dabrock | Parameters Affecting Solvent Production by Clostridium pasteurianum[END_REF]). An acidic pH affects the activity of the hydrogenase enzyme while it is one of the important parameters for the inhibition of methanogenic activities in a mixed culture system (Khanal et al., 2003;Li and Fang, 2007a).

The optimum pH range for biohydrogen production varies from pH 4.5 (Khanal et al., 2003) to 9 [START_REF] Lee | Effect of pH on microbial hydrogen fermentation[END_REF] in DF of sucrose. Table 2.8 provides optimum operating pH ranges in different studies. The possible explanations for the disagreements in optimum pH in the various studies can be differences in inoculum sources, inoculum enrichment methods, substrate types and applied OLR (Wang and Wan, 2009).

The operational pH influences the metabolic by-products and biohydrogen yields. In most of the studies, acetate and butyrate are the major end products of favorable hydrogen synthesis (equations 2. 1, 2 and 7). Table 2.8 shows that a neutral operational pH favors the acetate pathways, while acidic pH conditions favor the butyrate pathways. However, Khanal et al. (2003) concluded the independence of the acetate and butyrate levels from different initial pH ranges studied (4.5-7.5). Similarly, Luo et al. (2010b) reported butyrate as a major VFA in the DF of cassava stillage in both BHP tests carried at the initial pH 5 and 7. [START_REF] Luo | Long-term effect of inoculum pretreatment on fermentative hydrogen production by repeated batch cultivations: homoacetogenesis and methanogenesis as competitors to hydrogen production[END_REF] found acetate as a major metabolic product when the operational pH was 7, while butyrate dominated at an initial pH 5.5 in the BHP tests carried under mesophilic (37 °C) conditions using an acid pre-treated inoculum. [START_REF] Luo | Long-term effect of inoculum pretreatment on fermentative hydrogen production by repeated batch cultivations: homoacetogenesis and methanogenesis as competitors to hydrogen production[END_REF] further reported the inhibition of homoacetogenesis can be achieved at pH 5.5

and thermophilic temperatures (55 °C).In a recent study of the DF of cheese whey from mozzarella production at different pH ranges (5.5-7.7) and a temperature of 39 °C, De Gioannis et al. ( 2014) reported pH 6 as the optimal pH and acetate levels were higher in all the tests except at pH 6.5 where butyrate and propionate levels exceeded those of acetate.

A lower pH (≤ 4.5) favors the solvent production (Van Ginkel and Logan, 2005). In the DF of glucose by Clostridium pasteurianum, a pH below 5 favors the butanol and acetone production [START_REF] Dabrock | Parameters Affecting Solvent Production by Clostridium pasteurianum[END_REF]. Selection of the operational pH is also substrate type and OLR dependent, which determines the VFA concentrations and thus the pH of the solution. The optimum temperature for DF processes varies with the substrates type and operational pH (Table 2.8). The optimum pH for organic food waste varies from 4.5 to 7, for lignocellulosic waste it varies from 6.5-7, whereas a neutral pH is optimal for animal manure (Guo et al., 2010). However, Tang et al. (2008) reported an optimum pH of 5.5 at 45 °C for the DF of cattle wastewater. Thus, it is important to determine the optimum pH conditions for DF of a selected substrate type at a particular loading rate and operational temperature.

A range of operational temperatures, i.e. mesophilic (35°C), thermophilic (55°C) and extreme thermophilic (>65°C) has been studied to determine its effect on the biohydrogen production [START_REF] Kongjan | Extreme thermophilic biohydrogen production from wheat straw hydrolysate using mixed culture fermentation: Effect of reactor configuration[END_REF]Shin et al., 2004;Valdez-vazquez et al., 2005). These studies have shown that the temperature can affect the metabolic pathways, thus shifting the composition of the by-products of DF (Table 2.8). Valdez-vazquez et al.

(2005) reported higher H2 yields for thermophilic fermentation than in the mesophilic temperature range. Also acetic acid was a dominant by-product in thermophilic digestion, whereas butyrate was in formed in a higher proportion during mesophilic digestion.

Similarly, results of the extreme thermophilic (70 °C) DF of household organic waste also showed acetic acid as the dominant by-product in DF tests conducted at pH 7 (Liu et al., 2008). In contrast, Shin et al. (2004) showed acetate as major end-product at mesophilic culture while butyrate levels and hydrogen production was higher by the thermophilic culture, obtained in DF of food waste carried at pH 5.5. In another study, Wang and Wan (2011) found the maximum substrate degradation efficiency, maximum H2 yield and production rate at 37.8 °C in DF of glucose. These studies suggest temperature influences biochemical pathways, although other factors such as culture pH, substrate types and loading rates are equally important.

The H2 yields depend on temperature as it affects the hydrolysis rate (Kim et al., 2006;Shin et al., 2004;Valdez-vazquez et al., 2005). Biomass such as agricultural residues require a high temperature to achieve a higher H2 yield because a better hydrolysis of lignocellulosic compounds is needed (Guo et al., 2010). [START_REF] Kongjan | Extreme thermophilic biohydrogen production from wheat straw hydrolysate using mixed culture fermentation: Effect of reactor configuration[END_REF] demonstrated biohydrogen production from extreme thermophilic DF of wheat straw hydrolysate. Similarly, thermophilic temperatures are favored in the DF of food waste (Shin et al., 2004). In contrast, easily biodegradable substrates prefer mesophilic temperatures for an optimal H2 yield. The difference between the optimum operational temperatures is due to the difference in the fraction of easily biodegradable compounds present in the feed substrate and the different inocula used. Table 2.8 reports ranges of optimum temperatures, which vary depending on the type of substrate and inoculum used.

There are some techno-economic studies done, which compare the mesophilic and thermophilic operation of DF processes. A thermophilic process seems to be more economical because of its higher yield and lower requirement of feedstock in comparison to mesophilic DF processes (Foglia et al., 2006). Foglia et al. (2006) reported a better economic performance for thermophilic DF in comparison to a two-step mesophilic process, converting sugars to hydrogen, CO2 and organic acids followed by a photoheterotrophic fermentation.

H2 partial pressure

The partial pressure of hydrogen inside a biohydrogen reactor can influence the dark fermentative biohydrogen production as a lower partial pressure in the head space of the reactors facilitates the mass transfer of hydrogen from the liquid to gas phase [START_REF] Bastidas-Oyanedel | Gas controlled hydrogen fermentation[END_REF][START_REF] Mandal | Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae[END_REF]. During the fermentation process, the hydrogenase is involved in reversibly oxidizing and reducing ferredoxin. If the hydrogen concentration in the liquid phase increases, the oxidation of ferredoxin becomes less favorable and the reduction of ferredoxin takes place (Chong et al., 2009), thus reducing the H2 production. [START_REF] Lee | Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy[END_REF] studied the effect of the reduced partial pressure on the hydrogen production in a CSTR reactor. Reduction in the partial pressure during the DF could lead to an improvement in H2 production. At a HRT of 6 h, they found an optimum hydrogen yield and hydrogen production efficiency of 4.50 mol H2/mol sucrose and 56.2%

respectively. Similarly, the reduced pressure of 380 mm Hg gave a higher yield than the partial pressure of 760 mm Hg in another study done by [START_REF] Mandal | Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae[END_REF].

In the AD process, the H2 and CO2 partial pressure is reduced by methanogens by their conversion into CH4. [START_REF] Jung | Bioreactor design for continuous dark fermentative hydrogen production[END_REF] reported strategies to remove dissolved H2 from the mixed liquor, including avoiding supersaturation by strong mixing, sparging with N2 and CO2 and application of a H2-permeable membrane to withdraw dissolved H2 from the mixed liquor. Similarly, the partial pressure of H2 could be reduced directly by decreasing the operating pressure in the reactor using a vacuum pump [START_REF] Lee | Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy[END_REF]. [START_REF] Mandal | Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae[END_REF] reduced the partial pressure of H2 in a methanogenic reactor by adjusting the saline level of the gas collector using a peristaltic pump. However, the use of vacuum pumps increases the cost of the process, while the sparging with N2 and CO2 might render the recovery of H2 difficult due to the dilution of the H2 stream. An effective way to reduce the H2 partial pressure would be to continuously collect the produced gas phase from the reactor. a mL H2/g VSremoved, b mL H2/g COD, c mL H2/g CODconsumed, d mL/g TS

Substrate pre-treatment for enhanced H2 yield

Fermentative biohydrogen production from lignocellulosic substrates is limited by biological hydrolysis (Monlau et al., 2013b). The complex organic substrates cited earlier, such as lignocellulosic biomasses, demand physical, chemical, biological or a combination of these pre-treatments to enhance the degradation process, system performance and biogas production [START_REF] Hendriks | Pretreatments to enhance the digestibility of lignocellulosic biomass[END_REF][START_REF] Mussoline | The Anaerobic Digestion of Rice Straw-A Review[END_REF]Taherzadeh and Karimi, 2008;[START_REF] Zheng | Pretreatment of lignocellulosic biomass for enhanced biogas production[END_REF]. These pre-treatment methods reduce the crystallinity of the cellulose and increase the surface area of the materials to improve the separation of the lignin and hemicellulose fractions [START_REF] Saratale | Outlook of biohydrogen production from lignocellulosic feedstock using dark fermentationa review[END_REF]. There have been some studies on the effect of the pre-treatment on fermentative biohydrogen production [START_REF] Chairattanamanokorn | Production of biohydrogen from hydrolyzed bagasse with thermally preheated sludge[END_REF][START_REF] Kongjan | Extreme thermophilic biohydrogen production from wheat straw hydrolysate using mixed culture fermentation: Effect of reactor configuration[END_REF]Pan et al., 2010;[START_REF] Zhu | Pretreatment by microwave/alkali of rice straw and its enzymic hydrolysis[END_REF]. These pre-treatment methods have in most cases a positive influence on the H2 yield, as the biohydrogen production depends on the soluble fraction of sugars or carbohydrates.

Physical pre-treatment methods which generally include mechanical comminution (chopping, grinding, milling), irradiation with gamma-rays, electro-beam or microwaves, hydrothermal treatment, high pressure steaming or pyrolysis are effective in breaking the crystallinity, increasing the accessible surface area and decreasing the degree of polymerization (Taherzadeh and Karimi, 2008). Chemical methods such as ozonolysis, acid or alkaline hydrolysis, solvent extraction, explosion with steam ammonia fiber or CO2 are effective in increasing the surface area, delignification and also decreasing the crystallinity and rendering the partial or complete hydrolysis of hemicelluloses. These physical and chemical treatment methods can be promising for industrial applications as they are rapid. However, these methods demand energy and chemical inputs. Moreover, lignocellulosic substrates can also be biologically treated with fungi and actinomycetes which provide delignification and partial hydrolysis of cellulose, while some enzymes (hemicellulase and cellulase) can aid in the hydrolysis and degradation of the lignocellulosic materials [START_REF] Mussoline | The Anaerobic Digestion of Rice Straw-A Review[END_REF].

The physical pre-treatment, especially the reduction of substrate particle size, has an effect on the biogas yield and process kinetics [START_REF] Esposito | Mathematical modelling of disintegration-limited co-digestion of OFMSW and sewage sludge[END_REF][START_REF] Esposito | Model calibration and validation for OFMSW and sewage sludge co-digestion reactors[END_REF][START_REF] Esposito | Model calibration and validation for OFMSW and sewage sludge co-digestion reactors[END_REF]. Chen et al. (2012) investigated the effects of the rice straw particle size and concentration on cumulative dark fermentative biohydrogen production.

They used a meshed rice straw concentration of 30 g TS/L with five particle sizes (<0. 297, 0.297-0.58, 0.58-1.19, 1.19-10 and >10 mm) as the substrate at an initial cultivation pH 6.5 and temperature of 55 °C. The results of the study showed that rice straw of a particle size <0.297 mm gave the highest cumulative H2 production (191 mL H2/L) with a H2 yield of 6.4 mL/g TS. The substrate with a larger particle size had an extended lag phase and lower hydrogen production. This can be explained by the fact that decreasing the particle size increases the substrate availability for microbial hydrolysis and fermentation. [START_REF] Kongjan | Extreme thermophilic biohydrogen production from wheat straw hydrolysate using mixed culture fermentation: Effect of reactor configuration[END_REF] pretreated wheat straw at 180 °C for 15 min to obtain a hydrolysate which mostly contained hemicellulose leaving the cellulose and lignin in solid form. The hydrolysate was used as the substrate for fermentative hydrogen production. Similarly, [START_REF] Zhang | Enhanced biohydrogen production from cornstalk wastes with acidification pretreatment by mixed anaerobic cultures[END_REF] reported the use of acid pretreated corn stalks for fermentative biohydrogen production. The biohydrogen yield from acid pretreated corn stalks was higher than of that of untreated waste. However, mostly physical pretreatment methods are applied in combination with chemical or biological pretreatment methods to obtain better and rapid hydrolysis of substrates (Table 2.9).

The effect of pre-treatment methods for different lignocellulosic substrates have a diverse effect on the hydrolysis of soluble sugars and release of inhibitory products [START_REF] Jönsson | Bioconversion of lignocellulose: inhibitors and detoxification[END_REF]Monlau et al., 2013a;[START_REF] Palmqvist | Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition[END_REF][START_REF] Parawira | Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review[END_REF]. This needs to be further investigated for the selection of suitable pretreatment methods. These studies have shown that the pre-treatment methods can enhance the system performance enhancing the biogas production. However, the selection of a pre-treatment process should be based on effectiveness, energy balance, economic feasibility and environmental sustainability (Ariunbaatar et al., 2014).

In addition, some studies have reported that during the pre-treatment of lignocellulosic biomass, various undesirable compounds are released which exert inhibitory effects on microorganisms [START_REF] Jönsson | Bioconversion of lignocellulose: inhibitors and detoxification[END_REF][START_REF] Palmqvist | Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition[END_REF][START_REF] Parawira | Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review[END_REF][START_REF] Quéméneur | Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures[END_REF]. The most commonly reported inhibiting substances which are released during the pre-treatment processes are furfural, hydroxylfurfural and phenolic substances. [START_REF] Quéméneur | Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures[END_REF] and Monlau et al. (2013c) investigated the inhibition and control of these inhibitors on the biohydrogen production.

Thus, the selection of pre-treatment methods for lignocellulosic substrates should also consider these aspects. a mL/g TS, b mL H2/g sugars, c mol H2/mol glucose, d mL H2/g COD, e mL H2/g dry grass

Addition of nutrients and trace elements

Microorganisms in fermentation processes require nutrients for bacterial activity and growth. Thus, nutrients such as nitrogen, phosphate, metal ions and other micronutrients are needed in fermentation processes for enzymatic activities and biomass growth, which affects the H2 production. Biomass rich in carbohydrates such as wheat wastes and palm oil effluents may be deficient in nutrients (such as nitrogen, phosphorous) or minerals (such as trace metals). Therefore, nutrients or micro nutrients must be provided as supplement for optimum microbial activities for biohydrogen conversion from carbohydrate rich substrates (Argun et al., 2008b;Lin andLay, 2005, 2004).

Nitrogen and phosphorous

Nitrogen has great significance for hydrogen producers, as it is an important component of proteins, nucleic acids and enzymes. Similarly, besides being an important nutrient, phosphate also serves in buffering the biochemical reactions (Wang and Wan, 2009). In the thermophilic DF of palm oil mill effluents (POME), [START_REF] O-Thong | Optimization of simultaneous thermophilic fermentative hydrogen production and COD reduction from palm oil mill effluent by Thermoanaerobacterium-rich sludge[END_REF][START_REF] O-Thong | Improvement of biohydrogen production and treatment efficiency on palm oil mill effluent with nutrient supplementation at thermophilic condition using an anaerobic sequencing batch reactor[END_REF] showed that supplementing iron (257 mg Fe 2+ /L), adjusting the C/N ratio from 95 to 74 (using peptone as nitrogen source) and the C/P ratio from 650 to 559 (using However, there are some disagreements in the carbon to nitrogen and phosphorous ratios.

Lin and Lay, ( 2004) achieved a 500% and 80% increased hydrogen yield and hydrogen production rate at a C/N ratio of 47 compared with the blank. Similarly, O-Thong et al.

( 2008,2007) attained an optimum hydrogen production and COD removal at a C/N ratio of 74 and a C/P ratio of 559. Several studies have used the integration of co-substrates as a strategy to maintain an appropriate C/N ratio, examples include the use of swine manure as a source of nitrogen in co-fermentation with vegetable waste (Tenca et al., 2011) and use of cassava starch in co-fermentation with the microalgae Chlorella pyrenoidosa [START_REF] Xia | Enhancement of energy production efficiency from mixed biomass of Chlorella pyrenoidosa and cassava starch through combined hydrogen fermentation and methanogenesis[END_REF].

Metal ions and micronutrients

Higher concentrations of metal ions exert inhibitory effects on the hydrogen producers (Li andFang, 2007a, 2007b;[START_REF] Lin | Heavy metal effects on fermentative hydrogen production using natural mixed microflora[END_REF]. However, trace amounts of some metal ions enhance the reactor performance [START_REF] Karadag | Enhancement of anaerobic hydrogen production by iron and nickel[END_REF]. [START_REF] Karadag | Enhancement of anaerobic hydrogen production by iron and nickel[END_REF] found that iron and nickel improved the reactor performance and H2 production was enhanced by 71%. O-Thong et al. [START_REF] O-Thong | Optimization of simultaneous thermophilic fermentative hydrogen production and COD reduction from palm oil mill effluent by Thermoanaerobacterium-rich sludge[END_REF] obtained the optimal hydrogen production when the substrate contained 257 mg Fe 2+ /L during the thermophilic DF of POME.

Inhibition due to heavy metals

Toxic heavy metals such as cadmium (Cd), chromium (Cr), zinc (Zn), copper (Cu), nickel (Ni), and lead (Pb) which may be present in industrial and municipal solid waste may lead to upset or ultimately failure of anaerobic reactors (Li and Fang, 2007a). [START_REF] Altaş | Inhibitory effect of heavy metals on methane-producing anaerobic granular sludge[END_REF] studied the inhibitory effect of heavy metals on methane producing anaerobic granular sludge. The order of toxicity for the individual heavy metals in decreasing order was: Zn (most toxic, 7.5 mg/L) >Cr (27 mg/L) >Ni (35 mg/L) ≈ Cd (least toxic, 36 mg/L). [START_REF] Lin | Heavy metal effects on fermentative hydrogen production using natural mixed microflora[END_REF] showed the relative toxicity of the heavy metals to fermentative hydrogen production was in the order of Zn>Cu>Cr. The maximum concentration of these metals that reduced the hydrogen producing activity by 50% was 4.5 mg Zn/L, 6.5 mg Cu/L and 60 mg Cr/L [START_REF] Lin | Heavy metal effects on fermentative hydrogen production using natural mixed microflora[END_REF]. However, Li and Fang (2007b) reported the relative toxicity to H2 production in the following order: Cu (most toxic)>>Ni~ Zn > Cr > Cd > Pb (least toxic). The bioactivity of the sludge was reduced to 50% of the control at 30 mg Cu/L, 1600 mg Ni and Zn/L, 3000 mg Cr/L, 3500 mg Cd/L and >5000 mg Pb/L.

Use of by-products

The low process yield and the incomplete conversion of organic biomass are two major bottlenecks for commercial dark fermentative biohydrogen production (Gómez et al., 2011;Ren et al., 2011). As overviewed in Section 4, dark fermentative biohydrogen can be enhanced by suitable substrate selection, inoculum enrichment strategies, and optimal operation of bioreactor or substrate pre-treatment. However, a single DF system cannot achieve beyond the highest yield of 4 moles H2 per mole hexose, as DF has a maximum yield of 33% (on sugars) (Gómez et al., 2011). Besides, DF residues mainly contain volatile fatty acids, major by-products of the DF process, which need to be utilized to achieve complete conversion of the organic biomass. Dual systems are integrated by the conversion of carbohydrates to organic acids in the first stage (DF) and the conversion of by-products in the second stage, either to H2 (photofermentation, bioelectrochemical cells) or CH4 (AD). Also, AD can be considered as the final stabilization stage to stabilize the residues of DF, photofermentation and bioelectrochemical cells. A number of studies have been carried out combining DF either with photofermentation [START_REF] Argun | Photo-fermentative hydrogen gas production from dark fermentation effluent of ground wheat solution: Effects of light source and light intensity[END_REF][START_REF] Chen | Biohydrogen production using sequential two-stage dark and photo fermentation processes[END_REF][START_REF] Hema | Production of Clean Fuel from Waste Biomass using combined dark and photo fermentation[END_REF][START_REF] Nath | Hydrogen production by Rhodobacter sphaeroides strain O.U.001 using spent media of Enterobacter cloacae strain DM11[END_REF][START_REF] Redwood | A two-stage, two-organism process for biohydrogen from glucose[END_REF][START_REF] Su | Hydrogen production from water hyacinth through dark-and photo-fermentation[END_REF]Su et al., , 2009aSu et al., , 2009b) ) or/and using bioelectrochemically assisted microbial reactors [START_REF] Cheng | Sustainable and efficient biohydrogen production via electrohydrogenesis[END_REF][START_REF] Jeremiasse | Microbial electrolysis cell with a microbial biocathode[END_REF][START_REF] Liu | Electrochemically assisted microbial production of hydrogen from acetate[END_REF][START_REF] Liu | Hydrogen generation in microbial electrolysis cell feeding with fermentation liquid of waste activated sludge[END_REF]Wang et al., 2011) for improving the biohydrogen yield or with the AD process for improving the economic viability [START_REF] Cavinato | Bio-hythane production by thermophilic two-phase anaerobic digestion of organic fraction of municipal solid waste: Preliminary results[END_REF]Lin et al., 2012;[START_REF] Liu | Hydrogen and methane production from household solid waste in the two-stage fermentation process[END_REF]Liu et al., 2013;Ruggeri et al., 2010;Venetsaneas et al., 2009).

Light dependent fermentative processes can be a good option for a second stage H2 production, because of their higher substrate conversion efficiency, and being less energy intensive and environmental friendly (Chen et al., 2010). On the other hand, bioelectrochemically assisted microbial fuel cells are also an option to treat the effluents from DF and increase the H2 yield [START_REF] Logan | Microbial Electrolysis Cells for High Yield Hydrogen Gas Production from Organic Matter[END_REF]. Likewise, the economic viability of the DF process can be enhanced by AD as a final step.

Photofermentation

Under anaerobic conditions, purple non sulfur photosynthetic bacteria carry out anaerobic photosynthesis using light as energy source for synthesizing hydrogen [START_REF] Adessi | Photobioreactor design and illumination systems for H2 production with anoxygenic photosynthetic bacteria: A review[END_REF]Eroglu and Melis, 2011). The purple non sulfur bacteria use the captured light energy to produce ATP and high energy electrons through reverse electron flow which reduces ferredoxin (Figure 2.6). Then, the ATP and reduced ferredoxin drives the proton reduction to hydrogen by nitrogenase (Hallenbeck and Ghosh, 2009). The research attention to these organisms is increasing because of their higher biohydrogen yield potential and better light utilization proficiency, as they are able to absorb and utilize both visible (400 -700 nm) and near infrared (700 -900 nm) light. Moreover, they are able to use a wide variety of substrates (Eroglu and Melis, 2011). Hallenbeck and Ghosh ( 2009))

The ability of purple non sulfur bacteria to convert the organic acids to biohydrogen makes photofermentation a good post treatment for biohydrogen production from DF effluents. An example of integrated dark and photofermentative conversion of acetic acid to biohydrogen is: acid pathway. Therefore, a theoretical biohydrogen potential of DF effluents containing acetate, propionate and butyrate can be written as (Barbosa et al., 2001;Han et al., 2012):

DF: C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2 (2.
Lactate: C3H6O3 + 3H2O → 6H2 + 3CO2 (2.10) Propionate: C3H6O2 + 4H2O → 7H2 + 3CO2 (2.11) Butyrate: C4H8O2 + 6H2O → 10H2 + 4CO2 (2.12)
Purple non sulfur species such as Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodobacter capsulatus and Rhodopseudomonas faecalis have been widely used in photofermentation studies for H2 production [START_REF] Adessi | Photobioreactor design and illumination systems for H2 production with anoxygenic photosynthetic bacteria: A review[END_REF]Eroglu and Melis, 2011), while some studies have been done with mixed cultures isolated from wastewater sludge [START_REF] Cheng | Combination of darkand photo-fermentation to improve hydrogen production from Arthrospira platensis wet biomass with ammonium removal by zeolite[END_REF]Venkata Mohan et al., 2008;Xia et al., 2013). Yangling et al. [START_REF] Yanling | Dynamic changes of microbial community diversity in a photohydrogen producing reactor monitored by PCR-DGGE[END_REF] evaluated the microbial community dynamics in a mixed photofermentative culture enriched from a digestate from the AD of pig dung and found the prevalence of mostly Rhodopseudomonas palustris.

Redwood et al. [START_REF] Redwood | Integrating dark and light bio-hydrogen production strategies: towards the hydrogen economy[END_REF] presented different integration strategies for combining two step dark and photofermentation processes. Nath et al. [START_REF] Nath | Hydrogen production by Rhodobacter sphaeroides strain O.U.001 using spent media of Enterobacter cloacae strain DM11[END_REF] studied the combined dark and photofermentation for biohydrogen production using glucose as the substrate. DF was carried out by Enterobacter cloacae strain DM11, followed by photofermentation by Rhodobacter sphaeroides strain O.U.001 using the spent medium from the DF, which mainly contained acetic acid. The combined hydrogen yield was higher than a single biohydrogen system, i.e. 1.86 mol H2/mol glucose in DF and 1.5-1.72 mol H2/mol acetic acid in the photofermentation. Similarly, combining the two fermentation processes, Chen et al. (Chen et al., 2010;[START_REF] Tao | High hydrogen yield from a twostep process of dark-and photo-fermentation of sucrose[END_REF] and Tao et al. [START_REF] Tao | High hydrogen yield from a twostep process of dark-and photo-fermentation of sucrose[END_REF] attained a total yield of 10.25 mol H2/mol sucrose and 6.63 mol H2/mol sucrose respectively. In a study by Su et al. (Su et al., 2009a), a yield of 4.16 mol H2 mol/mol glucose was obtained from photofermentation of DF effluents using glucose as the substrate, which increased the total yield to 5.48 mol H2/mol glucose.

Other researchers have used effluents from DF of diverse substrate types in photofermentative biohydrogen production. Argun et al. [START_REF] Argun | Photo-fermentative hydrogen gas production from dark fermentation effluent of ground wheat solution: Effects of light source and light intensity[END_REF] used the DF effluent of a ground wheat solution with a H2 yield of 781 ml/g total VFA. In another studies by Su et al. [START_REF] Su | Hydrogen production from water hyacinth through dark-and photo-fermentation[END_REF](Su et al., , 2009b)), cassava starch and water hyacinth were used as the substrates in DF and its effluent was utilized successfully for photofermentative biohydrogen production. The studies reported the increase in total H2 yield from 240.4 mL H2/g starch to 402.3 mL H2/g starch (Su et al., 2009b) and 76.7 to 596.1 mL H2/VS [START_REF] Su | Hydrogen production from water hyacinth through dark-and photo-fermentation[END_REF] using Rhodopseudomonas palustris. These studies have shown that combined dark and photofermentation is a potential technology for biohydrogen production using diverse substrates.

Some drawbacks of photofermentative systems include the inherent high energy demand associated with the nitrogenase enzyme, lower solar conversion efficiencies and economic issues of anaerobic photobioreactors covering large areas [START_REF] Hallenbeck | Biological hydrogen production; fundamentals and limiting processes[END_REF]. However, these inefficiencies can be overcome by developing an efficient photobioreactor [START_REF] Dasgupta | Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production[END_REF][START_REF] Gebicki | Comparison of two reactor concepts for anoxygenic H2 production by Rhodobacter capsulatus[END_REF]. Besides the presence of light conditions, the culture medium of photofermentation should be under ammonia limitation and oxygen should be absent, as both inhibit the nitrogenase activity (Argun et al., 2008a;[START_REF] Eroglu | Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor[END_REF]Koku et al., 2003) . Higher ammonia concentrations (in excess of 2-5 mmol) can be detrimental to hydrogen production (Argun et al., 2008a;Lee et al., 2011). Thus, the effective removal of ammonia from DF residues can be a bottleneck in coupling photofermentation with DF processes. Therefore, substrates with a high C/N ratio seem more suitable for H2 conversion in these systems.

Nonetheless, several ammonia removal strategies such as stripping, natural zeolites and selective membranes can be applied which could facilitate the coupling of the two processes (Androga et al., 2012a;Redwood et al., 2012b).

In addition to biohydrogen production, accumulation of poly-hydroxybutyrate (PHB) could raise future interests, as it possesses economic value as a precursor of biodegradable polymer (Koku et al., 2002). Thus, energy recovery and economic sustainability of the commercial development of DF also depends on the development of post-treatment processes like photofermentation.

Microbial Electrolysis Cells

Biohydrogen production from DF residues is also possible through an emerging technology known as electrohydrogenesis or biocatalyzed electrolysis or microbial electrolysis (Chookaew et al., 2014;Das and Veziroglu, 2001;Gómez et al., 2011;Li et al., 2014;[START_REF] Liu | Hydrogen generation in microbial electrolysis cell feeding with fermentation liquid of waste activated sludge[END_REF]Moreno et al., 2015). Electrochemically assisted Microbial Fuel Cells (MFCs), Microbial Electrolysis Cell (MECs) or Bioelectrochemical Systems (BES) use microorganisms to catalyze the biochemical reactions at the anode and/or cathode, producing protons and electrons from the oxidation of organic matter [START_REF] Jeremiasse | Microbial electrolysis cell with a microbial biocathode[END_REF][START_REF] Liu | Electrochemically assisted microbial production of hydrogen from acetate[END_REF][START_REF] Logan | Microbial Electrolysis Cells for High Yield Hydrogen Gas Production from Organic Matter[END_REF]. MECs should not be confused with MFCs, the former is an electrolysis reactor which produces hydrogen, while a MFC is a fuel cell that produces electricity [START_REF] Logan | Microbial Electrolysis Cells for High Yield Hydrogen Gas Production from Organic Matter[END_REF]. In MECs, on oxidizing acetate under standard biological conditions (25 °C, 1 bar pressure and pH 7)

H2 can be produced at the cathode by applying a small circuit voltage, theoretically 0.14

V [START_REF] Logan | Microbial Electrolysis Cells for High Yield Hydrogen Gas Production from Organic Matter[END_REF] (Figure 2.7). Some exoelectrogenic microorganisms which are capable of electron transfer to an electrode (anode) include the genera Geobacter, Shewanella and Pseudomonas sp. [START_REF] Liu | Electrochemically assisted microbial production of hydrogen from acetate[END_REF][START_REF] Logan | Microbial Electrolysis Cells for High Yield Hydrogen Gas Production from Organic Matter[END_REF]Moreno et al., 2015), while the function and the community composition of the microorganisms at the cathode are not known [START_REF] Logan | Microbial Electrolysis Cells for High Yield Hydrogen Gas Production from Organic Matter[END_REF]. The evolution of hydrogen in BESs can be represented in the following reactions:

C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2 (2.13) Anode: CH3COOH + 2H2O →2CO2 + 8e -+ 8H + (2.14) Cathode: 8H + + 8e -→ 4H2 (2.15)
A minimum theoretical voltage required to produce hydrogen at pH 7 is -410 mV (Normal Hydrogen Electrode). However, the anode potential produced by the oxidation of organic matter is approximately -300 mV. Thus, hydrogen can theoretically be produced at the cathode by applying a circuit voltage higher than -110 mV (i.e. Vapplied = Vanode -Vcathode = -410-(-300) mV), though it has been found that a minimum applied voltage of more than 250 mV is needed due to ohmic resistance and electrode overpotential (Das and Veziroglu, 2008;[START_REF] Liu | Electrochemically assisted microbial production of hydrogen from acetate[END_REF]. This applied voltage required is considerably lower than -1210 mV, the theoretical voltage needed for hydrogen production via electrolysis of water at neutral pH conditions [START_REF] Liu | Electrochemically assisted microbial production of hydrogen from acetate[END_REF].

Anaerobic digestion

Anaerobic Digestion (AD) is a proven biological waste treatment method for volume reduction, waste stabilization and biogas recovery (CH4) from organic waste (Esposito et al., 2012). The AD process can be combined with DF to achieve further conversion of end products of DF and the residues from photofermentation and MECs systems (Figure5). Photofermentation requires a clear medium for efficient light utilization. Thus, the residue from the filtration of DF effluents, microbial biomass produced in photofermentation and the residues from MECs (if any) can be utilized in AD for the final stabilization. The two stage processes, combining biohydrogen production in the first stage and AD in the second stage, not only increase the sustainability of the process, but also guarantee the complete treatment of the organic waste (Gómez et al., 2011). A number of studies have been done on dual systems [START_REF] Cavinato | Bio-hythane production by thermophilic two-phase anaerobic digestion of organic fraction of municipal solid waste: Preliminary results[END_REF][START_REF] Lin | Hydrogen-methane production from pulp & paper sludge and food waste by mesophilic and thermophilic anaerobic co-digestion[END_REF][START_REF] Liu | Hydrogen and methane production from household solid waste in the two-stage fermentation process[END_REF]Z. Liu et al., 2013;Ruggeri et al., 2010;Venetsaneas et al., 2009;Wieczorek et al., 2014). DF followed by AD (Figure 2.8) has shown technical and economic feasibility of the integrated process up to pilot scale (Cavinato et al., 2012;[START_REF] Lee | Bioproduction of hydrogen from food waste by pilotscale combined hydrogen/methane fermentation[END_REF]. [START_REF] Wang | A bench scale study of fermentative hydrogen and methane production from food waste in integrated two-stage process[END_REF]

53

H2/g VS and 520 L CH4/g VS using OFMSW. Similarly, [START_REF] Antonopoulou | Biohydrogen and Methane Production from Cheese Whey in a Two-Stage Anaerobic Process[END_REF] showed the feasibility of a two stage hydrogen-methane process using cheese whey. [START_REF] Jung | Comprehensive study on a two-stage anaerobic digestion process for the sequential production of hydrogen and methane from cost-effective molasses[END_REF] showed that two stage H2-CH4 conversion from molasses is economically feasible. Ruggeri et al. (2010) used the energy balance as a tool to determine the sustainability of integrated DF and AD, which showed the positive energy gain.

Similarly, [START_REF] Schievano | Can two-stage instead of one-stage anaerobic digestion really increase energy recovery from biomass[END_REF] reported 8%-43% increment in energy production in two stage systems in comparison to a single stage AD. Thus, in light of recent popularity of two stage AD processes for treating high strength wastewater or concentrated solids, the former stage can be modified to be used for hydrogen production (Guo et al., 2010).

Also, the DF process can be seen as a pre-treatment stage if the organic waste of interest is subjected to complete stabilization [START_REF] Wang | A bench scale study of fermentative hydrogen and methane production from food waste in integrated two-stage process[END_REF]. Thus, in order to improve the economic competence of commercial DF, AD could provide an attractive solution [START_REF] Ljunggren | Techno-economic evaluation of a two-step biological process for hydrogen production[END_REF].

Other applications

Besides the conversion of volatile fatty acids and other reduced carbon sources to biomethane or biohydrogen in biological processes, VFAs can be used in various applications: biological nutrient removal from wastewater (Elefsiniotis et al., 2004;[START_REF] Lim | Volatile fatty acids production from food wastes and its application to biological nutrient removal[END_REF], sulfur and sulfate reduction [START_REF] Finke | Acetate, lactate, propionate, and isobutyrate as electron donors for iron and sulfate reduction in Arctic marine sediments, Svalbard[END_REF]Sørensen et al., 1981), biopolymer (such as polyhydroxybutyrate) production (Ntaikou et al., 2009) and microbial lipids production [START_REF] Fei | The effect of volatile fatty acids as a sole carbon source on lipid accumulation by Cryptococcus albidus for biodiesel production[END_REF][START_REF] Fontanille | Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica[END_REF]. [START_REF] Lim | Volatile fatty acids production from food wastes and its application to biological nutrient removal[END_REF] studied the use of volatile fatty acids produced from food waste as carbon sources in the removal of nitrogen and phosphorous from municipal wastewater in a sequential batch reactor (SBR) [START_REF] Lim | Volatile fatty acids production from food wastes and its application to biological nutrient removal[END_REF]. Similarly, Elefsiniotis et al. (2004) studied the denitrification process (20 to 200 mg NO3 --N/L) using VFA generated from the AD of starch rich industrial and municipal wastewater as a carbon source in batch reactors.

Similarly, a "carboxylate platform" or third biorefinery platform has been introduced to generate a mixture of carboxylates as intermediates for the production of complex fuels utilizing waste biomass [START_REF] Agler | Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform[END_REF]. Ntaikou et al. (2009) investigated the combined production of biohydrogen and biopolymers from the DF of olive mill wastewater and the use of DF effluents which mostly contained VFAs in SBR using polyhydroxyalkanoates (PHAs) producing bacteria culture.

Pilot scale applications

Most DF studies have been carried out at laboratory scale batch, semi-continuous or continuous reactors. To the best of our knowledge, no studies have reported the DF process at industrial or full scale. Limited studies have been done on pilot-scale applications of DF processes (Cavinato et al., 2012;[START_REF] Jayalakshmi | Bio hydrogen generation from kitchen waste in an inclined plug flow reactor[END_REF]C. M. Lee et al., 2010;[START_REF] Lee | Bioproduction of hydrogen from food waste by pilotscale combined hydrogen/methane fermentation[END_REF][START_REF] Lin | A pilot-scale high-rate biohydrogen production system with mixed microflora[END_REF][START_REF] Ren | Biohydrogen production from molasses by anaerobic fermentation with a pilot-scale bioreactor system[END_REF].

Challenges and future prospects

Modeling and simulation

Several researches have been proposed to integrate DF processes with AD, photofermentation or bioelectrochemical systems to utilize the VFAs produced to increase its viability. Modeling of kinetic parameters and biohydrogen production becomes important for the design, analysis and operation of the fermentative processes.

Also, the predictive capacity of the model for end products helps to design the downstream processes. Several models have been proposed to describe the biohydrogen production, growth of hydrogen fermenters, substrate consumption and intermediate biochemical processes [START_REF] Arudchelvam | Predicting VFA formation by dark fermentation of particulate substrates[END_REF]Gadhamshetty et al., 2010;Wang and Wan, 2009). With increasing research on DF, the modeling of the biohydrogen production process could be of primary interest to achieve a better understanding of the DF pathways and control of the process.

Parameters such as substrate concentration, pH, temperature, and HRT affect the H2 yield and production rate and the nature of the end products (See section 4). Wang and Wan (Wang and Wan, 2009) reviewed existing mathematical models such as the Modified Gompertz model for product formation (H2 production), the Logistic model for biomass growth [START_REF] Mu | Kinetic modeling of batch hydrogen production process by mixed anaerobic cultures[END_REF], substrate utilization based on Monod Kinetics, the Arrhenius model for temperature effects, pH inhibition models based on the IWA Anaerobic Digestion Model no. 1 (ADM1) [START_REF] Batstone | The IWA Anaerobic Digestion Model No 1 (ADM1)[END_REF] and the Modified Luedeking-Piret models for the formation of by-products [START_REF] Mu | Kinetic modeling of batch hydrogen production process by mixed anaerobic cultures[END_REF].

There is, however, a need to upgrade the different kinetic models, including complex biochemical processes, which involve the fermentative biohydrogen production such as hydrolysis, acidogenesis and H2 production from complex substrates (Figure2). The IWA ADM1 has been used extensively to model AD processes [START_REF] Batstone | The IWA Anaerobic Digestion Model No 1 (ADM1)[END_REF][START_REF] Blumensaat | Modelling of two-stage anaerobic digestion using the IWA Anaerobic Digestion Model No. 1 (ADM1)[END_REF][START_REF] Esposito | Mathematical modelling of disintegration-limited co-digestion of OFMSW and sewage sludge[END_REF][START_REF] Esposito | Model calibration and validation for OFMSW and sewage sludge co-digestion reactors[END_REF]. ADM1 is a structured mathematical model based on the COD balance of composite substrates and includes a number of biochemical processes involving disintegration of substrates such as hydrolysis, acidogenesis, acetogenesis and methanogenesis, biomass growth and decay processes and the physical interaction of the gas-liquid phases. Because of the similarity of some initial biochemical and physical processes, a modified ADM1 has been proposed

to model dark fermentative biohydrogen production processes [START_REF] Arudchelvam | Predicting VFA formation by dark fermentation of particulate substrates[END_REF]Gadhamshetty et al., 2010;[START_REF] Peiris | ADM1 simulations of hydrogen production[END_REF]. Nonetheless, a model that can simulate the process and predict the formation of all the major intermediates and biohydrogen considering all influencing parameters is a necessity.

Energy balance and COD conversion

The net energy gain in DF processes is an important issue that has been addressed by few researchers [START_REF] Perera | Fermentative biohydrogen production: Evaluation of net energy gain[END_REF]Ruggeri et al., 2010;[START_REF] Tommasi | Energy valorisation of residues of dark anaerobic production of Hydrogen[END_REF]. The energy balance is an important factor for the process sustainability. Higher culture temperatures have been suggested in the literature [START_REF] Cavinato | Optimization of two-phase thermophilic anaerobic digestion of biowaste for hydrogen and methane production through reject water recirculation[END_REF]Chen et al., 2012;Lee et al., 2008;Shin et al., 2004;Valdez-vazquez et al., 2005) for maximizing H2 yield, without considering the net energy gain [START_REF] Perera | Fermentative biohydrogen production: Evaluation of net energy gain[END_REF]. Some studies [START_REF] Perera | Fermentative biohydrogen production: Evaluation of net energy gain[END_REF]Ruggeri et al., 2010)

pH control

Unlike AD processes where the production of acidity from VFAs generation is balanced by alkalinity of the systems, DF processes are unstable because of the continuous production of acidity (VFAs production). As discussed earlier (Section 4.2.3), a very low pH can inhibit the hydrogen production, while the acidic range (5-6) favors H2 production depending on the type of substrate. The use of an excessive amount of buffers, acids or base to maintain the pH acidic can decrease the economics and sustainability of the process as well as increase the salt concentration of the DF effluents.

One of the sustainable solutions could be to explore substrates with a higher pH or alkalinity to equilibrate the system. (Choi and Ahn (2013) suggested the use of substrates with a high pH to replace the use of buffers. Life Cycle Analysis (LCA) of two-step thermophilic DF followed by photofermentation of potato peels, showed that most of the impact was generated by the use of the phosphate buffer during the process [START_REF] Ochs | Life cycle inventory analysis of biological hydrogen production by thermophilic and photo fermentation of potato steam peels (PSP)[END_REF]. To provide natural buffering, [START_REF] Cavinato | Optimization of two-phase thermophilic anaerobic digestion of biowaste for hydrogen and methane production through reject water recirculation[END_REF] recycled the reject water (effluent) from the AD step in the two-step DF and AD. The AD reject water provided alkalinity to maintain the pH in the DF step around 5.5, giving a H2 yield of 51 L/kgVS of food waste fed with a H2 content of 37% in the biogas. However, the major concern with the recirculation of the AD reject water is the activity of methanogens present in the reject water, which can affect the purity of the biohydrogen produced in the DF step. In addition, inhibition of H2 production due to higher levels of ammonia present in the reject water could be another concern as reported in a study by Cavinato et al. (Cavinato et al., 2012). Thus, long term studies to assess the effect of reject water recirculation from the AD step on the H2 content in biogas produced from DF could open further doors to ensure the sustainability of DF systems.

Solid State Dark Fermentation (SSDF)

Anaerobic reactors are generally categorized into wet (<10% TS), semi-dry (10-20% TS)

and dry (>20% TS) processes [START_REF] Karthikeyan | Bio-energy recovery from high-solid organic substrates by dry anaerobic bio-conversion processes: a review[END_REF]. However, some categorized wet digestion for low (<15% TS) substrates and dry digestion of high solids (>15% TS) processes (Motte et al., 2013). By increasing the TS content, dry fermentation processes can be operated at a high OLR with little water addition, which offers advantages such as smaller reactor volume, easy handling of the digestate residues and technical simplicity [START_REF] Karthikeyan | Bio-energy recovery from high-solid organic substrates by dry anaerobic bio-conversion processes: a review[END_REF]Motte et al., 2013). This could be attractive for commercialization of these processes. However, the drawbacks of SSDF are the low H2 yields due to mass and energy transfer limitations, which affects the product formation (Robledo-Narváez et al., 2013).

Using agro-industrial wastes (70% sugarcane bagasse, 15% of pineapple peelings and 15% of waste activated sludge) under mesophilic conditions, Robledo-Narváez et al.

(2013) found a decrease in H2 yield (3 mmol H2/g TS) at a TS content higher than 18 %TS in a tested TS content range from 15-35%. Similar results were obtained by Valdez-Vazquez and Poggi-Varaldo (2009), where the highest H2 productivity and yield (463.7

NmL/kg/d and 54.8 N mL/g VS removed, respectively) was obtained at a TS of 20.9 % using organic solid waste (paper (40%) and food (60%) wastes) for the tested TS range from 20.9 -35.1 %TS. Likewise, Motte et al. (2014) also reported 19 %TS as the limit to achieve higher H2 production performance during the DF of wheat straw, as metabolic pathways shifted towards lactic acid formation at higher TS content. Further research is required on SSDF in order to elucidate the mechanisms involved during dark fermentation at high TS contents. This research could provide practical solutions for biohydrogen production from organic solid waste.

Conclusions

DF technology has an excellent future potential for biohydrogen production as renewable biomass can be used as a feedstock and the integration with other systems could foster a higher H2 yield and economic feasibility. The economic considerations and production at industrial scale recommend a continuous bioprocess. Thus, more research on continuous DF processes needs to be carried out to demonstrate the long-term operational feasibility of continuous processes. Microbial community of hydrogen producers and innovative substrates needs to be explored. The use of spent dark fermentation residues in photofermentation and or electrochemical systems as a secondary step could pave the way towards sustainable biohydrogen production in up-scaled systems. Finally, anaerobic digestion is required to further stabilize the residues generated from the upstream processes. The future design and configuration of industrial scale dark fermentative processes is expected to be similar to anaerobic digestion processes, with some modifications in process parameters. Existing two stage methane-producing plants can be modified for dark fermentation, while SSDF fermentation opens new opportunities for biohydrogen production from renewable biomass.

Valorization of dark fermentation effluents via photo fermentative production of biohydrogen and biopolymers

This chapter presents the research advances in utilization of dark fermentation effluents, which mainly contain volatile fatty acids (VFAs), to produce biohydrogen (H2) and biopolymers by photofermentation (PF) processes. The recent and past studies of PF of organic substances, mainly organic acids, using the purple non-sulfur bacteria (PNSB), are presented. The different laboratory and pilot scale PF studies carried out and the conditions necessary for optimal H2 production and/or synthesis of biopolymer, polyhydroxybutyrate (PHB), using PNSB strains are reported. This review also focuses on the design considerations of the photobioreactors and economics of production. In the context of increasing application of PF process for waste valorization via H2 and PHB production, this work provides a state of art of the technology in operational parameters such as bacterial strains, substrate types, light intensity, concentration, culture pH and temperature and the design consideration for photobioreactors for the valorization of dark fermentation effluents by the application of PNSB as future reference.

Introduction

Most energy fuels, chemicals and raw materials in our daily lives are derived from petroleum based refineries. However, depleting fossil fuel reserves and increasing greenhouse gas emissions and severe pollution problems as the consequence of byproducts from fossil fuel utilization is driving interests towards biorefineries for the production of energy and useful chemicals (Cherubini 2010;Menon and Rao 2012). In the energy and environmental sector, hydrogen (H2) has gained considerable interests owing to its higher specific energy content (122 MJ/kg) as well as water and energy being the sole by-products [START_REF] Balat | Hydrogen from biomass -Present scenario and future prospects[END_REF]. At present, H2 production for industrial applications is mainly derived from thermo-catalytic and gasification processes, which are highly dependent on fossil fuels. In comparison to the energy intensive physicochemical routes for H2 production, biological processes can be operated at ambient conditions and are advantageous as they can utilize renewable biomass (Ghimire et al. 2015;Das and Veziroglu 2001).

Based on the light dependency as an energy source for the biochemical reactions, biological H2 production pathways can be broadly categorized into light dependent and independent processes (Das & Veziroglu 2008;Hallenbeck & Ghosh 2009). The light dependent photo-hydrogen production systems can be further classified into i) direct photolysis, where water is broken down into H2 and O2 gas by algae and cyanobacteria, ii) indirect photolysis in which cyanobacteria or cyanophytes synthesize H2 in the presence of light and inorganic carbon, and iii) photofermentation (PF), carried out by photosynthetic bacteria where photodecomposition of organic compounds occurs. The light independent processes include i) dark fermentation (DF), which involves fermentative hydrogen production from carbohydrate rich organic biomass, and ii) H2

from bio-electrochemical systems or microbial electrolysis cells.

DF is a well studied biological route for the production of hydrogen from organic biomass, including waste, owing to its higher H2 production rates than light dependent processes (Ghimire et al. 2015). However, due to the thermodynamic constraints, dark fermentative conversion of carbohydrate rich organic biomass offers lower H2 yields and gives incomplete conversion of organic biomass, i.e. organic acids and alcohols remain as major fermentation by-products. On the brighter side, the PF processes can convert these dark fermentative by-products to biohydrogen. Moreover, PF processes have higher H2 yields and generate less residues compared to DF processes [START_REF] Li | Heterotrophic Photo Fermentative Hydrogen Production[END_REF]Lo et al. 2010). A dual system can integrate the conversion of carbohydrates to organic acids in the first stage (DF) and the utilization of its by-products in the second stage (PF) [START_REF] Redwood | Integrating dark and light bio-hydrogen production strategies: towards the hydrogen economy[END_REF].

Using light as a source of energy, purple non-sulfur bacteria (PNSB) synthesize H2 by carrying out an anaerobic photosynthesis. In PNSB, this takes place in the presence of the nitrogenase enzyme and light, with reduced carbon sources such as organic acids. In addition, under certain operating conditions, PNSB also synthesize cell reserve materials or biopolymers, i.e. polyhydroxybutyrate (PHB) molecules (Khatipov et al. 1998;De Philippis et al. 1992).

An example of PF is the conversion of acetic acid to biohydrogen and/or biopolymers. It can be expressed by the following equations (2.16 and 2.17):

2CH3COOH + 4H2O Moreover, the biopolymer production can add an economic value to the PF process.

However, photofermentative production of H2 and PHB are competing processes (Wu et al., 2012;Khatipov et al. 1998). Nonetheless, a concomitant production of H2 and PHB is also possible, as shown in a study by Montiel-Corona et al. (2015). The photofermentative H2 and PHB production depends on several operating conditions, such as nutrients availability (carbon to nitrogen ratio (C/N) ratio), PNSB strain (mixed or pure culture), pH, light intensity and presence of physical-chemical stress, for example the presence of inhibitors of H2 formation such as ammonium in the culture medium [START_REF] Adessi | Photobioreactor design and illumination systems for H2 production with anoxygenic photosynthetic bacteria: A review[END_REF][START_REF] Chen | Perspectives on cultivation strategies and photobioreactor designs for photo-fermentative hydrogen production[END_REF][START_REF] Li | Heterotrophic Photo Fermentative Hydrogen Production[END_REF].

The ability of PNSB to convert reduced carbon sources such as organic acids and alcohols to H2 and PHB makes PNSB based PF a good post treatment process for dark fermentation effluents (DFE) [START_REF] Cheng | Hydrogen production using amino acids obtained by protein degradation in waste biomass by combined dark-and photo-fermentation[END_REF]Chookaew et al. 2015;[START_REF] Dipasquale | Introducing capnophilic lactic fermentation in a combined dark-photo fermentation process: a route to unparalleled H2 yields[END_REF][START_REF] Nasr | Continuous biohydrogen production from starch wastewater via sequential darkphoto fermentation with emphasize on maghemite nanoparticles[END_REF]Rai et al. 2014). Moreover, the potential of the PF process to be operated as stand alone system for wastewater treatment has also been reported [START_REF] Li | Heterotrophic Photo Fermentative Hydrogen Production[END_REF][START_REF] Eroğlu | Effect of clay pretreatment on photofermentative hydrogen production from olive mill wastewater[END_REF][START_REF] Hülsen | Phototrophic bacteria for nutrient recovery from domestic wastewater[END_REF]. With the increasing application of DF processes for H2 production, the integrated DF-PF process can enhance H2 yields, thus providing sustainability to scaled-up biohydrogen production processes. Likewise, the potential of PF processes for the production of biopolymers can give further economic gain.

This chapter aims to summarize the state of the art of PF processes for H2 production by overviewing existing understanding of the microbiology of the PF process, different photobioreactor (PBR) design, conversion efficiencies of different PNSB strains, process operational parameters such as pH, temperature, nutrient requirements. This competence can be applied for the valorization of DFE and wastewater through H2 and PHB production. In addition, this work presents current approaches of the mathematical modeling of PF as well as highlights the economics of the process.

Microbiology and phototrophic metabolism of PNSB

Bacterial photosynthesis

Bacterial photosynthesis can be divided into two types depending on the presence or absence of oxygen for the metabolism of bacteriochlorophyll, a bacterial photosynthetic pigment. Oxygenic photosynthesis is carried out by cyanobacteria and prochlorophytes, whereas anoxygenic photosynthesis can be generally mediated by purple bacteria, green sulfur bacteria, heliobacteria and others [START_REF] Kim | Bacterial Physiology and Metabolism[END_REF]. Photosynthetic anoxygenic bacteria are a very diverse groups of bacteria which carry out bacteriochlorophyll dependent photosynthesis as a metabolic process [START_REF] Mcewan | Photosynthetic electron transport and anaerobic metabolism in purple non-sulfur phototrophic bacteria[END_REF].

The anoxygenic phototrophic bacteria can be broadly grouped into different classes (Figure 2.9), based on their photosynthetic pigments and electron donors [START_REF] Kim | Bacterial Physiology and Metabolism[END_REF][START_REF] Mcewan | Photosynthetic electron transport and anaerobic metabolism in purple non-sulfur phototrophic bacteria[END_REF]. Depending on the electron donors used, purple bacteria can be further divided into purple sulfur bacteria (use sulfur compounds as electron donors) and non-sulfur bacteria (use organic substances as electron donor).

Some drawbacks of this photofermentative system as pointed by [START_REF] Hallenbeck | Biological hydrogen production; fundamentals and limiting processes[END_REF] include inherent high energy demand associated with the nitrogenase enzyme, lower photo conversion efficiencies and economic issues of anaerobic photobioreactors covering large areas. These drawbacks can be overcome by effective design and operation of the photobioreactors (PBRs) and selecting proper strains or enrichment of PNSB for an efficient conversion to photo-H2.

Purple non-sulfur bacteria (PNSB)

Among the anoxygenic bacteria, the PNSB exhibit very diverse morphological, biochemical and metabolic properties [START_REF] Imhoff | Rearrangement of the Species and Genera of the Phototrophic Purple Nonsulfur Bacteria[END_REF]. PNSB are gram-negative photo-heterotrophs, which normally carry out photosynthesis under anaerobic conditions.

Although PNSB are facultative anaerobes, they can also grow chemotropically under oxygenic conditions using oxygen as electron acceptor [START_REF] Mcewan | Photosynthetic electron transport and anaerobic metabolism in purple non-sulfur phototrophic bacteria[END_REF]. Different from purple sulfur bacteria, which use elemental sulfur as the electron donor, PNSB typically use organic electron donors such as organic acids, however, they can also use hydrogen gas as electron donor [START_REF] Kim | Bacterial Physiology and Metabolism[END_REF].

PNSB can utilize various types of carbon sources such as short-chain organic acids and glucose. The theoretical photofermentative conversion of different organic acids, typically present in DFE, to H2 can be expressed by the reactions presented in Eqns. 2.9 -2.12.

However, the conversion ability of different PNSB for different substrates varies (Barbosa et al. 2001;Bianchi et al. 2010). Some species prefer a certain sole carbon source, while H2 yields seem to be higher with mixed sources of carbon (Han et al. 2012).

The variation in H2 production from different carbon sources can be explained by differences in their reduction states and the associated metabolism for the assimilation of different carbon sources [START_REF] Kars | Towards a super H2 producer: Improvements in photofermentative biohydrogen production by genetic manipulations[END_REF]Han et al., 2012;[START_REF] Wang | Photofermentative hydrogen production from mixed substrate by mixed bacteria[END_REF].

Similarly, when the carbon source is acetate, most of the reducing power of the PNSB is utilized for the synthesis of PHB rather than H2 (Hustede et al.,1993;[START_REF] Kars | Towards a super H2 producer: Improvements in photofermentative biohydrogen production by genetic manipulations[END_REF].

Photosystem of PNSB

The photosynthetic apparatus of PNSB is simple as it contains only one photosystem (PS), unlike the two PS in algae and cyanobacteria. PNS bacterial cells contain bacteriochlorophyll α or β located on cytoplasmic membrane. The PS of PNSB contains the light harvesting complexes that absorb photons initiating a charge (electron-hole) separation through excitation (Figure 2.10). Electrons that are liberated from organic acids are transported around through a number of electron carriers, i.e. the cytochrome C2 complex, cytochrome bc1 complex (Cyt bc1) and quinone Q (Figure 2.10). The transfer of electrons across the membranes creates a large proton gradient which drives the synthesis of ATP from ADP by ATP synthase (Figure 2.10) [START_REF] Akkerman | Photobiological hydrogen production: photochemical efficiency and bioreactor design[END_REF][START_REF] Hu | Photosynthetic apparatus of purple bacteria[END_REF]. The extra energy in the form of ATP will be used to reduce ferredoxin-fd.

Then, the ATP and reduced ferredoxin drives the proton reduction to hydrogen by nitrogenase (Hallenbeck and Ghosh 2009). Thus, as a result of anoxygenic photosynthesis, conversion of organic substances into H2 takes place. Nitrogenase and hydrogenase are the two enzymes that strongly influence hydrogen production: nitrogenase promotes its production, whereas hydrogenase consumes hydrogen (Figure 2.10). Besides the light conditions, the PF culture medium should be under nitrogen limitation and oxygen should be absent, as their presence inhibits the nitrogenase activity (Koku et al. 2002;[START_REF] Li | Heterotrophic Photo Fermentative Hydrogen Production[END_REF][START_REF] Kars | Towards a super H2 producer: Improvements in photofermentative biohydrogen production by genetic manipulations[END_REF]. The activity of the nitrogenase enzyme is of fundamental importance for efficient photo-H2 production [START_REF] Hallenbeck | Biological hydrogen production; fundamentals and limiting processes[END_REF]. Equations 2.18 and 2.19 explain the effect of N2 on the metabolism of PNSB (Das and Veziroglu 2001):

With dinitrogen: N2 + 8H + + 8e -+ 16ATP → 2NH3 + H2 + 16ADP + 16Pi (2.18) Without dinitrogen: 8H + + 8e -+ 16ATP → 4H2 + 16ADP + 16Pi (2.19)
The presence of nitrogen, either in gaseous form or in the culture medium, can thus inhibit the activity of the nitrogenase enzyme that synthesizes molecular H2. Therefore, substrates with a high C/N ratio are more suitable for H2 conversion in these systems.

PHB accumulation by PNSB

PNSB accumulate poly-β-hydroxybutyrate (PHB), an intracellular storage of carbon and energy formed under physiological stress, particularly, at high carbon to nitrogen (C/N) ratio, higher ammonia concentration or sulphur deprived conditions (Khatipov et al. 1998;Eroglu and Melis 2011;Waligórska et al. 2009). The production of PHB and H2 functions as the way to dissipating the excess reducing power and the PHB synthesis competes with the H2 production (Figure 2.10). Thus, depending on the aim of the process, the PF can be directed towards H2 production by suppressing the PHB synthesis through genetic engineering of the PNSB (Kim et al. 2011). [START_REF] Kars | Towards a super H2 producer: Improvements in photofermentative biohydrogen production by genetic manipulations[END_REF] reviewed the different genetic manipulation strategies to improve photofermentative biohydrogen production. They proposed to modify the acetate assimilation pathways that share the common biosynthetic route of PHB.

After the deletion of the PHB producing gene from R. sphaeroides KD131, the H2 production rate was increased from 36.1 ml H2/l/h to 43.8 ml H2/l/h (Kim et al. 2011), in accordance with the study of Hustede et al. (1993) who observed an increase in cell growth and H2 production when eliminating the gene for PHB synthesis in Rhodobacter sphaeroides.

In addition, PNSB produce light harvesting bacterial pigments (bacteriochlorophylls and carotenoids) that can be of commercial interests [START_REF] Venil | Bacterial pigments and their applications[END_REF]. This ability of PNSB has been highlighted in a few older studies and need to be explored again [START_REF] Schmidt | Carotenoids of purple nonsulfur bacteria[END_REF][START_REF] Cohen-Bazire | Kinetic studies of pigment synthesis by non-sulfur purple bacteria[END_REF]. [START_REF] Akkerman | Photobiological hydrogen production: photochemical efficiency and bioreactor design[END_REF] suggested three parameters to evaluate the photo-H2 production process: H2 production yield, the yield coefficient of H2 produced relative to the carbon source consumed and the photochemical efficiency (PE). Table 2.11 compares PF and DF systems in terms of H2 yields from substrate conversion and production rate. PF systems are superior in terms of substrate to H2 conversion, while they have slower H2 production kinetics than DF systems. Considering the theoretical conversion of substrate to H2 from Eqns. 2.9 -2.12 and the experimental results reported in past studies (Table 2.11), PNSB have a very versatile metabolism and high substrate to H2 conversion efficiency (Bianchi et al. 2010;[START_REF] Mcewan | Photosynthetic electron transport and anaerobic metabolism in purple non-sulfur phototrophic bacteria[END_REF][START_REF] Rupprecht | Perspectives and advances of biological H2 production in microorganisms[END_REF]). Their PF system lacks oxygen sensitivity issues that are encountered in biophotolysis. Moreover, their light utilization proficiency is high, as PNSB can absorb and utilize both visible (400 -700 nm) and near infrared (700 -900 nm) light. Also, PNSB use a wide variety of substrates (Eroglu & Melis 2011). The application of PNSB can be promising for PF systems, as they not only give a higher substrate to product conversion and higher H2

Photo-hydrogen conversion efficiencies

yield, but also benefit in their capability to reduce pollution loads, e.g. treatment of effluents (organic acids) from DF, with the added economic benefit in the form of PHB production, a valuable biopolymer. The photofermentative H2 production efficiency can also be measured as photochemical efficiency (PE), which is an efficiency parameter with which the light is utilized to produce energy stored as hydrogen in a PF process. The PE depends on the photosynthetically active radiation (PAR) range, which determines the light energy absorbed by the photofermentative species. For example, green algae have a PAR range of 400-700 nm, while the range for PNSB is 400-950 nm (Figure 2.11). [START_REF] Akkerman | Photobiological hydrogen production: photochemical efficiency and bioreactor design[END_REF] reported the PE values vary between 3 to 10% in green algae. Redwood et al. (2012) achieved a 71% increase in combined photosynthetic activity by illuminating both Rhodobacter sphaeroides and Arthrospira (Spirulina) platensis by dividing a single beam of simulated sunlight using a dichroic mirror. [START_REF] Akkerman | Photobiological hydrogen production: photochemical efficiency and bioreactor design[END_REF] In addition to H2 yield and the other parameters mentioned before, the performance of a PF process can be evaluated by the light conversion efficiency. Light or solar energy conversion efficiency can be calculated as the ratio of total energy produced, i.e. stored in the form of H2, to the total energy input to the bioreactor (energy as photons in case of solar conversion efficiencies). The light conversion efficiency (η) can be calculated with an empirical formula, i.e. the ratio of the total energy (heat of combustion) value of hydrogen to energy input to the PBR by solar radiation (Koku et al. 2002). The η can be evaluated as

η (%) = [33.61 • ρ H 2 • V H 2 ] [I • A • t] • 100 (2.20)
Where, VH2 is the volume of produced H2 in l, Sunlight intensity ρH2 is the density of the produced hydrogen gas in g/l, I is the light intensity in W/m 2 , A is the irradiated area in m 2 and t is the duration of hydrogen production in hours. Koku et al. (2002) reported a varying range of light conversion efficiencies between 1-5% on the average for different strains of Rhodobacter sphaeroides. According to the best of knowledge of the authors, a PE higher than 10% has not been reported so far. However, a wide range of approaches to increase the PE has been suggested in the literature, including the design of efficient PBR and improving lighting conditions [START_REF] Adessi | Photobioreactor design and illumination systems for H2 production with anoxygenic photosynthetic bacteria: A review[END_REF][START_REF] Chen | Perspectives on cultivation strategies and photobioreactor designs for photo-fermentative hydrogen production[END_REF], genetic modifications of PNSB for enhancing nitrogenase activity [START_REF] Ozturk | Hydrogen production by using Rhodobacter capsulatus mutants with genetically modified electron transfer chains[END_REF], reduction of the pigment content for higher light uptake [START_REF] Kondo | Enhancement of hydrogen production by a photosynthetic bacterium mutant with reduced pigment[END_REF], deletion or inactivation of the genes responsible for PHB synthesis (Kim et al. 2011;[START_REF] Franchi | Metabolically engineered Rhodobacter sphaeroides RV strains for improved biohydrogen photoproduction combined with disposal of food wastes[END_REF]) and developing hydrogenase deficient (hup -) mutant PNSB strains responsible for H2 uptake [START_REF] Franchi | Metabolically engineered Rhodobacter sphaeroides RV strains for improved biohydrogen photoproduction combined with disposal of food wastes[END_REF][START_REF] Uyar | Hydrogen production by hup-mutant and wild-type strains of Rhodobacter capsulatus from dark fermentation effluent of sugar beet thick juice in batch and continuous photobioreactors[END_REF].

Operating conditions of PF

PNSB inoculum

PNSB are widely distributed in nature and prefer aquatic environments with low oxygen concentrations, significant amounts of soluble organic matter, moderate temperatures and weak as well as stronger light conditions (Imhoff et al. 2005). Besides freshwater, members of the PNSB group can also be found in marine and hypersaline environments and even in sediments that are exposed to light. These organisms can also thrive in thermal springs and alkaline soda lakes (Imhoff et al., 2005). An eutrophic lake is an example of a favorable habitat for members of these genera (Imhoff et al. 2005;Bianchi et al. 2010).

Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodobacter capsulatus and others are the most studied PNSB stains. However, PNSB strains capable of utilizing the substrates and light at higher conversion efficiencies are of research interest. Many studies have successfully isolated H2 producing PNSB strains from different mixed consortia.

Some examples of isolated H2 producing PNSB and their H2 yields and production rates are presented in Table 2.12. (2015), the H2 yields from enriched mixed PNSB cultures was higher (1478 ± 17 mL H2/L) than from pure R. capsulatus cultures (1252 ± 20 mL H2/L).

Inoculum Age

The selection of inoculum culture age can be critical to obtain a higher performance of PF systems. It has been found that the PNSB inoculum from the exponential phase of the growth curve is suitable for better performance of PBRs for biohydrogen production [START_REF] Basak | The Prospect of Purple Non-Sulfur (PNS) Photosynthetic Bacteria for Hydrogen Production: The Present State of the Art[END_REF]. Koku et al. (2003) found vast differences in total H2 production, H2 production rates and the overall substrate conversion rates when Rhodobacter sphaeroides O.U. 001 of two different inoculum ages were used in the PF of malic acid.

The inoculum harvested from the mid-exponential phase gave a higher total gas production (357 mL H2), gas production rate (0.009 mL H2/L/h) and overall substrate conversion rate (35%) than from an inoculum harvested at the stationary phase, which gave a lower total gas production (236 mL H2), gas production rate (0.003 mL H2/L/h) and overall substrate conversion rate (24%).

In a study by [START_REF] Sasikala | Environmental regulation for optimal biomass yield and photoreduction of hydrogen by Rhodobacter sphaeroides O.U.001.pdf[END_REF] on the effect of culture age on the photo-production of hydrogen by R. sphaeroides O.U. 001, the inoculum with a 20 hour culture period gave the highest H2 evolution (60 mL H2/L reactor), while it was lower for a short (4 h) or long (38 h) culture period. A range of optimal inoculum ages has been reported in the literature.

Akroum-Amrouche et al. ( 2011) reported an optimum inoculum age of 36 -48 hours in PF using Rhodobacter sphaeroides CIP 60.6, while [START_REF] Liu | Optimization of Photo-Hydrogen Production by Immobilized Rhodopseudomonas Faecalis RLD-53[END_REF] reported an inoculum age of 24 hours for Rhodoseudomonas faecalis RLD-53 as optimum.

The aged inoculum can give poor performance in terms of H2 production and large retention times may shift the metabolic pathways to accumulation of poly-βhydroxybutyrate (PHB) (Koku et al. 2003). They also reported that a repeated culture of PNSB might lead to loss of H2 production capacity due to a decline in the activity of the electron carrier ferredoxin.

Cell immobilization

Studies have used different cell immobilization techniques in order to have the advantage of operating the PF process in the exponential growth phase for an infinite period of time and protect the culture strains from the inhibitory effects of chemicals which might be present in influent [START_REF] Chen | Enhancing phototropic hydrogen production by solid-carrier assisted fermentation and internal optical-fiber illumination[END_REF][START_REF] Liu | Optimization of Photo-Hydrogen Production by Immobilized Rhodopseudomonas Faecalis RLD-53[END_REF]Zhu et al. 1999a;Zhu et al. 1999b). However, a major limitation in cell immobilized PF systems is the penetration and transmission of light through the immobilization media. Also, the cell immobilization technology might not be practical when the PNSB cells are required to be harvested for PHB production.

Carbon sources and nutrients sources

The substrate types and their concentration used in PF can influence the H2 production rates and yields. Han et al. (2012) The results of the study showed that the H2 production for the mixed substrates is higher (794 mmol H2/mol substrate for 2.02 g/L lactate and 2.0 g/L succinate) than using a single substrate (424 mmol H2/mol substrate for 0.8 g/L sodium propionate). This makes PF prominent for the application in the treatment of DFE that typically contains more than one organic acid [START_REF] Nasr | Continuous biohydrogen production from starch wastewater via sequential darkphoto fermentation with emphasize on maghemite nanoparticles[END_REF]Rai et al. 2014).

Effect of OLR and HRT

Similarly, the OLR and HRT could affect the performance of PBRs as they determine the substrate degradation efficiency and the hydrogen production rate. Mohan et al. (2008) studied the effect of different OLRs on photo-H2 production and substrate degradation efficiency. The synthetic wastewater gave the maximum substrate degradation efficiency (1.4 kg COD/m 3 /day) at an OLR of 2.45 kg COD/m 3 /day, while higher specific H2 production (19.29 mol H2/kg CODremoved) was achieved at an OLR of 1.4 kg COD/m 3 /day with 45% COD removal. In another study, [START_REF] Tawfik | Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent[END_REF] studied the effect of varying OLR (3.2 to 16 kg COD/m 3 /day) using mixed PNSB cultures, which resulted in maximum H2 production at an OLR of 6.4 kg COD/m 3 /day. Increasing OLR caused VFAs accumulation, which might inhibit the PNSB. Therefore, inhibition of the nitrogenase activity resulted in decreasing H2 production when the OLR was higher than 6.4 kg COD/m 3 /day [START_REF] Tawfik | Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent[END_REF]. This is supported by another PF study carried out with acid hydrolyzed wheat starch and a pure culture of Rhodobacter sp. [START_REF] Kapdan | Bio-hydrogen production from acid hydrolyzed wheat starch by photo-fermentation using different Rhodobacter sp[END_REF].

The results of the study showed that, upon increasing the initial sugar concentration from 2.2 to 13.0 g/L, the H2 yield (H2Y) increased, with a maximum H2Y achieved at 5 g/L (143.5 mL H2/g COD).

A range of optimum HRT, varying from 2.5 h [START_REF] Tawfik | Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent[END_REF] to 3 days (Ozmihci and Kargi 2010) has been reported in the literature for achieving higher photo-H2 production in a continuous reactor. [START_REF] Tawfik | Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent[END_REF] found an optimum HRT at 2.5 h (0.97 ± 0.12 LH2/gCODremoved/d), when studying a range of HRT from 0.9 to 4.0 h. They also observed the improvement in removal efficiency of butyrate and lactate when the HRT was increased. Similarly, another study carried out with mixed PNSB by Zhang et al. (2015) showed that varying HRTs from 12 to 72 h significantly affected the H2Y with the highest H2Y of 482.4 mmol H2/L obtained at a HRT of 36 h. In contrast, [START_REF] Ozmihci | Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal[END_REF] obtained the highest H2Y and production rate at an HRT of 72 h during PF of DFE using Rhodobacter sphaeroides. The differences in optimum HRT may be attributed to differences in PNSB strains, substrate concentration, carbon to nitrogen ratio (C/N) and other operating conditions such as pH, temperature and light intensity.

Effect of C/N ratio

The carbon to nitrogen ratio plays an important role in the growth of PNSB, photo-H2 and PHB production. However, higher levels of nitrogen inhibit H2 production while higher C/N ratios enhance the production of PHB [START_REF] Eroglu | Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor[END_REF]Koku et al. 2003;Argun et al. 2008;Waligórska et al. 2009). A low C/N ratio can result in the accumulation of ammonia, which inhibits the nitrogenase and thus the H2 production process. Therefore, it is always desirable to have nitrogen-limited conditions in the PBR. Due to the nitrogen requirements for bacterial photosynthetic metabolism and inhibition of nitrogenase at higher ammonium concentrations, there is a tradeoff between the minimum amount of nitrogen for bacterial growth and non-inhibiting levels.

A range of C/N ratios has been reported in the literature, i.e. from as low as 8 to as high as 120. [START_REF] Eroglu | Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor[END_REF] 2008) reported the optimum total VFAs and NH + 4-N concentrations of 2350 mg/L and 47 mg/L, respectively, for increasing the H2 production by Rhodobacter sphaeroides strains. In another study [START_REF] Eroğlu | Comparison of physicochemical characteristics and photofermentative hydrogen production potential of wastewaters produced from different olive oil mills in Western-Anatolia, Turkey[END_REF], the highest H2 production potential of 19.9 m 3 H2/m 3 was obtained from olive mill wastewater with the highest C/N molar ratio of 73.8. Waligórska et al. (2009) found that accumulation of PHB increased by 30 fold when the C/N ratio increased from 6 to 120 in R. sphaeroides. However, the amount of PHB accumulation mainly depends on the PNSB strains and the other process operational conditions (De Philippis et al., 1992;Montiel-Corona et al., 2015). As PHB biosynthesis is a H2 competing pathway, its concomitant production with H2 could raise future interests, as PHB possesses economic value as a biodegradable polymer (Koku et al. 2002). Some of the results from previous studies on H2 and PHB production in PF processes are summarized in Table 2.13.

Micronutrients

Microorganisms need different micronutrients such as iron and nickel for their metabolism and growth. The PF process relies on the photosynthetic electron transport systems from which bacteria obtain their energy (Figure 2.10). The constituents of the electron transport systems such as cytochromes are Fe protein complexes and PNSB strains have 24 Fe atoms in each nitrogenase [START_REF] Zhu | Effect of ferrous ion on photo heterotrophic hydrogen production by Rhodobacter sphaeroides[END_REF]. Another electron carrier, ferrodoxin, also contains Fe. Thus, Fe limitation can influence the metabolism of PNSB and production of H2. Uyar et al. (2009) found that the hydrogen yield increases from 0.3 to 1.0 L/Lculture when iron was added to micronutrient. They suggested 0.1 mM of ferric citrate as optimum concentration for hydrogen production. Similarly, [START_REF] Zhu | Effect of ferrous ion on photo heterotrophic hydrogen production by Rhodobacter sphaeroides[END_REF] studied the effect of ferrous ion (0 -3.2 mg/l) on PF using Rhodobacter sphaeroides and found that the photo-H2 production was significantly suppressed when Fe 2+ was limited. The H2 production increased when increasing the Fe 2+ concentration and reached the maximum at the concentration of 2.4 mg/l. In another study, Rai et al. (2014) studied the effects of Ni 2+ , Fe 2+ and Mg 2+ on the PF of cheesewhey for H2 production, and showed significant effects of Ni 2+ and Fe 2+ supplementation on H2 yields. However, the presence of nickel might also enhance the hydrogenase activity, which takes up the H2 produced by the nitrogenase activity, thus decreasing the net H2 production yield [START_REF] Li | Heterotrophic Photo Fermentative Hydrogen Production[END_REF].

Presence of bicarbonate

Some studies have shown that addition of bicarbonate and carbonate ions enhances the H2 production in PF (Montiel-Corona et al., 2015;Takabatake et al., 2004). Bicarbonate and carbonate function as electron acceptors and enhance the utilization of butyric and propionic acids, while their absence unbalances the oxidation-reduction potential resulting in decreased H2 production. Takabatake et al. (2004) reported that the presence of carbonate improves assimilation of ammonium (NH4 + ) and VFAs. They also observed that the uptake of acetate releases carbonate, however it was not enough to promote butyrate and propionate consumption, which are more oxidative than bacterial cells. For PNSB growth on butyrate, each mole of butyrate requires 0.7 mol of CO2 (Montiel-Corona et al. 2015).

Effect of light intensities and wavelength

The light conversion efficiency (η) varies for different PNSB strains because of their different light harvesting antenna pigments, thus they have a different photosynthetically active radiation (PAR) range. However, η also depends on the light intensity, illuminated area of the PBR, reactor design and other operational conditions of the PF process.

Generally, the intensity of light has a positive influence on the H2 production. There are some studies dedicated to assess the effect of the light intensity on growth and H2 production by PNSB (Koku et al. 2002;[START_REF] Uyar | Effect of light intensity, wavelength and illumination protocol on hydrogen production in photobioreactors[END_REF][START_REF] Sevinç | Kinetic analysis of photosynthetic growth, hydrogen production and dual substrate utilization by Rhodobacter capsulatus[END_REF][START_REF] Androga | Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710[END_REF][START_REF] Akman | Investigation of the effects of initial substrate and biomass concentrations and light intensity on photofermentative hydrogen gas production by Response Surface Methodology[END_REF]. [START_REF] Uyar | Effect of light intensity, wavelength and illumination protocol on hydrogen production in photobioreactors[END_REF] studied the effect of intensity of light, light wavelength and illumination protocol on the growth and H2 production by Rhodobacter sphaeroides O.U. 001 in photobioreactors (Figure 2.12). The hydrogen production increased with increasing the light intensity and the highest production was reached at 270 W/m 2 . The results also showed the decrease in photoproduction of hydrogen by 39% when there is a lack of infrared light (750-950 nm wavelength). The substrate conversion efficiency was increased and hydrogen production was stimulated when the light was illuminated after inoculation and no hydrogen was produced during the dark periods. [START_REF] Sevinç | Kinetic analysis of photosynthetic growth, hydrogen production and dual substrate utilization by Rhodobacter capsulatus[END_REF] studied the effect of temperature (20, 30 and 38 °C) and light intensity (1500, 2000, 3000, 4000 and 5000 lux) on the kinetic parameters and hydrogen production in PF of acetic and lactic acid using Rhodobacter capsulatus. The results of the study reported the maximum hydrogen production at 5000 lux for 20 °C and 3000 lux for 30 and 38 °C. In a more recent study, [START_REF] Androga | Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710[END_REF] established an optimal light intensity and temperature of 287 W/m 2 (4247.6 Lux) and 27.5 °C, respectively, in PF tests carried out using R. capsulatus DSM 1710 in a medium containing acetate, lactate and glutamate. In another recent study, [START_REF] Akman | Investigation of the effects of initial substrate and biomass concentrations and light intensity on photofermentative hydrogen gas production by Response Surface Methodology[END_REF] reported an optimum light intensity of 263.6 W/m 2 (3955 lux) in a PF study carried out with acetate as the carbon source and R. capsulatus, which is in accordance with the study from [START_REF] Androga | Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710[END_REF], that established 287 W/m 2 as optimum light intensity in PF carried out using R.

capsulatus.

Future development of PF systems requires an economical solution to provide the sources of light, so that outdoor systems utilizing natural sunlight become a practical option.

Therefore, research interests have been growing to exploit the natural sunlight in PF processes (Androga et al. 2012a;Montiel-Corona et al. 2015;[START_REF] Avcioglu | Biohydrogen production in an outdoor panel photobioreactor on dark fermentation effluent of molasses[END_REF][START_REF] Androga | Factors affecting the longterm stability of biomass and hydrogen productivity in outdoor photofermentation[END_REF]. Even though sunlight cannot ensure continuous light conditions, there are some studies that have shown that the dark and light cycles might not have significant effects on photo-H2 production [START_REF] Li | Effects of light/dark cycle, mixing pattern and partial pressure of H2 on biohydrogen production by Rhodobacter sphaeroides ZX-5[END_REF] or have positive effects on H2 production depending on the exposure duration of the light and dark conditions [START_REF] Sargsyan | Light-dark duration alternation effects on Rhodobacter sphaeroides growth, membrane properties and bio-hydrogen production in batch culture[END_REF]. Montiel-Corona et al. (2015) reported a 40.25% reduction in H2 yields during PF using mixed PNSB in comparison to indoor conditions. However, H2 yields obtained from outdoor reactors can be comparable to those under indoor conditions [START_REF] Androga | Factors affecting the longterm stability of biomass and hydrogen productivity in outdoor photofermentation[END_REF]). In addition to the type of light source, photofermentative H2 production also depends on other operating conditions of the PBRs, such as mixing conditions that affects the distribution of light, culture temperature and pH. Furthermore, harnessing the natural light in upscale applications of PF might reduce the cost of long-term PBRs operation.

Culture temperature and pH

The operating temperature of a culture is one of the important parameters that affects the bacterial metabolism or metabolic pathways as well as substrate conversion efficiency and thus H2 production. [START_REF] Basak | The Prospect of Purple Non-Sulfur (PNS) Photosynthetic Bacteria for Hydrogen Production: The Present State of the Art[END_REF] W/m 2 ) as optimum culture temperature for a higher H2 yield. Moreover, culture pH affects the biochemical reactions as it determines the ionic form of the active sites for enzymatic activity [START_REF] Chen | Perspectives on cultivation strategies and photobioreactor designs for photo-fermentative hydrogen production[END_REF]. PF studies have been carried out in the pH range varying between 5.5 to 7.5 (Table 2.12 and 2.14). Akroum-Amrouche et al. ( 2011) reported an optimum pH of 7.5 (± 0.1) for the H2 production by Rhodobacter sphaeroides, while Nath and [START_REF] Das | Advances in biohydrogen production processes: An approach towards commercialization[END_REF] have reported an optimum H2 production at pH 6.5 for the same PNSB species. This difference of change in optimum pH can be attributed to the difference in substrate type used in PF experiments as lactate was used as a sole carbon source in the former, while DF spent medium was used in the latter study. In another study, Koku et al. (2002) reported an optimum pH of 7.1 -7.3 for the activity of the nitrogenase enzyme, while the range of 6.5 to 7.5 is optimum for the activity of the hydrogenase enzyme.

During most of the PF tests, pH has shown an increasing trend which could be due to PHB production (Khatipov et al. 1998;[START_REF] Nath | Effect of light intensity and initial pH during hydrogen production by an integrated dark and photofermentation process[END_REF]. [START_REF] Eroglu | Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor[END_REF] reported a slight decrease in pH during the bacterial growth phase and pH increase during H2

production. The effluents from DF are generally in the acidic pH range (Ghimire et al. 2015), and are required to be adjusted to a pH range 6.5 -7.5 to ensure the optimum operating conditions in the PF process. However, the range of optimum pH seems to be dependent on the PNSB species. Some studies by [START_REF] Tawfik | Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent[END_REF] and Tao et al. (2008) have shown the feasibility of H2 production by mixed PNSB at pH 5.5 -6.0, which is generally an ideal pH range of DFE obtained from DF processes.

Effect of mixing

Mixing is required in PBRs to keep the PNSB biomass suspended and uniformly distribute the substrates and nutrients in the culture medium. Moreover, mixing ensures the uniform distribution of light throughout the PBRs, avoiding light gradients. It also helps to maintain sufficient mass transfer, which generally includes the exchange of gases, i.e. H2 and CO2. Akroum-Amrouche et al. ( 2011) found unstable H2 production with a 13.0% and 60.8% reduction of the average and maximum H2 production rate when mixing was stopped during the exponential phase of PF. In another study, [START_REF] Li | Effects of light/dark cycle, mixing pattern and partial pressure of H2 on biohydrogen production by Rhodobacter sphaeroides ZX-5[END_REF] reported that mixing during the H2 production phase of the PNSB stationary growth phase as vital for higher H2 yields than during the exponential cell growth phase. Moreover, the type of mixing system may also affect the photo-H2 production performance. Zhang et al. (2015) showed that baffled PBRs can outperform magnetic-stirred PBRs as supported by higher H2 yields as well as faster cell growth and substrate conversion. This higher H2

production can be attributed to enhanced gas transfer and distribution of light in the PBRs due to well mixing conditions.

Inhibition of photo-H2 production

Nitrogenase plays an important role in the hydrogen generation. Thus, the presence of chemical substances that disrupt the nitrogenase activity decreases the photo-H2 production. Koku et al. (2002) reported that the presence of N2 and NH4 + inhibit the H2 production. Also CO, EDTA and O2 are likely to inhibit the nitrogenase activities.

Similarly, an elevated level of CO2 inside the reactor inhibits the photo-H2 production, while lower levels (4 -18% w/v) favor the growth phase of PNSB and thus H2 production (See Carbon sources and nutrients requirements). Furthermore, a lower C/N ratio does not favor photo-H2 production as it could result in the accumulation of ammonium and inhibition of nitrogenase in a PF process for H2 production.

PBR systems

PBR reactor configurations

The design considerations of PBRs for photo-H2 production are similar to those of PBRs for algal biomass production. However, anaerobic conditions are required for the PF process using PNSB. Most of the published reviews on the design of PBRs for biohydrogen production are based on bioreactors for algal biomass production [START_REF] Akkerman | Photobiological hydrogen production: photochemical efficiency and bioreactor design[END_REF][START_REF] Dasgupta | Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production[END_REF][START_REF] Carvalho | Microalgal reactors: a review of enclosed system designs and performances[END_REF]. In some more recent

Tubular reactors

Tubular PBRs contain a long transparent tube with a length ranging from 10 to 100 meters and diameters ranging from 3 to 6 cm [START_REF] Akkerman | Photobiological hydrogen production: photochemical efficiency and bioreactor design[END_REF]. These PBRs are one of the most suitable reactors for outdoor conditions. Generally, tubular PBRs are constructed with transparent glass or plastic tubes. The culture is recirculated with a mixing system (such as a pump) to provide efficient mass transfer and equal light distribution. One of the major problems during the scaling up of tubular PBRs is the decrease in illumination surface to volume ratio because of the increase in diameter of the tube. This causes a decrease in light intensity (light shading effect) for the cells at the lower part of the tube, which negatively affects the cell growth. However, a good mixing system provides also an efficient light distribution [START_REF] Ugwu | Design of static mixers for inclined tubular photobioreactors[END_REF].

Vertical-column reactors

Vertical-column reactors have been subject of research for algal biomass production as they are compact, have low cost and are easy to operate [START_REF] Ugwu | Photobioreactors for mass cultivation of algae[END_REF]. Bubble columns, airlift reactors and annular column reactors are common vertical-column PBR configurations [START_REF] Posten | Design principles of photo-bioreactors for cultivation of microalgae[END_REF]. Bubble column reactors have a larger diameter than tubular reactors and are frequently used indoor (at a larger lab scale) or outdoor. Because of the larger diameters in these reactors, darker zones are created at the center of the column, which might be disadvantageous for photosynthetic bacterial growth. Besides these three major reactors types, laboratory scale PF research has been carried out in internally illuminated reactors (Chen et al. 2010). [START_REF] Dasgupta | Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production[END_REF] have briefed the possibility of using different configurations such as torus shaped and helical reactors.

The concept of an annular column reactor aims to overcome the problem associated with the central darker zones in bubble column reactors [START_REF] Posten | Design principles of photo-bioreactors for cultivation of microalgae[END_REF]. The major advantages of this reactor configuration are high mass transfer rate, good mixing conditions with less shear on bacterial cells, low energy consumption and potential for industrial application.

However, the small illumination surface makes this configuration less competitive than other counterparts.

Comparison between panel and tubular PBRs

Table 2.15 compares studies done in various configurations of PBRs. Flat panel and tubular reactors have the highest theoretical efficiencies and have been used at pilot scale under outdoor conditions [START_REF] Boran | Biological hydrogen production by Rhodobacter capsulatus in solar tubular photo bioreactor[END_REF][START_REF] Eroglu | Hydrogen production by Rhodobacter sphaeroides O.U.001 in a flat plate solar bioreactor[END_REF][START_REF] Gebicki | Comparison of two reactor concepts for anoxygenic H2 production by Rhodobacter capsulatus[END_REF]).

These studies opened perspectives for scaling up of these two promising PBRs for photo-H2 production using PNSB cultures. In some lab scale studies, higher H2 productivities were obtained with flat panel PBRs, while some studies with tubular PBRs have shown good performance under outdoor light conditions. Moreover, tubular PBRs are easier to manage and scale-up.

Photo-H2 production through PF can be a promising technology for clean energy recovery. In addition, recovery of PHB can be of further interest. To establish PF as post treatment of DFE, more research needs to be performed for improving the system efficiency through optimization of different operating parameters. The system efficiency can be improved by providing optimum culture conditions and bioreactor design. The PF systems have been presented as the bottlenecks in the integrated DF-PF process because of their higher production cost. Thus, innovative low-cost mixing, heating and cooling systems need to be explored and PBR designs for improving the surface area to volume (A/V) ratio require future research. Moreover, PHB can add economic value to the PF process. Using mixed PNSB to utilize the conversion efficiencies of different microbial consortia can give an economic advantage by the reducing cost of H2 production. [START_REF] Tredici | Efficiency of Sunlight Utilization: Tubular Versus Flat Photobioreactors[END_REF] a Photosynthetic efficiency of the cultures was calculated by multiplying the reactor productivity by the mean enthalpy value of the biomass of A. platensis M2 cultivated outdoors (21.56 kJ g -1 ) and divided by the mean visible solar energy input on the culture surface (14.08 MJ/d). Other photosynthetic efficiency was calculated using equation 2.20.

Design considerations for PBRs

In addition to the physical parameters such as quantity of light penetrating into the bioreactor, a good PBR design should consider various physiochemical parameters such as pH, temperature, dissolved oxygen and CO2, shear due to agitation, C/N ratio, carbon sources and availability of nutrients. As mentioned earlier, these parameters influence various biochemical pathways and ultimately the H2 production in PBRs.

A general consideration to achieve a good design of PBRs as reported by [START_REF] Dasgupta | Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production[END_REF] includes the following physicochemical parameters which affect the performance of PBRs: high light penetration into PBRs high surface area to volume ratio (higher illumination area) temperature and pH control good mixing system better gas exchange or mass transfer transparency and durability of the materials

Surface area to volume (A/V) ratio

The amount of light absorbed by a reactor system is a limiting factor in PBR systems.

Surface area to volume ratio is one of the important parameters to be considered during the design of PBRs as it determines the amount of light entering into the system. The higher the A/V ratio, the larger will be the surface area for receiving light for growth and metabolism. Therefore, the A/V ratio can be directly co-related with cell concentration and the volumetric productivity of the system [START_REF] Dasgupta | Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production[END_REF]).

Gebicki et al. (2009) compared hydrogen productivities of a flat panel (A/V ratio of 20

m -1 ) and an inclined horizontal tubular (A/V ratio of 15.38 m -1 ) PBR with respect to illuminated surface area and ground area occupied by the reactor. The mean hydrogen productivity of the flat panel reactor was 1250 mlH2/(m 2 illuminated surface/day), while that of the tubular reactor was 1100 mlH2/(m 2 illuminated surface/day). The illuminated area per unit ground area occupied by the panel reactor was 8.9 times higher than that of the tubular reactor, which gives the economic edge of the comparison. However, a fenced type tubular PBR (Figure 2.13) could be a research interest in the future as this reactor configuration occupies less space compared to inclined horizontal tubular PBR.

Mixing systems

Mixing systems in PBRs could include pumping, mechanical stirring and airlift mixers. [START_REF] Ugwu | Design of static mixers for inclined tubular photobioreactors[END_REF] proposed a static mixer for tubular bioreactors. The selection of the type of mixing system is important as the pumps used for mixing or recirculation exert shear forces that might be harmful to PNSB. Another disadvantage of the mixing system is the additional cost due to the required energy for its operation.

Construction materials

Selection of materials during the construction of PBRs not only determines the economy, but also the performance of the system. Several factors should be considered while selecting the construction materials. PBRs can be constructed from glass, polyvinyl chloride (PVC) material, low-density polyethylene (LDPE), poly-methyl methacrylate (PMMA) and fiberglass. [START_REF] Dasgupta | Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production[END_REF]

Mathematical modeling of growth and product kinetics of PNSB

Knowledge on the kinetics of the biological process becomes vital to have a better design and control of the process. The strong influence of operational parameters such light intensity and substrate concentrations on photofermentative H2 and PHB synthesis has been demonstrated [START_REF] Uyar | Effect of light intensity, wavelength and illumination protocol on hydrogen production in photobioreactors[END_REF][START_REF] Androga | Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710[END_REF]Hustede et al. 1993;Han et al. 2012;[START_REF] Wu | Correlation between bio-hydrogen production and polyhydroxybutyrate (PHB) synthesis by Rhodopseudomonas palustris WP3-5[END_REF]). However, very limited work has been done on the kinetic analysis of the photofermentation process [START_REF] Gadhamshetty | Photofermentation of malate for biohydrogen production-A modeling approach[END_REF]Zhang et al. 2015;Koku et al. 2002).

Biomass growth

Few mathematical models have been proposed to study growth kinetics of PNSB cultures.

A theoretical cell growth rate can be expressed as:

dX dt = μX -𝑚𝑋 (2.21)
Where: X is the cell dry weight concentration (g/L), m is maintenance coefficient for biomass (decay rate) and μ is the specific growth rate (h -1 ). [START_REF] Gadhamshetty | Photofermentation of malate for biohydrogen production-A modeling approach[END_REF] proposed the Monod equation to provide the expression for μ to model the growth curve in a batch PBRs with the assumptions that sufficient light and optimal C/N ratio is available under stressful nitrogen concentrations. The proposed model simulates the biomass growth under substrate-limited conditions as:

dX dt = μX = ( μ m S K s + S ) X (2.22)
where: the specific growth rate μ (hr -1 ) depends on both maximum specific growth rate μm (hr -1 ) and the half saturation constant KS (mg/l).

However, the growth curve obtained for R. sphaeroides O.U. 001 deviated from the Monod model (Koku et al., 2003). The Equation 2.22 needs to include the substrate inhibition and inhibition due to higher biomass concentration. Moreover, the inhibition from higher substrate levels could be due to osmotic stress and/or the presence of one or more unknown inhibitors such as pigments [START_REF] Gadhamshetty | Photofermentation of malate for biohydrogen production-A modeling approach[END_REF]). Besides higher biomass concentration reduces the light intensity inside the PBR, causes self-shading effects and limits the substrate diffusion, which in turn affects the hydrogen production.

Thus, the specific growth rate (μ) in Equation 2.22 is modified in Equation 2.23 to include the two inhibitory effects:

𝜇 = 𝜇 𝑚 𝑆 𝐾 𝑠 + 𝑆 + 𝑆 2 𝐾 𝑋𝑖 (1 - 𝑋 𝑋 𝑚 ) (2.

23)

The inhibitory effect due to biomass concentration is provided by a Logistic model. The term "Xm" is the maximum cell dry mass concentration at which growth will cease. The specific growth rate in the Equation 2.23 is further modified to include the effect of the light exposure on PNSB. The modification included the declining effect of excess light on biomass growth as the surplus absorbed light energy may results in damage and degradation of the reaction center involved in the photosynthetic process. The final equation is expressed as:

𝜇 = 𝜇 𝑚 𝑆 𝐾 𝑠 + 𝑆 + 𝑆 2 𝐾 𝑋𝑖 (1 - 𝑋 𝑋 𝑚 ) ( 𝐼 𝐾 𝑋𝐼 + 𝐼 + 𝐾 𝐼 𝐼 2 ) (2.24)
The smaller the value of KI, the larger is the inhibition effect of light on PNSB growth.

Consumption of substrate

The Contois model can be used to describe the consumption of the substrate: where: 'Pi' is the concentration of the product 'i' and 'YPi/X' is the yield of product 'i' with respect to biomass 'X'.

𝑑𝑆 𝑑𝑡 = - 𝜇 𝑚 𝑆 𝑌(𝑆 + 𝐾 𝑆 𝑋) 𝑋 (2.25) or 𝑑𝑆 𝑑𝑡 = 1 𝑌 𝑆/𝑋 𝜇𝑋 (2.26)

Relation between biomass growth and product formation

Similarly, the formation of products with respect to consumption of substrate can be written as:

𝑑𝑃 𝑖 𝑑𝑡 = -𝑌𝑃 𝑖 𝑆 𝑑𝑆 𝑑𝑡 (2.

29)

where: 'Pi' is the concentration of the product 'i' and 'Ypi/s' is the yield of product 'i' with respect to substrate 'S'.

The growth of biomass can be expressed in relation to the substrate consumption as:

𝑑𝑋 𝑑𝑡 = -𝑌𝑋 𝑆 𝑑𝑆 𝑑𝑡 (2.30)
where: 'X' is the concentration of the biomass and 'YX/s' is the yield of biomass with respect to the substrate 'S'.

On integrating Equation 2.30 from initial concentration (S0) to final substrate concentration (S) and product (from initial concentration of 0 to final product concentration Pi), it is possible to write the following equations: with i = H2 and PHB.

𝑑𝑃 𝑖 = -
These relationships can be applied to model the kinetics of substrate consumption, PNSB growth and products formation (H2 and PHB) in the PF process.

Future perspectives

Economics

There are very few studies aimed at determining the economics of photo-H2 production [START_REF] Benemann | Feasibility analysis of photobiological hydrogen production[END_REF]HYVOLUTION 2011[START_REF] Benemann | Feasibility analysis of photobiological hydrogen production[END_REF] presented an economic analysis of a conceptual two-stage process where microalgae are used to produce a carbohydrate rich biomass cultivated in large open ponds and hydrogen will be produced in tubular photobioreactors. The paper reported the estimated overall total hydrogen production costs of 9.5 $/GJ.

An integrated process of biohydrogen combining thermophilic dark fermentation followed by photofermentation had a biohydrogen production cost of 10 €/GJ (~13.42 $/GJ), i.e. 1.21 €/kgH2 (~1.62 $/kgH2) based on the lower heating value of H2 (HYVOLUTION, 2011). In the integrated concept, the total cost per kilogram of biohydrogen is 56 € (~75.15 $), which is much higher than that of hydrogen from coal (0.36 -1.83 $/kgH2) and natural gas (2.48 -3.17 $/kgH2) [START_REF] Bartels | An economic survey of hydrogen production from conventional and alternative energy sources[END_REF]HYVOLUTION, 2011). The standalone thermophilic dark fermentation process costs 21

€/kgH2 which is lower than the combined system, thus the PF needs improvement to lower the per unit production cost. In their study, HYVOLUTION (2011) estimated the capital cost of commercial scale tubular and flat panel photofermenters at 91 and 332 million € (122.11 and 445.51 $), respectively. However, the capital cost depends on several factors such as reactor size, material and labor cost.

The light conversion efficiencies of the PF play an important role in determining the economics of photo-hydrogen production. In addition, the substrates and the PNSB strains are also crucial factors. The selection of PBRs also influences the capital and operational cost and in the end, the unit cost of the photo-hydrogen production (HYVOLUTION 2011).

Integration with dark fermentation

PF can be applied as a post treatment stage on DFE, which mostly contains organic acids and alcohols (Figure 2.14). The integrated DF-PF process has been demonstrated by several studies (Rai et al. 2014;[START_REF] Tawfik | Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent[END_REF]Yang et al. 2015). DF has the unique capability to utilize a wide range of complex waste biomass that can ensure the future supply of feestock, and combining the two processes (DF + PF) can provide the complete conversion of organic substrate in addition to enhanced H2 yields. Typical chemical reactions of conversion of organic acids produced in mixed type fermentation to photo-H2 are presented in Equations 2.9 -2.12. [START_REF] Redwood | Integrating dark and light bio-hydrogen production strategies: towards the hydrogen economy[END_REF] reviewed different possible integration strategies for coupling DF-PF processes. In general, DF-PF systems can be integrated in three possible ways; i) utilizing DFE produced in PF systems, ii) cultivating dark and photofermentative microorganisms in one reactor system [START_REF] Chandra | Single-Stage Operation of Hybrid Dark-Photo Fermentation to Enhance Biohydrogen Production through Regulation of System Redox Condition: Evaluation with Real-Field Wastewater[END_REF][START_REF] Liu | Bio-hydrogen production by mixed culture of photo-and dark-fermentation bacteria[END_REF] or iii) separating the two systems by a physical barrier such as a membrane (Redwood et al. 96 2011; [START_REF] Liu | Simultaneous hydrogen and ethanol production from cascade utilization of mono-substrate in integrated dark and photo-fermentative reactor[END_REF]. DF followed by photo-H2 production is well studied by many researchers (Ghimire et al. 2015).

Depending on the process operating parameters such as pH, substrate loading and substrate type, DFE generally has an acidic pH (< 6.0) and inhibiting levels of ammonia and organic acids. Therefore, the DFE requires pre-treatment such pH adjustment, dilution and removal of ammonia before feeding into a PF process. Ammonia concentrations exceeding 2 -5 mM inhibit the photo-H2 production (Lee et al. 2011;Argun et al. 2008). Therefore, substrates with a higher C/N ratio are usually preferred for PF. Depending on the DFE requirements, several ammonia removal strategies such as stripping, treatment with natural zeolites and membrane processes can be applied (Androga et al. 2012b;Redwood et al. 2012). However, most continuous dark fermentative processes lack high ammonia levels due to incomplete conversion of proteins or amino acids present in the substrates, making them ideal substrates for the PF processes.

Evaluation of methods for obtaining H2 producing seed inoculum for dark fermentation

This section summarises the results of the study performed on the enrichment of microbial communities for enhancing hydrogen production in dark fermentation (DF) using mixed culture, which considerably affect the overall performance. This work evaluates the following pre-treatment methods: acid treatment, heat shock (at 95 °C and 105 °C) and load shock pre-treatment, keeping into account scaling-up of DF systems. Further insights are also provided on the safety aspects concerning the production and storage of H2, and on the importance of operational costs and feasibility of the pre-treatment methods.

Introduction

The progressive running down of fossil fuel reserves coupled with the need of reducing the greenhouse gas (GHG) emissions in the atmosphere has made the development of new, renewable and environmental friendly energy sources very crucial. Hydrogen (H2) biologically produced from organic wastes seems to be really promising, due to its efficient hydrogen to power conversion coefficient (3.0 kWh/Nm 3 ), high energy density (142 MJ/kg) and harmless combustion by-products [START_REF] Cardoso | Hydrogen Production by Dark Fermentation[END_REF]. To this aim, either photo fermentation (PF) or dark fermentation (DF) processes have been successfully used to biologically produce H2 from organic sources. However, DF is usually preferred to PF due to lower operational costs and process conditions at ambient temperature and pressure (Das and Veziroglu, 2008).

The biological conversion of organic sources into H2 is obtained by using biomasses either consisting of pure cultures or composed of mixed cultures. Mixed systems are generally less performing in terms of H2 yields, but are easier and less expensive to handle as they do not require any asepsis procedure and can be fed with several different substrates, as reported in previous studies (Valdez-vazquez et al., 2004). Here it is worth noting that mixed bacteria communities with the ability of producing H2 are intrinsically present in soils, sediments, sludge from wastewater treatment plants, compost, cow dungs, municipal organic solid wastes (Wong et al., 2014). Hence, these communities can be enriched by appropriate pre-treatment methods, although higher H2 production rate can be only obtained if H2 consuming organisms such as methanogens and homoacetogens are inhibited (Wang and Wan, 2009).

The most commonly and successfully used biomass pre-treatment methods include heat (Wang and Wan, 2008), acid (Wang and Wan, 2008), base (Zhu and Beland, 2006), and load shock (Luo et al,. 2010) as well as aeration [START_REF] Giordano | Evaluation of aeration pretreatment to prepare an inoculum for the two-stage hydrogen and methane production process[END_REF]. These methods are based on the observation that when the biomass experiences hostile environmental conditions, H2 producers survive due to their ability in forming spores (e.g. Clostridium)

that protect them from the adverse conditions, hence returning to be effective again when the environmental conditions turn to be favourable as the spores germinate (Li and Fang, 2007). Besides, the H2 consumers may not survive unless with same capacity.

The effectiveness of these pre-treatments on H2 production depends on nature of biomass, which in turn can cause the occurrence of inconsistency in results from lab scale experiments (Wang and Wan, 2009). Therefore a deeper knowledge of the effects that pre-treatment methods have on H2 production from DF is necessary before operating the scaling up of these methods as well as, being H2 highly flammable and explosive, safety aspects in large-scale reactors are also a primary concern.

The aim of this paper is to evaluate the effectiveness of the following pre-treatment methods (i) acid shock treatment, (ii) heat shock treatment and (iii) load shock pretreatment on H2 production through bio-H2 potential DF batch (BHP) tests. The evaluation has been done by analysing the following parameters from the BHP tests: (i) cumulative H2 production; (ii) H2 production rate; (iii) length of the lag phase; and (iv) production of process intermediates. Furthermore, this study also deals with the safety aspects concerning the production and storage of H2 (USEPA, 2011) and highlights the relevance of operational cost, feasibility and complexity of the pre-treatment methods in scaled up systems.

Materials and methods

Biomass used to perform the BHP tests was collected from the anaerobic digester treating dairy waste produced by the factory "La Perla del Mediterraneo" located in Capaccio (Salerno, Italy). The total solids (TS) and volatile solids (VS) content of biomass were 2.79 ± 0.05 % (w/w on wet mass) and 67.2 ± 0.4 % (w/w on dry mass). The sludge was stored at 4 °C before being used. The BHP tests were fed with glucose.

All BHP tests were carried out in 1,000 mL transparent borosilicate glass bottles GL 45 (Schott Duran, Germany) used as DF batch reactors and placed in a water bath maintained at 34 ± 1 °C by a thermostat (ALEAS AL 2201, 150 W). In the batch reactors, airtight conditions were provided with caps sealed with silicon. Each bottle was equipped to sample the internal mixture and spill out the gas. BHP tests were carried out in duplicates at the initial pH of 7.

Heat shock treatments were carried out by heating the biomass at 105 °C for 4 h (HST-105°C) and at 95 °C for 45 min (HST-95°C); acid shock treatment (AST) was performed by adjusting the pH of the biomass at pH 3 using 1 M HCl for 24 h and then turning pH back at 7 using 1 M NaOH; load shock (LST) treatment was carried out by feeding the batch reactors with 85 g COD/L of glucose followed by acidification process for 4 days and finally extracting the supernatant after a settlement process and replacing the extracted liquid volume with distilled water. A substrate to biomass ratio of 0.85 g COD glucose/g VS biomass was maintained in all BHP tests. Once the cumulative H2 production in the reactors reached a stable value (Load I), the reactors were furthermore fed with 4.5 g of glucose (Load II).

The volume of gas produced from each BHP tests was measured on daily basis by acid solution (1.5 % HCl) displacement method. The biogas volumes were corrected for moisture at 0°C and 1 atm (NmL) and reported as the daily average. H2, CO2 and CH4 content in gas were measured with Varian Star 3400 gas chromatograph equipped with ShinCarbon ST 80/100 column provided with a thermal conductivity detector and argon as carrier gas. Samples of the digesting mixture collected from each reactor to measure the volatile fatty acids (VFAs) content and their composition were preliminarily extracted at 80⁰C according to the head space-solid phase micro-extraction technique (HS-SPME) [START_REF] Abalos | Development of a headspace solidphase microextraction procedure for the determination of free volatile fatty acids in waste waters[END_REF] and subsequently analysed with gas chromatograph equipped with mass spectrometry provided with helium as carrier gas. The pH was measured with a pH meter (WTW, inolab, pH level 2). The TS and VS content of biomass and organic wastes were determined according to Standard Methods (APHA, 2005).

The modified Gompertz relationship (equation 3.1) was used to model the H2 production from BHP tests (Wang and Wan, 2008). The equation contains 3 parameters: i) cumulative H2 production potential Ho (mL), ii) H2 production rate R (mL/h), iii) lag time λ (h). Ho, R and λ were estimated from BHP test by using the Curve Fitting Toolbox in MATLAB®.

H(t) = H o • exp {-exp [ R.e H o ] (λ -t) + 1} (3.1)
Where t is the time.

Results and discussions

The results from BHP tests are shown in Figures 3.1 and3.2 andTables 3.1 and3.2. In Figure 3.1, the effects of different biomass pre-treatment methods are represented by plotting the average cumulative H2 production, whereas in Tables 3.1 and 3.2, the same effects are evaluated comparing the specific H2 production and the parameters calibrated by using equation 3.1. production performance with highest specific H2 production (143.5 ± 13.2 NmL/g glucose), H2 production rate (9.4 NmL/h) and a lag phase slightly longer (0.53 h) than HST-95°C, while AST showed the lowest lag time (13.57 h). AST also gave good cumulative H2 production (373.1 NmL) whereas BHP in the tests with HSTs was low.

From the analysis of methane content in biogas, it can be concluded that there were negligible methanogenic activities in the tests with LST, HST-105°C and AST whereas the BHP tests with HST-95°C was unable to completely inhibit the methanogenic microorganisms, which could explains the lower H2 production. After the batch reactors were fed with a second load of glucose (Load II), the H2 yield decreased in the BHP tests with LST, HST-105⁰C and AST whereas it increased in tests with HST-95C (Figure 3.1). In Table 3.2 the specific H2 production obtained from the first (Load I) and the second (Load II) feeding operation as well as the respective pH values at the beginning and at the end of the BHP tests are compared. Figure 3.2 shows the major fermentative products accumulated at the end of the BHP tests. The production of intermediates (VFAs) and pH values were monitored in order to evaluate the performance of DF process. A possible reason for the lower H2 yield than expected when a LST was performed could actually be explained with the occurrence of the inhibiting effect due to the high butyric acid accumulation in the reactor, as indicated in the study published by Van Ginkel and Logan (2005), whereas a low pH (3.7±0.44) could be the cause of the lower H2 production in AST during Load II. In order to select and set up a method to pre-treat the biomass in full scale reactor, several parameters need to be considered: the operational costs, the feasibility and complexity of the method as well as the time required to enrich the biomass with H2 producing bacteria (safety will be considered in the next section) Table 3.3 shows a simple evaluation of the parameters based on this study and literature data for the four pre-treatment methods investigated in this paper. HSTs show a high energy demand, which makes them less attractive in a full scale application. AST requires large amounts of acid and base solutions. LST is more feasible to be used in a full-scale reactor due to lower operational costs compared with the other methods. The H2 production and process performance are strongly influenced by many factors such as physico-chemical properties of substrate and co-substrates, type of biomass sources, reactor configuration, and operational conditions. Luo et al. (2010) actually evaluated the effects of different pre-treatment methods on mixed culture for H2 production using cassava stillage as substrate and found differences in H2 yields only when DF was performed in batch reactors, whereas no difference was noticed in continuous DF processes.

Safety considerations on scale-up

Several accidents can be found in the literature due to severe reactivity of biogas. Hence, specific analyses are due for this mixture for the correct design of prevention and mitigation systems (e.g. venting, suppression), and for the structural design of the reactors, including auxiliary and transportation systems (USEPA, 2011).

When batch reactors are adopted, the isochoric-isotherm option should be considered for the hazard of hydrogen mixture. By using the ideal gas equation, the calculated maximum pressure in the lab reactors varied from 2.11 to 2.15 bar, considering a reactor head space 540 mL, reactor temperature of 35 °C and ambient conditions 25 °C and 1 bar for the measurement of the biogas. Quite clearly, due to anaerobic conditions, the reactors are flushed with nitrogen and no hazards are predicable unless oxygen (air) leakage due to rapid depressurisation and oxygen (air entrance). On the other hand, the continuous operations adopted in large-scale reactors are normally operated under ambient conditions and air. Hence, a deflagration or even a detonation of the mixture of hydrogen possibly mixed with several other oxidation components that are typical in large-scale biomass operation as CO, CO2, methane and other low-weight gases, including toxic H2S, may occur.

The literature on the safety characterisation of complex biogas mixtures is very scarce and mainly based on experimental observations [START_REF] Cammarota | Journal of Loss Prevention in the Process Industries Combined effects of initial pressure and turbulence on explosions of hydrogen-enriched methane / air mixtures[END_REF], as no additive methodologies are applicable for the definition of flammability limits, burning velocity, and for the definition of occurrence of dramatic scenarios as deflagration to detonation transition or combustion-induced Rapid Phase Transitions [START_REF] Salzano | Explosion behavior of hydrogen-methane/air mixtures[END_REF].

In large-scale reactors, H2 might ranges between 40 % to 50 % v/v however with inerts as CO2 (50 -60 % v/v) and water vapour (1 -5 %) and operation are conducted under thermophilic temperature ranges (55 -60 °C) in comparison or mesophilic reactors (35-40 °C). For ambient conditions, may be adapted the analysis reported in (Di Benedetto et al., 2009), that clarified the effect of CO2 on H2 burning, which is essentially thermal, and the ranges of adiabatic flame temperature (i.e. adiabatic pressure) and laminar burning velocity for the given mixtures obtained by means of both experimental and numerical analysis. Stable flames (for the use in combustion equipment) or, conversely, flame extinguishing (for fire and explosion safety) are obtained, at ambient temperature, for CO2 larger than 40 % v/v in air, hence in the presence of N2. The effect at higher temperature has to be defined in future works.

Conclusions

The evaluation of results from the BHP tests and the analysis of different pre-treatment methods suggest that LST of biomass can favour the development and growth of an efficient H2 producing bacteria community to start-up and handle up-scaled DF systems.

Moreover, monitoring of metabolites production and pH can give useful information on process performance and its reliability, thus helping to prevent VFAs accumulation and the subsequently occurrence of inhibition phenomena affecting the H2 producing biomass activity. Also, safety aspects need to be taken into consideration in the up-scaled DF systems during H2 production, storage and application.

Effects of operational parameters on dark fermentative H2 production

This section presents the findings on the effect of initial pH, combination of food to microorganism ratio (F/M) and initial pH, substrate pre-treatment and different inoculum sources on the dark fermentative H2 yields obtained using three model complex waste biomass: food waste, olive mill wastewater (OMWW) and rice straw. The cumulative H2 production, H2 production rate, lag time for H2 production and accumulation of metabolites were used as comparison parameters to determine the optimal conditions for H2 production carried out in a series of batch tests.

Introduction

Dark fermentation (DF) of organic waste is one of the promising technologies for biohydrogen (H2) production. The DF processes are usually preferred over other light dependent, photofermentation or biophotolysis processes because of the high bioreactor productivities and the potential to utilize a wide range of organic wastes as feedstock (Hallenbeck et al., 2009;Urbaniec and Bakker, 2015). In addition, the associated production of organic acids and alcohols, among others, can be either used in sidestream processes like anaerobic digestion for methane or photofermentative H2 production for energy recovery, or can be used for the production of platform molecules (Bastidas-Oyanedel et al., 2015;[START_REF] Sarma | Hydrogen biorefinery: Potential utilization of the liquid waste from fermentative hydrogen production[END_REF].

Waste biomass is abundant and can sustain DF processes in scaled-up applications. Easily degradable food waste (the organic fraction of municipal solid waste (OFMSW)), more slowly degradable agricultural residues (i.e. rice straw) as well as agro-industrial waste such as olive mill wastewaters (OMWW) can serve as sustainable feedstock sources for dark fermentative H2 production (Guo et al., 2010;Kapdan and Kargi, 2006;Ntaikou et al., 2010;Show et al., 2012). A major bottleneck in the utilization of these low cost waste biomasses is the rather low H2 yields observed in the DF processes (Ghimire et al., 2015a;Urbaniec and Bakker, 2015). Nevertheless, H2 yields and process kinetics can be enhanced by optimizing operating parameters, such as pre-treatment of inocula, food to microorganisms (F/M) ratio (also substrate to inoculum ratio), pre-treatment of substrates, culture temperature and pH (De Gioannis et al., 2013;Guo et al., 2010;Ntaikou et al., 2010;Wang and Wan, 2009). During recent years, extensive experimental research has been devoted to establish the optimal operational conditions for maximizing H2 production, with a special focus on operational pH, temperature and substrate utilization (De Gioannis et al., 2013;Ghimire et al., 2015a;Wong et al., 2014).

A wide range of optimal pH values have been reported for different substrates to enhance H2 yields: an initial pH of 6.5 for food waste (Cappai et al., 2014), initial pH of 8.0 for food waste (Kim et al., 2011), a controlled pH of 7.0 for vegetable kitchen waste (Lee et al., 2008), an initial pH of 6.5 for rice straw (Chen et al., 2012), an initial pH of 6.0 for cheese whey (De Gioannis et al., 2014) and an initial pH of 4.5 for sucrose and starch (Khanal et al., 2004). This considerable variability in culture pH is mainly due to differences in temperature, substrate type and concentration (F/M ratio), inoculum types and their pre-treatment methods.

H2 yields in DF of organic waste are strongly affected by the operational temperature as it can influence the rate of hydrolysis and the production of volatile fatty acids (VFAs)

and thus the final pH of the fermentation (De Gioannis et al., 2013;Ghimire et al., 2015a).

A thermophilic temperature has been reported to favor the dark fermentative H2 production (Shin et al., 2004;Valdez-vazquez et al., 2005). Likewise, the physicochemical characteristics of the substrates, and most importantly the biodegradability or bioavailability (can also be defined as the fraction of easily accessible carbohydrates for fermentative conversion) crucially affects the H2 production (Monlau et al., 2013a).

Therefore, several studies have established a strong correlation between H2 yields and the initial carbohydrate fraction (soluble sugars in some cases) present in the substrates (Alibardi and Cossu, 2015;Guo et al., 2013;Monlau et al., 2012).

In this context, alkaline pre-treatment methods have been popularly adopted for the saccharification of lignocellulosic biomass (plant stalks, rice and wheat straw), which could enhance the production of H2 in DF and CH4 in DF coupled to anaerobic digestion, respectively and could thus give economic credentials [START_REF] Monlau | Alkaline pretreatment to enhance one-stage CH4 and two-stage H2/CH4 production from sunflower stalks: Mass, energy and economical balances[END_REF](Monlau et al., , 2013c;;[START_REF] Sambusiti | A comparison of different pre-treatments to increase methane production from two agricultural substrates[END_REF]. Alkaline pre-treatment of lignocellulosic biomass has been reported to be carried out at different concentrations of alkaline agents (2 -12% NaOH, weight basis), temperature (40 -190 °C) and treatment period (30 minutes -24 hours), with varying level of effectiveness in terms of increase in biogas yields (H2 and CH4) with consequent higher net energy recovery and economic return [START_REF] Monlau | Alkaline pretreatment to enhance one-stage CH4 and two-stage H2/CH4 production from sunflower stalks: Mass, energy and economical balances[END_REF](Monlau et al., , 2013b;;[START_REF] Sambusiti | A comparison of different pre-treatments to increase methane production from two agricultural substrates[END_REF]. However, alkaline agents (i.e. Na + from NaOH) might exert inhibitory effects on dark fermentative microbial communities (Kim et al., 2009).

Consequently, an investigation of selected alkaline pre-treatment conditions for a particular substrate type becomes vital to study the conditions that enhance the H2 production.

H2 production from organic waste is influenced by the presence of an effective hydrolyzing, H2 producing microbial community, which depends on the inoculum source and inoculum pre-treatment method [START_REF] Abreu | Inoculum type response to different pHs on biohydrogen production from larabinose, a component of hemicellulosic biopolymers[END_REF][START_REF] Bellucci | Inoculum pretreatment affects the fermentative activity of hydrogen-producing communities in the presence of 5-hydroxymethylfurfural[END_REF]Chen et al., 2012;[START_REF] Pakarinen | Batch dark fermentative hydrogen production from grass silage: The effect of inoculum, pH, temperature and VS ratio[END_REF]. [START_REF] Abreu | Inoculum type response to different pHs on biohydrogen production from larabinose, a component of hemicellulosic biopolymers[END_REF] and Chen et al. (2012) showed that the H2 yields mainly depend on the inoculum sources. However, the response of fermentative microorganisms towards the presence of inhibiting substances present in a substrate can influence the DF process. In a recent study, [START_REF] Bellucci | Inoculum pretreatment affects the fermentative activity of hydrogen-producing communities in the presence of 5-hydroxymethylfurfural[END_REF] reported a varying response of fermentative microbial communities for H2 production, when the inhibitor 5hydroxymethylfurfural (HMF) was added. This was linked to the difference in inoculum pre-treatment methods applied. Likewise, the presence of polyphenolic compounds in substrates such as OMWW can exhibit inhibitory effects on fermentative microbial communities and H2 yields [START_REF] Hamdi | Toxicity and biodegradability of olive mill wastewaters in batch anaerobic digestion[END_REF]Ntaikou et al., 2009). Subsequently, investigating the effect of the inoculum source on H2 production performance from substrates like OMWW is fundamental to reach an optimum in H2 production.

Despite some studies attempted to establish the optimal operational conditions of initial pH, F/M ratio, alkaline pre-treatment of substrate and inoculum selection, dissimilarities in H2 production exist due to the differences between substrate types and experimental conditions. Therefore, it becomes essential to investigate the optimum initial pH for food waste under thermophilic DF conditions. So far, only few studies have considered the combined effects of F/M ratio and initial pH on thermophilic DF of food waste [START_REF] Ginkel | Biohydrogen production as a function of pH and substrate concentration[END_REF]Pan et al., 2008). [START_REF] Ginkel | Biohydrogen production as a function of pH and substrate concentration[END_REF] revealed a profound impact of the concentration of substrate and pH on the H2 yields in sucrose DF of, with an optimum pH and substrate concentration at pH of 5.5 and 7.5 g COD/L, respectively. In other study, Pan et al. (2008) established a F/M ratio of 6.0 as optimum for thermophilic DF of food waste, without the consideration of initial pH. Similarly, past studies on pre-treatment of substrates seemed more focused on maximizing the methane yields in anaerobic digestion by adopting higher concentrations of alkaline agents and treatment temperature (Monlau et al., 2013a). Therefore, optimum conditions of alkaline pre-treatment for dark fermentative H2 production need to be investigated for lignocellulosic agricultural residues such as rice straw. Finally, different inoculum sources can be explored to study the effect on H2 production from a typical poorly biodegradable feedstock such as OMWW, which contains polyphenolic compounds (Ntaikou et al., 2009).

The present study aims to investigate the effects of i) the initial pH and combined pH and F/M ratio on food waste, ii) alkaline substrate pre-treatment on dark fermentative H2 production from rice straw and iii) the effect of inoculum source and pre-treatment on H2 production from OMWW. Cumulative H2 production, H2 yields, H2 production rates, lag phase and accumulation of DF metabolites (mainly organic acids and ethanol) were used to evaluate the efficiency of these various strategies to improve the H2 production performance from these complex organic wastes.

Materials and methods

Inoculum

Two types of inoculum, i.e. anaerobic digested sludge (ADS) and waste activated sludge (WAS) were used in the experiments. ADS was collected from the effluent of an anaerobic digestion plant of a dairy farm located in Capaccio (Salerno, Italy). The plant features include a 100 m 3 CSTR operating at a hydraulic retention time of 24 days and operating within a pH and temperature range of 7.4 -7.5 and 52 -56 °C, respectively.

The plant is continuously fed with buffalo manure, cheese whey of buffalo milk and sludge from an industrial wastewater treatment plant. WAS was collected from a secondary clarifier unit at the Nola Municipal Wastewater Treatment Plant located in Naples (Campania, Italy). The characteristics of the ADS and WAS before pre-treatment are presented in Table 3.4. The inocula were stored at 4 °C until used. The WAS and ADS underwent a heat shock treatment (HST) at 105 °C for 1.5 and 4 hours, respectively, in order to enrich spore forming Clostridium sp. and inhibit methanogens [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF]. WAS had a shorter time for HST than ADS because it was obtained from an aerobic activated sludge process.

Preparation of feedstock

Three Three types of waste as reference models of complex waste biomass with different characteristic biodegradability, were used in this study: i) food waste, representative of moderately biodegradable organic waste was selected to study the effect of initial pH and substrate concentration on H2 yields, ii) rice straw as a representative of slowly degrading lignocellulosic agricultural residues was used to study the technical feasibility of substrate pre-treatment on biohydrogen production and iii) OMWW was used to study the effect of the inoculum type and its adaptation to toxicants, as OMWW contains phenolic compounds and long chain fatty acid that can affect microbial growth [START_REF] Hamdi | Toxicity and biodegradability of olive mill wastewaters in batch anaerobic digestion[END_REF]Ntaikou et al., 2009). Food waste was a mixed waste with a composition similar to the one reported by VALORGAS (2010) for European countries as (% by weight): fruit and vegetables: 72%, cooked pasta and rice: 10%, bread and bakery: 5%, dairy products (cheese): 2%, meat and fish: 8% and snacks (biscuits): 3%. To prepare the food waste, food was bought fresh from municipal markets in Naples (Italy), shredded with a blender (120 W Black and Decker, Kitchen Blender) for 5 minutes without adding water and immediately stored at frozen conditions (-20 ⁰C) to avoid acidification. The rice straw was harvested from rice fields in Pavia (Italy) in 2012 and stored inside an airtight plastic bag at room temperature. Rice straw was reduced with the help of general paper scissors to a particle size of less than 2 mm (sieved with sieve size of 2mm by 2mm). OMWW was collected from a pressure olive mill of the Frascati area (Lazio, Italy) in autumn 2013

and was stored at < 4 ⁰C until use. The characteristics of the feedstocks are presented in Table 3.4.

Experimental set-up

Batch tests were carried out in one-liter borosilicate glass bottles (Simax, Czech Republic) maintained in thermophilic conditions (55 ± 2°C) with a thermostat in a water bath. The operating reactor volume in all experiments was 600 mL. The batch reactors were sealed with airtight caps having ports for sampling soluble metabolites and gas. The tests were carried out in duplicates with 30 reactors in total. The different sets of experiments were carried out to study the effect of the different operational parameters using the three selected model substrates (Table 3.5).

Effect of Initial pH and F/M ratios on H2 yield

The effect of initial pH and F/M ratio on biohydrogen production was studied with food waste and pretreated heat treated ADS as seed inoculum. The effect of the initial pH (4.5, 5, 5.5, 6.0, 6.5 and 7.0) was studied at a F/M ratio 0.5 and under thermophilic conditions (55 ± 2 °C). Another set of experiments was performed at F/M ratios 0.5, 1.0 and 1.5 with the two initial pH values of 5.0 and 6.5. The F/M ratios and two initial pH values were selected due to the fact that they are less affected by acidification at higher F/M ratios and the culture pH in the tests was not buffered with external alkalinity source. In addition, pH 6.5 was previously reported as optimal for food waste by Cappai et al. (2014), and thus considered for investigation in this study. The F/M ratios 0.5, 1.0 and 1.5 were obtained by adding 10 g, 18 g and 27 g food waste respectively, with a 190 g inoculum required to obtain the aimed F/M ratio. The final volume of the mixture was made up to 600 mL by adding distilled water. The initial pH was adjusted once, initially with 1 M

HCl and 1 M NaOH prior to the start of the tests.

Effect of alkaline substrate pre-treatment on H2 yield

Direct conversion of lignocellulosic biomass to biohydrogen is often limited due to their low biodegradability (Monlau et al., 2012;Pan et al., 2010). Biological hydrolysis is one of the limiting factors in DF. The evaluation of the effect of alkaline pre-treatment on H2 yields was performed on rice straw. This study investigated an alkaline pre-treatment with 4 % NaOH (4 g/100g TS) and 8 % NaOH (8 g/100g TS) at a solid liquid ratio of 1:5 (w/v).

This mixture was kept at 55 (± 2) °C for 24 hours in a one-liter borosilicate glass bottle (Simax, Czech Republic). The results were compared with untreated rice straw at thermophilic DF using 200 g of heat-treated WAS as inoculum. The concentration of rice straw was 45 gTS/L and the initial pH was adjusted to 6.5 during the batch tests that gave the optimal dark fermentative H2 performance for rice straw as reported by Chen et al. (2012).

Effect of inoculum sources and adaptation using OMWW on H2 yield

Heat shocked WAS and ADS was used as inoculum in a DF of OMWW carried out in batch tests and operated under thermophilic conditions (55 ± 2°C). The F/M ratio was fixed at approximately 1 gVS substrate/gVS inoculum in all sets of batch tests using 200 g of OMWW and a respective volume of ADS and WAS. The initial pH was adjusted to pH 6.0 in all experiments.

Analytical methods

Hydrogen was quantified with a gas chromatograph (VARIAN STAR 3400, USA) equipped with a ShinCarbon ST 80/100 column and a thermal conductivity detector.

Argon was used as carrier gas with a front and rear end pressure of 20 psi. The duration of analysis was 14 minutes. The gas volume was measured with a volumetric displacement method. The biogas was passed through acidic water (1.5 % HCl) and the volume was quantified by water displacement (Ghimire et al., 2015c). The volume of hydrogen was calculated from the gas composition. Fermentation end products (lactic, acetic, propionic and butyric acids) were quantified by High Pressure Liquid Chromatography (HPLC) (Chromatography Oven LC 25 Model, Dionex, USA) equipped with a Synergi 4u Hydro RP 80A (size 250×4.60mm) column and an UV detector (AD25 Model, Dionex, USA). Gradient elution consisted of 20% methanol, 10% acetonitrile in 5 mM H2SO4 pumped at a rate of 0.9 mL/min by using a gradient pump (GP 50 Model, Dionex, USA). The elution time was 18.5 minutes. Ethanol and caproic acid were determined with an Aminex HPX-87H column (300 mm on 7,8 mm, Bio-rad), using 5 mM H2SO4 as an eluent at a flow rate of 0.4 mL/min. pH was measured with a pH meter (WTW, inolab, pH level 2). The COD of the food waste was measured as reported by Noguerol-Arias et al. (2012). The total lipid content was measured by the Bligh and Dyer chloroform/methanol total lipid extraction method (Bligh and Dyer, 1959). as described in the Standard Methods [START_REF] Apha | Standard Methods for the Examination of Water and Wastewater[END_REF].

Measurements and data analysis

The biogas accumulated in the reactors was measured daily, except at the starting period of the experiments, i.e. 1 -3 days, where it was measured twice a day, until the H2 production completely ceased. The biogas volumes were normalized at 0 C and 1 atm (NmL) and reported as a daily average. The average values were considered for the evaluations, while the data range based on the duplicate samples is provided and indicated by "±". H2 yields were calculated by dividing the final cumulative recovery of H2 by the amount of VS added at the start of the experiment.

De Gioannis et al. ( 2013) defined a parameter "t95" as the time required to achieve 95%

of the maximum H2 yield. This parameter was used to compare the kinetics associated to different BHP tests, and to evaluate the effect of the experimental conditions.

t 95 = H o R. e (1 -ln(-ln0.95)) + λ (3.2)
Equation 3.2 corresponds to a rearranged form of the modified Gompertz equation 3.1, that has been widely used to model biohydrogen production kinetics (Gadhamshetty et al., 2010;Wang and Wan, 2009). This empirical formula gives biohydrogen production trends and includes five major parameters: i) cumulative biohydrogen production (or potential) (Ho, mL/g VS), ii) biohydrogen production rate (R, mL/h), iii) e is 2.71828, iv) lag time (λ, hours) and v) total cultivation time (t, hours). The cumulative biohydrogen production is a non-linear curve and in the present study, the parameters Ho, R and λ were estimated using the Curve Fitting Toolbox in MATLAB ® (Version MATLAB R2012b, Curve Fitting Toolbox 3.3) with an associated 95% confidence limit. The total cumulative production, hydrogen production rates and lag phase time were used as parameters to compare the characteristics of the biohydrogen production systems. R software (OSX version 3.1.3) with the package Rcmdr (OSX version 2.1.7) was used for the statistical analysis of data obtained from the experiments. The p value was set at 0.05 and the significance of the results tested with p values: * < 0.05; ** < 0.01; *** < 0.001; while not significant results were with p > 0.05.

Results

Effect of the initial pH and combined effect of F/M ratio and pH on H2 yields

The H2 yields and the time required to achieve 95% of the maximum H2 yield were plotted against the initial pH values (Figure 3.3). The H2 yields showed a decreasing trend to the increasing pH. Figure 3.3 confirmed that H2 production was favoured at the acidic pH range, i.e. at initial pH 4.5 and 5.0 with H2 yields of 60.6 (± 9.0) and 50.7 (± 1.0) N mL H2/g VS, respectively. This result is in agreement with the study reported by Khanal et al. (2004). The fermentative H2 production patterns at the various pH values investigated are described by a modified Gompertz equation, as presented in Table 3.6 (Modeled plot is provided in Supplementary information S1). The different initial pH values in the tests were characterized by the differences shown in cumulative H2 production, H2 production rates and lag phase (Table 3.6). H2 production rates (R, mL/h) were high at initial pH 7.0, however, higher rates were not co-related with higher H2 yields (Figure 3.3 and Table 3.6).

Unsurprisingly, the lag phase decreased when increasing the initial pH, which represents the time required for spore forming H2 producers present in heat-treated ADS to germinate or adapt a sudden change of their environment [START_REF] Ferchichi | Influence of initial pH on hydrogen production from cheese whey[END_REF]Kim et al., 2011). Figure 3.3 shows the time required to achieve 95% of the maximum H2 yield decreased by increasing the initial pH, while the rate of H2 production was higher at initial pH 7.0 (Table 3.6). H2 production started faster at higher pH and lasted for a short time while it continued for longer time during the tests at lower pH. Thus, a decreasing lag phase did not correspond to an increase in H2 yields. This can be explained by the methanogenic activities which started at higher initial pH, that was confirmed by the presence of methane in the biogas produced when H2 production ceased completely. The final pH at the end of the tests was mainly lower than the initial pH (Table 3.6), which is mainly due to the production of VFAs (Table 3.6). As exception, the final pH in the batch tests with initial pH 4.5 was higher than the initial pH (Table 3.6), which could be due to the higher alkalinity of the inoculum (ADS) and the lower substrate concentration (F/M 0.5) used to avoid the use of chemical buffer. The final pH in all the tests was lower than 5.5, except for tests with initial pH 7.0 where the final pH was 6.6. This can be due to the higher alkalinity (buffering capacity) of the ADS inoculum (Table 3.4). The concentrations of the main accumulated metabolites at the end of the tests are summarised in Table 3.6. Results confirm that different fermentation pathways occurred.

The presence of propionate and ethanol generally does not indicate H2 favorable pathways (Kim et al., 2011). The concentration of ethanol was comparatively higher in the tests with initial pH range 6.0 -7.0, that could be linked to the low H2 yields. In particular, the butyric to acetic acid ratio (B/A, mM:mM) co-related with the H2 yields (Figure 3.4). This observation is consistent with a study by Kim et al. (2006), which reported a higher corelation between B/A ratios (1.6 -9.3) and H2 yields. However, this ratio might not always give a good indication of high H2 production. Guo et al. (2013) reported that the homoacetogenic activities can influence the concentration of end-metabolites due to acetate production from H2 and CO2. The presence of acetate in higher concentrations The results of the batch tests carried out at F/M ratios 0.5, 1.0 and 1.5 at two initial pH values (5.0 and 6.5) are presented in Table 3.7. Table 3.7 shows the major metabolites accumulated at the end of the tests. At the initial pH 5.0 and F/M ratios of 0.5, 1.0 and 1.5, H2 yields were 50.7 (± 0.8), 60.3 (± 5.0) and 49.3 (± 12.2) mL H2/g VS, respectively.

Likewise, in tests carried out with an initial pH 6.5, respective H2 yields of 28.2 (± 4.2), 43.2 (± 2.0) and 54.1 (± 4.4) mL H2/g VS were obtained. An ANOVA analysis confirmed the significance of difference in H2 yields at pH 5.0 and 6.5 for an F/M ratio of 0.5 (p value <0.05). However, it was not significant for F/M ratios 1.0 and 1.5 at both initial pH values tested. Likewise, at initial pH 5.0, the differences in H2 yields were not significant for all the three tested F/M ratios. Interestingly, the differences in H2 yields were significant (p value <0.05) at an initial pH of 6.5 for F/M ratios 0.5 and 1.5. This implies a combined influence of the F/M ratios and initial pH on dark fermentative H2 production.

The result also suggests that the comparable H2 yields can be achieved through a combination of pH and F/M ratios by maximizing the utilization of substrates.

The different metabolites yields measured at the end of the batch tests explain the differences in H2 yields (Table 3.7). The presence of different metabolites suggests a typical mixed type fermentation that can occur in complex substrates like food waste.

Acetate yields were higher at initial pH 6.5 compared to pH 5.0, which was also 3.6). Similarly, higher ethanol yields were obtained at increasing F/M ratios and initial pH. High levels of butyrate yield at pH 6.5 and F/M ratios 1.0 and 1.5 can be associated to higher H2 yields obtained in respective tests, as the production of butyrate is generally co-related to H2 production (Kim et al., 2011).

Effect of substrate alkaline substrate pre-treatment on H2 yields

Figure 3.5 shows the effects of alkaline substrate pre-treatment on biohydrogen production. The results illustrate that biohydrogen production can be significantly improved with alkaline pretreatment of rice straw. As expected, the alkaline pre-treatment enhanced the saccharification of sugars from rice straw, which increased along with the concentration of NaOH. The COD values of hydrolysate after pre-treatment with 4% and 8% NaOH were 7.3 (± 0.8) and 8.3 (± 0.7) g/L, respectively, in comparison to the untreated rice straw with 3.8 (± 0.1) g/L soluble COD (determined with solid liquid ratio of 1:5). The results of end-product accumulation (Table 3. [START_REF] Wu | Continuous biohydrogen production from liquid swine manure supplemented with glucose using an anaerobic sequencing batch reactor[END_REF] show that higher H2 yields corresponded to higher B/A ratios (mM:mM), irrespective of the concentration of acids accumulated at the end of the tests.

Effect of inoculum sources on H2 yields

The cumulative H2 yields and accumulation of end metabolites during the application of two heat treated inoculum sources on biohydrogen production from OMWW is depicted in Figure 3.6 and Table 3.9, respectively. The differences observed when using two inoculum types, i.e.

ADS and WAS, at thermophilic temperature gave an indication of the level of inhibition of the polyphenols present in the OMWW on the microorganisms [START_REF] Hamdi | Toxicity and biodegradability of olive mill wastewaters in batch anaerobic digestion[END_REF][START_REF] Paraskeva | Technologies for olive mill wastewater ( OMW ) treatment : a review[END_REF]. The initial lag phase observed in Figure 3.6 can give evidence for the adaptation of H2 producing fermentative microbial communities to phenolic compounds present in OMWW. The maximum H2 yield from OMWW with WAS was almost 2 fold higher than with ADS. In addition, WAS sludge required less heat-shock pre-treatment time to inhibit hydrogen consuming methanogens and showed a shorter lag phase (Figure 3.6, Table 3.9). This

shows that heat-shocked WAS is an appropriate inoculum for DF of OMWW for higher H2 recovery.

The lower H2 yield obtained from OMWW in tests inoculated with ADS is further supported by the analysis of the metabolic pathways (Table 3.9), which showed an accumulation of lactic acid. Metabolic pathways leading to lactic acid are not favorable to H2 production (Hawkes et al., 2007), which explains the lower H2 yields observed in the batch tests inoculated with ADS.

Likewise, the higher levels of acetate in the tests carried out with WAS than ADS can explain the higher H2 yields from OMWW, as acetate pathways generally yields to more H2 per mole of glucose than the butyrate pathways (Hawkes et al., 2007).

Discussion

Effect of the pH and F/M ratio on H2 yield

This study showed that higher H2 yields can be achieved from easily biodegradable organic waste like food waste, when compared to other complex substrates such as rice straw (Table 3.10). This is mainly a result of the high fraction of easily degradable carbohydrates contained in food waste, as already suggested by Guo et al. (2013). The combination of initial pH and substrate concentration is important to avoid inhibition of H2 producers through elevated VFA accumulation and consequent pH depletion, and high hydrogen partial pressure [START_REF] Ginkel | Biohydrogen production as a function of pH and substrate concentration[END_REF]. This is likely the case of substrates like food waste which generally show faster hydrolysis kinetics compared to lignocellulosic biomass such as rice straw (Table 3.10), that requires higher optimal substrate concentrations or F/M ratios compared to food waste.

Table 3.10 compares the results of the H2 yields observed in this study with literature data reported under similar conditions. The highest H2 yields observed at initial pH 4.5 and 5.0 (60.6 ± 9 and 50.7 ± 1 mL H2/ g VS food waste, respectively) in this study were in contrast with Cappai et al. (2014), who obtained the highest H2 yield (56.2 mL H2/ g VS food waste) at pH 6.5. This difference in optimum initial pH might be due to the higher substrate concentrations used by Cappai et al. (2014) (Table 3.10). Furthermore, two possible explanations can be given for the relationship between initial pH (4.5 and 5.0) and the higher H2 production: (i) a selection of hydrogen producers at pH range (4.5 -5.0) and (ii) an inhibition of H2 consuming methanogens. In addition, the differences in metabolic products accumulating at different initial pH ranges might support the growth of different microbial communinities which can influence the H2 production as reported in the studies from Fang and Liu (2002) and Lee et al. (2008). Khanal et al. (2004) reported that a microbial shift to solventogenesis did not occur at a pH range 4.5 -6.5, which provides further evidence of the importance of the initial microbial community and pH to reach higher H2 yields. In addition, native microorganisms present in the food waste might also influence the DF process in real conditions (waste type and storing conditions).

In this study, the storage of food waste at freezing conditions might have impacted native microorganisms. Nevertheless, the comparison of the results between the tests operated at different initial pH remains unaffected as uniform substrates were used.

At lower F/M ratios (0.5 and 1.0), an initial of pH 5.0 favored the H2 production whereas it was the inverse at a F/M ratio 1.5 and initial pH 6.5. At the initial of pH 5.0 and F/M 1.5, a lower H2 yield was observed, which might be due to the shock load on the microbial systems. This was also confirmed in the study of [START_REF] Ginkel | Biohydrogen production as a function of pH and substrate concentration[END_REF], who reported an inhibition of H2 production at higher substrate loading rates due to shock loads. The conversion of substrates to metabolic products at pH 5.0 and F/M 1.5 is lower than at F/M ratios 0.5 and 1.0, which can be due to an inhibition of the substrate conversion. In addition, a low final pH (4.5 ± 0.1) at the end of the test at pH 5.0 and F/M 1.5 (Table 3.7) suggests that H2 production might be inhibited due to a 'load shock'. This can be supported by the time required to achieve 95% of the maximum H2 yield (t95 = 47 days) (Table 4). Pan et al. (2008) reported that a F/M ratio of 6.0 as appropriate for thermophilic (50 ± 2 °C) fermentation of food waste (Table 3.10). However, the initial pH in their study varied from 6.2 to 6.7. Therefore, in the DF systems where initial pH is not buffered, H2

production is a combined function of suitable F/M ratio and initial pH. Likewise, an optimal operational pH range could be maitained through subsequent substrate feeding strategies which can garantee higher H2 production and avoid the H2 consuming activities i.e. methanogens and homoacetogens.

Effect of alkaline substrate pre-treatment on H2 yield

The alkaline pre-treatment method applied in this study aimed at improving hydrolysis and solubilization of the organic matter that limit the dark fermentative substrate conversion [START_REF] Monlau | Alkaline pretreatment to enhance one-stage CH4 and two-stage H2/CH4 production from sunflower stalks: Mass, energy and economical balances[END_REF](Monlau et al., , 2013b)). However, the level of effectiveness of the different pre-treatment methods depends on the nature of the substrate (Ariunbaatar et al., 2014;[START_REF] Carlsson | The effects of substrate pre-treatment on anaerobic digestion systems: a review[END_REF]. In the study of Monlau et al. (2013c), H2 yields from sunflower stalks increased from 2.3 (± 0.9) to 4.4 (± 2.6) mL H2/g VS, while in our study an increase from 0.3 (± 0.1) to 6.6 (± 0.1) from mL H2/g VS from rice straw as the substrate was achieved under similar conditions of thermo-alkaline pre-treatment (Figure 3.5 and Table 3.8). Meanwhile, H2 yields further increased to 15.7 (± 1.0) mL H2/g VS when 8 % w/w NaOH was applied (Figure 3.5). This H2 yield is lower than the value reported by Chen et al. ( 2012) with untreated rice straw, i.e. 24.8 mL/g TS at a substrate concentration of 90 g TS/L, whereas, it is 2.2 fold higher when the substrate concentration was 30 g TS/L (i.e. 7.1 mL H2/g TS). This disagreement might be due to physico-chemical properties of the lignocellulosic substrates, such as particle sizes, soluble carbohydrates content and/or substrate concentration (Monlau et al., 2013a). Chen et al. (2012) reported an increasing trend of H2 yields, when the particle size of rice straw decreased from 10 mm to < 0.297 mm. In their study, a maximum H2 yield was obtained with a particle size of < 0.297 mm (6.4 mL H2/g TS) at a substrate concentration of 30 g TS/L.

The effects of the chemical agents applied (NaOH) and or by-products formed (furfural, phenols) during the pre-treatment process and the response on the dark fermentative microbial community should be taken into consideration while selecting appropriate pretreatment method. Kim et al. (2009) reported a decrease in H2 yields when the Na + concentration in a continuous DF reactor gradually increased from 0.27 to 21.00 g Na + /L while the acclimatized fermentative community maintained their activity up to 6.00 g Na + /L. Nonetheless, in this study, the H2 yields increased when 8 % w/w NaOH was applied compared to 4 % w/w NaOH (Figure 3.5). Moreover, under similar pre-treatment conditions, 12 % w/w NaOH (i.e. 5.40 g Na + /L) might either enhance the H2 yields or exert effect on fermentative microbial community, depending on the inocula type and adaptation to Na + concentration. However, the application of pre-treatment methods should be based on the substrate type (biodegradability or bioavailability of easily fermentable carbohydrates), their practicability and economy viability.

associated with the optimization of the DF process. Several studies have suggested the integration of DF with processes such as photofermentation (H2), bioelectrochemical systems (H2) and anaerobic digestion (CH4) for further energy recovery and production of platform molecules of economic interest, such as biopolymers (Bastidas-Oyanedel et al., 2015;[START_REF] Elmekawy | Bioelectro-catalytic valorization of dark fermentation effluents by acetate oxidizing bacteria in bioelectrochemical system (BES)[END_REF]Ghimire et al., 2015c;Xia et al., 2013)

Conclusion

This study aimed to investigate the optimal operational parameters in the thermophilic DF of three types of complex waste biomass with varying biodegradability, i.e. food waste, rice straw and OMWW. The DF applied to food waste was favored in the acidic pH range (4.5 -5.0), though an appropriate substrate concentration must be considered while selecting an acidic pH range. F/M ratios of 0.5 and 1.0 at an initial pH of 5.0 gave, respectively, 1.8 and 1.4 folds higher H2 yields than at initial pH 6.5. Likewise, F/M ratios and pH can be optimized to achieve higher substrate utilization and H2 yields. During the tests, higher B/A ratios (mM:mM) were associated with higher H2 yields, a B/A ratio equivalent to 1.5 was related to the optimal H2 yield. Similarly, pre-treatment of rice straw with 4% NaOH and 8% NaOH at 55 °C for 24 hours increased the H2 yield by 26 and 57 fold, respectively. Furthermore, WAS showed adaptability to OMWW containing phenols and gave a nearly 2 fold higher H2 yield when compared to ADS. In conclusion, the selection and application of the optimal operational parameters for the optimization of H2 production rely mainly on the substrate biodegradability. Therefore, these parameters should be optimized for each particular type of substrate prior to application in scaled-up DF systems. during this period and it was shown that the butyric acid pathway was mainly responsible of the H2 production. A transitory accumulation of lactic and/or propionic acids was observed when the OLR (increment) or the HRT (decrement) was changed, causing a decrease in H2 production. Monitoring of soluble metabolites provided a reasonable indication of DF process performances.

Introduction

Over the past few decades, anaerobic digestion has gained a lot of interest since it constitutes a promising technology for producing an energy-rich biogas from renewable waste and biomass resources such as wastewaters, municipal organic waste, agricultural residues, etc. The inherent characteristics of biohydrogen (H2) such as higher energy content (143 GJ per ton), energy and water as the only by-products generated from its combustion and the ability to be produced biologically, makes H2 as a very interesting alternative of future sustainable biofuels (Kotay and Das, 2008). In particular, dark fermentation (DF) systems have the potential to be one of the prominent technologies for H2 production from renewable waste biomasses (Ghimire et al., 2015a;Urbaniec and Bakker, 2015).

Low cost renewable waste biomasses such as agricultural residues, organic fraction of municipal waste (OFMSW), agro-industrial wastes, etc. might give a competitive economic advantage for the future supply of sustainable feedstock, aiming at the industrial development of DF systems with biological treatment of waste as added benefit (Chong et al., 2009;De Gioannis et al., 2013;Kapdan and Kargi, 2006;Ntaikou et al., 2010;Wong et al., 2014). In contrast, the use of simpler feedstock sources such as pure carbohydrates (e.g. sucrose and glucose), although it presents higher H2 conversion rates, could make DF processes less economically competitive (Ren et al., 2011).

OFMSW which is mainly composed of food waste is receiving lot of attention because of its high biodegradability and its potential to be utilized for the production of biofuels and other platform chemicals [START_REF] Uçkun Kiran | Bioconversion of food waste to energy: A review[END_REF]. Every year, about 1.3 billion tons per year of food get wasted, which is approximately one-third of the food produced for human consumption (Gustavsson Jenny, Cederbery Christel, Sonesson Ulf, Van van Otterdijk Robert, 2011). Food wastes are generated from the agricultural production, to industrial manufacturing processes and final consumption in households. In the European Union, the total annual production of food waste is estimated at 89.3 Mt, comprising 37.7

Mt generated from household consumption alone (European Commission, 2010). The food waste content in volatile solids ranges from 21 to 27% which shows the high content of organic carbon which can be valorized, and in particular for H2 production by DF (VALORGAS, 2010). During the past few years, several researches have shown the high potential of food waste to be used as a feedstock in DF processes for H2 production (Cavinato et al., 2012;Elbeshbishy et al., 2012;Faloye et al., 2013;Han and Shin, 2004;[START_REF] Han | Biohydrogen production by anaerobic fermentation of food waste[END_REF]Shin and Youn, 2005;Sreela-or et al., 2011;Valdez-vazquez et al., 2005;Xiao et al., 2013).

With the advantage of a steady operation, continuous DF processes are preferred and scaling-up is more viable in comparison to batch processes which involves regular downtime periods of maintenance (Hawkes et al., 2007). However, stable operation of continuous DF of food waste is mostly influenced by the bioreactor operating parameters such as the pH, temperature, organic loading rate (OLR) and hydraulic retention time (HRT) [START_REF] Davila-Vazquez | Fermentative biohydrogen production: trends and perspectives[END_REF]De Gioannis et al., 2013;Guo et al., 2010;Ntaikou et al., 2010;Wang and Wan, 2009). These factors also influence the microbial communities and thus the biochemical pathways that can affect the total H2 yields in mixed cultures (Li and Fang, 2007).

Hydrogen production rates and total H2 yield are mainly a function of substrate types and OLRs applied (Ghimire et al., 2015a). A varying range of optimal OLR values has been reported for FW for H2 conversion carried out in thermophilic DF processes (Ghimire et al., 2015a). Shin et al. (Shin and Youn, 2005) found an optimal hydrogen yield of 126.25 L H2/kg VS at an OLR of 8 kg VS/m 3 /d while the H2 production decreased when the OLR was increased to 10 kg VS/m 3 .d. The authors reported 8 kg VS/m 3 .d, 5 days and pH of 5.5, respectively, as optimal OLR, HRT and culture pH. In a study coupling DF and AD, Cavinato et al. (Cavinato et al., 2012) reported 66.7 L H2/kg VS added at an optimum OLR of 16.3 VS/m 3 .d, a HRT of 3.3 days and for a pH maintained in the range of 5-6 through the recirculation of AD effluent. Generally, HRTs in a range of 2-6 days have been reported as optimum for DF of organic FW in a CSTR process (Ghimire et al., 2015a). This range of HRTs is similar to the first stage of two-stage AD process (Aslanzadeh et al., 2014).

Unlike OLR, the HRT is also a function of the substrate types and bioreactor operation parameters. It is well understood that DF processes generate acidic microbial metabolites.

Therefore, high OLRs are often responsible for a decrease in pH due to the accumulation of volatile fatty acids (VFAs) present in the DF effluent (DFE). Thus, most of the DF systems require the addition of external alkalinity sources such as alkaline chemicals (NaOH or KOH) or buffering agents (bicarbonate or phosphate buffers) (Shin and Youn, 2005). Meanwhile, few studies have reported the use of recycle water from AD as a solution to reduce the use of external alkaline chemicals (Gottardo et al., 2013;[START_REF] Jung | Conversion of organic solid waste to hydrogen and methane by two-stage fermentation system with reuse of methane fermenter effluent as diluting water in hydrogen fermentation[END_REF]. Moreover, there are additional concerns regarding the decrease in H2 yields due to hydrogen consuming activities of methanogens or propionic producing bacteria [START_REF] Jung | Conversion of organic solid waste to hydrogen and methane by two-stage fermentation system with reuse of methane fermenter effluent as diluting water in hydrogen fermentation[END_REF].

Moreover, thermophilic temperature is mainly favored in DF of food waste since higher H2 yields are usually observed (Shin et al., 2004;Valdez-vazquez et al., 2005). Moreover, a thermophilic process seems to be more economically interesting owing to its higher yield and less requirement of feedstock in comparison to mesophilic dark fermentation processes (Foglia et al., 2006). Foglia et al. (2006) reported better economic performances of thermophilic DF in comparison to mesophilic operation when the process was operated to convert sugars into hydrogen, CO2 and organic acids that were further used in a second photofermentation process.

Most of the past studies on continuous and/ or semi-continuous dark fermentative H2 production were carried out at controlled culture pH with chemical buffering agents such as K2HPO4, NaHCO3, Na2HPO4 (Carrillo-Reyes et al., 2012;Valdez-vazquez et al., 2005). Likewise, even in a recent pilot scale application of DF has shown the dependency on buffering agents for stable H2 production (Elsamadony and Tawfik, 2015). The high amount of chemical buffering agents needed to maintain the operable acidogenic pH (higher than 4.5 -5.5), which might effect the operational cost of DF bioreactors.

Moreover, the effect of use of high concentrations of buffering agents on downstream processes like anaerobic digestion, photofermentation, bioelectrochemical systems is uncertain.

The current study aims at investigating thermophilic DF of food waste for continuous H2 production at varying low OLR and HRT. The study also considers the recycling of DF effluents to investigate its effect on the performance of bioreactors, which has never been reported in past studies, to knowledge of authors. Moreover, a major aim is to demonstrate a long-term feasibility of continuous H2 production at varying operational conditions of the bioreactor. The performances of the DF reactor were evaluated by daily monitoring of H2 and metabolites production rates.

Materials and methods

Preparation of feedstock

An average mix waste composition as found in European countries was prepared at the laboratory paccording to (VALORGAS, 2010). The waste mixture composed of (in % by weight); fruit and vegetables: 72%, cooked pasta and rice: 10%, bread and bakery: 5%, dairy products (cheese): 2%, meat and fish: 8% and snacks (biscuits): 3%. The food waste ingredients were freshly brought from municipal markets in Naples (Italy), shredded with a blender and immediately stored at -20 ⁰C to avoid acidification. The food waste had a pH of 4.37 ± 0.01 and the characteristics are presented in Table 4.1.

Analytical methods

Hydrogen was quantified with a Varian Star 3400 gas chromatograph equipped with ShinCarbon ST 80/100 column and a thermal conductivity detector. Argon was used as carrier gas with a front and rear end pressure of 20 psi. The duration of analysis was 14 minutes. The fermentation products (lactic, acetic, propionic and butyric acids) were quantified by High Pressure Liquid Chromatography (HPLC) (Dionex LC 25

Chromatography Oven) equipped with Synergi 4u Hydro RP 80A (size 250×4.60mm) column and UV detector (Dionex AD25 Absorbance Detector). Gradient elution consisted of 20% methanol, 10% acetonitrile in 5 mM H2SO4 pumped at a rate of 0.9 ml/min, using Dionex GP 50 Gradient pump. The elution time was 18.5 minutes. COD of food waste was measured according to a method described elsewhere (Noguerol-Arias et al., 2012). The carbohydrate content was determined according to the Dubois method (DuBois, M., Gilles, K., Hamilton, J., Rebers, P., & Smith, 1956). Total lipids was measured following a Bligh and Dyer chloroform/methanol total lipid extraction method (Bligh and Dyer, 1959). The TS, VS of seed sludge and TKN were determined according to the Standard Methods [START_REF] Apha | Standard Methods for the Examination of Water and Wastewater[END_REF].

Water out

Water in

(3) (2) (6) (7) (1) (4)
(5)

Data analysis

Hydrogen production rates (HPR) were expressed in L H2/m 3 /d while the H2 yields (HY)

were determined considering the total daily organic load fed to the reactor and expressed as L H2 /kg VS added. Average and deviations for daily production were determined during the steady state reached after 3 -4 days operation. The H2 Production Stability Index (HPSI) was evaluated by considering the ratio of standard deviation and average HPR as reported by Tenca et al. (2011):

(4.1)

A HPSI index closer to 1 represents a stable hydrogen production.

FactoMineR, an extension on R software, was used for multivariate analysis of metabolites distribution from the different experimental periods in relation to the hydrogen yields and co-relation circles of the major metabolites are generated.

Results and discussion

Effect of operational parameters on quality and quantity of H2

The HPR, HY, H2 content in gas produced and HPSI during the eight experimental conditions (Table 4.2) are summarized in Table 4.3. Figure 4.1 shows HPR and pH trends during the experimental period. The reactor was operated for 253 days to demonstrate the long-term operation feasibility operation continuous H2 production in a semi-continuous thermophilic DF reactor. The effect of varying operational conditions of OLR and HRT was investigated. The culture pH was experiment aimed at reducing the amount of chemical buffering agents that are used to maintain an acid pH. Initial pH of the influent was 7 and the pH was not regulated and adjusted itself according to the fermentative activity with the aim at reducing the dependency on chemical buffering agents.

HPR trends showed the increases in H2 yield with the increase in OLR, the change in HY was not significant (Table 4.3 and Figure 4.2 a). The range of HRT (12 -4 d) studied does not show a significant effect on HY, as seen from the comparison of HYs during the experimental period IV and V (Table 4.3). PCA analysis, presented in Figure 4.3 (a-b)

showed that range of OLR studied (1 -2.5 gVS/L/d) has more effects on HY than the HRT (12 -4 d).

HPSI =1 S.D.(HPR)

Avg.HPR

Effects on production of overall metabolic products

The concentration of major by-products, i.e. lactate, acetate, propionate, butyrate and ethanol monitored during the fermentation period of 253 days are summarized in Table 5. These metabolites are generally present in the DF of complex substrate using mixed cultures (Guo et al., 2013). PCA was done to understand the relationship between the OLR, by-products and H2 production (Figure 4.3). It can be seen that H2 production is more correlated with the butyrate as explained by variable Dim 2. However, the presence of other metabolites does not show a clear relationship to H2 evolution, while their proximity can suggest that these metabolites can be expected under the DF of complex substrate by mixed consortia. The accumulation of lactate or propionate does not represent the H2 favorable pathways, which can clarify the lower production of H2 during the period VII, whereas, the production of butyric in a mixed fermentation pathway indicates higher H2 yields. The presences of these metabolites for H2 favorable and unfavorable pathways are further supported by the biochemical reactions shown in the equations 2-9 (Table 4.6).

Although the presence of ethanol and acetate might indicate H2 production pathways (eqn.

2 and 4). However, from the PCA, they do not show clear relation with the evolution of H2. Moreover, there might be a biochemical pathways that could favors the production of propionate and acetate which are catalyzed by propionic acid bacteria, shown in equation 7 [START_REF] Tyree | The production of propionic acid from sugars by fermentation through lactic acid as an intermediate[END_REF]. This fact can be supported by an increase in production of acetic and propionic acid (8.3 ± 3.4 and 23.7 ± 12.6 mM) during the experimental period VII, when HRT increased from 4 days to 8 days. Likewise, no clear co-relation was found between HY and butyric to acetic acid (B/A) ratio as suggested by Hawkes et al. (2007) (data not presented here) (equation 5). Therefore, the presence of acetate might not always give an indication of H2 production. It can be seen from the results presented in Table 4.5 that the sudden change in OLR or/and HRT could change the metabolic pathways to lactate and propionate production. However, the pathways can be reversible, when the HRT was changed back to 4 days from 8 days the reactor stated to H2 production. The monitoring of soluble metabolites can aids in an operational management of the DF bioreactors.

a ΔG ' 0 values are adapted from (Kim et al., 2006;[START_REF] Thauer | Energy conservation in chemotrophic anaerobic bacteria[END_REF] Figure

Conclusion

The paper shows the long-term feasibility of continuous H2 production in semicontinuous reactor using food waste as substrate. The low OLRs have been taken to maintain the culture pH at operable conditions that showed the comparable

Co-fermentation of cheese whey and buffalo manure for pH control

This section presents investigation on the effect of buffalo manure (BM) addition on pH control and H2 production stability during the dark fermentation of cheese whey (CHW).

Dark fermentation (DF) processes are often favored at acidic pH ranging 5.0 -6.0 depending on type of substrates and bioreactor operating conditions. H2 production in DF process is inhibited at lower culture pH (<4.0) and becomes unstable due to lack of production of buffering capacity like in anaerobic digestion for methane. The cofermentation with substrates contributing to alkalinity such as animal manure can highly reduce dependency on chemical buffering agents for maintaining the optimal pH conditions. CHW and BM, the abundant waste by-products from agro-industrial activities (mozzarella cheese industries) in the Campania Region, Italy, were used as substrates for continuous H2 production in a semi-continuous thermophilic DF reactor operated at various organic loading rates (OLR). At CHW to BM ratio of 4 g VS/gVS, the maximum H2 yield, production rate and H2 content in the biogas of 152.2 ± 43.9 mL H2/g VS, 215.4 ± 62.1 mL H2/L•d and 58.01 ± 4.8%, respectively, were achieved at an OLR of 2.1 g VS/L/d of CHW at a hydraulic retention time (HRT) of 12 days. BM addition aided to maintain culture pH around 4.8 -5 in the dark fermentation reactor. The use of BM as co-substrate improved the H2 production stability and can give economic sustainability to DF systems in scaled-up applications.

Introduction

Activities associated with dairy industries, either related to livestock farming for milk production or processing of milk products such as cheese, generates large amount of waste (De Gioannis et al., 2014). In 2013, out of the 144 million tonnes of whole milk collected in European Union (EU-28 countries), 36.2 % was used for production of cheese, butter (28.1%), drinking milk (12.1%), cream (12%), milk powder (3.2%) and other uses (8.4%) (Eurostat, 2013). Cheese manufacturing industries generate liquid waste by-products, mainly cheese whey (CHW) [START_REF] Carvalho | Cheese whey wastewater: Characterization and treatment[END_REF]Venetsaneas et al., 2009). Simultaneously, livestock activities also produce large quantities of solid animal manure waste, fodder waste (which generally contains a lignocellulosic fraction)

and wastewater which includes urine and feces which can pose threats to the atmospheric and aquatic environment due to pathogens and high nitrogen (ammoniacal nitrogen) contents (Cantrell et al., 2008). DF of waste biomass can be one of the very promising technologies, which can provide environmental credentials from recovery of renewable energy in the form of biohydrogen (H2), as well as organic waste treatment.

Generally, the waste biomass rich in carbohydrates is considered to be most suited for DF processes (Azwar et al., 2014;Ghimire et al., 2015a;Guo et al., 2010;Monlau et al., 2013;[START_REF] Yasin | Food waste and food processing waste for biohydrogen production: a review[END_REF]. CHW can be very suitable feedstock for DF processes as it is characterized by high organic loads, comprising mainly soluble form of carbohydrates (lactose), protein and lipids [START_REF] Marone | Optimization of substrate composition for biohydrogen production from buffalo slurry co-fermented with cheese whey and crude glycerol, using microbial mixed culture[END_REF]Moreno et al., 2015;Teli et al., 2014).

Although animal manure is not considered as suitable substrate for DF processes, it can be used as co-substrate. It has been suggested in previous studies that animal manure can provide macro and micronutrients such as NH3, P, K and trace metals required for bacterial growth as well as it can act as buffering agent to maintain the alkalinity [START_REF] Lateef | Biohydrogen production from co-digestion of cow manure and waste milk under thermophilic temperature[END_REF][START_REF] Marone | Optimization of substrate composition for biohydrogen production from buffalo slurry co-fermented with cheese whey and crude glycerol, using microbial mixed culture[END_REF][START_REF] Perera | Evaluation of dairy cattle manure as a supplement to improve net energy gain in fermentative hydrogen production from sucrose[END_REF]. In anaerobic digestion processes for methane production, the production of acidity from VFAs generation is balanced by production of alkalinity from ammonia and bicarbonate [START_REF] Michael | The microbiology of anaerobic digesters[END_REF][START_REF] Redzwan | Supplementation of ammonium bicarbonates for the treatment of fruit cordial wastewater by anaerobic digestion process[END_REF]. However, DF processes are not stable due to continuous production of VFAs, lowering the process pH.

Culture pH plays an important role in the biochemical pathways and H2 yields. Depending on the type of substrates, often an acidic range (5.0-6.0) favors H2 production while a very low pH can inhibit the hydrogen production (Ghimire et al., 2015a;Khanal et al., 2003). DF processes require nutrient supplements and adequate pH buffering agents to maintain optimal DF conditions, which can inevitably impede the economic sustainability of the DF process in scaled-up applications (Gottardo et al., 2013;Tenca et al., 2011). Choi and Ahn (2013) have suggested the use of substrates with a high pH to replace chemical buffers. There have been very few studies carried out using animal manure as a buffering agent and nutrient amendment in order to get higher H2 yields [START_REF] Marone | Optimization of substrate composition for biohydrogen production from buffalo slurry co-fermented with cheese whey and crude glycerol, using microbial mixed culture[END_REF]Tenca et al., 2011;Wu et al., 2009). In particular, reports on the effect of animal manure addition on long-term continuous H2 production is very scarce (Tenca et al., 2011).

The aim of this work is to study the use of buffalo manure (BM) as a co-substrate for continuous H2 production using CHW as a main substrate in thermophilic DF process.

The study also aims at maximizing the H2 yields along with optimal process stability. In addition to quantity and quality of daily H2 productions, major soluble metabolites, culture pH, total alkalinity, and the ammonium (NH4 + -N) concentrations were monitored to assess the process performances.

Materials and methods

Start-up, inoculum and feedstock

The heat shocked anaerobic digested sludge collected from an anaerobic digestion plant described elsewhere [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF]

Semi-continuous reactor and operating conditions

A continuously stirred tank reactor of 1500 ml working volume and 700 ml headspace was setup with continuous biogas measurement (Figure 4.1). The reactor was fed with CHW and the effluent extracted manually on daily basis. The produced total volume of gas was measured with volumetric displacement method passing through acidic water (1.5 % HCl) and the volume of H2 was confirmed by the analysis of gas composition.

Based on the different feeding strategy of BM and operational conditions of the reactor, the operation periods were divided into seven experimental periods as shown in Table 4.8.

Analytical methods

The biogas composition was quantified by Varian Star 3400 gas chromatograph equipped with ShinCarbon ST 80/100 column and a thermal conductivity detector. The duration of analysis was 14 minutes. Argon was used as carrier gas with front and rear end pressure of 20 psi. The major fermentation products (lactic, acetic, propionic and butyric acids)

were quantified by High Pressure Liquid Chromatography (HPLC) (Dionex LC 25

Chromatography Oven) equipped with Synergi 4u Hydro RP 80A (size 250×4.60mm) column and UV detector (Dionex AD25 Absorbance Detector), as described elsewhere [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF]. Ethanol was also quantified by HPLC (Aminex HPX-87H column (300 mm on 7,8 mm, Bio-rad), as described elsewhere [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF].

COD was determined according to a method described elsewhere (Noguerol-Arias et al., 2012). The carbohydrates were determined by Dubois method (DuBois et al., 1956) and total lipids were measured by Bligh and Dyer chloroform/methanol total lipid extraction method (Bligh and Dyer, 1959). TS, VS of seed sludge and TKN were determined according to Standard Methods [START_REF] Apha | Standard Methods for the Examination of Water and Wastewater[END_REF].

Data analysis

Biohydrogen production rates (HPR) were expressed in mL H2/L/d while the H2 yields (HY) were determined considering the total daily CHW and BM fed to reactor and expressed as mL H2 /g VS added. Average values and corresponding standard deviations were calculated after allowing the 3 -4 days of time to achieve the steady state. The H2 Production Stability Index (HPSI) was evaluated by considering the ratio of standard deviation and average HPR, reported previously by Tenca et al. (2011) given in equation 4.1. Principal Component Analysis (PCA) analysis was carried in statistical analysis was carried in FactomineR extension R Commander (Version 2.1-7 OS X) included in R software (Version 3.1.1 OS X).

Results and discussion

Effect of CHW:BM ratio on HY, HPR and HPSI

The HY, HPR and HPSI during seven experimental periods of 110 days are summarized in Table 4.9 and Figure 4.5. The addition of buffalo manure improved the H2 production stability to 0.66 and 0.71 during the experimental periods III and IV, respectively. The HY during the start-up of the reactor, i.e. periods I and II increased for some days, however the production of H2 was not sustained due to pH depletion as a result of VFA accumulation. The HY decreased at the end of the period IV, even though the pH was stable around 4.8 ± 0.1. This might be attributed due to the increase in total metabolites concentration in the reactor. One of the best strategies to avoid VFA accumulation is facilitating its removal from the reactor by decreasing the HRT without washing out of the microbial biomass.

During the experimental period V, the H2 production decreased further when the HRT of the reactor was decreased from 12 to 8 days. This might be due to washing out of the biomass which was evident by the decreased in fermentative acitivities seen from lower yields in other metabolic by-products (Table 4.10). Moreover, another reason for low HY can be the sudden decrease in the culture pH. Consequently, BM fraction in the feed was increased (Table 4.8) as a strategy to increase alkalinity in the reactor. The H2 production increased for a while, however it did not lasted longer. This might be due to proliferation of H2 consumers such as methanogens present in the BM (Cheong and Hansen, 2006;Wu et al., 2009). As a control strategy CHW:BM ratio was increased again, decreasing the BM in the influent feed. This eventually increased the H2 production (Figure 4.5).

Effect of BM addition on pH, alkalinity and ammonia concentration

The trends of alkalinity, ammonia and pH during the different BM feeding strategies are presented in Figure 4.6. The production of organic acids that followed the DF are responsible for decreases in the culture pH. The culture pH has profound impact on the selection and growth of fermentative microbial communities and thus their metabolic pathways (H2 production). Therefore, it is necessary to maintain the culture pH in the DF process below the inhibitory levels of 4.5, which favours solventogenesis. The limitation in the use of animal manure might be a need for physical or chemical treatment for inhibiting methanogenic activities which consume H2 (Cheong and Hansen, 2006;Wu et al., 2009). However, this study did not consider any pre-treatment for BM before feeding. Moreover, another, limitation in the use of BM could be an inhibition of the H2 production due to higher ammonia content in BM. The animal manure such as swine, poultry and dairy manure have a low C/N ratio (C/N ratio of swine manure: 12.8) (Yin et al., 2014) and higher levels of ammoniacal nitrogen (cattle slurry: 1040 -1925 mg/l and chicken manure 7000 -12,800 mg/L) (Callaghan et al., 2002) that might cause inhibition of microbial community. Cavinato et al. (2012) reported the decrease in H2 production at total ammoniacal nitrogen concentration higher than 2 g N/L. However, ammonia levels in our study were lower than the inhibitory levels reported in literature.

Metabolites production

Major

Conclusion

DF of acidic wastewater could be challenging due to decrease in pH by the consequent production of organic acids during the DF process without source of alkalinity. A longterm continuous production of H2 has been demonstrated in this work using CHW and BM. HY of 131.8±38.0 mL H2/g VS was obtained with HPSI of 0.71 when the CHW to BM ratio was 3.4 g VS/gVS. However, use of BM characterized by higher alkalinity could be applied as co-substrate for maintenance of operable pH during the DF process around 4.8 -5. Therefore, addition of BM can aid in the stability of the continuous dark fermentative H2 production and remove the dependency on chemical-buffering agents.

Furthermore, BM can provide the source of nutrients (nitrogen) during the DF of carbohydrate rich substrates like CHW. Hence, a co-fermentation of CHW with BM could give economic sustainability in scaled-up applications of DF processes that use locally available feedstock sources.

Integration of dark-photo fermentation and anaerobic digestion for enhanced energy yields

This sections presents the results of the study aimed at maximizing the energy yields from food waste in a three-step conversion scheme coupling dark fermentation (DF), photofermentation (PF) and anaerobic digestion (AD). The DF effluents mainly contained volatile fatty acids (VFAs) and alcohols as metabolites and un-hydrolyzed solid residues.

The supernatant, after separation, was used to recover H2 in a PF using Rhodobacter sphaeroides. The solid residual fraction along with PF effluent was converted into methane by anaerobic digestion.

Introduction

The inherent characteristics of hydrogen (H2), such as higher energy content (142 MJ per kg), energy and water as the only by-products generated from its combustion, application in fuel cells for electricity generation and the ability to be produced biologically, makes H2 a very interesting alternative future sustainable energy carrier (Kotay and Das, 2008).

Among several biological technologies proposed for H2 production, dark fermentation (DF) is emerging as one of the prominent options, shown by the increasing research interests in this technology (Ghimire et al., 2015a). The advantages such as the flexibility to operate under different conditions of temperature and pressure, higher production rates, possibility to use renewable waste biomass as feedstock and the treatment capability make the DF process attractive. Waste biomass such as agricultural residues, the organic fraction of municipal solid waste (OFMSW) and agro-industrial wastes are economically competitive when considering a supply of sustainable feedstock, aiming at the industrial development of DF systems for biological treatment of waste (Chong et al., 2009;De Gioannis et al., 2013;Ntaikou et al., 2010).

It has been well documented that dark fermentative H2 production is generally due to the conversion of the initial soluble fraction of carbohydrates present in the complex organic biomass, that will lead to accumulation of volatile fatty acids (VFAs) and alcohols in DFEs (Guo et al., 2013;Monlau et al., 2012). Some recent studies have shown the potential of these DFEs to be utilized in PF processes for H2 production (Chookaew et al., 2015;Rai et al., 2014). Combining DF with PF, Su et al. [START_REF] Su | Hydrogen production from water hyacinth through dark-and photo-fermentation[END_REF] achieved an increase in H2 yield from 76.7 to 596.1 L H2/kg VS from water hyacinth. Meanwhile, Rai et al. (Rai et al., 2014) achieved 43% higher volumetric H2 yields from acid hydrolyzed sugarcane bagasse in two step DF-PF systems. However, during the conversion of complex organic biomass like FW, a part of the unhydrolyzed solid residues will remain that can be further valorized in AD systems producing methane (CH4) in a three steps conversion scheme (Figure 5.1). Xia et al. (Xia et al., 2013a(Xia et al., , 2013b) ) reported that a threestep conversion of algal biomass combining DF-PF-AD can achieve 1.7 and 1.3 times higher energy yields in comparison to a two-stage DF-AD and an one stage AD process, respectively. High OLRs are often responsible for a decrease in culture pH due to the accumulation of VFAs present in DFE. Thus, most of the continuous DF systems utilizing acidic substrates such as food waste require constant addition of external alkalinity sources such as alkaline chemicals (NaOH or KOH) or buffering agents (bicarbonate or phosphate buffers) (Elsamadony and Tawfik, 2015;Shin and Youn, 2005;Valdez-vazquez et al., 2005)

Materials and methods

Preparation of feedstock

An average composition of waste, as found in European countries, was prepared as cited elsewhere (VALORGAS, 2010). The waste mixture was prepared at the laboratory and was composed of (in % by weight): fruit and vegetables 72%, cooked pasta and rice 10%, bread and bakery 5%, dairy products (cheese) 2%, meat and fish 8% and snacks (biscuits) DFE were collected from the outlet of the fermenter and had a pH of 4.5 ± 0.1. After undergoing settling for 30 minutes and centrifugation at 4500 rpm for 20 minutes, the supernatant was collected. The DFE characteristics are presented in Table 5.1. The DFE was supplemented with KH2PO4, 3 g/L; NaHCO3, 0.7 g/L; ferric citrate 24.5 mg/L and 10 mL of a trace metals solution (for composition, see below). pH was adjusted to 6.5 and then the DFE medium was autoclaved at 121 ⁰C for 20 minutes. The solid residues left after settling and centrifugation of DFE along with the PF effluents mainly containing photofermentative biomass were used as feed for AD. The characteristics of the solid residues generated from solid-liquid separation was comprised of undigested FW which had a pH of 4.5 ± 0.1 and solid DF residue with a content of: COD 2.64 ± 0.4 g/kg residue; TS 2.42 ± 0.02% and VS 2.31 ± 0.02%. The PF effluent had a pH of 7.26 ± 0.01; and contained a soluble COD of 1407.7 ± 109 mg/L; with 0.71 ± 0.01 % TS and 0.28 ± 0.01 % VS contents.

Experimental setup and operational conditions

Dark fermentation bioreactor

The experimental set-up and start-up of DF reactor is explained in Section 4.1.2. The reactor was operated in semi-continuous mode with three different HRTs and four OLRs in six different operational conditions (Table 5.2).

Photofermentation bioreactor

Rhodobacter sphaeroides AV1b (kindly provided by professor Roberto De Philippis, University of Florence, Italy) was previously isolated from the Averno lake in Naples (Italy) as described elsewhere in Bianchi et al. (Bianchi et al., 2010) and was used as inoculum for PF. R. sphaeroides AV1b was first grown in a medium as previously described by Bianchi et al. (Bianchi et al., 2010), which was composed of (in g/L): DLmalic acid, 2; sodium glutamate, 1.7; K2HPO4 , 0.5; KH2PO4, 0. silica and flushed with argon to ensure anaerobic conditions and eliminate the nitrogen gas (N2) from the headspace since N2 can inhibit the activity of the nitrogenase enzyme responsible for photofermentative H2 production (Koku et al., 2002). The H2 production was quantified as in DF process.

AD of residues from DF-PF process

A batch test was carried out in 1 liter transparent borosilicate serum glass bottles (Simax, Czech Republic) and was maintained at 34 ± 1°C in a water bath. The working volume of the reactor was 600 mL with an initial S/X ratio of 0.5 with a substrate concentration of 4.5 g VS/L. A low S/X ratio 0.5 was selected to assess the biomethane potential of the feed used. Based on the substrate type, a range of S/X ratio 0.5 -2.3 gVS substrate/gVS inoculum is suggested to prevent the acidification of the AD reactor (Esposito et al., 2012). The source of inoculum used in the tests was the same as the start up inoculum used in the semi-continuous DF reactor. The characteristics of the inoculum were (in g/L): TS, 23.71 ± 0.17; VS, 14.55 ± 0.11; ammonium (NH4 + -N), 0.46 ± 0.02; and had a pH 8.2 ± 0.1. The tests were carried out in duplicates.

Results and discussion

Effect of operational parameters on H2 production rate and yield

The results in terms of H2 yields (HY), hydrogen production rates (HPR) and H2

Production Stability Index (HPSI) during the different OLRs and HRTs investigated in the six operation periods (Table 5.2) are summarized in Table 5.3. (Tables 2 and3). Meanwhile, the overall HY increased from 54.1 ± 41.3 N L/kg VSadded to 105.1 ± 14.9 N L H2/kg VSadded. During the experimental period IV, the H2 production had a comparatively better stability as shown by a HPSI of 0.86. However, no significant effect was observed on the total HY and HPR when the HRT changed to 4 days during operational period V (Table 5.3). When the OLR was changed from 2 to 2.5 kg VS/m 3 /d during period VI, both HY and HPR increased. However, the H2 production was not stable, supported by a lower value of HPSI of 0.63. This instability could be explained by the accumulation of acids and a subsequent decrease in pH to 4.4 ± 0.1, which might have affected the microbial community.

During a short operation period (at the end of period IV), the culture pH inside the reactor was regulated manually to an initial culture pH 5.5 with 1 M NaOH, during feeding, with the objective to assess the influence of pH on the H2 production performance (Figure 5.2 b). However, pH regulation did not show any effect on the HPR (Figure 5.2 a).

Nevertheless, the increased HPSI (Table 5.3) showed that H2 production was stable during that period in comparison to the experimental period when the culture pH was uncontrolled. The percentage of H2 and CO2 in the gas averaged 59 ± 6% and 39 ± 6%, respectively, when the H2 production stabilized. However, the H2 production performances in experimental period IV (HPR: 210. shaded region represents the experimental period when the culture pH inside the reactor was adjusted daily to pH 5.5 during the feeding operation.

A comparison of previous studies on dark fermentative H2 production from FW with the results from this study (Table 4.4) suggests that comparable results in terms of H2 production can be achieved even at low OLRs and without pH control. Nonetheless, the characteristics of FW can also affect the overall HY as H2 production is mainly function of the soluble fraction of carbohydrates present in the substrate (Guo et al., 2013). The OLRs reported in the past studies were higher than in this study, and thus a source of alkalinity to balance the pH conditions at optimum was required. Valdez-Vazquez et al. (Valdez-vazquez et al., 2005) used NaHCO3 and K2HPO4 to maintain the optimum pH at 6.4, while Lee et al. (Lee et al., 2010) used NaOH and H3PO4 to maintain the culture pH at 6. Thus, this pH decrease resulting from the production of acids can be minimized by the use of lower OLRs. Higher OLRs can exert detrimental effects on the microbial community, and thus H2 production, by decreasing the pH due to the accumulation of metabolites ( Van Ginkel and Logan, 2005).

Metabolic intermediates

Lactate, acetate, propionate, butyrate and ethanol were the main metabolic intermediates observed during the different experimental periods. Such a mixture of intermediates is characteristic of mixed fermentation pathways occurring with complex substrates (Guo et al., 2013). Average concentrations of the main metabolites during the six different experimental periods are summarized in Table 5.4. There can be a number of possible H2 production pathways during mixed type fermentation, as represented by equations 2 -5 (Table 4.6), whereas H2 consuming or unfavorable pathways presented in equations 6

-9 might exist at the same time (Hawkes et al., 2007;Li and Fang, 2007). The presence of ethanol, acetate and butyrate are evidences for the presence of an ethanol-acetate or butyrate-acetate pathway for H2 production in the DF of the FW investigated. On the other hand, the presence of lactate or propionate can be attributed to fluctuations in H2 production resulting in low H2 yields. 4.6) in a DF with glucose as model substrate. However, the pathways leading to ethanol-acetate also yield H2, as shown in Equation 4 [START_REF] Hwang | Anaerobic bio-hydrogen production from ethanol fermentation: the role of pH[END_REF][START_REF] Lin | Enhancement of fermentative hydrogen/ethanol production from cellulose using mixed anaerobic cultures[END_REF]. Nonetheless, Figure 5.3 shows that the ethanol is not correlated with acetate. Therefore, most of the H2 yields can be attributed to the butyrate-acetate pathway, which showed a good correlation and is explained in Dim 1. The variable Dim 3 is mostly explained by lactate concentrations (Figure 5.3 b), which correlated oppositely with HY and is an orthogonal and independent variable. The proximity of butyrate, ethanol and propionate suggests that these metabolites can be expected from DF by mixed microbial consortia. This is also supported in a study by [START_REF] Hwang | Anaerobic bio-hydrogen production from ethanol fermentation: the role of pH[END_REF] who obtained butyrate, ethanol and propionate as the major metabolites during the DF at a pH range of 4-4.5, 4.5-5.0, 5.0-6, respectively. 5.1) were both non-inhibiting levels for photofermentative H2 production. Han et al. (Han et al., 2012) reported that concentrations equal to 9.8 mM, 10.9 mM and 4.2 mM, respectively, for acetate, butyrate and propionate gave the optimum H2 yield using R. sphaeroides. However, concentrations up to 30 mM of acetate have been reported by Hustede et al. (Hustede et al., 1993). Similarly, the ammonium concentration was at non-inhibitory levels, as only a concentration higher than 2 -5 mM of NH4 + -N has been reported to inhibit the photofermentative H2 production (Argun et al., 2008;Lee et al., 2011). The PF of spent DFE yielded a cumulative production of 365.6 ± 3.2 NmL H2, corresponding to a volumetric yield of 914 ± 8 N L H2/m 3 and a substrate yield of 427 ± 6 N L H2/kg COD consumed. The batch experiments were carried out for 40 days until the H2 production completely ceased (Figure 5.4 a). This is longer than any H2 production time reported elsewhere (Rai et al., 2014;Xia et al., 2013b). The long lag phase (9 days) can partly explain this result. The final effluents were analyzed for COD, VFAs and biomass concentration which showed a COD reduction of 60.1%, while more than 98 ± 1% of VFAs were removed to reach a final biomass concentration of 1.6 g TSS/L.

Theoretical COD removal calculated from the VFA concentration in final effluents showed a COD removal efficiency of 99.2%. However, the production of biomass and other bacterial carotenoids increased the final total COD of the PF effluent and thus reduced the total COD removal efficiency. This was evident by the reddish brown color of the effluent. The maximum percentage of H2 in the biogas was 89% with 8.9% of CO2.

The volumetric H2 production obtained in this study (914 ± 8 N L H2/m 3 ) is higher than the study of Rai et al. (2014) using Rhodopseudomonas BHU 01 with a volumetric H2 yield of 755 L H2/ m 3 . In another study by Uyar et al. (2009) using Rhodobacter capsulatus (DSM 155) as biomass and DFE of Miscanthus hydrolysate as substrates, a volumetric yield of 1000 L H2/ m 3 was obtained, which is slightly higher than in this study. The present study showed the potential of an integrated DF-PF system to achieve higher H2 yields. Thus, the combined DF-PF processes can help in the industrial development of DF processes using FW. The residues generated from the downstream of these processes can, nevertheless, still be treated with anaerobic digestion in order to provide additional conversion of organic matter to further recover energy.

AD of DF-PF waste stream

The solid residues generated by the coupled DF-PF process can be ideal for AD as the undigested FW residues from the DF process and the PF effluent containing biomass generated from the PF can be converted to methane in a biorefinery model (Figure 5.1).

The result of the average cumulative methane production trends during the biomethane potential test using the waste stream generated from the DF-PF process is presented in previously reported in the literature (Table 5.5). The energy yields of hydrogen and methane from the stand alone DF as well as the two stage DF-PF and DF-AD was calculated based on the heating values of H2 (242 kJ/mol) and methane (801 kJ/mol).

These calculated energy yields represent the energy gain from the conversion of substrates by biological processes. However, the net energy gain can be estimated by considering the energy input in the processes, which is not representative in lab scale reactors and thus not calculated in this study. By coupling DF with PF and AD processes, an additional 4.4 MJ/kg VS of energy yield can be achieved from food waste, which is higher than the coupled DF -AD process or stand alone DF processes (Table 5.5). Out of the overall energy recovered from the threestage conversion (DF-PF-AD) of food waste, H2 contributes only 35.8% out of 5.55 MJ/kg VS. However, this may be a positive add-on to the overall economic return compared to CH4 productivity only. Therefore, the three-step process can definitely increase the recovered energy yield. Moreover, it is a very good solution for waste treatment as a higher FW conversion was accomplished. Table 5.5 shows that the energy yield of DF and PF from the study of [START_REF] Zong | Efficient hydrogen gas production from cassava and food waste by a two-step process of dark fermentation and photo-fermentation[END_REF] is higher than the energy yield reported in this study. This is likely because of the difference in H2 yield achieved in these studies. In other studies by Xia et al. (2013aXia et al. ( , 2013b) ) and Wang et al. (2015), although the overall energy yields obtained from the respective three and two step conversion were high, the pre-treatment of the substrate required an energy input. Therefore, the overall energy yields obtained from the coupling of various processes depends on the H2 and CH4 yields and production rates in individual processes, which are mainly a function of process operational conditions such as pH, temperature, HRT and OLR as well as carbohydrate content and nature of the feedstock. Moreover, the coupling of the PF and AD processes in the downstream process is not only advantageous from the energy point of view, but it also provides biological treatment of the waste stream generated by the DF processes (COD and pathogen removal) [START_REF] Ward | Optimisation of the anaerobic digestion of agricultural resources[END_REF].

Conclusion

This study has shown the long-term feasibility of continuous H2 production as well as the possibility to further recover energy through integration of PF and AD using FW as the substrate. In addition, the viability of H2 production at low OLRs without the culture pH control can minimize the excessive use of chemical buffering agents for pH control. The integration of DF with PF can increase the overall H2 yield 1.75 fold. On the other hand, applying AD for the post treatment of waste streams generated by the coupling of the DF-PF processes can further increase the overall energy yield by 5.55 MJ/kg VS of food waste, adding a synergistic effect to the overall energy recovery during the conversion of food waste.

H2 and biopolymer production by phototofermentation

This section presents results of the study of concomitant production of biohydrogen and polyhydroxybutyrate (PHB) from photofermentation (PF) using spent medium produced from thermophilic dark fermentation (DF) of food waste mainly containing volatile fatty acids (VFAs) and alcohols as soluble metabolites. This study showed that DF-PF coupling not only yields energy and economic benefits in terms of H2 and PHB productions but it also provides post treatment of residues by removal of COD.

Introduction

Biological hydrogen (bio-H2) processes have gained much interest as they could lead to low cost and renewable hydrogen production technologies which are environmentally benign (Das and Veziroglu, 2008). Biological hydrogen production processes can be categorized into light dependent processes such as biophotolysis and light independent processes such as dark fermentation (DF) and bioelectrochemical systems or microbial electrolysis cells (Ghimire et al., 2015a). In light dependent processes, water is broken down into H2 and O2 gas by algae and cyanobacteria. Alternatively, cyanobacteria or cyanophytes can also synthesize H2 from water and inorganic carbon. Moreover, photofermentation (PF) is carried out by photosynthetic bacteria, where photodecomposition of organic compounds into H2 occurs (Das and Veziroglu, 2008;Hallenbeck and Ghosh, 2009).

DF systems are a promising biological route for H2 production due to its mild operational requirements (ambient temperature and pressure), higher conversion rates to H2 and wide range of complex low cost waste biomass that can be used as feedstock (Ghimire et al., 2015a;Guo et al., 2010). However, dark fermentative conversion of complex organic biomass to H2 produces by-products, mainly volatile fatty acids (VFAs), lactic acids and alcohols as soluble metabolites and un-hydrolyzed solid residues, leaving incomplete conversion of the organic biomass (Xia et al., 2013). Dark fermentative biohydrogen production is strongly correlated with the initial soluble carbohydrate fraction present in the substrates (Alibardi and Cossu, 2016;Guo et al., 2013;Monlau et al., 2012).

Nonetheless, the soluble metabolites (organic acids and alcohols) present in DF residues can be further converted to biohydrogen through PF (Chookaew et al., 2015;[START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF]Rai et al., 2014).

operational conditions (De Philippis et al., 1992;Montiel-Corona et al., 2015). In R.

sphaeroides, Waligórska et al. (2009) found that accumulation of PHB increased 30 fold when the C/N ratio rose from 6 to 120. Although PHB biosynthesis is a H2 competing pathway, its concomitant production with hydrogen raises future interests, as PHB possesses economic value as a precursor for biodegradable polymers (Koku et al., 2002).

Use of a mixed culture of PNSB is important for practical applications, as it reduces the asepsis costs involved when waste residues from DF systems are utilized. PF by pure cultures using spent DF residues generated from complex waste biomass has been reported in a few studies, i.e. sugarcane bagasse (Rai et al., 2014), glycerol (Chookaew et al., 2015) and cassava [START_REF] Zong | Efficient hydrogen gas production from cassava and food waste by a two-step process of dark fermentation and photo-fermentation[END_REF]. However, there are limited studies that have been conducted using mixed PNS cultures for DFE conversion to H2 (Montiel-Corona et al., 2015;[START_REF] Tawfik | Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent[END_REF]. In a recent study, [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF] reported the 1.75 fold increase in H2 yield from the integration of DF and PF processes using adapted R.

sphaeroides cultures as inoculum. However, a long lag phase for H2 production was observed, which was attributed to the initial PHB accumulation [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF].

The aim of this study was to investigate the concomitant production of H2 and PHB from DFE (with and without dilution) obtained from the thermophilic DF of food waste, using adapted pure and mixed PNSB cultures under sterile and non-sterile conditions, respectively. H2 production, PHB quantification and COD removal efficiency were the major parameters taken into consideration during this study of DFE valorization. Other hydrogen production performance parameters such as lag phase and time required to achieve 95% of the maximum H2 production were considered for the evaluation of the photofermentative H2 production performance.

Materials and methods

Dark fermentative H2 production

A thermophilic DF process, described elsewhere by [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF], was set-up for continuous hydrogen production from food waste. A semi-continuous stirred 2.0 L serum bottle with a 1.5 L working volume and 500 mL headspace was used as DF reactor.

The culture pH was 4.5 (± 0.2). The H2 yields and production rates were 104 (± 17 NmL) H2/g VS and 208 (±35) NmL H2/L/d at organic loading rates (OLRs) of 2 gVS/L/d and hydraulic retention time (HRT) of 4 days (described in section 4.1).

uptake of propionic and butyric acid due to unbalances in the oxidative and reductive potential (Montiel-Corona et al., 2015). The pH of the DFE medium was adjusted to 6.5

with 1 M NaOH.

PF experiments

Three sets of experiments were conducted to assess effect of dilution and use of the pure and mixed PNSB culture for H2 and PHB production by PF of DFE medium. Transparent borosilicate glass bottles (Simax, Czech Republic) with a 500 mL capacity and a 400 mL working volume were used as photofermentative batch reactors. The batch reactors were maintained at a room temperature 24 (± 2) °C (April-June) under the luminance of approximately 4000 Lux (20 W compact florescent light) and positioned on a continuous stirrer (250 rpm). A long lag phase was observed during the H2 production in the study of [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF]. The PHB concentration was analyzed in the samples collected every 3-5 days during the tests. The reactors were provided with arrangements for sampling of gas and culture medium. The bottles were flushed with argon to provide the anaerobic conditions and eliminate the nitrogen from the headspace.

Analytical methods

Hydrogen was quantified by a Varian Star 3400 gas chromatograph equipped with ShinCarbon ST 80/100 column and a thermal conductivity detector. Argon was used as carrier gas with of 20 psi front and rear end pressure. The duration of analysis was 14 minutes. The fermentation products were quantified by High Pressure Liquid Chromatography (HPLC) (Dionex LC 25 Chromatography Oven) equipped with a Synergi 4u Hydro RP 80A (size 250×4.60 mm) column and UV detector (Dionex AD25 Absorbance Detector) as described by [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF]. Gradient elution consisted of 20% methanol and 10% acetonitrile in 5 mM H2SO4, pumped at a rate of 0.9 ml/min by using a Dionex GP 50 Gradient pump. The elution time was 18.5 minutes.

For PHB analysis, samples were vacuum dried and the polymer was extracted according to [START_REF] Oehmen | Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems[END_REF]. PHB was quantified by gas chromatography (GC) equipped with a mass spectrometer (MS) and HP 5MS (Agilent) column and helium as the carrier gas.

The light intensity was measured with a light meter (Lutron-LX-107).

The COD was determined by the Closed Reflux method and Total Kjeldahl Nitrogen (TKN) by macro-Kjeldahl as described in the Standard Methods [START_REF] Apha | Standard Methods for the Examination of Water and Wastewater[END_REF]. Biomass growth was quantified by spectrophotometric measurements of the Optical Density at 660 nm (OD660) (Photolab Spektral, WTW, Germany). Dry Cell Weight (DCW) was determined after filtering 20 mL of PNSB culture samples on GF/F Whatman filters dried at 105 ºC for 24 hours. DCW was correlated to the OD660 measurements using the calibration curves OD660 = 3.6876*DCW (R = 0.99823) and OD660 = 3.1839*DCW (R = 0.99865), respectively, for R. sphaeroides AV1b and mixed PNSB cultures.

Data analysis

The H2 production was quantified with water (acidified with 1.5% HCl) displacement, and was normalized at standard conditions described else where in [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF].

The modified Gompertz equation (5.2) allowed to compare the kinetics associated to different PF tests, and to evaluate the effect of the experimental conditions. The empirical equation (5.2) gives five major parameters: i) cumulative biohydrogen production (or potential) (Ho, mL), ii) bio-H2 production rates (R, mL/h), iii) e = 2.71828, iv) lag time (λ, hours) and v) total cultivation time (t, hours). The equation 5.2 can be rearranged to equation 5.3 in order to calculate the time required to produce 95% of the maximum H2 production (t95). The parameters Ho, R and λ were estimated using the Curve

Fitting Toolbox in MATLAB ® with an associated 95% confidence limit.

Results and discussions

Concomitant production of H2 and PHB

Undiluted versus diluted DFE When the VFAs were completely degraded (Figure 5.6 B), a decrease in PHB accumulation was observed with a final PHB accumulation of 32.5 (± 3%) of DCW. This trend of PHB accumulation is in accordance with the literature, as PNSB can accumulate PHB as cell reserve material, which they use during a famine stage when substrate is depleted, as explained in the "feast-famine" theory [START_REF] Johnson | Enrichment of a mixed bacterial culture with a high polyhydroxyalkanoate storage capacity[END_REF]. [START_REF] James | Poly-3-Hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments[END_REF] reported that microorganisms use PHB as an energy source for survival during the low nutrient environments. Therefore, R. sphaeroides AV1b might have used PHB for their growth and metabolism when VFA depleted (Figure 5.6 A and B). This phenomenon can be supported by a decrease in PHB concentration and small increment in H2 production after day 35, when the VFAs were completely depleted (Figure 5.6 B).

However, this small increment in H2 production can be also due to conversion of more complex or recalcitrant organic matter, i.e. carbohydrates, that might be present in the DFE medium as reported also by Montiel-Corona et al. (2015). The cumulative H2 production from RS-I tests (914 ± 8 mL H2/L) is comparable to that of Uyar et al. (2009), who obtained a maximum H2 production of 1000 mL H2/L from the DFE obtained from DF of Miscanthus hydrolysate using Rhodobacter capsulatus. The H2 yield is a function of reactor operational parameters, PNSB species used and substrate type (Eroglu and Melis, 2011). The average biomass concentration was 1.6 (± 0.1) g TSS/L at the end of the test. 5.7 show that the lag phase decreased to half when the DFE was diluted. Moreover, the time required for achieving 95% of the maximum production also decreased by 4 -6 days. This might be due to the lower biomass concentration in the culture during the RS-D tests (Figure 5.7 B) compared to RS-I tests (1.6 g TSS/L). The biomass concentration strongly influences the availability of light for H2 producing activity by PNSB (Koku et al., 2003). This can be supported by the 33.6 % decrease in the H2 yield in RS-D tests.

This shows that the H2 production performance was compromised by dilution. However, this decrease in the H2 yield can be due to the decrease in carbon source (COD) concentration in the culture medium i.e. DFE (Table 5.6). Besides, the H2 production activity in R. sphaeroides AV1b might decrease with time during batch cultures due to decline in the activity of the electron carrier ferredoxin (Koku et al., 2003). Therefore, a continuous PF reactor can be adopted to eliminate the issues with a longer lag phase and reduction in the H2 production activity of the PNSB cultures to maintain optimal H2 production performance.

Pure culture versus mixed PNSB culture) The trends of PHB production showed that the maximum PHB concentration is reached close to the fermentation time period when 95% of the maximum H2 production was achieved (Figure 5.6 and Table 5.7). In all experiments (Figure 5.7 and Table 5.7), it can be seen that the PHB concentration decreases when all VFAs are depleted in the medium.

Thus, the bacterial biomass should be harvested during this period to recover the maximum amount of PHB. The H2 and PHB yields for pure cultures are higher than those of mixed PNSB, this might be attributed to the absence of effective PNSB species that are responsible for H2 production or gives lower PHB yields, i.e. R. palustris have a comparatively lower capacity to accumulate PHB compared to R. sphaeroides (Montiel-Corona et al. 2015).

Substrate conversion efficiency and COD removal

The theoretical photofermentative conversion of organic acids, typically present in DF residues, to H2 and PHB can be expressed by equations 3 -7 in Table 5.8 (Barbosa et al., 2001;De Philippis et al., 1992;Han et al., 2012). The conversion ability of different PNSB varies on difference in substrate types (Barbosa et al., 2001;Bianchi et al., 2010).

Identical to H2 production (Table 5. 5.6). Therefore, the production of PHBV in significant amounts was not expected.

Depending on the operational parameters such as C/N ratio, pH and substrate concentration, the hydrogen production in PNSB competes with PHB production (Hustede et al., 1993). Montiel-Corona et al. (2015) reported a negative correlation between the H2 and PHB production in photofermentation using DFE obtained from DF of fruit and vegetable wastes. Nevertheless, they also reported that the conditions such as substrate type, concentration, argon flushing and alkaline culture pH can induce PHB accumulation along with H2 production. Likewise, this study shows that the concomitant production of hydrogen and PHB is possible through PF of DFE. (2015). In addition, the enrichment of PNSB cultures that determine a healthy population of H2 producers can strongly influence H2 yields. Therefore, the differences in H2 and PHB yields might be attributed mainly to differences in substrates types and concentration and PNSB cultures. In R. sphaeroides, PHB yields are higher when acetate is a sole substrate in PF medium (Hustede et al., 1993) (Table 5.9). Similarly, the competitive nature of H2 and PHB can be clearly seen from the fact that the higher H2 yields are obtained when PHB yields are lower (Table 5.9). On the contrary, studies conducted with complex substrates such as DFE (mixed organic acids) and wastewater have shown the concomitant H2 and PHB production (Montiel-Corona et al., 2015;Yiǧit et al., 1999), which is in agreement to this study.

In addition to production of valued added products, PNSB can efficiently remove COD from the DFE, which makes PF process attractive for environmental engineering applications. Considering the removal of major VFAs (acetate, butyrate and propionate)

and lactate from the DFE, the total VFAs removal efficiencies exceeding 99, 95 and 85%, respectively, were achieved in the tests with RS-I, RS-D and PM-D, respectively.

However, the biomass in the final PF medium, evidenced by the reddish brown color of the effluent due to the presence of colloidal bacterial pigments, contributes to a fraction of the final total COD of the effluent. Nevertheless, concentrated PNSB biomass can be used as feedstock for the anaerobic digestion processes for the recovery of methane, as

shown by [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF].

Conclusions and future perspective

Concomitant H2 and PHB production was demonstrated using undiluted and diluted DFE by pure and mixed PNSB cultures. Higher H2 and PHB yields were obtained from R.

sphaeroides AV1b with undiluted DFE. H2 and PHB yields from mixed PNSB cultures were lower than R. sphaeroides AV1b cultures. Moreover, the use of mixed cultures could be more appropriate for the treatment of DFE in scaled-up applications, as it can give high COD removal efficiency, save the associated asepsis costs and a wide range of waste biomass can be used. Nonetheless, pure R. sphaeroides cultures can be applied for PHB production.

Solid State Dark Fermentation (SSDF) can serve as biological pre-treatment for the utilization of feedstock in a biorefinery concept. A SSDF process can offer several advantages over conventional wet processes, which are usually operated under the low total solids (TS) contents (often less than 10%). A commercial dry AD process is usually operated at TS content higher than 20%. The operational advantages include high substrate loading rates and low water addition. Therefore, SSDF can offer i) economic benefits by reducing the reactor volume and specific energy requirements, (ii) an efficient handling of digestate and (iii) a higher technical simplicity.

The past studies have shown that an increase in TS content impacts the substrate degradation and biogas production (Abbassi-Guendouz et al., 2012;Motte et al., 2013). [START_REF] Fernández | Effect of substrate concentration on dry mesophilic anaerobic digestion of organic fraction of municipal solid waste (OFMSW)[END_REF] reported decrease in degradation of OFMSW by 17% in SS-AD when the TS content increased from 20 to 30%. In fact, high-solids processes can be restricted by mass transfer limitations that impact the biogas yields as well as the microbial metabolic pathways (Abbassi-Guendouz et al., 2012;Abbassi-Guendouz et al., 2013;Bollon et al., 2013;Liotta, et al., 2014). The mass and energy transfer limitations are driven by the low water content of the system (Motte et al., 2014;Valdez-Vazquez & Poggi-Varaldo, 2009). Thus, microbial activity can be impacted by the transport of soluble components (i.e. substrates, intermediate and end-metabolites). Some studies have shown the dependency of H2 production on TS content (Motte et al., 2013;Motte et al., 2014;Robledo-Narváez et al., 2013;Valdez-Vazquez & Poggi-Varaldo, 2009).

During the study of the effect of TS content on H2 production from DF of wheat straw (WS), Motte et al. (2013) reported significant decrease in H2 production at 19 % TS along with the decrease in substrate conversion. In another study, Motte et al., (2014) showed the reduction in H2 yields, in addition to the favouring the growth of lactic acid producing microbial community in WS. However, very few studies (Valdez-Vazquez & Poggi-Varaldo, 2009) have addressed the issues of effect of increasing TS content in the DF of food waste (FW).

Moreover, in a recent study, Cazier et al. (2015) showed an inhibition of biomass hydrolysis in (SS-AD) anaerobic digestion due to a high hydrogen partial pressure (pH2) in WS. However, the effect of high TS content on acidogenesis and H2 yields is rather unknown, but inhibition of substrate hydrolysis by high local H2 partial pressure in SSDF process is probable. The impact of these parameters under SSDF of FW is not well studied. It is important to understand the limitation of SSDF of organic waste to increase its potential and to open new paths for its industrial application for the production of biofuels and biochemicals.

The present study aims to investigate the effect of the TS content on organic waste conversion in SSDF using FW and WS as representatives of substrates with high and low biodegradability, respectively. In addition, the particular effect of pH2 was also studied to investigate the effect of pH2 in biomass hydrolysis and metabolic pathways.

Materials and methods

Inoculum source and feedstock

Experiments were designed to study the effect of TS content and pH2 on substrate degradation and biochemical pathways in batch SSDF tests. FW was prepared in the laboratory with the composition similar to the one described in [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF].

Heat shocked (90 °C, 15 min) waste activated sludge obtained from a municipal wastewater treatment plant in Limoges (France) was used as inoculum. This inoculum was centrifugated (at 6500 rpm for 20 min, 4 °C) to obtain 11% total solids (TS) and 9%

volatile solids (VS) content. Similarly, WS with TS and VS content of 95% and 97%, respectively, was used as a representative of lignocellulosic biomass.

Experimental set-up

Effect of TS content on H2 production and substrate conversion

Batch tests in triplicates were designed at 10%, 15%, 20%, 25% and 30% TS content to investigate the effect of TS on substrate conversion of FW. In each 600 ml flask, 53.4 g of digestate (38.87 % final TS content), composed of FW (20.3 g) and inoculum (4.1 g) in a ratio of 10 g VS substrate/g VS inoculum, i.e. S/X, 16.0 g 2-(N-morpholino) ethanesulfonic acid (MES) buffer, 12 ml 3.2 % NaOH and 1 ml of trace metal solution (containing FeCl2 2g/L, CoCl2 0.5 g/L, MnCl2 0.1 g/L, NiCl2 0.1 g/L, ZnCl2 0.05 g/L, H3BO3 0.05g/L, Na2SeO3 0.05g/L, CuCl2 0.04 g/L, Na2MoO4 0.01g/L) were added. This mixture had an initial pH of 5.5. The amount of distillated water to be added was calculated with a mass balance on TS contents including substrate, inoculum, buffer and other solution addition to obtain the final TS content of 10.0 ± 0.01, 14.98 ± 0.03, 19.89 ± 0.04, 24.92 ± 0.02, 30.0 ± 0.07 %TS, in each set of experiments. The batch tests were then incubated at 37 ± 1 °C for 14 days.

Effect of partial pressure of H2 on substrate conversion

To study the effect of pH2, batch tests were carried out in four replicates with FW and WS at a S/X ratio 10 and final TS content of 25 ± 1 %. The tests were carried out with a thin layer of digestate (<1 cm), approximately 22 ± 2 g, in order to minimize the effect of gas diffusion (Cazier et al., 2015). H2 was initially added in the headspace of the 600 ml serum bottles in two sets of tests; in one set pH2 was equivalent to 542 ± 32 mbar (33 ± 2 % H2 in the headspace, named as "A") and in the other set it was 1087 ± 29 mbars (66 ± 1 % H2 in the headspace, named as "B"). A control with only N2 in headspace was carried out (named as "C") and the final total pressure at the start of the tests for all the conditions was 1500 mbars. The initial culture pH was maintained at 5.5 with MES buffer and the culture was incubated at mesophilic temperature (37 ± 1 °C) for two fermentation periods of 14 and 21 days.

Analytical methods

Gas composition was measured by gas chromatograph (Perkin Clarus 580) equipped with a thermal conductivity detector at 150°C and an injector heated at 250°C and two capillary columns heated at 60°C. The first column was an RtUbond for the CO2 while the second column was an RtMolsieve used for the detection of the O2, H2, N2 and CH4. Argon at pressure of 350 kPa and flow rate 31.8 mL/min was used as carrier gas. The gas production was monitored with increase in gas pressure, which was measured with a digital manometer (2000, Leo2 Keller).

5.0 g of digestate were diluted in 5 g of deionized water, mixed during 30 minutes, centrifuged at 18,000 rpm during 20 min at 4°C and then filtrated at 0.2 µm with a nylon membrane. The liquid was then used to measure VFAs, others metabolites and soluble sugars. Dark fermentation metabolites in the digestate were measured at the beginning and end of the experiments. VFAs were quantified with gas chromatograph (Perkin Clarus 580) and Elite FFAP crossbond ® carbowax ® 15 m column connected to a flame ionization detector at 280°C and N2 as carrier gas at the flow rate of 6 mL/min, described elsewhere (Cazier et al., 2015). High performance liquid chromatography (HPLC) was used to quantify other metabolites and soluble sugars, that comprised of Aminex HPX-87H column (300 mm on 7.8 mm, Bio-rad), a pre-column to filter residues (Micro guard cation H refill cartbridges, Bio-rad) and an automatic sampler (Water 717). Sulfuric acid 0.005 M was used as eluent at the flow rate of 0.4 ml/min.

Data analysis

Substrate degradation was estimated computing a theoretical chemical oxygen demand (COD) mass balance by calculating the difference in metabolic end-products (accumulated in both gaseous and liquid phase) at the initial and final state. The COD measurements of the complex organic residues such as lignocellulosic biomass and FW may vary more than 10 % while the overall ubstrate degradation during the process might be lower than 10% COD, thus direct measurement of COD was not considered in this study (Cazier et al., 2015). Therefore, total substrate degradation is calculated as the amount of COD produce from the DF of substrate estimated per kg of TS added initially and calculated as: Where, A H 2, f is the amount of H2 remaining at the end in the headspace, A met,f the final amount of metabolites accumulated, A GC the total amount of gas (H2) sampled for analyses, A H 2, i the initial amount of H2 added and A met,i the initial amount of metabolites in the medium.

𝐓𝐨𝐭𝐚𝐥 𝐒𝐮𝐛𝐬𝐭𝐫𝐚𝐭𝐞 𝐃𝐞𝐠𝐫𝐚𝐝𝐚𝐭𝐢𝐨𝐧 = COD of
R software (OSX version 3.1.3) with the package Rcmdr (OSX version 2.1.7) was used for the statistical analysis of data obtained from the experiments. The P value was set at 0.05 and the significance of the results tested with P values: * < 0.05; ** < 0.01; *** < 0.001; while not significant results were with P >0.05.

Results

Influence of TS content

Figure 5.9 shows the effect of TS content on substrate degradation after 14 days of fermentation period. The substrate conversion decreased and biohydrogen production was significantly inhibited when the initial TS content increased and concomitant shift in the metabolic pathways was observed (Figure 5.9 b). The maximum and minimum substrate degradation of 134.44 ± 22 and 51.45 ± 3 g COD/kg TS was achieved at 10 and 30 % TS, respectively. The H2 production decreased drastically when the TS content increased more than 15%

TS and the metabolic pathways favored the lactic acid production, which can be attributed to insignificant amount of H2. H2 was produced only in the TS content 10% and 15%. On the basis of biohydrogen production and nature of end-metabolites, two distinct behaviors were observed at TS 15%. Only one of the three replicates (named TS15a) showed similar nature behavior of DF as TS 10% while the other two replicates showed comparable nature of fermentation as in higher TS content (Figure 5.9 a and b). At TS content higher than 15%, the metabolic pathways mainly shifted towards lactic acid conversion that might explain the decrease in H2 production and substrate conversion (Figure 5.9 b).

Effect of pH2

The different initial pH2 during the SSDF process was tested to investigate its effect on substrate conversion of FW and WS at higher TS content (25 %TS). was observed at 25% TS as in SS-AD (Cazier et al., 2015). This was further evident in metabolic products accumulated at the end of the experimental periods, which show no noteworthy shift (Fig 5.11 b). Substrate degradation slightly increased with fermentation time, and lactic acid and ethanol as the major metabolites were observed in all the tested pH2 with FW.

These results are in contrast with the study of Cazier et al. (2015), which reported an inhibition of H2 on the hydrolysis of WS during SS-AD. In this work, such an inhibition started at pH2 > 742 mbars and substrate degradation decreased from 90 ± 10 to 20 ± 10 g COD/kg TS in the controls and at pH2 1555 mbars, respectively, followed by the decrease in production of methane and acidogenic metabolic products. The effect of pH2 on substrate conversion was not clearly evident during SSDF of readily degradable substrate like FW. The culture pH decreased sharply regardless of adjusting the initial pH with a buffering agent (i.e. MES). Therefore, further tests were carried out with WS, a representative of substrate with low biodegradability, under the similar experimental conditions with an objective to confirm the results obtained with FW.

Nonetheless, the pH did not significantly decrease at the end of the experimental period.

However, the effect of pH2 was not evident, as seen from Figure 5.12 a and b, which was further verified with ANOVA (P-values >0.05). Obviously, the H2 production was inhibited in the tests with higher pH2 (tests A and B). Similarly, lactic acid or ethanol was not present in the metabolic products as in the tests with FW. Interestingly, the substrate degradation of WS during the control tests, i.e. 22.4 ± 2 g COD/kg TS (at pH 5.4), is similar in the study of Cazier et al. (2015), i.e. 20 ± 10 g COD/kg TS, when the maximum inhibition of hydrolysis occurred at pH2 1555 mbars at pH 8-9.

Discussion

The results of the SSDF tests carried with FW at different TS content have shown that the H2 production is impacted by the increase in TS contents, which was in accordance with the earlier studies (Motte et al., 2014;Valdez-Vazquez & Poggi-Varaldo, 2009). In particular, the results from the study of the effect of TS content (Figure 5.9 a and b) further suggest that the limiting effect of TS content starts between 15 and 20% as in agreement with Motte et al. (2014), that reported a metabolic shift at 19% TS with WS as substrate.

The metabolites in all the tested % TS were analysed for the possible biochemical pathways.

Theoretically, presence of acetate and butyrate in metabolic by-products are generally correlated with hydrogen production pathways (Ghimire et al., 2015;Guo et al., 2013); however, in this study, the H2 production was only correlated with butyrate production as shown by the PCA correlation plot in Figure 5.10 (a). Similarly, Figure 5.10 (b) showed that the H2 production followed substrate degradation, while lactate production is not well correlated with conversion of substrate. Two possible explanations for lactate production at higher TS content are the following: i) lactic acid bacteria (LAB) are more adaptable to harsh environmental conditions which enable them to inhabit in moisture limited conditions at higher TS content [START_REF] Sikora | Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence[END_REF]; ii) a decrease in pH related to the higher substrate concentration and production of VFA can affect the microbial community structure. However, pka of lactic acid is 3.86 in comparison to 4.75 and 4.78

for acetate and butyrate. Therefore, the decrease in pH is most likely due to the production of lactic acid. This shift to LAB at higher TS contents has been also shown in a study by Motte et al. (2014), regardless of the pH which was maintained constant at 5.50. This further strengthens the fact that the moisture lacking conditions create harsh environmental conditions, which trigger the growth of LAB. In addition, from the decrease in pH in this study, it can be concluded that the alkalinity requirements at high solids systems are higher than in the wet conditions.

Furthermore, Abbassi-Guendouz et al. ( 2012) showed significant inhibition of methane yields at 30% TS due to accumulation of intermediates such as organic acids and dissolved hydrogen. Thus, pH2 might impact the substrate conversion in SSDF, with the accumulation of H2 in the medium, as reported in a recent study by Cazier et al. (2015) in SS-AD. However, it has been confirmed from this study that the accumulation of H2 does not impact on the hydrolysis of substrate under SSDF (Figure 5.11 and 5.12), in contrast to the results obtained by Cazier et al. (2015) under SS-AD. This could be due to the fact that the substrate degradation is already under limitation under DF conditions, which is supported by the relationship established between H2 production (or substrate conversion)

and soluble carbohydrates present in the substrates, as reported by Guo et al. (2013) and Monlau et al. (2012). In addition, the hydrolysis of substrate seems to be also a function of culture pH as shown by Veeken et al. (2000).

Moreover, this difference in results can be also attributed to lower operational pH in the present study (pH 3.7 -5.5) compared to SS-AD (pH 8 -9) as reported by Cazier et al. (2015). The pH might also affect the conversion of substrates and metabolic products.

Veeken et al. (2000) reported the decrease in hydrolysis of complex substrates with the decrease in culture pH. The hydrolase enzyme of hydrolyzing bacteria functions at an optimal neutral pH (Parawira et al., 2005). [START_REF] Lin | Effects of initial cultivation pH on fermentative hydrogen production from xylose using natural mixed cultures[END_REF] reported that xylose removal decreased from 85% to 37% when the culture pH decreased from 8 to 5. Similarly, Fang and Liu (2002) also reported the decrease in glucose degradation by 10% when pH decreased from 5.5 to 4. VFAs yields obtained in this work are compared with the anaerobic fermentation studies reported in the literature (Table 5.10). All the SSDF tests carried out in this study shown lower VFAs yields (Table 5.10), which can be explained by the difference in operating conditions during the fermentative studies. The production of VFAs under fermentative conversion process is significantly affected by operating parameters such as culture pH, temperature and substrate concentration (Cho et al., 2015;[START_REF] Wang | Photofermentative hydrogen production from mixed substrate by mixed bacteria[END_REF]Wang et al., , 2015)). Most of the studies reported in Table 5.10 are carried out at pH 6.0 and higher under controlled pH conditions. This could explain the higher conversion of the waste biomass into fermentative products reported in these studies, compared to the results of this study where the hydrolysis was inhibited due to low pH conditions (3.7-5.1). Likewise, under dry dark fermentative process, the conversion of substrate was dependent on culture pH and TS content. However, the effect of pH2 was not clearly evident on substrate hydrolysis. This might be due to the fact that substrate conversion in the tests with applied pH2 was already under inhibited conditions due to high TS content (25% TS). In addition the culture pH was in the range of 5 -5.5 with WS and (3.7 -5.3) with FW. In addition, the accumulation of lactic acids during these limiting conditions suggests that LAB are dominant in harsh and nutrient rich environment such as FW at higher % TS and not in WS [START_REF] Sikora | Lactic Acid Bacteria in Hydrogen-Producing Consortia: On Purpose or by Coincidence[END_REF]. Likewise, the lactic acid fermentation pathways is not favourable for conversion of substrates.

Conclusion and future perspectives

This study highlights the effect of limiting parameters on substrate conversion during SSDF. Initial TS content has shown significant effect on the substrate degradation and metabolic by-products. The biohydrogen production ceased at TS content higher than 15%, therefore the TS content in the SSDF has to be maintained lower than 15 %, if the process is aimed at biohydrogen production. The investigation of the pH2 effect on hydrolysis of FW and WS showed that accumulation of H2 as gaseous product does not have inhibitory effect on hydrolysis of organic substrates in SSDF. However, in general, the hydrolysis of substrate seemed to be limited under DF conditions due to low pH conditions.

Introduction and objectives

Several factors such as greenhouse gas emission and pollution problems from the byproducts of combustion of the fossil fuels are driving biobased economy for the production of bioenergy and useful chemicals (Cherubini, 2010;Menon and Rao, 2012).

In this regards, current research technologies need to be directed towards biorefinery, based on renewable sources such as waste biomass. Specifically, creation of hydrogen (H2) based economy could hold the potential for future supply of energy. Moreover, production of H2 from the biological pathways that utilize the renewable resources such as organic waste biomass can be promising and could ensure sustainable production of H2. Among the different biological technologies, dark fermentation (DF) is one of the potential technologies for H2 production and valuable by-products such as organic acids and alcohols. These by-products can be either recovered or further converted to other valuable biofuels and platform chemicals in physical, chemical and or biological systems (Bastidas-Oyanedel et al., 2015, Bonk et al., 2015).

DF processes utilizing waste biomass in scaled-up application are limited by low H2 yields and use of process by-products (Ghimire et al., 2015). The H2 production from complex waste biomass by dark fermentative mixed culture is strongly influenced by physico-chemical properties of the substrate and co-substrates, types of inocula, food to microorganism (F/M) or substrate/inoculum (S/X) ratio, substrate concentration, organic loading rates (OLR) in continuous bioreactors, pre-treatment of substrates, culture temperature, pH reactor configuration and hydraulic retention times (HRT) (De Gioannis et al., 2013;Ghimire et al., 2015a;Guo et al., 2010;Ntaikou et al., 2010;Urbaniec and Bakker, 2015;Wang and Wan, 2009). The H2 yield and production rates from DF process can be enhanced by the optimization of these parameters (Figure 2.4). Furthermore, byproducts from DF processes, which mostly include organic acids, alcohols and unhydrolyzed residues can be utilized in other biological systems for their valorization by energy recovery (Figure 2.5). The DF effluents (DFEs) could be utilized in photo fermentation (PF) processes, which could increase the total H2 yields from the substrate (Figure 2.14). In addition, biopolymer (polyhydroxybutyrate or PHB) can be produced concomitantly via PF processes, while the waste stream generated from coupling of DF-PF processes can be utilized in anaerobic digestion (AD) for further energy recovery as methane (Figure 5.1). Likewise, Solid State Dark Fermentation (SSDF) processes which methods for starting-up a DF process. Three types of inoculum pre-treatment methods that are commonly reported in literature studies, namely acid treatment, heat shock treatment (conducted at 95 °C and 105 °C) and load-shock treatment were applied to anaerobic digestate obtained from an anaerobic digester treating buffalo manure and cheese whey (Chapter 3, Section 3.1). The effectiveness of the inoculum pre-treatment methods was evaluated for H2 production performance parameters such as cumulative H2 production, H2 production rate, length of the lag phase and process intermediates production in biohydrogen potential (BHP) tests fed with glucose (Figures 3.1 and 3.2, Table 3.1). Moreover, further evaluations were done based on operational costs and feasibility of the inoculum pre-treatment methods for scaled-up application of DF (Table 3.3). The results shown that load shock on anaerobic digestion can favor higher H2 yields.

This can be due to development and growth of an efficient H2 producing bacteria community as reported by O-Thong et al. (2009). Therefore, load shock pre-treatment can be effective to prepare start-up inoculum for up-scaled DF systems. However, it should be taken into account that in a continuous DF reactor the selection of H2 producing communities is a function of reactor operating conditions rather than only inoculum preparation or pre-treatment methods (Li and Fang, 2007). Nonetheless, this load shock method can be applied to adapt the biomethanation process for dark fermentative H2

production. This could have application for the two-stage anaerobic digestion plants for biohythane (biohydrogen and methane) production (Figure 2.8).

Another set of BHP tests were carried to investigate the effects of initial culture pH (Figures 3.3,3.4 and Table 3.6), combination of food to microorganism ratio (F/M) and initial culture pH (Table 3.7), substrate pre-treatment (Figure 3.5 and Table 3.8) and type of inoculum source (Figure 3.6 and Table 3.9) on the dark fermentative H2 yields. Three model organic wastes, i.e. food waste, olive mill wastewater (OMWW) and rice straw were used as representative of readily, moderarately and slowly biodegradable substrates, respectively. BHP tests with food waste and heat treated anaerobic digestate have shown that a decrease of initial culture pH from 7.0 to 4.5 and 5.0 can increase the H2 yields by 4.2 fold (60.6 ± 9 mL H2/gVS) and 3.5 fold (50.7 ± 1 mL H2/gVS), respectively.

Furthermore, BHP tests carried out at pH 5.0 and 6.0 with F/M ratios 0.5, 1 and 1.5 have shown that the lower F/M ratios (0.5-1) at the initial pH 5 favored H2 production in comparison to pH 6.5. Moreover, raw rice straw with alkaline treatment with 4% and 8%

NaOH at 55 ⁰C for 24 hours increased the H2 yields by 26 and 57 fold, respectively.

4.8 -5, during the DF process. The use of co-substrate aided in the stability of the continuous dark fermentative H2 production (Table 4.9). This can give economic sustainability for a DF process inscaled-up applications, as it helps to achieve stability of H2 production in an economical way, removing the dependency on chemical-buffering agents. Moreover, the co-fermentation can provide the biological treatment of waste that otherwise can pose environmental threats in places like Campania Region of Italy where cheese whey and buffalo manure are abundant by-products of agro-industrial activities (mozzarella cheese industries) [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF].

Integration of dark fermentation in a biorefinery concept

In addition to low process H2 yields, an incomplete conversion of organic biomass adds another bottleneck in the commercialization of dark fermentative H2 production (Gómez et al., 2011;Ren et al., 2011). Chapters 3 and 4 have shown that the higher H2 yields and process stability can be achieved by optimizing the different operational parameters.

However, higher substrate conversion of complex waste biomass cannot be achieved with a sole DF system and thus demands downstream process/es (Figure 2.5) (Gómez et al., 2011). Similarly, there have been increasing interests in incorporating DF into biorefinery concept utilizing it as a biological pre-treatment step (Bastidas-Oyanedel et al., 2015;Motte et al., 2015;[START_REF] Sambusiti | Algae as promising feedstocks for fermentative biohydrogen production according to a biorefinery approach: A comprehensive review[END_REF]Venkata Subhash and Venkata Mohan, 2014).

Recently, Motte et al. (2015) have reported higher substrate conversion of lignocellulosic biomass through integration of SSDF in a biorefinery approach. SSDF is advantageous in terms of higher process yields, due to its operation at high substrate loading rates and low water addition. Benefits, such as reduced reactor volume and specific energy requirements, simplicity in operation and handling of digestate, result in economic advantages of the SSDF process. In this context, Chapter 5 covers these aspects, which have been addressed by few studies.

Section 5.1 (Chapter 5) investigated the influence of integrating DF, PF and AD on total energy yields from three-step conversion of food waste (Figure 5.1 and Table 5.5). The supernatant, after separation of DFE, was used to recover H2 from a PF process using

Rhodobacter sphaeroides that increased H2 yield from the food waste by 1.75 fold. The solid residual fraction of DFE along with PF effluent was converted into methane by AD, increasing the total energy yield from 1.13 to 5.55 MJ/kg VSfoodwaste added. The threestage conversion can achieve the higher energy yields compared to stand-alone DF or DF-

Figure 1 . 1 -

 11 Figure 1.1 -A general schematic representation of the thesis study ...............................

Figure 2 . 1 -

 21 Figure 2.1 -Biological pathways to produce hydrogen ................................................

Figure 2 . 2 -

 22 Figure 2.2 -Number of peer reviewed publications on DF published in the last decade (Google Scholar, 2014; Scopus, 2014) .......................................................

Figure 2 . 3 -

 23 Figure 2.3 -Biodegradation and microbiological pathways involved in the fermentative breakdown of waste biomass (Adapted and modified from Peiris et al. (2006) ........................................................................................................

Figure 2 . 4 -

 24 Figure 2.4 -Strategies to enhance the biohydrogen yield in DF of organic biomass .....

Figure 2 . 5 -

 25 Figure 2.5 -Different strategies for integrating DF with post treatment processes for improved biofuel production ......................................................................

Figure 2 . 6 -

 26 Figure 2.6 -Schematic presentation of photofermentation (adapted and modified from Hallenbeck and Ghosh (2009))...................................................................

Figure 2 .

 2 Figure 2.7 -Schematic diagram of two chambered MEC separated by a proton exchange membrane and power supply (adapted and modified from Liu et al. (2005)).......................................................................................................

Figure 2 . 8 -

 28 Figure 2.8 -Two-stage process for hydrogen and methane production from organic waste .........................................................................................................

Figure 2 . 9 -

 29 Figure 2.9 -Classification of anoxygenic photosynthetic bacteria................................

Figure 2 .

 2 Figure 2.10 -Schematic representation of mechanisms of photofermentative H2 and PHB production in PNSB (Adapted and modified from Adessi and De Philippis (2014); Akkerman et al. (2002); Kars and Gündüz (2010)) ..........

Figure 2 .Figure 2 .

 22 Figure 2.11 -Sunlight and light absorption by purple bacteria (Akkerman et al., 2002) Figure 2.12 -Effect of light intensity on biohydrogen production by Rhodobacter sphaeroides O.U. 001 (Uyar et al., 2007) ...................................................

Figure 2 .

 2 Figure 2.13 -Schematic representation of the potential PBRs for PF ...........................

Figure 2 .

 2 Figure 2.14 -Sequential DF-PF process ......................................................................

Figure 3 . 1 -

 31 Figure 3.1 -Average Cumulative H2 Production in BHP tests ...................................

Figure 3 . 2 -

 32 Figure 3.2 -Major fermentative end products in the BHP tests with different biomass pre-treatment methods during (a) Load I and (b) Load II ..........................

Figure 3 . 3 -

 33 Figure 3.3 -Effect of initial pH on H2 yield and time required for H2 production to achieve 95% of the maximum yield during the DF of food waste at F/M ratio 0.5 and thermophilic temperature (55 ± 2 °C) using ADS ........................

Figure 3

 3 Figure 3.4 -H2 yields and B/A ratio as a function of pH in the thermophilic DF of food waste at F/M ratio 0.5 ..............................................................................

Figure 3 . 5 -

 35 Figure 3.5 -Effect of alkaline pre-treatment of rice straw on H2 yields ......................

Figure 3 Figure 4 . 1 -

 341 Figure 3.6 -Effect of inoculum sources on cumulative H2 production from the DF of OMWW using ADS (anaerobic digested sludge) and WAS (waste activated sludge) .....................................................................................................

Figure 4 . 2 -

 42 Figure 4.2 -(a) HPR (mL H2/L/d) (b) pH trends in semi-continuous thermophilic DF bioreactor (the shaded region in Figure 4.2 (a) represents the experimental period when the DF residues were recycled back to the reactor and 1 (b) represents the period when pH was adjusted at pH 5.5) ............................

Figure 4 . 3 -

 43 Figure 4.3 -Correlation circle of pH, HRT, OLR, HPR and HY formed by the first three principle components dim1, Dim 2 and Dim 3 representing 53.25, 23.26 and 15.15 % of the total variance, respectively (a) Projections according to the first two factors (Dim 1 and Dim 2). (b) Projects according to the first and third factors (Dim 1 and Dim 3) ...............................................................

Figure 4 . 4 -

 44 Figure 4.4 -Correlation circle of six metabolites, pH and OLR formed by the first three principle components dim1, Dim 2 and Dim 3 representing 33.29, 16.81 and 16.54 % of the total variance, respectively (a) Projections according to the first two factors (Dim 1 and Dim 2). (b) Projects according to the first and third factors (Dim 1 and Dim 3) ...............................................................

Figure 4

 4 Figure 4.5 -Daily H2 yields during the different buffalo manure feeding strategies in semi-continuous DF reactor using CHW as main substrate and BM as cosubstrate ..................................................................................................

Figure 4

 4 Figure 4.6 -CHW:BM ratio, total alkalinity (as mg CaCO3/L), and ammonium concentration (as NH4 + N/L) (A) and pH trends (B), during the different buffalo manure feeding strategies in a semi-continuous DF reactor ..........

Figure 4

 4 Figure 4.7 -Total/Alkalinity ratio and HPSI during the different operational strategies..................................................................................................

Figure 4 . 8 -

 48 Figure 4.8 -Correlation circle of six metabolites formed by the first two principle components dim1 and Dim 2 representing 26.56 and 23.65 % of the total variance, respectively. ..............................................................................

Figure 5 . 1 -

 51 Figure 5.1 -Schematic of the three-stage conversion of FW to hydrogen and methane. ..................................................................................................

Figure 5 . 2 -

 52 Figure 5.2 -HPR (L H2/m 3 /d) (a) and pH trends in semi-continuous thermophilic reactor (b); shaded region represents the experimental period when the culture pH inside the reactor was adjusted daily to pH 5.5 during the feeding operation. .................................................................................................

Figure 5 . 3 -

 53 Figure 5.3 -Correlation circle of five metabolites and HY formed by the first three principle components Dim1, Dim 2 and Dim 3 representing 35.00, 18.03 and 16.54 % of the total variance, respectively. Projections according to the first two (Dim 1 and Dim 2) (a) and first and third factors (Dim 1 and Dim 3) (b) ............................................................................................................

Figure 5 . 4 -

 54 Figure 5.4 -Cumulative hydrogen production (a) and depletion of major VFAs (acetate, propionate and butyrate) (b) in a PF tests using DFE and R. sphaeroides AV1b. ......................................................................................................

Figure 5

 5 Figure 5.5 -Methane yields from mesophilic AD of waste stream generated in the coupled DF-PF processes .........................................................................

Figure 5 . 7 -

 57 Figure 5.7 -Cumulative hydrogen production (A, D) and biomass and PHB concentrations (B, E) and depletion of major VFAs (C, F) in RS-D (left) and PM-D (right) tests ....................................................................................

Figure 5 . 8 -

 58 Figure 5.8 -PHB, H2 and biomass yield per gram of COD and soluble COD removal (%) in different PF experimental runs ......................................................

Figure 5 . 9 -

 59 Figure 5.9 -Substrate degradation (in g COD per kg of initial TS) (a); End metabolites accumulation (mM per kg of initial TS) (b); at different TS content .........

Figure 5 .

 5 Figure 5.10 -Principal component analysis correlation circle plot (a) Hydrogen and major metabolic by-products production. (b) Substrate degradation and metabolic products. ..................................................................................

Figure 5 .

 5 Figure 5.11 -Substrate degradation using FW (in g COD per kg of initial TS) (a); Substrate degradation using FW (in g COD per kg of initial TS) (b); at the end of two fermentation times (14 ≥14 d, 21 ≥ 21 d) and different pH2 of of A = 532 ± 33 mbar, B = 1,086 ± 29 mbar and C = 0 mbar at 25 % TS content .....................................................................................................

Figure 5 .

 5 Figure 5.12 -Substrate degradation in WS (in g COD per kg of initial TS) (a); Substrate degradation using WS (in g COD per kg of initial TS) (b); at the end of two fermentation times (14 ≥14 d, 21 ≥ 21 d) and different pH2 of A = 552 ± 31 mbar, B= 1,087 ± 30 mbar and C = 0 mbar at 25% TS .............................

  pleasant time together. This desertation is a part of the Erasmus Mundus Joint Doctoral Program Environmental Technologies for Solids, Soils and Sediments (ETeCoS 3 ) developed in collaboration between the University of Cassino and Southern Lazio (Italy), University of Paris Est (France) and UNESCO-IHE (the Netherlands) and several other partner institutions. I am thankful for Erasmus Mundus comminssion for the funding support. Likewise, it has been a great opportunity to carry out my resaserch activites at the Department of Civil, Architectural and Environmental Engineering, University of Naples Federico II (Naples, Italy) and INRA, Laboratoire de Biotechnologie de l'Environnement (Narbonne, France). First of all, I am thankful to my supervisor Prof. Ing. Giovanni Esposito for his support throughtout my doctoral studies. I am equally grateful to my co-supervisors Prof. Dr. Ir. P.N.L. Lens and Prof. Dr. Ing. Francesco Pirozzi for their constant support and guidance during the PhD desertation. I would like to thank Dr. R. Escudié and Dr. E. Trably for their guidance and care during my stay at INRA, LBE (Narbonne, France). Moreover, I do not have enough words to thank my mentor Dr. Ing. Luigi Frunzo for being supportive and who have become one of my closest friends at the end of my PhD. Similarly, I would like to thank all my colleagues from the laboratories in Naples, Cassino and Narbonne with whom I have spent quality time at work and outside. I am thankful to Vincenzo, Alberto, Maria Rosaria, Marco, Fabio, Serena, Ester, Amelia and Martha from Naples for the joyful time in the laboratory in Naples (Italy). I am thankful to all the collegues from LBE, INRA, Narbonne (France) for their immense help and companionship during my stay there. Finally, I would like to thanks my parents Madav and Kalpana and my brothers Anup and Anil for their continuous inspirations and support throughout all these years.

Figure 1 . 1 -

 11 Figure 1.1 -A general schematic representation of the thesis study

Figure 2 . 1 -

 21 Figure 2.1 -Biological pathways to produce hydrogen

Figure 2 . 2 -

 22 Figure 2.2 -Number of peer reviewed publications on DF published in the last decade (Google

Figure 2 .

 2 3 gives a schematic representation of the different steps and biochemical pathways involved in the DF of complex organic biomass.

Figure 2 . 3 -

 23 Figure 2.3 -Biodegradation and microbiological pathways involved in the fermentative breakdown of waste biomass (Adapted and modified from[START_REF] Peiris | ADM1 simulations of hydrogen production[END_REF]

Figure 2 . 4 -

 24 Figure 2.4 -Strategies to enhance the biohydrogen yield in DF of organic biomass

  Na2HPO4•2H2O) could enhance H2 production. In these studies, the hydrogen production rate increased by 60% (O-Thong et al., 2008) and COD removal efficiencies improved from 35.5±9.8 % to 62.2±2.8% (O-Thong et al., 2007) compared to raw POME without nutrient supplementation. Likewise, Argun et al. (2008b) studied the effects of the C/N and C/P ratio on the hydrogen yield and specific H2 production rate in DF of wheat powder solution by supplementing nitrogen and phosphorous. The results of the study showed the highest H2 yield of 281 NmL H2/g starch were obtained at a C/N ratio of 200 and C/P ratio of 1000.

Figure 2 .

 2 5 shows an example of different possibilities of integrating DF to other post treatment processes.

Figure 2 . 5 -

 25 Figure 2.5 -Different strategies for integrating DF with post treatment processes for improved biofuel production

Figure 2 . 6 -

 26 Figure 2.6 -Schematic presentation of photofermentation (adapted and modified from

  Photofermentation: CH3COOH + 2H2O Light energy 4H2 + 2CO2(2.9)

Figure 2 . 7 -

 27 Figure 2.7 -Schematic diagram of two chambered MEC separated by a proton exchange membrane and power supply (adapted and modified from Liu et al. (2005))

Figure 2 . 8 -

 28 Figure 2.8 -Two-stage process for hydrogen and methane production from organic waste

  [START_REF] Tuna | Hydrogen gas production by electrohydrolysis of volatile fatty acid (VFA) containing dark fermentation effluent[END_REF] used the volatile fatty acids produced in DF processes for hydrogen production by electrohydrolysis. Hydrogen was generated by applying a low voltage in the range of 1-3 V DC current to DF effluents of wheat powder containing different VFAs concentrations. The applied voltage of 2 V and 10.85 g/L of total VFA gave the highest energy efficiency (56%).

  [START_REF] Ren | Biohydrogen production from molasses by anaerobic fermentation with a pilot-scale bioreactor system[END_REF] studied a 1.48 m 3 continuous flow anaerobic reactor for 200 days at an kg COD/m 3 /d fed with molasses. The maximum hydrogen yield was 26.13 mols H2/kgCOD removed in the OLR range of 35-55 kg COD/m 3 /d and a maximum production rate of 5.57m 3 H2/m 3 reactor/d was reached.[START_REF] Jayalakshmi | Bio hydrogen generation from kitchen waste in an inclined plug flow reactor[END_REF] worked with a plug-flow inclined DF reactor of volume 0.15 m 3 with kitchen waste as the substrate. The reactor gave a H2 yield of 72 mL H2/gVS added.Another reported long term pilot-scale study was carried out at Fen Chia University (Taiwan), comprising of two feedstock storage tanks (0.75m 3 each), a nutrient storage tank (0.75m 3 ), a mixing tank (0.6 m 3 ), an agitated granular sludge bed fermenter (working volume 0.4 m 3 ), a gas-liquid-solid separator (0.4 m 3 ) and a control panel. A pilot-scale high-rate reactor was operated for a period of 67 days under mesophilic conditions (35 °C) at an OLR of 40-240 kg COD/m 3 /d with sucrose as the substrate. An OLR of 240 kg COD/m 3 /d gave a hydrogen production rate of 15.59 m 3 /m 3 d and a hydrogen yield of 1.04 mol H2/mol sucrose. In another study, Cavinato et al.(Cavinato et al., 2012) carried out a two-stage pilot-scale thermophilic DF and AD of food waste for the production of, respectively, biohydrogen and methane with recirculation of AD effluents to DF to control the pH (5-6). The organic loading rate of 16.3 kgTVS/m 3 d was maintained with a HRT of 3.3 days in the DF stage, yielding 66.7 L H2/kg TVS.

Figure 2

 2 Figure 2.9 -Classification of anoxygenic photosynthetic bacteria

Figure 2 .

 2 Figure 2.10 -Schematic representation of mechanisms of photofermentative H2 and PHB production in PNSB (Adapted and modified from Adessi and De Philippis (2014); Akkerman et al. (2002); Kars and Gündüz (2010))

Figure 2 .

 2 Figure 2.11 -Sunlight and light absorption by purple bacteria[START_REF] Akkerman | Photobiological hydrogen production: photochemical efficiency and bioreactor design[END_REF]

  [START_REF] Afsar | Hydrogen productivity of photosynthetic bacteria on dark fermenter effluent of potato steam peels hydrolysate[END_REF] carried out PF studies using different PNSB strains, which showed the PF efficiency highly depends on the effluent composition and bacterial strain used. The PF was carried out using the effluents from the thermophilic DF of glucose and potato steam peel hydrolysate as carbon source under indoor batch conditions. The PNS strains, such as Rhodobacter capsulatus (DSM1710), Rhodobacter capsulatus hup -(YO3), Rhodobacter sphaeroides O.U.001 (DSM5864), Rhodobacter sphaeroides O.U.001 hup - and Rhodopseudomonas palustris, were used in the study. The results showed that Rb.sphaeroides gave the highest amount of hydrogen from PF of glucose dark fermentation effluents, while Rb. capsulatus produced better results on effluents from the dark fermentation of potato steam peels hydrolysate.However, the use of pure cultures of bacterial strains demands maintenance of sterile conditions in the bioreactors. The varying PF efficiencies of different PNS bacterial strains on different substrates suggests, for substrates such as DFEs which contain mixed organic acids, the use of mixed consortia of PNSB bacteria in order to exploit the substrate utilization capacity of different PNS bacterial strains. In a study byMontiel-Corona et al.

  photofermentative hydrogen production from tofu wastewater using Rhodobacter sphaeroides.

Figure 2 .

 2 Figure 2.12 -Effect of light intensity on biohydrogen production by Rhodobacter sphaeroides O.U. 001[START_REF] Uyar | Effect of light intensity, wavelength and illumination protocol on hydrogen production in photobioreactors[END_REF]

Figure 2 . 13 -

 213 Figure 2.13 -Schematic representation of the potential PBRs for PF

  [START_REF] Boran | Biological hydrogen production by Rhodobacter capsulatus in solar tubular photo bioreactor[END_REF] successfully developed and demonstrated a pilot scale (80 L) tubular PBR for photofermentation of acetate using Rhodobacter capsulatus in outdoor operating conditions (during winter seasons) in Ankara (Turkey). The PBR gave an average molar productivity of 0.31 mol H2/m 3 /h during daylight hours and the gas contained 99% hydrogen and 1% carbon dioxide by volume. The system provided an overall hydrogen yield of 0.6 mol H2/mol acetate and the H2 production with respect to the total illuminated surface area amounted to 0.112 mol H2/m 2 /day.[START_REF] Ugwu | Photobioreactors for mass cultivation of algae[END_REF] reported some limitations that tubular PBRs face during scale up: difficulty in operational temperature control fouling and growth on the walls of the tubes large space requirements

  reported the following considerations for the selection of the construction material for PBRs: high transparency durable and low cost non-toxic to PNS strains and resistant to chemicals and metabolites produced by the PNS strains high weathering resistant and easiness in cleaning The results of the Net Energy Analysis (NER) of three different materials, viz. glass, LDPE and PMMA, done by Gebicki et al. (2010) suggests the use of LDPE for the construction of tubular and panel PBRs.

  [START_REF] Mu | Kinetic modeling of batch hydrogen production process by mixed anaerobic cultures[END_REF] used the Modified Luedeking-Piret model to establish the relationship product (Pi) formation, substrate (S) degradation and biomass (X) growth for the DF hydrogen production by mixed anaerobic cultures. The following Luedeking-Piret model could be used to describe the relationship between three parameters. The Luedeking-Piret model and its modified form can describe the relationship between formation of H2 and PHB as products and biomass:

Figure 2 . 14 -

 214 Figure 2.14 -Sequential DF-PF process

Figure 3 . 1 -

 31 Figure 3.1 -Average Cumulative H2 Production in BHP tests

Figure 3 . 2 -

 32 Figure 3.2 -Major fermentative end products in the BHP tests with different biomass pretreatment methods during (a) Load I and (b) Load II

  TS and VS concentrations were determined by the Method 2540 (Part 2000), alkalinity by titration (Method 2320, Part 2000) and TKN by macro-Kjeldahl (Method 4500-Norg, Part 4000)

Figure 3 . 3 -

 33 Figure 3.3 -Effect of initial pH on H2 yield and time required for H2 production to achieve 95% of the maximum yield during the DF of food waste at F/M ratio 0.5 and thermophilic temperature (55 ± 2 °C) using ADS

  5 -7.0 might indicate the prevailance of an homoacetogenic activity responsible of lower H2 yields.

Figure 3 .

 3 Figure 3.4 -H2 yields and B/A ratio as a function of pH in the thermophilic DF of food waste at F/M ratio 0.5

  tests carried out earlier at different initial pH (Table

Figure 3 . 5 -

 35 Figure 3.5 -Effect of alkaline pre-treatment of rice straw on H2 yields

Figure 3 . 6 -

 36 Figure 3.6 -Effect of inoculum sources on cumulative H2 production from the DF of OMWW using ADS (anaerobic digested sludge) and WAS (waste activated sludge)

  H2/kg total organic carbon; b MWWS: Municipal wastewater plant sludge; c PMS: Paper Mill Sludge; d CDS: Cow Dung Compost; e mL H2/g TS

Figure S1 .

 S1 Figure S1. Cumulative H2 production at different initial pH values using food waste at a F/M ratio 0.5 and ADS as inoculum (dotted lines represents the results from a modified Gompertz model)

Figure 4 . 1 -

 41 Figure 4.1 -Schematic description of semi-continuous reactors setup for H2 production 1-Magnetic Stirrer; 2-Thermophilic water circulation bath maintained at 55 ± 2 C; 3-2 L Serum bottle as CSTR reactor; 4-VFA sampling and substrates feeding ports; 5-Gas delivery pipe; 6-1 L serum bottle with 1.5% HCl; 7-Graduated cylinder or bottle for collecting displace acidic water from (6)

Figure 4 . 2 -Figure 4 . 3 -

 4243 Figure 4.2 -(a) HPR (mL H2/L/d) (b) pH trends in semi-continuous thermophilic DF bioreactor (the shaded region in Figure 4.2 (a) represents the experimental period when the DF residues were recycled back to the reactor and 1 (b) represents the period when pH was adjusted at pH 5.5)

  Figure 4.4 -Correlation circle of six metabolites, pH and OLR formed by the first three principle components dim1, Dim 2 and Dim 3 representing 33.29, 16.81 and 16.54 % of the total variance, respectively (a) Projections according to the first two factors (Dim 1 and Dim 2). (b) Projects according to the first and third factors (Dim 1 and Dim 3)

  HPR and HY in relation to past studies. The optimum HPR, HY and HPSI of 208.0 ± 34.8 NmL H2/L/d, 104.0 ± 17.4 NmL H2/g VS and 0.83, repectively, was obtained at the OLR of 2.5 g VS/L/d at the HRT of 4 days. The change in operating conditions can affected the metabolic pathways and thus the H2 production as seen from the inhibition of H2 production from the accumulation of lactate and propionate in the bioreactor. The recirculation of solid residues present in DFE does not significantly affect the H2 yield so it can be used feedstock in the anaerobic digestion process for further energy conversion in the form of methane.

Figure 4 . 5 -

 45 Figure 4.5 -Daily H2 yields during the different buffalo manure feeding strategies in semicontinuous DF reactor using CHW as main substrate and BM as co-substrate

  Figure4.7 (b) tried to establish the relationship between the Total alkalinity (Tak) to Total acids (Tac) ratio and HPSI. The higher HPSI was obtained when the Tak/Tac ratio is between 3 -4. This suggests requirement of constant alkalinity source to maintain a stable culture pH and H2 production during the DF process.

Figure 4

 4 Figure 4.6 -CHW:BM ratio, total alkalinity (as mg CaCO3/L), and ammonium concentration (as NH4 + N/L) (A) and pH trends (B), during the different buffalo manure feeding strategies in a semi-continuous DF reactor

Figure 4 . 8 -

 48 Figure 4.8 -Correlation circle of six metabolites formed by the first two principle components dim1 and Dim 2 representing 26.56 and 23.65 % of the total variance, respectively.

Figure 5 . 1 -

 51 Figure 5.1 -Schematic of the three-stage conversion of FW to hydrogen and methane.

  Photofermentation

  Figure 5.2 shows the HPR (a) and pH trends (b), over the operation period of 193 days. The results show an increase in HPR when OLRs were increased. During the operating periods II, III and IV at a constant HRT of 6 days, the HPR increased from 54.1 ± 41, to 109.5 ± 33 and 210.2 ± 30 N L/m 3 /d, when the OLR was increased from 1 to 1.5 and 2 g VS/m 3 /d, respectively

  2 ± 29.8 N L/ m 3 /d and HY: 105.1 ± 14.9 N L/kg VSadded at a HRT of 6 days and OLR 2 kg VS/m 3 /d) were comparable to experimental period V (HPR: 208.0 ± 34.8 N L/m 3 /d and HY: 104.0 ± 17.4 N L/kg VSadded at a HRT of 4 days and OLR of 2 g VS/L/m 3 /d). Thus, the operational conditions of period V were considered as ideal for the DF of FW in thermophilic semi-continuous reactors, 201 as a lower HRT are generally more economically efficient in terms of bioreactor design and operation.

Figure 5 . 2 -

 52 Figure 5.2 -HPR (L H2/m 3 /d) (a) and pH trends in semi-continuous thermophilic reactor (b);

Figure 5 .

 5 Figure 5.3 shows the plot of correlation circles of the five major metabolites and the HY.

Figure 5

 5 Figure 5.3 (a) shows that the butyrate and acetate concentration is well correlated with the HY values. Not surprisingly, propionate, lactate and ethanol are in the Dim 2 and are not correlated with the HY, which is supported by equations 6 -9 (Table4.6) in a DF

Figure 5 . 3 -

 53 Figure 5.3 -Correlation circle of five metabolites and HY formed by the first three principle components Dim1, Dim 2 and Dim 3 representing 35.00, 18.03 and 16.54 % of the total variance, respectively. Projections according to the first two (Dim 1 and Dim 2) (a) and first and third factors (Dim 1 and Dim 3) (b)

Figure 5 . 4 -

 54 Figure 5.4 -Cumulative hydrogen production (a) and depletion of major VFAs (acetate, propionate and butyrate) (b) in a PF tests using DFE and R. sphaeroides AV1b.

Figure 5 . 5 .

 55 Figure5.5. The cumulative CH4 production stabilized after 50 days and the average cumulative CH4 production was 871 ± 16 mL, corresponding to a total average yield of 324 ± 6 N L CH4/kg VS added (feed) and 0.9 kg COD/kg VS removed (calculated from

Figure 5 . 5 -

 55 Figure 5.5 -Methane yields from mesophilic AD of waste stream generated in the coupled DF-PF processes

Figure 5

 5 Figure 5.6 A presents bio-H2 production (Fig 5.6 A) and concomitant depletion of VFAs and PHB production (Figure 5.6 B) in photofermentation of DFE medium (RS-I) using R. sphaeroides AV1b. A cumulative volumetric yield of 914 (± 8) N mL H2/L was obtained at the end of the 40 days of incubation (Figure 5.6 A). The maximum composition of H2 and CO2 in the biogas reached 89.0% and 8.9%, respectively. TheVFAs concentration decreased gradually, in particular, the acetate and propionate concentration decreased sharply until 10 days, while butyrate concentration decreased steadily until 30 days. The decrease in VFA concentration was followed by the increase in PHB concentration (Figure5.6 B). The maximum PHB concentration of 1864.5 (± 76.4) mg/L, corresponding to 39.2 ± 9 % DCW, was obtained after 33 days from the reactor.

Figure 5 .

 5 Figure 5.6 -Cumulative H2 production (A) and VFAs depletion and PHB concentration (B) in the reactor during the test using RS-I medium and Rhodobacter sphaeroides AV1b

Figure 5 .

 5 Figure 5.7 compares the results of volumetric H2 production, biomass growth, PHB accumulation and major VFAs depletion in the RS-D (A, B and C) and PM-I (D, E and F) tests, respectively. The mixed PNSB culture gave lower H2 yields in comparison to the pure R. sphaeroides AV1b cultures. The lower H2 yields of the mixed PNSB might be due to the absence of a H2 producing PNSB population in the mixed PNSB culture. Likewise, the opportune microorganisms present in unsterilized DFE medium (PM-D) during application of mixed PNSB culture might consume available COD which is supposed to be utilized by PNSB for H2 and / or PHB production. Montiel-Corona et al.(2015) reported a H2 yield of 591.2 N mL H2/g COD by an mixed PNSB culture. Thus, the H2 yields of DFE via a mixed PNSB culture depends on the enrichment of H2 producing PNSB in a mixed culture. The continuous or semi-continuous operation of PF processes in different reactor types, such as CSTR, tubular and flat panel reactors could efficiently enrich the mixed community of H2 producing PSNB. In addition, bioaugmentation with pure PNSB cultures can be considered as an option to increase the H2 yields from PF of DFE.[START_REF] Nasr | Continuous biohydrogen production from starch wastewater via sequential darkphoto fermentation with emphasize on maghemite nanoparticles[END_REF] reported a H2 yield of 166.83 (± 27.8) mL H2/g CODremoved from a continuous photofermentative reactor, in comparison to this study which gave a lower H2 yield of 96.8 (± 6) NmL H2/g CODremoved in a batch process inferring an effect of the enrichment of an active mixed PNSB culture and reactor operational conditions.

Figure 5 . 7 -

 57 Figure 5.7 -Cumulative hydrogen production (A, D) and biomass and PHB concentrations (B, E) and depletion of major VFAs (C, F) in RS-D (left) and PM-D (right) tests

  [START_REF] Wu | Continuous biohydrogen production from liquid swine manure supplemented with glucose using an anaerobic sequencing batch reactor[END_REF], PHB yields also depend on the type of VFAs present in the DFE. During the cultivation of R. sphaeroides in aerobic dark conditions,[START_REF] Kemavongse | Effect of cosubstrate on production of poly-beta-hydroxybutyrate (PHB) and copolymer PHBV from newly identified mutant Rhodobacter sphaeroides U7 cultivated under aerobicdark condition[END_REF] reported that the addition of propionate (40 mM) and valerate (40 mM) to acetate (40 mM) in the substrate can induce the production of poly-β-

  2 to 16.0 g COD/L/day. However, H2 production increased to an OLR of 6.4 g COD/L/day and decreased gradually on further increasing the OLR. This is due to inhibition due to VFAs accumulation present in the DFE medium. Likewise, Montiel-Corona et al. (2015) correlated the decrease in H2 yields at higher COD concentrations (at 9.0 and 13.6 g/L) and the associated higher nitrogen content of the DFE to the interference in light penetration because of higher biomass concentration.

Figure 5 . 8 -

 58 Figure 5.8 -PHB, H2 and biomass yield per gram of COD and soluble COD removal (%) in different PF experimental runs

Figure 5 . 9 -

 59 Figure 5.9 -Substrate degradation (in g COD per kg of initial TS) (a); End metabolites accumulation (mM per kg of initial TS) (b); at different TS content

Figure 5

 5 Figure5.9 (b) presents the molar yield (mmol/kg TS) of all the major metabolic end products after 14 days of fermentation at different TS content. The highest substrate conversion of 2901.13 ± 143 mM/KgTS was obtained at wet TS conditions (10%) while the lowest value of 1435.2 ± 13 mmol/kg TS was obtained at TS 30% (Figure5.9 b). PCA correlation plot of metabolites and hydrogen production is presented in Figure5.10.

Figure 5 .

 5 Figure 5.10 -Principal component analysis correlation circle plot (a) Hydrogen and major metabolic by-products production. (b) Substrate degradation and metabolic products.

  Figure 5.11 (a) shows the total substrate degradation values (expressed as g COD/kg TS) after 14 and 21 days of DF at different pH2 using FW. The level of inhibition of pH2 on substrate hydrolysis was determined based on difference in level of substrate degradation (Figure5.11 a). No significant effect of initial pH2 on hydrolysis of biomass (ANOVA test, P-values > 0.05)

Figure 5 .

 5 Figure 5.11 -Substrate degradation using FW (in g COD per kg of initial TS) (a); Substrate degradation using FW (in g COD per kg of initial TS) (b); at the end of two fermentation times (14 ≥14 d, ≥ 21 d) and different pH2 of of A = 532 ± 33 mbar, B = 1,086 ± 29 mbar and C = 0 mbar at 25 % TS content

Figure 5

 5 Figure 5.12 -Substrate degradation in WS (in g COD per kg of initial TS) (a); Substrate degradation using WS (in g COD per kg of initial TS) (b); at the end of two fermentation times (14 ≥14 d, 21 ≥ 21 d) and different pH2 of A = 552 ± 31 mbar, B= 1,087 ± 30 mbar and C = 0 mbar at 25% TS

Table 2 .

 2 1 -Dark fermentative biohydrogen potential of different waste biomass under varying operating conditions ......................................................................

Table 2 .

 2 2 -Composition of typical agricultural waste (% of dry matter) ......................

Table 2 .

 2 3 -Biohydrogen production studies using pure culture....................................

Table 2 .

 2 4 -Pre-treatment methods used to enriching hydrogen producing microorganisms in anaerobic sludge ..........................................................

Table 2 .

 2 5 -Comparison of various inoculum pre-treatment methods for enriching hydrogen producing inocula .......................................................................

Table 2 .

 2 6 -Evaluation of inoculum pre-treatment methods to enhance the DF capacity of the inoculum sludge ...............................................................................

Table 2 .

 2 7 -Examples of innovative continuous DF bioreactors ...................................

Table 2 . 8

 28

-Effects of operational temperature and pH on fermentative hydrogen production .................................................................................................

Table 2.9 -Examples of different pre-treatment methods applied to complex substrates used in DF ................................................................................................. Table 2.10 -Examples of operational conditions and system performances of integrated DF systems ................................................................................................ Table 2.11 -Comparison of photo and fermentation dark systems for biohydrogen production ................................................................................................. Table 2.12 -Comparison of photo-H2 production by different isolated and mixed PNSB strains from various inoculum sources ....................................................... Table 2.13 -Comparison of hydrogen and PHB production by different isolated strains and enriched mixed cultures of PNS via photofermentation of various carbon sources ...................................................................................................... Table 2.14 -Variation of different operational parameters in PF studies ...................... Table 2.15 -Comparison of Tubular and Plate PBRs under outdoor conditions ........... Table 3.1 -Effects of biomass pre-treatment methods on biohydrogen production performance during Load I ......................................................................

Table 3 .

 3 2 -Comparison between Load I and Load II feeding operations....................Table 3.3 -Evaluation of biomass pre-treatment methods for DF process .................. Table 3.4 -Characteristics of the substrates and inocula used in this study ................ Table 3.5 -Experimental conditions applied in the DF batch tests of the tested substrates ................................................................................................. Table 3.6 -Effects of initial pH on H2 production performance and characteristics of accumulated end products ........................................................................

Table 3.7 -Effects of initial pH and F/M ratio on H2 production performance and characteristics of accumulated end products in DF of food waste ............. Table 3.8 -Effect of substrate pre-treatment on biohydrogen production performance measured by the modified Gompertz model ............................................. x

Table 3 .

 3 9 -Effects of inoculum source on H2 production performance measured by the modified Gompertz model and characteristics of accumulated end products in DF of OMWW.....................................................................................Table 3.10 -Summary of various strategies to improve the H2 yields from the substrate with different biodegradability ................................................................. Table 4.1 -Characteristics of food waste ................................................................... Table 4.2 -Experimental design used for the operation of semi-continuous reactor ... Table 4.3 -H2 production rate, yields and production stability from FW by mixed anaerobic cultures .................................................................................... Table 4.4 -Comparison of H2 production from food waste by mixed cultures ........... Table 4.5 -Characteristics of influent and effluents DF of FW during different experimental periods ................................................................................

Table 4.6 -Reaction stoichiometry in dark fermentation of glucose ........................... Table 4.7 -Characteristics of cheese whey and buffalo manure ................................. Table 4.8 -Operational conditions and buffalo manure feeding strategies during different experiments runs ....................................................................... Table 4.9 -H2 production performance during the dark fermentation at different CHW:BM ratio ........................................................................................ Table 4.10 -Characteristics of effluents from the DF of CHW with BM as co-substrate during different experimental periods ...................................................... Table 5.1 -Characteristics of the DFE used in PF experiments. ................................. Table 5.2 -Experimental design used for the operation of semi-continuous reactor ... Table 5.3 -H2 production rate, yields and production stability from FW by mixed anaerobic cultures .................................................................................... Table 5.4 -Characteristics of influent and effluents from DF of FW during different experimental periods ................................................................................ Table 5.5 -Comparison of energy yields from gaseous biofuels produced out of FW as feedstock using stand alone or coupling of different technologies ............

Table 5 .

 5 6 -Characteristics of substrates used in photofermentative experiments ........Table 5.7 -Summary of photo-H2 performance estimated by modified Gompertz model ...................................................................................................... Table 5.8 -Possible photofermentative pathways ...................................................... Table 5.9 -Comparison of hydrogen and PHB production by different isolated strains and enriched mixed cultures of PNS via photofermentation of various carbon sources .................................................................................................... Table 5.10 -Production of VFA from different types of fermentation .......................

	xi

Table 2 .

 2 1 -Dark fermentative biohydrogen potential of different waste biomass under varying operating conditions

	Substrate type	Microbial inoculum source	Reactor type	Temperature (°C)	pH	Maximum H2 yield VSadded) (mL H2/g	Maximum H2 rate production	H2 in (%) biogas	Reference
	Food waste	Heat shock	Leaching	37	5.5 -7	310	151.25 mL	10-55	(Han and
		treated anaerobic	Bed Reactor				H2/L/h		Shin, 2004)
		sludge
	Food waste	Thermophilic	Batch	55	4.5	46.3	3 mL H2/g	23	(Shin et al.,
		acidogenic					VSS/h		2004)
		culture
	Vegetable kitchen	Kitchen waste	Intermittent-	55	6.0	38 a	1.0 L H2 /L/d 40	(Z.-K. Lee et
	waste	compost	CSTR						al., 2010)
	Food waste and	Anaerobic	Batch	35	5.0-6.0 122.9 a	111.2 mL	-	(Kim et al.,
	sewage sludge	digester sludge					H2/g VSS/h		2004)
	OFMSW	Anaerobic	Semi-	55	6.4	360 b	-	58	(Valdez-
		digestate	continuous						vazquez et al.,
			CSTR						2005)
	OFMSW	Non-anaerobic	Packed bed	38	5.6	99 b	-	47	(Alzate-
		inocula (soil, pig	reactor						Gaviria et al.,
		excreta)							2007)
	Wheat straw	Cow dung	Batch	36	6.5	68.1	10.14 ml H2/g	52	(Fan et al.,
		compost					VS/h		2006)
	Rice straw	Wastewater	Batch CSTR 55	6.5	24.8 c	-	-	(Chen et al.,
		treatment plant			(initial)				2012)
		sludge
	Corn stalk wastes	Enriched cow	Batch CSTR 50	7	149.69	7.6 mL H2/h	45-56	(Zhang et al.,
	with acidification	dung composts			(initial)				2007)
	pre-treatment
	Rice slurry	Anaerobic	Batch	37	4.5	346 d	2.1 L/g	45-56	(Fang et al.,
		digester sludge			(initial)		VSS/d		2006)
	Cheese whey	Adapted	Batch	55	7	111 e	3.46 mL H2/	-	(Kargi et al.,
		anaerobic sludge			(initial)		L/h		2012a)

Table 2 .

 2

	2 -Composition of typical agricultural waste (% of dry matter)
	Component	Rice straw a (%)	Wheat straw b (%)	Barley straw c (%)	Corn stalk c (%)	Corn cob d (%)
	Cellulose	38.6	44.1	37.2	36.7	35.3
	Hemicellulose	19.7	36.0	24.4	26.2	37.1
	Lignin	13.6	6.9	16.1 e	16.9 e	16.4 e
	Ash	-	6.1	6.4	4.9	1.5

a Data obtained from

[START_REF] Zhu | Pretreatment by microwave/alkali of rice straw and its enzymic hydrolysis[END_REF]

on wet basis; b Data obtained from

(Motte et al., 2013)

; c Data obtained from

[START_REF] Panagiotopoulos | Fermentative hydrogen production from pretreated biomass : A comparative study[END_REF]

; d Data obtained from

[START_REF] Panagiotopoulos | Exploring Critical Factors for Fermentative Hydrogen Production from Various Types of Lignocellulosic Biomass[END_REF]

); e Acidinsoluble lignin

Table 2

 2

	.3

Table 2

 2

	.3 -Biohydrogen production studies using pure culture
						Optimum H2
	Culture	Substrate	Culture type	Temp. (°C)	pH	yield (mol H2/mol	Reference
						glucose eqv.) a
	Enterobacter cloacae IIT-BT08	Glucose	Batch	36	6	2.2	(Kumar and Das, 2000)
	Clostridium thermolacticum DSM 2910	Lactose	Continuous	58	7	1.5	(Collet et al., 2004)
	Enterobacter cloacae DM 11	Malt, yeast extract & glucose	Continuous	37	6	3.9	(Mandal et al., 2006)
	Caldicellulosiruptor saccharolyticus DSM 8903	Hydrolyzed potato steam peels	Batch	70	6.9 3.4	(Mars et al., 2010)
	Thermotoga neapolitana DSM 4349	Hydrolyzed potato steam peels	Batch	80	6.9 3.3	(Mars et al., 2010)
	C. thermocellum
	DSM 1237and C. thermopalmarium	Cellulose	Batch	55	7	1.36	(Geng et al., 2010)
	DSM 5974
	Clostridium thermocellum 7072	Corn stalk	Continuous (100 Liters)	55	7.2 1.2	(Cheng and Liu, 2011)
	a mol H2/mol glucose equivalent was calculated based on the information provided from references at
	Standard Temperature and Pressure (STP) (0°C and Pressure 1 atm)

Table 2

 2

	.4 -Pre-treatment methods used to enriching hydrogen producing microorganisms in anaerobic sludge
	Treatment	Description	Inoculum source	Reference
	Heat	100 °C for 15 min	Anaerobic digested sludge	(Wang and Wan, 2008)
	Heat	80°C, 90°C and 100°C for 15-30 min	Anaerobic sludge	(Wang et al., 2011)
	Heat	Heating in boiling water bath for 10-30 min	Anaerobic granular sludge	(Mohammadi et al., 2011)
	Heat	105 °C for 4 hour	Anaerobic granular sludge	(Giordano et al., 2011)
	Heat	Incubation at 90 °C for 1 hour	Anaerobic granular sludge	(Luo et al., 2010a)
	Heat	100-105 °C in oven for 2 hour	Cow dung compost	(Fan et al., 2004)
	Acid	pH to 2 for 24 h and increasing pH to 5.5 by adding a 2 N NaOH solution	Anaerobic digested sludge	(Lee et al., 2009) (Mohammadi et al., 2011)
	Acid	pH 3 with 2 N HCl for 24 hours	Anaerobic digested sludge	(Luo et al., 2011)
	Acid	pH to 3 with 1 N HCl for 30 min	Anaerobic digested sludge	(Zhu and Beland, 2006)
	Acid	pH 3 with 0.1 N HCl solution for 24 hours and adjusting back to pH 7	Anaerobic granular sludge	(Hu and Chen, 2007)
	Base	pH of the sludge to 3 with 1 mol/L of NaOH for 24 hours	Anaerobic digested sludge	(Wang and Wan, 2008)
	Base	pH 8, 9 and 10 with 1 mol/L of NaOH for 3 hours Anaerobic sludge	(Wang et al., 2011)

Table 2

 2

	.6 -Evaluation of inoculum pre-treatment methods to enhance the DF capacity of the
	inoculum sludge
	Pretreatment Method	Energy Requirement	Chemical Requirement	Economic cost	Scale-up application
	Heat shock treatment + + +	+	+ + +	+ +
	Acid treatment	+	+ + +	+ +	+ + +
	Chemical Treatment	+	+ + +	+ + +	+ +
	Aeration	+ + +	+	+ +	+ + +
	Load shock treatment + +	+ +	+	+ + +
	+ Less intensive; ++ Moderately intensive; +++ Very intensive

Table 2

 2

	.7 -Examples of innovative continuous DF bioreactors
	Major substrate	Biomass retention system	Reactor type	Optimum HRT (hours)	Optimum Organic Loading Rate (OLR)	Optimum H2 production index	Reference
	Glucose	Granule	CSTR	0.5	10 g glucose/L	H2 yield 1.81 mol H2/mol glucose	(Show et al., 2007)
	Cheese whey	Granule	UASB reactor	6	20 g COD/L/d	H2 Production Rate 0.36-0.38 L H2/L/d	(Carrillo-Reyes et al., 2012)
	Food waste	Biofilm	Batch pilot scale up-flow rector (packed with coir pith)	0.50 m/day a	50 g COD/L	H2 Production Rate 9.67 LH2/L/h	(Pasupuleti et al., 2014)
	Food waste (pre-treated with alkali)	Suspended	ASBR (fill: 0.5 h; reaction: 8 h; settle: 3 h & discharge: 0.5 h)	36	30 g COD/L	H2 yield0.69 mol H2/mol hexoseadded	(Kim et al., 2010)
	Tequila vinasse	Suspended	ASBR (fill: 3 min; reaction: 5.33 h; settle: 30 min & discharge: 7 min) with 50% volumetric exchange rate	12	3 g COD/L	H2 Production Rate 50.5 mL H2/L/h	(Buitrón and Carvajal, 2010)
	Kitchen waste	Suspended	Inclined plug-flow reactor (inclined at 20°)	168	6.5 kgVS/m 3 /d	H2 yield 72 mL H2/g VS	(Jayalakshmi et al., 2009)
	Municipal food waste & kitchen wastewater	Suspended	Anaerobic baffled reactor (ABR)	38.4	29 g CODtotal/L/d	H2 Production Rate 6 L H2/d	(Tawfik and El-Qelish, 2012)
	Glucose	Suspended	Anaerobic membrane bioreactor (MBR)	12 (SRT b )	5.8 g glucose/L	H2 Production Rate 640 mL H2/h	(S.-E. Oh et al., 2004)
	a Up-flow velocity, b SRT: Solid Retention Time

Table 2 .

 2

	8 -Effects of operational temperature and pH on fermentative hydrogen production
	Substrate type	Microbial inoculum	Optimum pH	Optimum Temperature (°C)	Reactor type	HRT (days)	Maximum H2 Yield (mL H2/g VS)	Major acid type produced	Reference
	Food waste Heat shock treated	6.3	35	Leaching Bed	25	310	Acetate	(Han and
		anaerobic sludge			Reactor				Shin, 2004)
	Cassava	Heat treated UASB sludge	7	60	Batch CSTR	3.5	53.8	Butyrate	(Luo et al.,
	stillage								2010b)
	Cassava	Heat treated UASB sludge	5	60	Batch CSTR	3.5	66.3	Butyrate	(Luo et al.,
	stillage								2010b)
	OFMSW	Untreated anaerobic	5.5	37	Semi-	21	165 a	Butyrate	(Valdez-
		digestate			continuous				vazquez et
					CSTR				al., 2005)
	OFMSW	Untreated anaerobic	6.4	55	Semi-	21	360 a	Acetate	(Valdez-
		digestate			continuous				vazquez et
					CSTR				al., 2005)
	Wheat	Cow dung compost	7.0	36	Batch CSTR	6.25	68.1	Acetate and	(Fan et al.,
	Straw							butyrate	2006)
	Vegetable	Enriched from kitchen	7.0	55	Batch CSTR	7.0	12.8 b	Butyrate and	(Lee et al.,
	kitchen	waste compost						lactate	2008)
	waste
	Cattle	Mixed wastewater sludge,	5.5	45	Batch CSTR	1.25	319 c	Butyrate	(Tang et al.,
	wastewater	cow dung compost, chicken							2008)
		manure compost, river
		sludge
	Rice straw	Heat treated wastewater	6.5	55	Batch CSTR	6.5	24.8 d	Acetate	(Chen et al.,
		sludge							2012)

Table 2 .

 2 9 -Examples of different pre-treatment methods applied to complex substrates used in

	DF
	Substrate	H2 yield	Pretreatment	Reactor	Temperature	Reference
		(mL H2/g	methods	mode	(°C)
		VS)
	Rice straw	24.8 a	Size reduction,	Bath	55	(Chen et al.,
			<0.297mm			2012)
	Wheat stalks 17.6	Size reduction, 1 mm	Batch	35	(Yuan et al.,
						2011)
	Wheat straw 212 b	Hydrothermal	Continuous	70	(Kongjan
			(180 °C for 15 min)	UASB reactor		and Angelidaki,
						2010)
	Corn Stover 2.84 c	Steam explosion, 190.220	Batch	35	(Datar et al.,
			•C for 3-5 min			2007)
	Corn Stover 3.0 c	Acidic steam explosion	Batch	35	(Datar et al.,
			(1.2% H2SO4), 180 and			2007)
			200 •C for 1-3 min
	Beet-pulp	66.7	Alkaline at pH 12 using 2	Batch	35±2	(Ozkan et
		±10.1 d	M NaOH for 30 min. +			al., 2011)
			Microwaves (170 °C for
			30 min)
	Bagasse	300	100 °C for 2 h +4%	Batch	55	(Chairattana
			NaOH (w/v) + cellulase			manokorn et
			(20 FPU/g)			al., 2009)
	Grass	72.2 e	4% HCl (w/v), boiled 30	Batch	35	(Cui and
			min.			Shen, 2012)
	Grass	39.5 e	4% NaOH (w/v), boiled	Batch	35	(Cui and
			30 min.			Shen, 2012)
	Corn stalks	209.8	1.5% H2SO4, 121 °C for	Batch	36±1	(Pan et al.,
			60 min+ 9.4 IU/g of			2011)
			cellulase 52 °C at pH4.8
			in 0.1 M sodium citrate
			buffer at 5% (w/v)
	Corncobs	107.9	100 °C, 30 min and 1%	Batch	36	(Pan et al.,
			HCl (w/w))			2010)

Table 2

 2

.10 gives some examples of integrated processes of DF combined with post treatments.

H

2 Production Photofermentation Microbial Electrolysis Cell Anaerobic Digestion H 2 Production CH 4 Production Dark Fermentation Reactor II Stage: Treatment of end products from Dark Fermentation Products of Dark Fermentation (Mainly Organic Acids) Application of CH 4 after cleaning H 2 Cleaning Application of H 2 Agricultural and Industrial Waste Physical-chemical pretreatment of feedstock I Stage: Conversion of Organic Biomass into Volatile Organic Acids

Table 2 .

 2 10 -Examples of operational conditions and system performances of integrated DF systems

	First Stage: DF

  suggested that DF processes have to be operated at ambient temperature in order to obtain a positive net energy. After evaluation of literature data on DF of different substrates,[START_REF] Perera | Fermentative biohydrogen production: Evaluation of net energy gain[END_REF] reported the net energy gain in dark

	products as VFAs and alcohols. Mohammadi et al. (2011) obtained 0.41 mmol H2/g COD
	from mesophilic DF of POME with a COD removal efficiency of 86%. In another study,
	O-Thong et al. (2008) obtained a COD removal efficiency of 55% with H2 yield of 0.142
	L H2/L POME. Nonetheless, it has been suggested from the studies (Table 10), that
	combining DF processes with AD, BES or PF will not only improve the energy recovery,

fermentative processes is positive when the process temperature is below 25 °C. In another study by

Ruggeri et al. (2010)

, the optimum working temperature of 20 °C has been recommended, which offers 20% of the available energy. However, these studies have suggested to couple DF processes with AD, microbial fuel cells, bioelectrochemical systems or photofermentation to obtain a more positive net energy balance from the recovery of energy from the DF end-products and residues.

[START_REF] Perera | Fermentative biohydrogen production: Evaluation of net energy gain[END_REF]

reported that DF combined with BES or DF with AD can result in a positive energy yield. Similarly,

Ruggeri et al. (2010)

found that the AD step after DF can deliver a positive net energy with 40-90% available energy.

Su et al. (2009b)

obtained a higher conversion efficiency of the heat value in DF from 13.3% to 46.0% when combined with photofermentation. This was due to an increase of the H2 yield from 1.59 to 5.48 mol H2/mol glucose.

Lower rates of COD reduction efficiencies are a concern if the DF process aims to treat waste biomass. The conversion of COD to hydrogen is low; theoretically 16 g of COD reduction is achieved per mole of H2 obtained. However, the COD remains in the bybut give higher COD reduction efficiencies and provide complete treatment of organic waste biomass.

  Photofermentative H2 production systems are attractive because of their higher H2 yield potential, i.e. 66.67 mmol H2/g COD (Eqn. 2.16) from PF systems compared to only 22.22 mmol H2/g COD from the DF process with acetate as sole by-product (Eqn. 2.1).

	Light energy 8H2 + 4CO2	(2.16)
	2CH3COOH + 2[H] → PBH-monomer + H2O	(2.17)

Table 2 .

 2 11 -Comparison of photo and fermentation dark systems for biohydrogen production

	Bio H2 systems (Microorganisms)	Carbon source	H2 production mL H2/L/h rate	H2 Yield COD a mL	H2/g	References
	Photofermentation
	R. palustris WP3-5	DFE	25.2	235.1		(Chen et al., 2010)
	R. sphaeroides RV	Succinate	16.5	158.7		(Han et al., 2012)
	Mixed culture	DFE	5.7	568.5		(Montiel-Corona et al., 2015)
	Dark fermentation
	Kitchen waste compost	Vegetable waste	1000	38		(Lee et al., 2010)
	Clostridium thermocellum 7072	Corn stalks	740	140		(Cheng and Liu, 2011)
	Klebsiella sp. TR17	Glycerol	48	128.6 b		(Chookaew al., 2015)	et
	a mL H2/g COD is calculated from the data provided in the publications
	b mL H2/g COD consumed

Table 2 .

 2 12 -Comparison of photo-H2 production by different isolated and mixed PNSB strains from various inoculum sources

	Microbial	Isolated PNSB	Highest H2 producing	Main	Temp.	pH	Light intensity	Maximum	Maximum H2	References
	Inoculum	members	stain	Carbon	°C			H2 yield	production
	sources			source					Rate
									(mL H2/L/h)
	Pig dung	Not reported	Rhodopseudomonas	Acetate	30	7	5,000 lx	660 ml at	-	Yanling et
			palustris					13th day		al., 2008
	Wastewater	Rhodobacter sps. Rhodobacter	Butyrate	30	6-9	4,000 lux	-	118	Tao et al.,
	ponds		sphaeroides ZX-5				(Tungsten			2008
							lamps)
	Water and lake	Not reported	Unidentified PNSB	Acetate	30	-	3,000 lux	1.85 mol	43	Suwansaar
	bed samples		strain TN1					H2/mol		d, et al.,
								acetate		2009
	Freshwater	Not reported	Rhodopseudomonas	Malate	35	7	4,000 lux	3.55 mol	25	Ren et al.,
	pond sludge		faecalis strain RLD-				(Incandescent	H2/mol		2009
			53				lamp)	acetate
	Lake water and	Rb. Capsulatus,	Rhodopseudomonas	Lactate	30	6.8	200 mmol	-	50.7	Bianchi et
	sediment	Rs. rubrum, Rb.	palustris stain AV33				(photons)			al., 2010
	samples	Sphaeroides, R.					m 2 /s
		palustris stain					(Incandescent
		AV33					lamp)
	Activated	R. palustris	Unidentified PNSB	DFE of	31	5.5	190 W/m 2	0.97 ± 0.1	120.8±7	Tawfik et
	sludge		mixed culture	starch			(Tungsten	L/g		al., 2014
				wastewater			lamps)	CODconsumed
	Activated	Not reported	Unidentified enriched	DFE	30	7.0	3000 lux (LEDs	1478 ± 17	5.7	Montiel-
	sludge		IZT PNSB				and halogen	mL H2/L		Corona et
							lamps)			al., 2015
	Silt sewage, pig	Not reported	Unidentified PNSB	Enzymatic	30	7.0	4000 lux (LED	11.5 L H2 /L 165	Zhang et
	manure, and		mixed culture	hydralysat			lamps)			al., 2015
	cow dung			e of
				corncob

  studied the effect of different carbon sources and their concentrations on the photo-H2 production using a batch culture of Rhodobacter sphaeroides RV. The substrates used were either individual substrates such as acetate, propionate, butyrate, lactate, malate, succinate, ethanol, glucose, citrate or sodium carbonate or mixed carbon sources such as malate and succinate, or lactate and succinate.

  reported the optimum C/N ratio of 15 mM to 2 mM (malic acid to glutamic acid) for the maximum hydrogen production rate. In another study, Boran et al. (2010) reported a C/N ratio of 45 with 40 mM of acetic acid and 2 mM of sodium glutamate in PF by Rhodobacter capsulatus in a solar tubular photobioreactor under outdoor conditions. Similarly, Argun et al. (

Table 2 .

 2 13 -Comparison of hydrogen and PHB production by different isolated strains and enriched mixed cultures of PNS via photofermentation of various carbon sources

	Microbial inoculum	Main carbon	C/N ratio	Light intensity PHB	Volumetric H2 yield	References
	sources	and nitrogen			(% Dry cell weight)	(mLH2/L)
		source
	Rhodobacter	30 mM acetate	8.6	1500 lux of	70	0	(Hustede et al.,
	sphaeroides 17023	and 7 mM		incandescent			1993)
	(wild type)	glutamic acid		light
	Rhodobacter	30 mM acetate	12.86	1500 lux of	24	2310	(Hustede et al.,
	sphaeroides 17023	and 7 mM		incandescent			1993)
	(wild type)	glutamic acid		light
	Enriched				5	1478 ± 17
	photoheterotrophic	DFE (11.61 g/L
	culture	butyric, L 1.76		3000 lux of			(Montiel-Corona
		g/L propionic	10.63	LEDS and			et al., 2015)
	Rhodobacter capsulatus	and 1.01 g/L		halogen lamps	29	1252 ± 20
		acetic acid and
		0.78 g/L total
		ammonia
	Rhodobacter	Sugar refinery	-	200 W/m 2	70.4	648	(Yiǧit et al.,
	sphaeroides O.U. 001	wastewater					1999)
	(DSM 5648)	(30% v/v in
		medium)
	Rhodobacter	40 mM Acetate	-	5000 lux	38	0	(Khatipov et al.,
	sphaeroides strain RV	only		incandescent			1998)
				light

  reported 31 to 36 °C as optimum temperature for Rhodobacter sp., whileAndroga et al. (2014) reported 26.8 °C (and 285

Table 2 .

 2 14 -Variation of different operational parameters in PF studies

	PNS strains	Carbon (& nitrogen)	Culture type	Culture	pH	Light intensity	Maximum H2	Maximum H2	References
		source	(Reactor type)	Temp.			yield	production rate
				°C
	Rhodobacter	Dark fermentation	Batch	30°C	buffer	150 -200 W/m 2	484 mmol H2/L	1.18 mmol	(Afsar et al.,
	sphaeroides O.U.001	effluents of glucose			6.4	(Tungsten lamp)	DFE	H2/L/h	2011)
	(DSM586)
	Rhodobacter	Dark fermentation	Batch	30°C	buffer	151 -200 W/m 2	117 mmol H2/L	0.5 mL H2/L/h (Afsar et al.,
	capsulatus	effluents of potato			6.4	(Tungsten lamp)	DFE		2011)
	(DSM1710)	steam peels
		hydrolysate
	Rhodobacter	Acetic acid	Continuous	<40°C	below 8 Natural sunlight	0.35 mol	0.40 mol	(Boran et al.,
	capsulatus (Hup _ )	(glutamate)	Tubular PBR			(Outdoor	H2/mol acetic	H2/(m 3 •h)	2012)
						conditions)	acid
	Rhodobacter	Lactate (glutamate) Batch	30°C	7	4,500-8,500 lux	-	39.88 L/m 3 /h	(Akroum-
	sphaeroides CIP 60.6					(Tungsten lamp)			Amrouche et al.,
									2011)
	Rhodobacter	Acetate (glutamate) Fed-batch	35°C	7	Natural sunlight	-	11.42 LH2/m 3 /h (Androga et al.,
	capsulatus YO3(hup -)		panel PBR			(Outdoor			2011)
						conditions)
	Rhodopseudomonas	Formic, acetic,	Continuous	28-35°C	6.8	4,000 -7,000 lux -	13.26 LH2/m 3 /h (Lee et al.,
	palustrisWP 3-5	butyric, lactic acid	Column PBR						2011)
		(glutamate)

Table 2 .

 2 15 -Comparison of Tubular and Plate PBRs under outdoor conditions

	PNS strains	Carbon	PBR type	Volume in	Operations	Maximum	Maximu H2	Productivity	Productivity	Light	References
		source		liters	conditions	H2 yield	production rate	per	per ground	conversion
				(Dimension)			LH2/m 3 /h	illuminated	area	efficiency
								surface area	LH2/(m 2 •d)
								LH2/(m 2 •d)
	Rhodobacter	Acetate,	Flat-panel	4×25 L	Summer	-	12.3	3.69	29.52	0.20%	(Gebicki et al.,
	capsulatus	sodium			(Aachen,						2010)
	DSM155	lactate and			Germany)
		glutamate
	Rhodobacter	Acetate,	Tubular	60 L (0.12	Summer	-	6.3	3.35	3.35	0.19%	(Gebicki et al.,
	capsulatus	sodium		m dia. &	(Aachen,						2010)
	DSM156	lactate and		0.65 m	Germany)
		glutamate		length)
	Rhodobacter	Acetate,	Tubular	80 L	Winter	15%	6.9	2.46	1.74	1%	(Boran et al.,
	capsulatus	lactate and			(Ankara,						2010)
	DSM 1710	glutamate			Turkey)
	Rhodobacter	Acetate and	Flat-panel	4 L	Summer	53%	11.4	1.5	4.93	-	(Androga et
	capsulatus	glutamate			(Ankara,						al., 2011)
	YO3 (Hup _ )				Turkey)
	Rhodobacter	Acetate and	Tubular	90 L	Outdoor	35%	0.4 mol H2/(m 3 •h)	0.432 mol	0.3 mol	0.20%	(Boran et al.,
	capsulatus	glutamate			Conditions			H2/(m 2 •d)	H2/(m 2 •d)		2012)
	YO3 (Hup _ )				(Ankara,
					Turkey)
	Arthrospira	CO2	Tubular	34 L	Summer	-	1.26 mol H2/(L•d)	32.95 mol	-	5.6% a	(Tredici &
	platensis M2				(Florence,			H2/(m 2 •d)			Zittelli, 1998)
	(cyanobacteria)				Italy)
	Arthrospira	CO2	Flat-panel	5.4 L	Summer	-	1.09 mol H2/(L•d)	30.65 mol	-	4.8% a
	platensis M2				(Florence,			H2/(m 2 •d)
	(cyanobacteria)				Italy)

Table 3 .

 3 1 -Effects of biomass pre-treatment methods on biohydrogen production performance

	during Load I
	Pre-treatment		Modified Gompertz model a
	method	Ho (Nml)	R (Nml/h)	(h)	R 2
	LST	657.8	9.40	69.94	0.9980
	HST-105 ⁰C	341.6	1.28	138.98	0.9880
	HST-95 ⁰C	238.9	2.44	69.41	0.9910
	AST	373.1	1.52	13.57	0.9953
	a The parameters were determined based on average cumulative daily H2 production during Load I

Table 3 .

 3

	2 -Comparison between Load I and Load II feeding operations
	Pre-treatment Method	NmL H2/g glucose (Load I)	mL H2/g glucose (Load II)	Initial pH	Final pH Load I	Final pH Load II
	LST	143.5±13.2	38.4±17.4	7±0.01	5.3±0.01	4.9±0.02
	HST-105 ⁰C	64.5±12.7	21.8±5.1	7±0.01	5.2±0.00	4.5±0.02
	HST-95 ⁰C	52.5 ±3.4	98.7±23.9	7±0.01	5.4±0.01	4.6±0.02
	AST	79.9±22.3	29.8±5.0	7±0.01	4.5±0.16	3.7±0.44
	± indicates data range based on duplicate samples

Table 3 .

 3 3 -Evaluation of biomass pre-treatment methods for DF process

	Pre-treatment	Energy	Chemical	Operational	Scale-up
	method	Requirement	Requirements	Costs	Application
	LST	+ +	+	+	+ + +
	HST-105 ⁰C	+ + +	+	+ + +	+ +
	HST-95 ⁰C	+ +	+	+ + +	+ +
	AST	+	+ + +	+ +	+ + +
	+ Less intensive; ++ Moderately intensive; +++ Very Intensive (Adapted and modified from Ghimire et al.,
	2015)

Table 3 . 4 -

 34 Characteristics of the substrates and inocula used in this study

	Characteristics	Food waste	OMWW	Rice Straw	ADS	WAS
	pH	4.4 ± 0.1	4.6 ± 0.1	NA	8.3 ± 0.1	7.0 ± 0.1
	Chemical Oxygen Demand (COD)	347.6 ± 47.0 g/kgfood waste	141.5 ± 13.0 g/LOMWW	NA	NA	NA
	Total solids	21.0 ± 0.1 %	4.7 ± 0.1 %	92.3 ± 0.2 %	2.33 ± 0.4 %	2.9 ± 0.2%
	Volatile solids	20.2 ± 0.1 %	3.1 ± 0.3 %	80.9 ± 0.6 %	1.93 ± 0.1 %	1.8 ± 0.1%
	Carbohydrate content	105.8 ± 0.7 g/kgfood waste	12.9 ± 0.2 g/LOMWW	NA	NA	NA
	Lipids	17.5 ± 1.0 g/kgfood waste	45.3 ± 4.0 g/LOMWW	NA	NA	NA
	TKN	6.4 ± 0.2 g/kgfood waste	0.5 g/LOMWW	NA	NA	NA
	NH4-N	NA	NA	NA	283.5 ± 11.0 mg NH4-N/L	203.1 ± 3.0 mg NH4-N/L
	Alkalinity	NA	NA	NA	1437.2 ± 14 mg CaCO3/L	2605.7 ± 70.0 mg CaCO3/L
	Total phenols	NA	1.16 ± 0.03 g/LOMWW	NA	NA	NA
	NA-Not Analyzed

Table 3 . 5 -

 35 Experimental conditions applied in the DF batch tests of the tested substrates

	Investigation	Substrate	Inoculum Initial pH	F/M
	Effect of initial pH	Food waste	ADS	4.5, 5.0, 5.5, 6.0, 6.5 and 7.0	0.5
	Combined effect of food waste and initial pH	Food waste	ADS	5.0 and 6.5	0.5, 1.0 and 1.5
	Effect of pre-treatment of substrate	Rice straw	WAS	6.5	7.0
	Effect of inoculum source and pre-treatment	OMWW	WAS and ADS	6.0	1.0

Table 3 .

 3 6 -Effects of initial pH on H2 production performance and characteristics of accumulated end products

	Initial	Parameters derived from modified Gompertz model			Characteristics of digestate at the end of DF
	pH	Ho (mL/gVS)	L (h)	R (mL/h) R 2	Average final pH	H2 (mM/kg VS)	Acetate (mM/kg VS)	Propionate (mM/kg VS)	Butyrate (mM/kg VS)	Ethanol (mM/kg VS)
	4.5	57.3	113.6	0.7	0.993	4.7 ± 0.1	1341.2 201.3 1854.6 114.0 964.5 99.1	2728.7 359.6	263.7 16.1
	5.0	50.9	68.1	1.0	0.999	4.9 ± 0.1	1121.3 17.2	1611.8 412	1686.7 253.3	3018.7 109.7	753.4 290.6
	5.5	20.3	41.2	0.4	0.995	5.2 ± 0.6	448.4 148.2	2830.2 381.0 1358.1 392.1	1973.7 374.9	623.7 53.8
	6.0	15.4	2.0	0.7	0.997	5.3 ± 0.1	308.0 26.8	3558.9 368.7 959.7 6.4	1992.0 238.1	2340.9 263.7
	6.5	11.2	3.3	0.8	0.995	5.5 ± 0.1	247.7 45.3	3900.2 838.5 260.0 34.8	2185.5 580.1	3056.7 32.3
	7.0	14.6	25.3	6.7	1.000	6.6 ± 0.1	322.6 80.7	5922.4 43.9	877.2 41.4	3255.6 308.1	1673.6 48.4
	R 2 represents the regression coefficient

Table 3 . 7 -

 37 Effects of initial pH and F/M ratio on H2 production performance and characteristics of accumulated end products in DF of food waste

			Parameters derived from modified Gompertz model			Characteristics of digestate at the end of DF
	pH	F/M	Ho (mL/g VS)	L (h)	R (mL/h)	t95 (day)	R 2	Average final pH	H2 (mM/kg VS)	Lactate (mM/kg VS)	Acetate (mM/kg VS)	Propionate (mM/kg VS)	Butyrate (mM/kg VS)	Ethanol (mM/kg VS)	Caproate (mM/kg VS)
		0.5	50.9	68.1	1.0	7.0	0.949	4.9 0.1	2264.9 34.8	17.5 8.1	1610.7 411.8	1687.0 253.3	3018.7 109.7	753.4 290.6	0.0 0.0
	5.0	1.0	58.5	81.9	1.4	9.7	0.997	4.7 0.1	2690.9 206.5	18.1 2.2	1264.0 27.1	3135.4 245.7	2959.9 35.2	1876.5 5.9	0.0 0.0
		1.5	54.2	87.9	0.3	46.5	0.991	4.5 0.1	2202.1 545.2	98 10.3	420.3 119.7	842.8 59.2	2638.1 202.9	1402.9 325.6	0.0 0.0
		0.5	11.2	3.4	0.8	1.2	0.995	5.5 0.1	1259.7 188.4	0.0 0.0	6043.0 357.2	830.3 38.9	2344.0 73.3	3056.7 32.3	0.0 0.0
	6.5	1.0	42.6	17.0	1.6	4.6	0.938	5.7 0.1	1928.7 89.3	126.3 124.2	1700.0 305.8	775.8 91.1	2062.9 169.1	3602.1 20.7	70.3 9.4
		1.5	56.9	2.3	1.8	7.0	0.944	5.3 0.1	2413.4 197.0	0.0 0.0	2364.5 216.1	655.5 166.3	2410.5 47.5	2206.0 63.1	263.3 23.1

Table 3 . 8 -

 38 Effect of substrate pre-treatment on biohydrogen production performance measured by the modified Gompertz model

	Pre-	Parameters derived from modified Gompertz model			Characteristics of digestate at the end of DF
	treatment method	Ho (mL/g VS)	L (h)	R (mL/h)	R 2	Average final pH	H2 (mM/kg VS)	Acetate (mM/kg VS)	Propionate (mM/kg VS)	Butyrate (mM/kg VS)	Ethanol (mM/kg VS)	B/A (mM:m M)
	Without treatment	0.3	37.3	0.1	0.958	4.7 ± 0.1 12.8 ± 4.1	462.6 ± 42.7	50.8 ±15.8	46.4 ±13.7	41.0 ± 7.2	0.10
	4% NaOH	6.7	23.9	2.9	0.999	4.9 ± 00	296.3 ± 19.2	775.0 ± 13.5	189.4 ±18.5	227.7 ± 38.5	129.4 ± 44.8	0.29
	8% NaOH	15.4	11.3	3.6	0.965	5.2 ± 0.6 699.4 ± 62.8	468.6 ± 84.4	55.6 ± 15.4	614.1 ±105.8 148.9 ± 11.8	1.31

Table 3 . 9 -

 39 Effects of inoculum source on H2 production performance measured by the modified Gompertz model and characteristics of accumulated end products in

	DF of OMWW
		Parameters derived from modified Gompertz model		Characteristics of digestate at the end of DF
	Inoculum type	Ho (mL/g VS)	L (h)	R (mL/h)	R 2	Average final pH	H2 (mM/kg VS)	Lactate (mM/kg VS)	Acetate (mM/kg VS)	Propionate (mM/kg VS)	Butyrate VS) (mM/kg	Ethanol VS) (mM/kg	B/A (mM:mM)
	ADS	106.1	101.0	1.0	0.996	5.6 ± 0.1	751.2 ± 15.2	1651.8 ± 573.4	1752.2 ± 510.9	269.5 ± 183.3	4293.5 ± 93.1	3423.2 ± 1104.2	1.95
	WAS	204.1	34.4	2.2	0.984	5.5 ± 0.2	1479.7 ± 46.3	0.0 ± 0.0	6823.0 ± 904.1	282.0 ± 217.1	5062.5 ± 131.0	3022.6 ± 0.8	0.44

Table 3 .

 3 10 -Summary of various strategies to improve the H2 yields from the substrate with different biodegradability

	Substrates	Optimization parameters	Optimal conditions	Substrate concentration (g VS/L)	Culture system	H2 Yield (NmL/g VSadded)	Reference
	Food waste	Initial pH (4.5-8.5)	pH 6.5	53.1 ± 0.9	Activated sludge, 39 °C, batch	56.2	(Cappai et al., 2014)

Table 4 .

 4

	1 -Characteristics of food waste
	Characteristics	g/kg Food waste
	Chemical Oxygen Demand (COD)	347.6±47.4
	Carbohydrate content	105.80±0.7
	TKN	6.4±0.18
	Lipids	17.50±1.19
	Total solids (%)	23.79±0.44%
	Volatile solids (%)	22.8±0.42%

Table 4 .

 4 3 -H2 production rate, yields and production stability from FW by mixed anaerobic

	cultures
	Exp. Period	HPR (N L/L/d)	HY (N L/kg VSadded)	H2 in biogas (%) HPSI
	I	116.9±40.1	116.9±40.1	52.8%±1%	0.66
	II	54.1±41.3	54.1±41.3	31.2%±1%	0.24
	III	109.5±32.8	73.0±21.9	43.8%±20%	0.70
	IV	210.2±29.8	105.1±14.9	59.4%±6%	0.86
	V	208.0±34.8	104.0±17.4	57.2%±6%	0.83
	VI	303.6±111.4	121.4±44.5	55.8%±10%	0.63
	VII	133.2±112.1	53.3±44.8	46.1%±28%	0.16
	VIII	408.8±97.6	163.5±39.0	59.4%±40%	0.76

Table 4 .

 4 4 -Comparison of H2 production from food waste by mixed cultures

						Maximum
	Substrate type	Reactor type	Temp. (°C)	pH	OLR VS/L•d) (g	assessed H2 (Nml H2/g yield	H2 in (%) biogas	Reference
						VSadded)
	FW	Batch	55	4.5 (initial)	6	46.3	23	(Shin et al., 2004)
	Vegetable kitchen waste	Intermitten t-CSTR	55	6.0	28 a	38.1 b	40	(Lee et al., 2010)
	FW and sewage sludge	Batch	35	5.0-6.0	-	122.9 c	-	(Kim et al., 2004)
	OFMSW	Semi-						(Valdez-
	(FW+pap	continuous	55	6.4	11 d	360	58	vazquez et
	er)	CSTR						al., 2005)
	OFMSW	Packed bed reactor	38±2	5.6±0.2 16 e	99	47	(Alzate-Gaviria et al., 2007)
	FW	Semi-continuous CSTR	55	4.5-5	2.5	104.0±17.4	57±6 %	This study

a gCOD/L•d, b mL H2/g COD, c mL H2/g carbohydrate COD, d g VS/kg wet mass reactor•d, e g VS/kg•d, FW=Food waste, OFMSW= Organic fraction of municipal solid waste

Table 4 .

 4 5 -Characteristics of influent and effluents DF of FW during different experimental

	periods
	Exp. Period	pH_IN	pH_OUT	Lactate (mM)	Ethanol (mM)	Acetate (mM)	Propionate (mM)	Butyrate (mM)
	I	7.00	4.7±0.3	0.1±0.2	4.8±0.2	13.1±3.6	3.8±2.2	10.4±2.8
	II	7.00	4.5±0.1	0.6±1.4	5.4±3.5	3.2±2.0	3.4±2.3	6.2±4.2
	III	7.00	4.5±0.2	4.0±9.1	8.7±2.7	4.9±0.6	6.0±2.2	11.0±1.6
	IV	7.00	4.9±0.4	0.0±0.0	17.2±8.6 8.5±1.8	9.6±2.9	12.0±2.9
	V	7.00	4.7±0.2	0.0±0.0	17.1±6.6 6.7±1.9	5.7±2.1	9.9±3.2
	VI	7.00	4.4±0.1	0.5±0.9	9.4±5.3	5.7±2.8	5.9±2.7	11.1±7.5
	VII	7.00	4.50±0.1	3.9±1.8	10.6±1.8 8.3±3.4	23.7±12.6 14.9±5.8
	VIII	7.00	4.47±0.9	0.2±0.2	6.1±2.2	9.0±6.5	8.8±7.7	16.4±10.5
	Table 4.6 -Reaction stoichiometry in dark fermentation of glucose
	Possible H2 producing pathways			Metabolic pathways ΔG ' 0	a	Eqn
								(kJ/mol)
	C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2		Acetate	-206.3	(2)
	C6H12O6 → CH3CH2CH2 COOH + 2CO2 + 2H2		Butyrate	-254.8	(3)
	C6H12O6 + 2H2O → CH3CH2OH + CH3COOH +		Ethanol & acetate -215.7	(4)
	2CO2 + 2H2
	4C6H12O6+2H2O→3CH3CH2CH2COOH +		Butyrate & acetate -254.0	(5)
	2CH3COOH + 8CO2 + 10H2
	Unfavorable and H2 consuming pathways
	C6H12O6 + 2H2 → 2CH3CH2COOH + 2H2O		Propionate	-359.6	(6)
	1.5 C6H1206 → 2C2H5COOH + CH3COOH +CO2 +	Propionate &	-310	(7)
	H2O					Acetate
	C6H12O6 → 2CH3CH2OH + 2CO2			Ethanol	-235.0	(8)
	C6H12O6 → 2CH3CHOHCOOH			Lactate	-198.1

  The CHW and BM collected from the cheese factory and buffalo farm in Salerno, Italy, were stored at <4 ⁰C for further use in the experiments. The waste composition used in the study is presented in Table4.7.

	was used as start-up inoculum. The total
	solids (TS), volatile solids (VS) and ammonia (NH +	4-N) content of the inoculum are
	29.54±0.22 gTS/L, 18.36±0.14 gVS/L and 283.47±10.8 gNH +	4/L, respectively. The pH
	of the inoculum was 8.3±0.1 and the total alkalinity was 1437.20±14.27 mg CaCO3/L.

Table 4 .

 4 7 -Characteristics of cheese whey and buffalo manure

	Characteristics	Cheese whey	Buffalo manure
	pH	4.88±0.01	8.05±0.01
	Total solids (%)	6.06±0.03%	5.67±0.04%
	Volatile solids (g/L)	50.54±0.22	42.17±1.35
	Total COD (g/L)	67.02±6	ND
	Soluble sugars (g/L)	12.88±0.34	ND
	TKN (g/L)	0.86±0	1.99±0.1
	Lactic acid (g/L)	2.52±0.172	ND
	Alkalinity (g/L)	0.5±0	4.37±1
	ND-Not Determined

Table 4 .

 4

	8 -Operational conditions and buffalo manure feeding strategies during different
	experiments runs
	Experimental	CHW:BM	Total OLR	OLR of CHW	HRT
	Periods	(gVS/gVS)	(gVS/L/d)	(gVS/L/d)	(d)
	I	0	0.7	0.7	12.0
	II	0	2.1	2.1	12.0
	III	1	4.2	2.1	12.0
	IV	4	2.6	2.1	12.0
	V	4	2.6	2.1	8.0
	VI	2	3.2	2.1	8.0
	VII	4	2.6	2.1	8.0

Table 4 .

 4 9 -H2 production performance during the dark fermentation at different CHW:BM ratio

	Exp. Periods	CHW:BM (gVS/gVS)	HY	HPR	HPSI	H2 %	CO2 %
	I	0	123.8±85.1	73.8±45.7	0.38	37.04±7.0%	40.13±10.4%
	II	0	95.3±64.1	134.9±90.7	0.33	46.69±7.1%	40.37±9.6%
	III	1	139.8±47.8	197.8±67.7	0.66	51.85±9.0%	44.43±6.7%
	IV	4	152.2±43.9	215.4±62.1	0.71	58.01±4.8%	39.13±4.6%
	V	4	51.8±29.3	73.4±41.4	0.44	38.47±12.6%	27.80±4.7%
	VI	2	76.2±76.1	183.0±107.7	0.41	38.38±21.2%	37.32±12.0%
	VII	4	131.7±44.6	186.3±63.7	0.66	51.10±6.3%	46.34±6.4%

Table 4 .

 4 10 -Characteristics of effluents from the DF of CHW with BM as co-substrate during different experimental periods

	Exp. Periods	Ammonia (mg NH4 + -N/L)	Total Alkalinity (mg CaCO3/L)	pH_OUT	Lactate (mM/gVS)	Ethanol (mM/g VS)	Acetate (mM/gVS)	Propionate (mM/gVS)	Butyrate (mM/gVS)	Hydrogen (mM/gVS)
	I	157.9±0.0	1019.3±145.0	5.6±0.3	0.00±0.0	17.27±4.2	21.19±11.6 3.43±2.9	14.80±7.3	7.44±4.5
	II	71.8±0.0	337.4±82.9	4.6±0.1	1.42±0.7	2.60±0.1	9.60±11.1	4.09±6.5	8.99±2.7	4.60±3.1
	III	389.3±59.2	1327.6±388.2	5.0±0.2	0.69±0.8	5.74±1.9	2.61±1.3	0.31±0.2	7.51±2.4	3.38±1.1
	IV	179.4±34.0	1184.4±373.6	4.8±0.1	0.29±0.5	10.45±3.3	4.18±2.1	0.51±0.4	14.12±6	5.88±1.7
	V	111.2±3.6	394.5±127.0	4.5±0.1	2.99±1.5	13.70±0.0	2.20±0.9	0.17±0.2	9.64±3.9	2.00±1.1
	VI	86.1±35.9	878.6±265.8	5.2±0.1	1.18±1.2	8.48±0.8	2.10±1.7	0.10±0.1	6.69±3.7	2.46±2.4
	VII	81.2±8.2	619.7±152.8	4.8±0.1	1.41±1.4	12.27±1.7	3.28±1.4	0.22±0.2	10.72±3.7	5.09±1.7

  metabolites such as lactate, acetate, propionate, butyrate and ethanol, produced during the different experimental periods are summarized in Table 4.10. With the different concentrations of organic acids and alcohols present in the dark fermentation effluent, H2 production can be related to more than one biochemical pathways presented in Table 4.6. The principal component analysis of co-relation circles presented in Figure

	4.8 suggests that the H2 production was mainly due to the butyrate and acetate pathways
	which are well co-related with the HY. Unsurprisingly, other metabolites such as
	propionate, lactate or ethanol, which normally do not represent H2 favorable pathways,
	were not well correlated.

  3%. The FW ingredients were freshly bought at municipal markets in Naples (Italy), shredded with a blender and immediately stored at -20 ⁰C to avoid acidification. The FW

	characteristics were (in g/kg FW): chemical oxygen demand (COD), 347.6 ± 47.4;
	carbohydrate content, 105.80 ± 0.7; total Kjeldahl nitrogen (TKN), 6.4 ± 0.18; lipids,
	17.50 ± 1.19; total solids (TS), 23.79 ± 0.44%; volatile solids (VS), 22.8 ± 0.42% and the
	pH was 4.4 ± 0.1.

Table 5 .

 5 1 -Characteristics of the DFE used in PF experiments.

	Parameters	Values (mg/L)
	Chemical Oxygen Demand (COD)	3561.8±131.1
	TKN	208.0±7
	NH4 + -N	1.14±0.3
	Phosphate	130.5±1
	Total iron (Total-Fe)	≤ 0.7
	Lactic Acid	33.0
	Acetic Acid	466.0
	Propionic Acid	449.6
	Butyric Acid	1075.4
	Ethanol	323.0

Table 5 .

 5 2 -Experimental design used for the operation of semi-continuous reactor

	Experimental periods	I	II	III	IV	V	VI
	OLR (kg VS/m 3 /d)	1	1	1.5	2	2	2.5
	HRT (d)	12	6	6	6	4	4
	Concentration (kg VS/m 3 )	12	6	9	12	8	10

  Lux and positioned on the top of the stirrers. Caps of the reactors presented two separate ports for biogas and culture medium sampling. The bottles were sealed with

	3; MgSO4.7H2O, 0.4;
	NaCl, 0.4; CaCl2.2H2O, 0.075; ferric citrate, 0.005; yeast extract, 0.4 and 10 mL of trace
	metals solution containing (in mg/L): ZnSO4.7H2O, 10; MnCl2.4H2O, 3; H3BO3, 30;
	CoCl2.6H2O, 20; CuCl2.2H2O, 1; NiCl2.6H2O, 2 and Na2MoO4.2H2O, 30.
	The R. sphaeroides AV1b pre-culture was grown again in a DFE supplemented with
	appropriate chemicals and autoclaved, as explained earlier. It was mainly composed of
	(in mg/L): acetic acid, 848; propionic acid, 457; butyric acid, 1184; NH4 + , 6; phosphate
	(as PO4 3-), 35.8 and total Fe 0.045. Ten mL of the culture (1.52 g TSS/L) that represents
	2.5 % V/V of the reactor working volume was used as inoculum in the PF experiments
	with DFE (Table 5.1).
	Transparent 500 mL borosilicate serum glass bottles (Simax, Czech Republic) with 400
	mL working volume were used as photofermentative batch reactor. The batch reactors
	were maintained at room temperature (24 ± 2 °C, April-May) under a luminance of about
	4000

Table 5 .

 5 3 -H2 production rate, yields and production stability from FW by mixed anaerobic

	cultures
	Exp. Period	HPR (N L/m 3 /d)	HY (N L/kg VSadded)	H2 in biogas (%) HPSI
	I	116.9±40.1	116.9±40.1	52.8%±1%	0.66
	II	54.1±41.3	54.1±41.3	31.2%±1%	0.24
	III	109.5±32.8	73.0±21.9	43.8%±20%	0.70
	IV	210.2±29.8	105.1±14.9	59.4%±6%	0.86
	V	208.0±34.8	104.0±17.4	57.2%±6%	0.83
	VI	303.6±111.4	121.4±44.5	55.8%±10%	0.63

Table 5

 5

	.4 -Characteristics of influent and effluents from DF of FW during different
	experimental periods
	Exp. Period	pH_IN pH_OUT	Lactate (mM)	Ethanol (mM)	Acetate (mM)	Propionate (mM)	Butyrate (mM)
	I	7.00	4.7±0.3	0.1±0.2	4.8±0.2	13.1±3.6	3.85±2.21	10.4±2.8
	II	7.00	4.5±0.1	0.6±1.4	5.4±3.5	3.2±2.0	3.44±2.33	6.2±4.2
	III	7.00	4.5±0.2	4.0±9.1	8.7±2.7	4.9±0.6	5.97±2.16	11.0±1.6
	IV	7.00	4.9±0.4	0.0±0.0	17.2±8.6	8.5±1.8	9.65±2.91	12.0±2.9
	V	7.00	4.7±0.2	0.0±0.0	17.1±6.6	6.7±1.9	5.70±2.15	9.9±3.2
	VI	7.00	4.4±0.1	0.5±0.9	9.4±5.3	5.7±2.8	5.89±2.70	11.1±7.5

Table 5 .

 5 5 -Comparison of energy yields from gaseous biofuels produced out of FW as feedstock using stand alone or coupling of different technologies

a The energy yield was calculated from the yield of biogas based on the heating values of hydrogen (242 kJ/mol and methane (801kJ/mol); b L H2/kg food waste; c Algal biomass pre-treatment by microwave heating with dilute H2SO4; d Algal biomass pre-treatment by steam heating with dilute H2SO4

Table 5

 5

	.6 -Characteristics of substrates used in photofermentative experiments
	Characteristics of substrates used in photofermentative experiments
	Characteristics	RS-I (mg/L)	RS-D (mg/L) a	PM-D (mg/L)
	Chemical Oxygen Demand (COD)	3561.8 ± 131.1	2182.2 ± 303.0	2400.9 ± 149.0
	Total Kjeldahl Nitrogen (TKN)	208.0 ± 7.0	189.1 ± 24.0 b	189.1 ± 24.0
	NH4 + -N	1.1 ± 0.3	1.6 ± 0.3	0.9 ± 0.3
	Lactic Acid	33.0	36.1	23.4
	Acetic Acid	466.0	277.0	288.1
	Propionic Acid	450.0	197.4	224.6
	Butyric Acid	1075.4	636.1	547.0
	a Analyzed after autoclaving the DFE
	b Analyzed before autoclaving the DFE

Table 5 .

 5 7 -Summary of photo-H2 performance estimated by modified Gompertz model Gompertz model is presented in Table5.7. A long initial lag phase of 9 days was observed and the 95% of the maximum production was reached after 26 days (Table5.7). The longer lag phase can be attributed to the time required for biomass growth or to the competitive nature of PHB and H2 production. The concentration of individual VFAs present in DFE for RS-I tests (Table5.6) was not in the inhibiting range as reported

	Tests	Volumetric Yield NmL H2/L	NmL H2/g CODadded	Kinetic model parameters of photo-H2 production Ho (mL) L (d) R (mL/L•h) t95 (d) R 2
	RS-I	914.1±8	256±2	368.8	9.3	2.2	26.0	0.9985
	RS-D	358.0±25	164.0±12	144.2	4.3	0.9	20.0	0.9984
	PM-D	168.7±14	71.3±6	69.8	2.1	0.4	22.3	0.9754
	The analysis of kinetics parameters of bio-H2 production in RS-I incubation obtained
	from the modified

by

Han et al. (2012)

. As a long lag phase was observed with the R. sphaeroides AV1b using undiluted DFE (RS-I), PF tests were carried out with diluted DFE (1:2 ratios with ultrapure water) as well. A pure R. sphaeroides AV1b and a mixed PNSB culture was incubated in the diluted DFE, RS-D and PM-D, respectively (Table

5

.6).

The results of the tests RS-I, RS-D and PM-D are presented in Table

5

.7. Figure

5

.7 and Table

Time (Days)

  PHBV), a copolymer. Moreover, the presence of valerate gave 4 times more PHBV than propionate. Nonetheless, in our tests the propionate concentrations were low and valerate was not present (Table

	hydroxybutyrate-co-β-hydroxyvalerate (
	F	Acetate
		Propionate
		Butyrate

Table 5 .

 5 Table5.9). Moreover, the different COD removal efficiency in RS-I (60.1 ± 1 %) and RS-D (80.2 ± 1%) tests can be due to varying initial COD concentrations, 3.6 ± 0.1 and 2.2 ± 0.3 g/L, respectively. This was supported in a study by[START_REF] Tawfik | Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent[END_REF], who reported a decrease in COD removal by mixed PNSB culture when the organic loading rate (OLR) increased from 3.

		8 -Possible photofermentative pathways
	Eqns.	Source of carbon	Possible photofermentative pathways	Major product
	(5.4)	Lactate	C3H6O3 + 3H2O → 6H2 + 3CO2	H2
	(5.5)	Acetate	C2H4O2 + 2H2O → 4H2 + 2CO2	H2
	(5.6)	Propionate	C3H6O2 + 4H2O → 7H2 + 3CO2	H2
	(5.7)	Butyrate	C4H8O2 + 6H2O → 10H2 + 4CO2	H2
	(5.8)	Acetate	2CH3COOH + 2[H] → PHB-monomer + H2O	PHB
	Figure 5.8 summarizes the major products yields from the conversion of DFE into H2,
	PHB and biomass per unit g COD added. Figure 5.8 also shows that the dilution slightly
	affects the soluble COD removal from DFE. The COD removal increased from 60.1 (±
	1) % to 80.2 (± 1) % and 73.6 (± 0) % for, respectively, R sphaeroides AV1b in RS-I
	DFE, RS-D DFE and mixed PNSB for PM-D. This COD removal efficiency is
	comparable to that reported by Montiel-Corona et al. (2015). COD removal from DFE
	depends on several parameters such as the type of PNSB culture, initial influent COD
	concentration, dilution factor or reactor operating conditions (Montiel-Corona et al.,
	2015). Nonetheless, initial COD concentrations and type of PNSB species applied seems
	to strongly influence the COD removal. With diluted DFE, the mixed PNSB cultures gave
	higher COD removal efficiencies compared to pure cultures (Table 5.9). The higher COD

removal efficiency can be due to the functioning of several microbial consortia present in the culture, supported by the results from

Montiel-Corona et al. (2015)

and this study (

Table 5 .

 5

	mL H2/L) is higher compared to Yiǧit et al. (1999) (648 mL H2/L), while the H2
	production with mixed PNSB (168.7 ± 14 mL H2/L) is lower compared to Montiel-
	Corona et al. (2015) (1478 ± 17 mL H2/L) (Table 5.9). The lower H2 production can be
	attributed to the lower initial COD concentration of 2.2 ± 0.3 g/L used in this study
	compared to 4.6 g/L in Montiel-Corona et al.

9 shows comparison of the H2 and PHB yields and COD removal obtained in this study with studies reported in the literature. The H2 yields obtained in RS-I tests (914 ± 8

Table 5 .

 5 9 -Comparison of hydrogen and PHB production by different isolated strains and enriched mixed cultures of PNS via photofermentation of various carbon sources

	Microbial Inoculum	Carbon and nitrogen	PHB	Volumetric	COD	References
	sources	source	(% DCW)	H2 Yield (mL H2/L)	Removal (%)
	Rhodobacter	30 mM acetate and 7	70	0	-
	sphaeroides 17023 (wild type)	mM glutamic acid 30 mM lactate and 7	24	2310	-	Hustede et al., 1993
		mM glutamic acid
	Enriched	DFE (11.61 g/L	5	1478 ± 17	89
	photoheterotrophic culture IZT	butyric, L 1.76 g/L propionic and 1.01 g/L				Montiel-Corona et
	Rhodobacter	acetic acid and	29	1252 ± 20	65	al., 2015
	capsulatus	0.78 g/L total ammonia
	Rhodobacter	Sugar refinery	70.4	648	-	Yiǧit et al.,
	sphaeroides O.U.	wastewater (30% v/v in				1999
	001 (DSM 5648)	medium)
	Rhodobacter	40 mM Acetate only	38	0	-	Khatipov et
	sphaeroides RV					al., 1998
	Rhodobacter	DFE (RS-I, Table 5.6)	32.5 ± 3	914.1 ± 8	60.1 ± 1	This study
	sphaeroides AV1a
	Enriched	DFE (PM-D, Table 5.6) 6.3	168.7 ± 14	73.6 ± 0	This study
	photoheterotrophic
	culture

  Final State -COD of Initial State 𝐓𝐨𝐭𝐚𝐥 𝐒𝐮𝐛𝐬𝐭𝐫𝐚𝐭𝐞 𝐃𝐞𝐠𝐫𝐚𝐝𝐚𝐭𝐢𝐨𝐧 = 𝐀 𝐇 𝟐 ,𝐟 + 𝐀 𝐦𝐞𝐭,𝐟 + 𝐀 𝐆𝐂

	𝐤𝐠 𝐓𝐒	-	𝐀 𝐇 𝟐 ,𝐢 𝐤𝐠 𝐓𝐒	(Equation 5.9)

Table 5 .

 5 10 -Production of VFA from different types of fermentation

	Feedstock	Inoculum	Operating conditions VFA Production	Reference
	Food waste	Anaerobic	Controlled pH 6.0,	799 g COD/kg	(Wang et al.,
		digested sludge	35 °C	VSadded	2015)
	Food waste	Anaerobic	Controlled pH 6.0,	918 g COD/kg	(Wang et al.,
		activated	30 °C	VSremoval	2014)
		sludge
	Kitchen	Waste	Controlled pH 8.0,	692.4 g COD/kg	(Chen et al.,
	waste	activated	37 °C	VSadded	2013)
		sludge
	Wheat straw Anaerobic	Initial pH >8.0, Final	140 ± 6 g	(Motte et al.,
		digestate	pH 5.2, 37 °C, 23 TS	COD/kg TSadded	2015)
			content, 64 days SRT
			(batch)
	Waste	No inoculum	Controlled pH 8, 55	368 g COD/kg	(Zhang et al.,
	activated	addition	°C, 9 days SRT	VSadded	2009)
	sludge		(batch)
	Food waste	Waste	Initial pH 5.5, Final	134.4 ± 22g	This study
		activated	pH 5.1, 37 °C, 10 %	COD/kg TSadded
		sludge	TS, 14 days SRT
			(batch)
	Wheat straw Waste	Initial pH 5.5, 37 °C,	22.3 ± 2 g	This study
		activated	25 % TS, 14 days	COD/kg TSadded
		sludge	SRT (batch)

/g VS)

from corn stalk by dark fermentation. International Journal of Hydrogen Energy, 37 [START_REF] Wu | Continuous biohydrogen production from liquid swine manure supplemented with glucose using an anaerobic sequencing batch reactor[END_REF], 6554-6561. Sørensen, J., Christensen, D., Srensen, J. A. N., & Jrgensen, B. O. B. (1981). Volatile fatty acids and hydrogen as substrates for sulfate-reducing bacteria in anaerobic marine sediment. Applied and Environmental Microbiology, 42 (1), 6-11.

Srikanth, S., Venkata Mohan, S., Prathima Devi, M., Peri, D., & Sarma, P. N. (2009). Acetate and butyrate as substrates for hydrogen production through photofermentation: Process optimization and combined performance evaluation.

International Journal of Hydrogen Energy,34(17), 7513-7522.

Su, H., Cheng, J., Zhou, J., Song, W., & Cen, K. (2009a). Combination of dark-and photo-fermentation to enhance hydrogen production and energy conversion efficiency. International Journal of Hydrogen Energy, 34(4), 1780-1786.

Su, H., Cheng, J., Zhou, J., Song, W., & Cen, K. (2009b). Improving hydrogen production from cassava starch by combination of dark and photo fermentation.

International Journal of Hydrogen Energy, 34(4), 1780-1786.

Su, H., Cheng, J., Zhou, J., [START_REF] Su | Hydrogen production from water hyacinth through dark-and photo-fermentation[END_REF]. Hydrogen production from water hyacinth through dark-and photo-fermentation. International Journal of Hydrogen Energy,35(17), 8929-8937.

Suwansaard, M., Choorit, W., Zeilstra-Ryalls, J. H., & Prasertsan, P. (2009). Isolation of anoxygenic photosynthetic bacteria from Songkhla Lake for use in a two-staged biohydrogen production process from palm oil mill effluent. International Journal of Hydrogen Energy,34(17), 7523-7529.

Tabatabaei, M., Zakaria, M. R., Rahim, R. A., Wright, A. G., Shirai, Y., Abdullah, N., … Hassan, M. A. (2009). PCR-based DGGE and FISH analysis of methanogens in an anaerobic closed digester tank for treating palm oil mill effluent. Electronic Journal of Biotechnology,12(3).

Taherzadeh, M. J., & Karimi, K. (2008). Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. International journal of molecular sciences, 9, 1621-1651.

Takabatake, H., Suzuki, K., Ko, I. B., & Noike, T. (2004). Characteristics of anaerobic ammonia removal by a mixed culture of hydrogen producing photosynthetic bacteria. Bioresource Technology, 95 (2), 151-158.

Tang, G.-L., Huang, J., Sun, Z.-J., Tang, Q.-Q., Yan, C.-H., & Liu, G.-Q. (2008).

Biohydrogen production from cattle wastewater by enriched anaerobic mixed consortia: influence of fermentation temperature and pH. Journal of Bioscience and Bioengineering, 106 (1), 80-7.

Tao, Y., Chen, Y., [START_REF] Tao | High hydrogen yield from a twostep process of dark-and photo-fermentation of sucrose[END_REF]. High hydrogen yield from a twostep process of dark-and photo-fermentation of sucrose, 32, 200-206. Tao, Y., Yangling, H., Wu, Y., Liu, F., Li, X., Zong, W., & Zhou, Z. (2008). Characteristics of a new photosynthetic bacterial strain for hydrogen production and its application in wastewater treatment. International Journal of Hydrogen Energy, 33(3), 963-973. Tawfik, A., El-Bery, H., Kumari, S., & Bux, F. (2014). Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent. Bioresource CHAPTER 3

OPERATIONAL STRATEGIES TO IMPROVE DARK FERMENTATIVE H2

PRODUCTION USING COMPLEX WASTE BIOMASS

The section 3.1 has been published as Ghimire, A., Frunzo, L., Salzano, E., Panico, A., Lens, P.N.L., Pirozzi, F., Esposito, G. (2015). Biomass enrichment and scale-up implications for dark fermentation hydrogen production with mixed cultures. Chem. Eng. Trans. 43, 2015, 391-396. The section 3.2 has been published as Ghimire, A., Sposito, F., Frunzo, L., Pirozzi, F., Escudie, R., Trably, E., Lens, P.N.L., Esposito, G. (2015). Effects of operational parameters on dark fermentative hydrogen production from biodegradable complex waste biomass. Waste management (In press)

Effect of inocula on H2 yield

The application of two different inoculum types for the DF of OMWW showed differences in response of ADS and WAS in terms of dark fermentative conversion to H2

and other metabolites (Figure 3.6 and Table 3.9). Comparatively, WAS exhibited better performances in terms of H2 production as shown by the H2 production yields and kinetics in Table 6. The difference in H2 yields might be a result of the effect of polyphenolic substances present in OMWW (total phenols in Table 3.4) on the fermentative communities present in ADS and WAS [START_REF] Hamdi | Toxicity and biodegradability of olive mill wastewaters in batch anaerobic digestion[END_REF]Ntaikou et al., 2009). Ntaikou et al. (2009) used diluted OMWW to avoid growth inhibition, whereas, [START_REF] Hamdi | Toxicity and biodegradability of olive mill wastewaters in batch anaerobic digestion[END_REF] observed an inhibition mainly on methanogens. Nonetheless, the difference in response of the two inocula could be also due to the difference in heat shock treatment time applied during the HST. ADS required a longer HST time to inhibit the activity of methanogens [START_REF] Ghimire | Biomass Enrichment and Scale-up Implications for Dark Fermentation Hydrogen Production with Mixed Cultures[END_REF] compared to WAS which has an aerobic origin. Therefore, the treatment time could have impacted the microbial communities that could contribute to fermentative H2 production.

The use of WAS as better inoculum is supported by the studies of Chen et al. (2012) and Kim et al. (2011). Chen et al. (2012) achieved higher H2 yields with a sludge originated from a municipal wastewater treatment plant when compared with other inoculum sources like cow dung, compost and paper mill sludge. The group attributed higher H2 yields to the presence of a potential hydrolytic and fermentative bacterial microbial community. Kim et al. (2011) hypothesized that such increase in H2 yields from sewage sludge addition was due to the presence of iron (Fe), calcium (Ca) and phosphorous (P) at much higher concentrations (no information on speciation was given). Further research on the nutrient and trace metal content in inocula and how these affect the DF rates is thus required.

The selection and application of various optimum operational parameters depends highly on the type of substrate, i.e. mainly its biodegradability. However, the improvement of dark fermentative H2 production should bear the cost of application of different optimal operational parameters in terms of net energy and economy gain. It should be taken into consideration that DF of waste biomass is not a complete conversion of organic waste, i.e. organic acids and alcohols accumulate in the effluent, for which a subsequent treatment needs to be provided. Valorization of these by-products can support the costs CHAPTER 4

LONG-TERM OPERATION OF HYDROGEN-PRODUCING CONTINUOUS

REACTORS

A part of section 4.1 of this chapter has been published as Ghimire, A., Valentino, S., Frunzo, L., Trably, E., Escudié, R., Pirozzi, F., Lens, P.N.L., Esposito, G. (2015).

Biohydrogen production from food waste by coupling semi-continuous darkphotofermentation and residue post-treatment to anaerobic digestion: A synergy for energy recovery. International Journal of Hydrogen Energy,40 (46): 16045-16055.

The section 4.2 of this chapter will be submitted as Ghimire, A., Frunzo, L., Pirozzi, F., Lens, P.N.L., Esposito, G., Continuous biohydrogen production from thermophilic dark fermentation of cheese whey using: use of buffalo manure for pH control and biohydrogen production stability.

Experimental setup and operational conditions

An anaerobic digested sludge was collected from an anaerobic digestion plant of the farm "La Perla del Mediterraneo" (Campania, Italy). The sludge was used as start-up seed inoculum, after a thermal pretreatment at 105 ⁰C for 4 hours to enrich the spore forming clostridium and inhibit the methanogens. The total solids (TS) and volatile solids (VS), ammonia content of the inoculum were 29.54±0.22 gTS/L, 18.36±0.14 gVS/L and 283.47±10.8 NH + 4/L respectively. The pH of the inoculum was 8.3±0.1 and the total alkalinity was 1437.20±14.27 mg CaCO3/L.

A semi-continuous stirred 2 L serum bottle (Simax, Czech Republic) with 1500 ml working volume and remaining headspace was setup in the laboratory (Figure 4.1). The reactor was fed with food waste and the effluent was manually extracted on a daily basis.

The varying operational conditions investigated during the experimental period are presented in the Table 4.2. Effluent and biogas samples from the reactor were daily analyzed for determining the metabolic intermediates, i.e. VFAs, and the gas composition (H2 and CO2). The total volume of gas was measured by volumetric water displacement.

The biogas was passed through acidic water (1.5 % HCl) and the volume of water displaced measured the volume of total biogas. Considering this volume and the gas composition analyzed, the volume of H2 produced was calculated. The effect of maintaining the culture at pH 5.5 exhibited only insignificant increase in HY, nevertheless, H2 production was stable during the experimental period (period IV,

shown by the shaded region in Figure 4.2 b), also shown by HPSI of 0.86. During the period, the percentage of H2 in the gas averaged between 59.4 ± 6% while CO2 averaged 39.1 ± 6%. The H2 production performances during experimental period V were nearly comparable to period IV. Furthermore, when the OLR was changed to 2.5 g VS/L/d in period VI, the HPSI decreased to 0.63, evident by the unstable HPR ( et al. (2012). Thus, this fraction of DF residues demands further treatment through anaerobic digestion. The H2 productions during the period V was compared with the previous studies conducted with similar feedstock (Table 4.4), showed that the results obtained from this study is comparable. Therefore, even at the low OLR 2 -2.5, the HY is comparable to that of the past studies. Additionally, the optimal operating conditions of OLR (2 -2.5 g VS/L/d) and HRT (4 -6 d), gives this DF system a potential flexibility to integrate with anaerobic digestion with two stage conversion to H2 and CH4 respectively (Aslanzadeh et al., 2014).

CHAPTER 5 INTEGRATION OF DARK FERMENTATION IN A BIOREFINERY CONCEPT

A part of section 5.1 of this chapter has been has been published as Ghimire, A., Valentino, S., Frunzo, L., Trably, E., Escudié, R., Pirozzi, F., Lens, P.N.L., Esposito, G. (2015). Biohydrogen production from food waste by coupling semi-continuous darkphotofermentation and residue post-treatment to anaerobic digestion: A synergy for energy recovery. International Journal of Hydrogen Energy, 40( 46) 16045-1605.

A section 5.2 of this chapter has been published as Ghimire, A., Valentino, S., Frunzo, L., Pirozzi, F., Lens, P.N.L., Esposito, G. (2016). Valorization of by-products from thermophilic dark fermentation of food waste via concomitant production of biohydrogen and polyhydroxybutyrate using adapted Rhodobacter sphaeroides and mixed photofermentative culture. Bioresource Technology (In press).

A section 5.3 of this chapter will be submitted as as Ghimire, A., Frunzo, L., Pirozzi, F., Trably, E., Lens, P.N.L., Esposito, G., Cazier, E.A., Escudie, R., Solid State Dark Fermentation for waste biomass valorization by production of biohydrogen and platform molecules: Effect of total solids contents and hydrogen partial pressure on substrate conversion.

Analytical methods

Hydrogen was quantified with a Varian Star 3400 gas chromatograph equipped with a ShinCarbon ST 80/100 column and a thermal conductivity detector. Argon was used as the carrier gas with a front and rear end pressure of 20 psi. The duration of analysis was 14 minutes. The fermentation products (lactic, acetic, propionic and butyric acids) were quantified by High Pressure Liquid Chromatography (HPLC) (Dionex LC 25

Chromatography Oven) equipped with a Synergi 4u Hydro RP 80A (size 250×4.60mm) column and UV detector (Dionex AD25 Absorbance Detector). The gradient elution consisted of 20% methanol and 10% acetonitrile in 5 mM H2SO4 pumped at a rate of 0.9 mL/min, using a Dionex GP 50 Gradient pump. The elution time was 18.5 minutes.

Ethanol was quantified by HPLC (Aminex HPX-87H column (300 mm on 7,8 mm, Biorad) using 5 mM H2SO4 as an eluent. The COD of the FW was measured as described elsewhere (Noguerol-Arias et al., 2012). The carbohydrate content was determined according to the Dubois method (DuBois, M., Gilles, K., Hamilton, J., Rebers, P., & Smith, 1956). Total lipids were measured following a Bligh and Dyer chloroform/methanol total lipid extraction method (Bligh and Dyer, 1959). The light intensity was measured with a light meter (Lutron-LX-107). The TS and VS of the seed sludge and TKN were determined according to the Standard Methods [START_REF] Apha | Standard Methods for the Examination of Water and Wastewater[END_REF].

Data analysis

Hydrogen production rates (HPR) were expressed in L H2/m 3 /d while the H2 yields (HY)

were determined considering the total daily organic load fed to the reactor and expressed as L H2 /kg VS added. Average and deviations for daily production were determined during the steady state reached after 3-4 days of operation. The H2 Production Stability Index (HPSI) was evaluated by considering the ratio of standard deviation and average HPR as reported by Tenca et al. (Tenca et al., 2011):

(5.1)

A HPSI index closer to 1 represents a stable hydrogen production.

FactoMineR, an extension on R software, was used for multivariate analysis of the metabolite distribution from the different experimental periods in relation to the hydrogen yields and co-relation circles of the major metabolites were generated.

HPSI =1 S.D.(HPR) Avg.HPR

Under anaerobic conditions, purple non-sulfur bacteria (PNSB) carry out an anaerobic photosynthesis using light as the energy source synthesizing bio-H2. In PNSB, this takes place with reduced carbon sources such as organic acids by the nitrogenase enzyme in the presence of light (Barbosa et al., 2001). Photofermentative bio-H2 production systems are attractive owing to their higher substrate to H2 conversion potential compared to dark fermentative systems (Han et al., 2012).

Moreover, a theoretical H2 potential of 12 moles of H2 per mole of hexose could be realized by integrating a PF process with DF systems (Han et al., 2012). Thus, the integration of DF-PF can provide a practical solution to H2 production along with the enhanced conversion of organic biomass. The integrated DF-PF process has been demonstrated by several studies (Rai et al. 2014;[START_REF] Tawfik | Use of mixed culture bacteria for photofermentive hydrogen of dark fermentation effluent[END_REF]Yang et al. 2015).

DF has the unique capability to utilize a wide range of complex waste biomass that can ensure the future supply of feestock, and combining the two processes (DF + PF) can provide the further conversion of organic substrate in addition to enhanced H2 yields.

The majority of the past studies carried out on combined DF-PF processes for H2

production have used synthetic pure substrates containing major VFAs and pure microbial cultures (Chen et al., 2010;[START_REF] Tao | High hydrogen yield from a twostep process of dark-and photo-fermentation of sucrose[END_REF]. However, low cost complex waste biomass such as agricultural residues, organic fraction of municipal solid waste (OFMSW) and industrial wastes are attractive substrates for economically sustainable scaled up applications of dark fermentative. A number of studies have recently shown the possibility of combined DF-PF processes using waste biomass as the substrate (Chookaew et al., 2015;Rai et al., 2014;[START_REF] Zong | Efficient hydrogen gas production from cassava and food waste by a two-step process of dark fermentation and photo-fermentation[END_REF]. In this scenario, the use of dark fermentation effluents (DFE) generated from DF of complex organic waste and the application of PNSB for its capability to produce H2 from DFE is attractive.

In addition to H2 production, PNSB can synthesize poly-β-hydroxybutyrate (PHB) under certain conditions of physiological stress, such as high Carbon/Nitrogen (C/N) ratio or sulfur deprivation (Eroglu and Melis, 2011;Waligórska et al., 2009). Similar to H2 production, PNSB synthesizes PHB as a way to dissipate the excess reducing power (Waligórska et al., 2009). PHB is a polyhydroxyalkanoate, an interesting biodegradable polymer having applications in bioplastics production and medicine [START_REF] Kemavongse | Poly-betahydroxyalkanoate production by halotolerant Rhodobacter sphaeroides U7[END_REF]. The amount of PHB accumulation depends on the PNSB strains and the process minutes to avoid the growth of opportune microorganisms. The inoculum was added after cooling while culture mediums for the mixed PNSB was not sterilized. Each photofermentative test was inoculated with 10 mL (1.5 g TSS/L, 2.5 % of working reactor volume) culture.

Preparation of PF medium

For each experimental test, the DFE was collected from the DF reactor during the 160-180 days operation period of the DF reactor described above, after settling for 30 minutes.

The supernatant was collected after centrifuging at 4500 rpm for 20 minutes and had a pH of 4.5 (± 0.2). The DFE was supplemented with KH2PO4, 3g/L; NaHCO3, 0.7 g/L; ferric citrate 24.5 mg/L and 10 mL of the trace metals solution. The DFE was supplemented with the above mentioned trace metals to provide all the necessary trace elements for the PF process (Bianchi et al., 2010;Montiel-Corona et al., 2015). Phosphate buffer (KH2PO4) was added to maintain the optimum pH around 6.5 -6.8. Moreover, sodium bicarbonate (NaHCO3) as it can act as an electron acceptor and can aid during the

Solid State Dark Fermentation for production of H2 and organic acids

This section presents the results of the investigations carried to evaluate the potential of Solid State Dark Fermentation for the production of biohydrogen and organic acids. The main aim is the assessment of the respective effects of total solids content and H2 partial pressure on substrate conversion, using food waste and wheat straw as model substrates.

Introduction

Dark fermentation (DF) is emerging as a potential biological pathway for production of hydrogen and useful by-products utilizing organic biomass (Liu et al., 2013;Azwar et al., 2014;Ghimire et al., 2015;Wang et al., 2015). Low cost renewable waste biomasses such as agricultural residues, organic fraction of municipal waste (OFMSW) and agroindustrial wastes might give competitive economic advantage for the future supply of sustainable feedstock which may be used industrially for DF systems with biological treatment of waste as an added benefit (De Gioannis et al., 2010;Ntaikou et al., 2010;Urbaniec & Bakker, 2015). OFMSW and lignocellulosic residues such a wheat and rice straws could be potential substrate sources for this purpose as their future supply is abundant and they do not compete with the food crops like the substrates used for first generation biofuels. Food waste has high volatile solids (VS) (21 to 27% VS) content and can be valorized by the concomitant production of biohydrogen and platform molecules as organic acids and alcohols (VALORGAS, 2010;[START_REF] Uçkun Kıran | Platform chemical production from food wastes using a biorefinery concept[END_REF]Wang et al., 2015). DF of food waste has several benefits, along with the production of H2 as clean energy carrier, volatile acids and alcohols as by-products, which can have wider applications. The soluble by-products of DF can be applied in i) wastewater treatment (Elefsiniotis et al., 2004), (ii) production of platform molecules such as biopolymers (Ntaikou et al., 2009), (iii) microalgal lipids production (Turon et al., 2015), (iv) H2

production by photo fermentation, (v) feed for microbial electrolysis cells for production of H2 and other value added chemicals [START_REF] Elmekawy | Bioelectro-catalytic valorization of dark fermentation effluents by acetate oxidizing bacteria in bioelectrochemical system (BES)[END_REF] and (vi) anaerobic digestion for energy recovery in the form of H2 and CH4 (Ghimire et al., 2015).

Recently, Motte et al. (2015) have propose to combine dry DF and mechanical pretreatment process as a measure to reduce the energy demands and effluents generation.

This configuration also enhances the overall substrate conversion, which makes it more plausible for lignocellulosic biomass to be applied in a biorefinery concept. Therefore, CHAPTER 6

DISCUSSION AND FUTURE PERSPECTIVE benefits from higher process yields and low energy and water requirements are attractive for biorefinery applications.

This study undertakes the aims to investigate the potential of DF of various complex waste biomasses for enhanced H2 production. The results presented in Chapter 3 elucidate the effects of various operating parameters in dark fermentative H2 production from a range of different waste biomass. The investigation on long-term operational feasibility of DF process for continuous H2 production and application of co-substrates to support stability in H2 production are demonstrated in Chapter 4. Moreover, a biorefinery concept is introduced in Chapters 5 by utilizing dark fermentation effluents in PF and AD for production of energy and biopolymers (PHB) and to investigate the limitations in the application of SSDF. The present chapter 6 summarizes and discusses the future implications of the major research findings in the application of DF processes for production of H2 and other valued by-products by using mixed culture and complex waste biomass as feedstock. Moreover, the significance of integration with other biological systems for valorization of DF by-products is discussed in a biorefinery framework.

Major findings and highlights

Effect of operational parameters on dark fermentative H2 yields

A number of studies have investigated the optimal operational conditions (e.g. culture pH, temperature, substrate utilization and inoculum enrichment) for maximizing H2 production in DF (Cappai et al., 2014;De Gioannis et al., 2013;Ghimire et al., 2015a;Luo et al., 2010;Wang and Wan, 2011;Wong et al., 2014). However, selection of optimal operating parameters depends higly on substrate type. Therefore, investigations becomes vital in order to establish optimal operating conditions in the dark fermentative H2 production from a particular feedstock type. Moreover, this study recommends that the biodegrability of the feedstock strongly influences the selection and application of various operating conditions (Chapter 3).

In a DF by mixed culture, the presence of H2 producing microbial communities is important to achieve higher H2 yields (Wong et al., 2014). In a scaled-up DF system utilizing waste biomass, mixed cultures are comparatively easier and less expensive to handle compared to pure cultures, as they do not require any asepsis procedure (Hawkes et al., 2007). This study evaluated the different H2 producing inoculum preparation Similarly, in the DF of OMWW, the H2 yield was doubled when heat-shock pre-treated activated sludge was used as inoculum in comparison to anaerobic digestate. The anaerobic digestate took longer time to adapt to OMWW, which could be due to phenolic compounds present in it (Figure 3.6). This study recommends that the selection and application of different operating parameters to maximize the H2 yields depends strongly on the biodegradability of the substrates (Table 3.10). These results have implications in the design of high rate DF reactors using complex waste biomass as substrate.

Continuous biohydrogen production

Continuous or semi-continuous processes are generally preferred for continuous H2 production as they are more viable for scaling-up. Therefore, the future development of DF process at industrial scale relies on the successful operation of continuous processes, that can offer the advantages of steady operation compared to batch processes which involves regular downtime periods of maintenance (Hawkes et al., 2007). This study established a semi-continuous thermophilic DF process for H2 production at low organic loading rates without controlling the culture pH. The continuous DF processes are not stable due to the decrease in culture pH as a result of the production of organic acids.

Therefore, they require sources of alkalinity to maintain the culture pH at non-inhibiting acidogenic pH range (4.5 -6). Most of the studies conducted on continuous and/ or semicontinuous dark fermentative H2 production relied on addition of chemical buffering agents such as K2HPO4, NaHCO3, Na2HPO4 (Carrillo-Reyes et al., 2012;Elsamadony and Tawfik, 2015;Valdez-vazquez et al., 2005). The high amount of chemical buffering agents might increase the operational cost of DF bioreactors at scaled-up production. It further adds uncertainty in the downstream processes applied for the treatment of DF effluent (DFE). This study has demonstrated a long-term feasibility of continuous H2 production at varying operational conditions of the DF reactor (Section 5.1, Table 5.3).

The optimal operational OLR equivalent to 2.5 g VS/L/d and HRT of 4 days have been established in a DF of food waste. These ranges of OLR and HRT values can be applied to the first stage of a two-stage AD process for the production of H2 and CH4, repectively (Aslanzadeh et al., 2014).

In another study presented in section 4.2, H2 production stability was investigated in the DF of cheesewhey with buffalo manure as co-substrates. The results showed that buffalo manure charaterised by higher alkalinity could be used to maintain a culture pH at a range PF systems. In addition, the integration provided the biological treatment of residues. This was supported in studies from Xia et al. (2013aXia et al. ( , 2013b)), who reported that a three-step conversion of algal biomass combining DF-PF-AD can achieve 1.7 and 1.3 times higher energy yields in comparison to a two-stage DF-AD and a one stage AD process, respectively.

Moreover, purple non-sulfur bacteria (PNSB) can concomitantly synthesize H2 and polyhydroxybutyrate (PHB) in PF process under certain conditions of physiological stress such as high Carbon/Nitrogen (C/N) ratio and sulfur deprivation (Waligórska et al., 2009;Eroglu and Melis, 2011). The capability of PNSB to utilize DFE generated from DF of complex organic waste for conversion to H2 and PHB could be of economical interest (Figure 5.8). The PF of DFE by enriched mixed culture of PNSB and adapted culture of Rhodobacter sphaeroides provided several benefits, e.g. treatment of effluent by COD reduction and recovery of H2 and PHB as added value products. Thus, integration of DF into a biorefinery concept can provide the economic sustainability to the scaled-up DF processes.

Furthermore, the last section of Chapter 5 dealt with the limitations of dry fermentation processes. SSDF processes are generally operated at total solids (TS) content higher than 15% and are constrained due to mass transfers limitations (Abbassi-Guendouz et al., 2012;Motte et al., 2013). Therefore, the TS content in SSDF could impact in conversion of feed to the desired fermentative products. In addition, accumulation of H2 could also limit the conversion of substrates, as revealed in a study by Cazier et al. (2015) during Solid State Anaerobic Digestion (SS-AD). The affect of TS content and accumulation of H2 on substrate conversion during SSDF was investigated with food waste and wheat straw as representative model substrates for readily and slowly degradable substrates.

During the SSDF of food waste, H2 production was inhibited at a TS content higher than 15%, resulting in a lactic acid accumulation (Figure 5.9). This suggests that the TS content plays a vital role and for the case of SSDF of food waste, TS content has to be less than 15% if the process is aimed at H2 production. Moreover, the accumulation of H2 as gaseous products does not exhibit inhibitory effects on hydrolysis of organic biomass during SSDF in contrast to SS-AD (Figures 5.11 and 5.12). This could be due to the operational culture pH of these two different processes. The lower hydrolysis during DF process can be due to the inhibition of hydrolase enzyme at acidic pH (Parawira et al., 2005). However, in general, the hydrolysis of substrate seemed to be limited under DF conditions as found in this study.

Future research prospective

The current study addressed several issues and potential in the application of DF of waste biomass interlinked in a biorefinery concept. However, more research needs to be directed at the pilot to full scale implementation of DF process based on real feedstock such as organic waste biomass [START_REF] Bonk | Converting the organic fraction of solid waste from the city of Abu Dhabi to valuable products via dark fermentation -Economic and energy assessment[END_REF]. Moreover, integration of DF with other physical, chemical and or biological systems can improve energy yields which could lead to reduction of the operational costs associated with DF and create possibility for revenues from the recovery of added value chemicals in side stream process.

Based on the local availability, novel feedstock sources, which do not compete with food and agriculture supply chain, should be utilized in DF process. The agricultural residues (straw, corn stover, manure, waste timber cuttings), municipal, agro-industrial waste and biomass sources such as micro and macro algae could serve as the future supply of feedstock for DF based biorefinery. A major difficulty in the utilization of this feedstock sources is poor biological hydrolysis that limits the complete conversion of biomass into intended products, as the efficient dark fermentative conversion depends on the presence of readily available depolymerized carbohydrates (monomeric sugars) (Guo et al., 2013;Monlau et al., 2012). However, different physical, chemical, biological and the combination of these pre-treatment methods can be applied to enhance biological hydrolysis depending on the physico-chemical characteristics of biomass [START_REF] Carrere | Review of feedstock pretreatment strategies for improved anaerobic digestion: From lab-scale research to full-scale application[END_REF]Monlau et al., 2013). More studies regarding technical and economical feasibilities of pre-treatment methods could elucidate their application to DF process.

The selection of optimum operational parameters, such as culture pH, temperature, substrate concentration, substrate, loading rate, food to microorganism ratio and reactor configuration during DF of waste biomass is strongly dependent on substrate type and source of inoculum (Tables 2.1, 2.5, 2.7 -2.9 and 3.9). Therefore, the selection of these parameters for a particular substrate type and experimental conditions needs investigations before full-scale implementation of the process. Moreover, additional research is required in process control with an aim to enhance the yield and recovery of other targeted metabolic by-products such as acetate, butyrate, propionate, ethanol, etc.

In recent years, DF was extensively reviewed and aimed for H2 production. However, dark fermentative H2 production accounts only 4% of the total products conversion with the maximum theoretical conversion, while 67% remains as by-products in liquid phase, i.e. acetic acid in a DF of glucose (Bastidas-Oyanedel et al., 2015).

Similarly, microbial community analysis during the varying operating conditions could further elucidate the existence of different fermentative communities responsible for yields of varying metabolites. The identification and enrichment of fermentative communities could be a precursor for future application of DF process in the production of targeted biomolecules in a biorefinery concept.

The residues generated from the DF process needs down stream process/es for the complete utilization of waste biomass. Biopolymer production in PF utilizing DFE can be economically interesting and requires investigations in process optimization for PHB production (Section 5.2). Moreover, investigations in the technologies for the economic recovery of H2 (gas cleaning and purification) and associated biochemicals (VFAs and alcohols) from DF fermentation will determine the future application and development of the DF process [START_REF] Bonk | Converting the organic fraction of solid waste from the city of Abu Dhabi to valuable products via dark fermentation -Economic and energy assessment[END_REF].

Compared to wet fermentation processes which require large reactor volume and have consequently higher energy requirements to treat same quantity of biomass, SSDF can offer benefits in terms of higher volumetric production rates due to higher substrate concentration (Elsamadony and Tawfik, 2015;[START_REF] Romero Aguilar | Effect of HRT on hydrogen production and organic matter solubilization in acidogenic anaerobic digestion of OFMSW[END_REF]. This research work investigated the technical limitations of SSDF (Section 5.3, Chapter 5).

However, additional research is essential in the operational control of the SSDF process towards intended metabolites production in SSDF, as this technology could hold greater promise in the creation of future biorefinery for the production of biohydrogen and biomolecules.

Xia, A., Cheng, J., Lin, R., Lu, H., Zhou, J., & Cen, K. (2013). Comparison in dark hydrogen fermentation followed by photo hydrogen fermentation and methanogenesis between protein and carbohydrate compositions in Nannochloropsis oceanica biomass. Bioresource Technology,138,