Simon Poirier
email: simon.poirier@epfl.ch

Jean-Philippe Steyer
email: jean-philippe.steyer@inra.fr

Nicolas Bernet
email: nicolas.bernet@inra.fr

Eric Trably
email: eric.trably@inra.fr

C Acetobutylicum

Mitigating the variability of hydrogen production in mixed culture through bioaugmentation with exogenous pure strains

Keywords:

Process stability is a key operational issue when operating dark fermentation with mixed microbial cultures for hydrogen production. This study aimed at mitigating the instability of hydrogen production by separately adding exogenous pure strains suspected to have key roles in fermentative cultures. Among them, Clostridium acetobutylicum, Clostridium pasteurianum and Lactobacillus bulgaris which became predominant within the mixed culture strongly reduced the spectrum of produced metabolites and H2 production variability.

Interestingly, Escherichia coli and Cupriavidus necator, which remained in minor abundance, maintained a high and stable H2 production while lowering the metabolic variability. 16S rRNA revealed that this could correlate to a simplification of the microbial diversity and the non-emergence of spore-forming competitors such as Sporolactobacillus sp. These results illustrate the potential beneficial role of minor OTUs as keystone species on H2-producing complex ecosystem and support the possibility of using them to engineer the ecosystem and maintain high and stable performances.

Introduction

Due to the current context of global warming as well as the growing scarcity of nonrenewable fossil fuels, more research efforts focusing on sustainable energy production are required. Among the possibilities for alternative energy development, hydrogen (H2) has attracted considerable attention over the past decade due to its high energetic yield (267 kJ.mol -1 ). Nowadays, H2 is mainly generated through energetically expensive thermochemical processes, such as natural gas reforming and water electrolysis. Therefore, development of clean, sustainable, and cost-competitive H2 production processes is of major concern [START_REF] Hosseini | Hydrogen production from renewable and sustainable energy resources: Promising green energy carrier for clean development[END_REF].

Encouraging alternatives to produce environmentally friendly H2 are driven by microbial processes that present the lowest environmental impacts [START_REF] Dincer | Review and evaluation of hydrogen production methods for better sustainability[END_REF].

Among the biological options, dark fermentation appears to be the least restrictive technology compared to biophotolysis and photofermentation since it can be operated as an intensive process and without any external supply of light [START_REF] Ghimire | A review on dark fermentative biohydrogen production from organic biomass: Process parameters and use of by-products[END_REF]. Additionally, strict anaerobic or facultative anaerobic bacteria involved in dark fermentation provide higher productivities than photo-fermentative bacteria [START_REF] Ghimire | A review on dark fermentative biohydrogen production from organic biomass: Process parameters and use of by-products[END_REF][START_REF] Manish | Comparison of biohydrogen production processes[END_REF]. Converting organic biomass in dark fermenters into H2 is basically a truncated anaerobic digestion process. Indeed, H2 is a key metabolic intermediate produced mainly during acidogenesis and is then immediately consumed to reduce by-products into mainly methane or acetate. A combination of suitable operational parameters such as moderately low pH and short solids retention time favor H2 production and suppress the principal consumers: the methanogens. Moreover, the ability of dark fermentation to generate H2 from a variety of renewable non-sterile and inexpensive feedstock's such as organic waste makes it suitable for P a g e 6 | 32 the demand of a sustainable development strategy [START_REF] Hosseini | Hydrogen production from renewable and sustainable energy resources: Promising green energy carrier for clean development[END_REF][START_REF] Łukajtis | Hydrogen production from biomass using dark fermentation[END_REF][START_REF] Wong | A review of sustainable hydrogen production using seed sludge via dark fermentation[END_REF].

Various pure cultures of fermentative bacteria having the ability to produce H2 from simple substrates have been identified [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: useful insights for driving the ecosystem function[END_REF][START_REF] Lee | Dark fermentation on biohydrogen production: Pure culture[END_REF]. As the most dominant H2-producing species found in microbial ecosystems, Clostridium sp. and Enterobacter sp. were the most widely hydrogen-producing bacteria (HPB) used as inoculum [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: useful insights for driving the ecosystem function[END_REF][START_REF] Mishra | Outlook of fermentative hydrogen production techniques: An overview of dark, photo and integrated dark-photo fermentative approach to biomass[END_REF]. Even though pure bacterial strains often exhibit better yields than mixed natural communities, bioprocess operation requires sterile conditions and well controlled conditions that make their development at industrial scale too costly.

Moreover, using pure cultures for producing H2 restricts feedstock's possibilities and does not take advantage of the wide metabolic possibilities inherent to the use of complex ecosystems.

Thereby, mixed cultures-based bioprocesses appear as a more suitable solution for H2 production from complex organic wastes. In theory, their robustness allows them to degrade a wide range of substrates, due to their higher resistance and resilience to environmental changes [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: useful insights for driving the ecosystem function[END_REF][START_REF] Chandrasekhar | Biohydrogen Production: Strategies to Improve Process Efficiency through Microbial Routes[END_REF].

However, complex microbial ecosystems present numerous interactions between the different communities involved in fermentation. Illustratively, and contrary to the competitive exclusion principle stating that microbial diversity could not be maintained in a chemostat at steady-state, it is regularly observed that low abundant bacterial species are always present.

Their roles remain unclear but many recent studies tend to consider them as keystone species [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: useful insights for driving the ecosystem function[END_REF]Rafrafi et al., 2013a). Despite their low abundance, they are supposed to significantly participate to ecosystem functioning through various interactions such as trophic competition. Ubiquitous microorganisms such as lactic acid bacteria (LAB) reduce H2 production yields due to substrate competition (replacement of H2 fermentation by lactic acid P a g e 7 | 32 fermentation) and to the excretion of bacteriocins [START_REF] Gomes | Bacteriocins of lactic acid bacteria as a hindering factor for biohydrogen production from cassava flour wastewater in a continuous multiple tube reactor[END_REF]. Homoacetogenic bacteria which grow on H2 and CO2 can also alter the performance of H2 bioreactors. Besides, a larger range of interactions can occur between microorganisms. Direct exchanges of molecules or electrons via cytoplasmic, pilis or nanotubes connections, with or without conductive materials have also been recently investigated in anaerobic environments (also called DIET for Direct Interspecies Electron Transfer [START_REF] Baek | Role and Potential of Direct Interspecies Electron Transfer in Anaerobic Digestion[END_REF]). Cellular communication between microorganisms called "quorum sensing" is also attracting more and more attention in fermentation [START_REF] Nguyen | Quorum sensing between Gram-negative bacteria responsible for methane production in a complex waste sewage sludge consortium[END_REF]. Whatever the type of interaction, fermentation patterns could be impacted thus redirecting the fermentation pathways towards H2 production [START_REF] Benomar | Nutritional stress induces exchange of cell material and energetic coupling between bacterial species[END_REF]. Hence, whether physical or chemical, intra or extracellular, all these interactions are evidences of a complex bacterial communication network in which all species, and even the least abundant, would be able to drive the functioning of the whole ecosystem.

Since structure and interactions within microbial communities are crucial for the efficiency of bioprocesses, many studies aimed at better managing complex ecosystems by inoculating exogenous functional microbial consortia or specific strains to mixed culture. This strategy, called bioaugmentation, has been used for several purposes such as withstanding bioprocess fluctuations, accelerating substrate degradation, restoring reactor performance or relieving toxicant inhibition [START_REF] Bartrolí | Bioaugmentation as a tool for improving the start-up and stability of a pilot-scale partial nitrification biofilm airlift reactor[END_REF][START_REF] Bouchez | Ecological study of a bioaugmentation failure[END_REF][START_REF] Fotidis | Bioaugmentation as a Solution To Increase Methane Production from an Ammonia-Rich Substrate[END_REF][START_REF] Herrero | Bioaugmentation and its application in wastewater treatment: A review[END_REF][START_REF] Jianlong | Bioaugmentation as a tool to enhance the removal of refractory compound in coke plant wastewater[END_REF][START_REF] Kotay | Novel dark fermentation involving bioaugmentation with constructed bacterial consortium for enhanced biohydrogen production from pretreated sewage sludge[END_REF][START_REF] Yang | Various additives for improving dark fermentative hydrogen production: A review[END_REF].

Bioaugmentation has notably been performed by several authors to enhance the hydrogen production as reviewed by [START_REF] Yang | Various additives for improving dark fermentative hydrogen production: A review[END_REF]. Interestingly, [START_REF] Goud | Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load[END_REF] demonstrated that bioaugmentation with three various acidogenic bacteria (Bacillus P a g e 8 | 32 subtilis, Pseudomonas stutzeri and Lysinibacillus fusiformis) can enhance hydrogen productivity from food waste despite overloaded substrate condition [START_REF] Goud | Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load[END_REF]. Moreover, [START_REF] Kumar | Enhanced biohydrogen production from beverage industrial wastewater using external nitrogen sources and bioaugmentation with facultative anaerobic strains[END_REF] showed that bioaugmentation with facultative anaerobes such as Escherichia coli XL1-Blue and Enterobacter cloacae DSM 16657 can improve hydrogen yield of a sewage sludge inoculum by creating strict anaerobic conditions for Clostridium species [START_REF] Kumar | Enhanced biohydrogen production from beverage industrial wastewater using external nitrogen sources and bioaugmentation with facultative anaerobic strains[END_REF]. The potentialities of bioaugmentation with hydrogen producing facultative anaerobes was also pointed by [START_REF] Marone | Hydrogen production from vegetable waste by bioaugmentation of indigenous fermentative communities[END_REF]. They reported that the addition of Rahnella sp. 10, Raoultella sp. 47 and Butiauxiella sp. 4 increased hydrogen production rate and yield compared to the indigenous ecosystem during the fermentation of vegetable food waste [START_REF] Marone | Hydrogen production from vegetable waste by bioaugmentation of indigenous fermentative communities[END_REF]. They also suggested synergistic interactions induced by the triple co-culture of these strains. Benomar et al., (2013) demonstrated that physical interactions, including an exchange of molecules such as proteins, between in a coculture of two anaerobic partners considered as competitors (Clostridium acetobutylicum and Desulfovibrio vulgaris Hildenborough) allowed a substantial increase in hydrogen production without requiring genetic engineering. Similarly, [START_REF] Laocharoen | Bioaugmentation of Lactobacillus delbrueckii ssp. bulgaricus TISTR 895 to enhance bio-hydrogen production of Rhodobacter sphaeroides KKU-PS5[END_REF] also indicated that the addition of a lactic acid-producing strain (Lactobacillus delbrueckii ssp. bulgaricus TISTR 895) could avoid lactic acid accumulation in the fermentation system and enhance hydrogen production by Rhodobacter sphaeroides KKU-PS5 [START_REF] Laocharoen | Bioaugmentation of Lactobacillus delbrueckii ssp. bulgaricus TISTR 895 to enhance bio-hydrogen production of Rhodobacter sphaeroides KKU-PS5[END_REF]. The importance of these non-hydrogen producing bacteria, often sub-dominant in bioreactors has also been highlighted by Rafrafi et al., (2013) (Rafrafi et al., 2013b).

In this framework, the aim of this study was to investigate the impact of the addition of seven exogenous strains, reported as keystone species, on H2 production performances and on the microbial ecosystem balance of a complex community. Based on the literature and on previous work, seven (Clostridium acetobutylicum, Clostridium pasteurianum, Escherichia P a g e 9 | 32 coli, Lactobacillus bulgaris, Enterococcus casseliflavus, Desulfovibrio vulgaris and Cupriavidus necator) were selected for their potential interaction with H2 producers.

Microbial dynamics were monitored with 16S rRNA gene sequencing. Inoculum originating from an industrial methanogenic reactor was tested. P a g e 10 | 32 2 Materials and methods

Experimental set-up

Experiments were carried out in 600 mL batch flasks with a working volume of 200 mL. Growth medium composition is detailed in Rafrafi et al. (2013). Main components were 2g/L of yeast extract, 10 g/L of glucose, 7.4 g/L MES Buffer, oligo-element solution 40 mL/L, H2O q.s.p 1 L. pH was initially adjusted to 6.0 using NaOH (5N). Bioreactors were sealed with a screw cap and a rubber septum and headspaces were flushed with N2 (purity > 99.99%, Linde gas SA) during 10 min in order to ensure strict anaerobic conditions. They were then autoclaved (121°C, 20 min) and stored at 4°C prior to inoculation.

An anaerobic inoculum was sampled from the outlet of a lab-scale methanogenic reactor fed with wine distillery wastewater (Ornaisons, France). To avoid hydrogen consumption by methanogenic populations, a heat-shock treatment (90°C, 10 min) was performed on this anaerobic sludge. It was then incubated at 35°C in a 1000 mL flask. A set of 24 anaerobic batch flasks were initially seeded with 10 mL of this anaerobic inoculum with a syringe through the rubber septum. One pure strain was added to each bioreactor. A set of seven pure strains were used for bioaugmentation assays on the basis of previous works, ie. Clostridium acetobutylicum, Clostridium pasteurianum, Escherichia coli, Lactobacillus bulgaris, Enterococcus casseliflavus, Desulfovibrio vulgaris and Cupriavidus necator [START_REF] Benomar | Nutritional stress induces exchange of cell material and energetic coupling between bacterial species[END_REF]Rafrafi et al., 2013). A total of 10 mL of each pure strain culture, corresponding to 10 7 CFU and 0.1% of the total biomass of the mixed culture, was sampled during their respective exponential growth phase and added to the bioreactor using a syringe through the rubber septum. Control flasks were supplemented with sterile water. Three replicates were carried out for each condition.

P a g e 11 | 32

Time zero (T0) samples were taken and all reactors were incubated at 37°C. Agitation was manually performed before and after each sampling campaign. Liquid samples (2 mL) were periodically taken through the septum and centrifuged at 10,000 g for 10 min. The pellets and supernatant obtained were stored separately at -20°C for analysis of biomass and chemical indicators respectively. The incubations were run up to 120 hours at 35 °C until all daily biogas productions decreased below 1 mL/h.

Analytical methods

Biogas production and composition (H2, CO2, O2, CH4 and N2 content) were analyzed using a gas chromatograph R3000 (SRA Instruments) as detailed in [START_REF] Chatellard | The type of carbohydrates specifically selects microbial community structures and fermentation patterns[END_REF].

The volume of produced biogas was normalized according to the ambient temperature and pressure.

To determine H2 production parameters, the cumulative H2 production (H (t)) data was fitted to a modified Gompertz equation (Eq. ( 1)):

( ) = × - × × ( -) + 1 (Eq.1)
where P is the maximum cumulative H2 production (mLH2/L), Rmax is the maximum H2 production rate (mLH2/L/day), λ is the lag-phase time (day), t is the incubation time (day) and e is exp (1). The cumulative H2 production was expressed in mL per L of culture taking into account the variations in volume due to gas and liquid sampling. The values of P, Rmax and λ were estimated using a non-linear regression algorithm developed with Matlab software (version 6.5, MathWorks). H2 production yield was calculated by dividing the maximum H2 production (P) by the molar amount of glucose consumed.

P a g e 12 | 32

Fermentation end products (lactate, ethanol) were quantified using high performance liquid chromatography (HPLC) coupled to a refractometer (Waters R410). The chromatograph was equipped with an HPX 87 column (Biorad) and the eluent corresponded to a H2SO4 solution (0.222 ml/L) under isocratic elution at 0.4 mL/min. The operating conditions were: temperature of column, 35°C; temperature of refractometer, 40°C.

H2 production dynamics

To accurately assess the H2 production performances, cumulative H2 production curves were fitted to a modified Gompertz equation for each batch experiment. Gompertz model showed a good fit with the experimental H2 production data, with determination coefficients r 2 over 0.99 for all batch tests. H2 production curves obtained with the mixed culture were decomposed to two distinct fitting curves, each corresponding to a different H2 production phase. Gompertz parameters determined for all incubations are presented in Table . 1.

Mixed culture (controls)

Cumulated H2 production dynamics obtained from the different replicates of the mixed culture are detailed in Fig. 1A. Kinetics evidenced a low reproducibility between the three replicates. H2 production started in all bioreactors after a lag time of 6 hours and consisted of 2 phases. The duration of this first production phase was variable between the three replicates.

It lasted respectively from 15 hours in replicates 2 and 3 ending with a plateau phase, up to 45 hours in replicate 1. The plateau phase duration ranged from 25 hours to 50 hours in replicates 3 and 2, respectively. Cumulated H2 production volumes at the end of this first phase varied between 180 mL and 280 mL. For all the replicates, a second H2 production phase followed the plateau whose duration was also variable from one replicate to another. Total H2 production was also not consistent, ranging from 70 mL to 280 mL during this phase.

Consequently, ultimate H2 production yield (Hmax) varied from 271 mL up to 562 mL. The variability observed in the analysis of H2 production kinetics could be explained by an instability of the bacterial communities during both growth phases that directly affected the P a g e 15 | 32 H2 production of the consortium as already described by a recent study [START_REF] Chatellard | The type of carbohydrates specifically selects microbial community structures and fermentation patterns[END_REF].

3.1.2 Pure strains behaviours H2 production was highly reproducible among each triplicate of hydrogen producing strains (Clostridium acetobutylicum, Clostridium pasteurianum and Escherichia coli). Data are respectively presented in presented in Supplementary Fig. S1-S3. H2 production dynamics revealed that Clostridium pasteurianum reached higher maximum production yields (514.4±32.5 mL, 2.02±0.04 molH2/mol glucose) than Clostridium acetobutylicum (463.1±26.6 mL, 1.94±0.14 molH2/mol glucose) and Escherichia coli (54.0±7.7 mL, 0.90±0.12 molH2/mol glucose). These yields are similar to those recovered in many studies [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: useful insights for driving the ecosystem function[END_REF].

Both Clostridia reached comparable H2 yield as those obtained with replicates 2 and 3 of the mixed culture, ie. 471 mL and 562 mL of H2, respectively. Pure bacterial strains such as

Clostridiales are known to give better yields when producing H2 than mixed natural communities suggesting that, despite a low reproducibility, two out of the three replicates of the mixed culture used for this study were able to reach good performances.

Confrontation between mixed culture and pure strains

The mixed culture confrontation with pure strains induced the establishment of new H2 production kinetics. Three tendencies were observed:

Interestingly, adding strains such as Clostridium acetobutylicum (Fig. 1B), Clostridium pasteurianum (Fig. 1C) and Lactobacillus bulgaris (Fig. 1D) generated the disappearance of the first plateau phase. In presence of exogenous Clostridies, the entire substrate was consumed after 60 hours of incubation which implied that the addition of these two pure P a g e 16 | 32 strains increased substrate consumption rate. However, lower H2 production yields were obtained. In presence of Clostridium acetobutylicum, a yield of only 271.1±42.4 mL was reached, which was still comparable to the Hmax obtained with one of the mixed cultures replicate. Clostridium pasteurianum led to higher Hmax values ranging between 330 mL and 370 mL of H2. Addition of Lactobacillus bulgaris within the mixed culture generated a strong reduction in H2 production. Only 15 hours after seeding this strain, H2 productivity reached zero. As a result, the global H2 yield reached only 169.5±18.1 mL. This suggested that Lactobacillus bulgaris addition caused trophic competition between this lactic acid bacteria (LAB) and the native ecosystem, likely favored by a release of bacteriocins in the medium as already reported by several authors [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: useful insights for driving the ecosystem function[END_REF][START_REF] Gomes | Bacteriocins of lactic acid bacteria as a hindering factor for biohydrogen production from cassava flour wastewater in a continuous multiple tube reactor[END_REF][START_REF] Noike | Inhibition of hydrogen fermentation of organic wastes by lactic acid bacteria[END_REF][START_REF] Palomo-Briones | Hydrogen metabolic patterns driven by Clostridium-Streptococcus community shifts in a continuous stirred tank reactor[END_REF]. Considering that H2 generation completely stopped after only 15 hours after this strain addition, this observation well supports the fact that Lactobacillus bulgaris outcompeted efficiently hydrogen-producing bacteria.

Overall, all pure strain bioaugmentations led to a decrease in H2 yields. However, these seeding generated an increase of the reproducibility of the H2 production dynamics when compared to the mixed culture control. It can be assumed that adding these pure strains led to new interactions in the global ecosystem. With regard to the hydrogen yields, it was presumed that trophic interactions could have taken place in the mixed culture leading to an increased competition for the substrate.

In addition, bioaugmentation of Escherichia coli (Fig. 1E) and Cupriavidus necator (Fig. 1F) did not prevent H2 production kinetics from being decomposed into two phases. However, it greatly reduced the variability of H2 production during the second phase. While the average production of this phase remained stable within these three replicates, the standard deviation dropped from 159.3 mL for the mixed culture alone down to 79.9 mL in presence of C.

P a g e 17 | 32

necator and even to 49.5 mL in presence of E. coli. The addition of these two strains may have led to new interactions taking place during the second phase of production and suggesting that they managed to remain active in the global ecosystem.

Finally, E. casseliflavus (Fig. 1G) and D. vulgaris (Fig. 1H) did not have any beneficial influence on the variability within the mixed culture during the second phase of H2 production. It could either indicate that both strains did not grow throughout the incubation or that their growth did not modify the interactions between the key hydrogen producers and consumers.

Metabolites accumulation

Fig. 2 presents the distribution of the soluble metabolites produced concomitantly with H2.

Mixed culture (controls)

Metabolite production kinetics by the mixed culture is presented in Supplementary Fig. Replicate 1 revealed a high lactate accumulation of 8.66 mmol during the second phase that consequently led to lower H2 production. In replicate 2, caproate and ethanol that were not P a g e 18 | 32 detected in the first phase, accumulated at a level of 2.32 mmol and 1.20 mmol respectively.

This result suggests that additional metabolic pathways were used for substrate conversion during the second phase. Interestingly, replicate 3 did not reveal any metabolic shift during the second phase. An increase in acetate (4.6 mmol) and butyrate (8.5 mmol) production was observed confirming the shape of the metabolite distribution present in the first phase. The difference observed in the three replicates indicated that the ecosystem was governed by a high variability in metabolic patterns with subsequent changes in metabolite distribution.

Compared to the other replicates, the metabolites distribution of replicate 3 was consistent between both phases and led to the highest H2 production. Instability found in replicates 1 and 2 could be the consequence of a trophic competition between the communities leading to different shifts in bacterial distribution of the ecosystem.

Pure strains

Final metabolite production of all pure strains is presented in supplementary Fig. S5. production. This strain addition could moreover limit the growth of bacterial population degrading lactate to butyrate, which apparently occurred in replicates 2 and 3 of the mixed culture. Clostridium pasteurianum promoted acetate production. However, the presence of ethanol and lactate in some replicates may indicate that some metabolic instability persisted in the ecosystem. Similarly, E. coli and C. necator induced an increase in butyrate production leading to higher and more stable H2 production levels. Contrastingly, E. casseliflavus and D.

P a g e 20 | 32 vulgaris, which do not produce H2 led to highly diverse metabolites production suggesting that they do not influence H2 production pathways.

The repeatability of the results obtained when adding these five strains contrasted with the variability of metabolites in the mixed culture replicates. This finding tended to imply that these strains were metabolically active in the ecosystem and that their inclusion would have a stabilizing effect on the overall consortium metabolism. Except when adding Lactobacillus, which seemed to activate lactate pathway while preventing the acetate and butyrate pathways, no trend appeared to emerge about the influence of Clostridium sp. on the metabolite patterns of the ecosystem. Indeed, the excessive variability of microbial communities observed during the mixed culture growth induced significant fluctuations in the production of metabolites which prevent us to conclude about a possible impact of adding Clostridium sp. on the choice of the ecosystem metabolic pathways.

Microbial population dynamics

Microbial community dynamics were investigated with 16S rRNA gene highthroughput sequencing. The objective was to identify key microorganisms involved in structural variations of the microbiota. A total of 15 samples were selected. One sample was used to characterize the initial composition of the mixed culture (inoculum). A second sample was collected after 15 hours of incubation of the mixed culture, during the first exponential H2 production phase. Due to the high variability observed within H2 production kinetics of the mixed culture, two samples per replicate were subsequently collected at the end of each plateau phase. For the incubation confronting the seven exogenous strains tested, one sample per triplicate, collected at the end of the H2 production was sequenced.

P a g e 21 | 32

Several thousands of sequences were identified for each sample. They were assembled into 493 bacterial OTUs. OTUs belonging to the same bacterial order were represented with the same color palette. Bacterial community composition dynamics were represented in histograms at the OTU level in Fig. 3.

Mixed culture instability

Inoculum bacterial composition was highly diverse with a total of 486 different OTUs.

For sake of clarity, OTUs for which relative abundance did not exceed 1% were aggregated and considered as minor OTUs.

Mixed culture was mainly composed of OTUs assigned to phyla Firmicutes (18%), Bacteroidetes (16%) and Proteobacteria (12%). Two H2 producing OTUs were notably predominant: Clostridium acetobutylicum (8%) and Clostridium butyricum (3%).

After 15 hours of incubation, Clostridium butyricum became highly predominant reaching a relative abundance of 81%. At the end of the first plateau phase, this OTU was equally represented within each replicate, with a relative abundance varying between 96% and 97%. This great microbial stability confirmed the good reproducibility observed within H2 production kinetics.

Interestingly, at the end of the second plateau phase, the microbial composition of the three replicates greatly varied. In all cases, Clostridium butyricum proportion dropped while different hydrogen producers took advantage on substrate consumption.

In the first replicate (R1), Clostridium acetobutylicum became predominant, representing 51% of the total OTUs, while Clostridium algifaecis and Clostridium sporogenes reached comparable proportions of 12-13%. Clostridium butyricum relative abundance dropped down to 19%. Moreover, two OTUs belonging to Sporolactobacillus genus became P a g e 22 | 32 detectable in the ecosystem with a total proportion of 4%. Their appearance can thus be directly correlated to the presence of lactate within the final metabolic pattern and to the inhibition of the second H2 production phase. Although, these lactic acid bacteria were not detectable in the inoculum, their ability to form spore could explain their resistance to the initial heat treatment.

In the second replicate, Clostridium butyricum proportion decreased down to 51% while two other hydrogen producers, Clostridium pasteurianum and Clostridium acetobutylicum emerged, reaching relative abundance of 29% and 19%, respectively. Meanwhile, a minor OTU assigned to Clostridium autoethanogenum also emerged. This genus has been reported

to perform medium-chain fatty acids synthesis via chain elongation, utilizing acetate and ethanol as main substrates. Its presence within the ecosystem could explain the production of caproate and ethanol in the final metabolites of this bioreactor.

Contrastingly, the third bioreactor was almost exclusively dominated by two OTUs assigned to Clostridium acetobutylicum (50%) and to Clostridium butyricum (45%). A small amount of lactate was produced and could be linked to the presence of Sporolactobacillus vinae (1%). However, contrary to the first replicate, this lactic acid bacterium did not seem to reach sufficient proportion to impact the H2 production.

The 16S rRNA sequences confirmed trends observed for H2 production and metabolites accumulation. It also evidenced the instability in microbial community composition and related metabolic patterns occurring during the second H2 production phase. This highlights the microbial rearrangements occurring between H2 producers and the emergence of competitors such as lactic acid bacteria. Considering their ability to form spores of Sporolactobacillus and Clostridiaceae, this instability could be explained by the fact that after P a g e 23 | 32 a certain lag phase, these bacteria managed to emerge and to create new competitions within the ecosystem.

Confrontation with exogenous pure strains

Three strains (C. acetobutylicum, C. pasteurianum, and L. bulgaris) completely annihilated the second H2 production phase, thus reducing the global performance variability of the microbiota. The 16S rRNA sequences revealed that after its addition in the bioreactor, C. acetobutylicum became ultra predominant in the final ecosystem, reaching a final proportion of 99%. Furthermore, the addition of C. pasteurianum induced equilibrium between this strain (59%) and the major H2 producer during the first production phase of the mixed culture, C. butyricum (39%). This result suggested that C. acetobutylicum outcompeted for the substrate against C. butyricum in higher extends than C. pasteurianum. However, in terms of performance, higher production yields were observed in presence of C. pasteurianum which tended to indicate that this co-culture consisted of two Clostridies was more efficient in producing as the single strain. L. bulgaris also became predominant within the ecosystem, which completely simplified the final composition of the microbiota. It was exclusively dominated by this lactic acid bacterium (45%) and C. butyricum (54%). Nevertheless, besides the metabolic stability of this triplicated reactors, L. bulgaris growth partly rerouted glucose fermentation towards lactate production, which greatly reduced the H2 production yields.

Thus, the annihilation of the second H2 production phase seemed to be explained by the extreme simplification of the ecosystem induced by the addition of good competitors for the substrate consumption.

Moreover, adding the two strains (E. coli and C. necator) significantly stabilized the performances during the second H2 production phase. Sequencing results evidenced that E.

coli slightly emerged (2%). Nevertheless, its presence seemed to induce beneficial effects P a g e 24 | 32 leading to a simplified microbiota quasi exclusively composed of C. acetobutylicum (67%)

and C. butyricum (27%). Similarly, while C. necator was not even detected in the final ecosystem, its addition in the mixed culture led to a great simplification of its composition tending towards a co-culture consisting of C. acetobutylicum (40%) and C. butyricum (54%).

These results suggested that the initial addition of these strains drove the microbiota towards a more stable composition similar to the one observed in the third replicate of the mixed culture alone. This type of ecosystem only consisted of two Clostridium sp. strains showed the best performance indicating that both strains had positive effects in terms of stability and final H2 production volumes.

In presence of E. casseliflavus or D. vulgaris, performance instability was not mitigated.

Samples were selected in bioreactors for which the second H2 production phase did not occur to evidence potential OTUs that could be responsible for the inhibition of this phase.

Sequencing results indicated that although E. casseliflavus reached a final proportion of 3%, its presence was also associated to the emergence of Sporolactobacillus which became predominant (28%). This lactic acid bacterium was also recovered in the first replicate of the mixed culture for which no second H2 production phase was observed. As a result, H2 Replicates 1 are represented in blue while replicates 2 are represented in red and replicates 3 in green. Replicate 1 is represented in blue while replicate 2 is represented in red and replicate 3 in green.

Figure S2: Cumulated H2 production (mL) over time (number of hours) of a pure culture of Clostridium pasteurianum.

P a g e 32 | 32

Replicate 1 is represented in blue while replicate 2 is represented in red and replicate 3 in green.

Figure S3: Cumulated H2 production (mL) over time (number of hours) of a pure culture of Escherichia coli.

Replicate 1 is represented in blue while replicate 2 is represented in red and replicate 3 in green. with the different pure strains after the total conversion of the substrate.

V max (mL H 2 .L -1 .h

2. 3

 3 Molecular analyses of microbial communities 2.3.1 DNA extraction and PCR amplification Total DNA was extracted from the pellet using the Wizard Genomic DNA Purification kit (Promega) according to the manufacturer's instructions. The purity and amount of DNA in the extracts were measured by spectrophotometry (Infinite NanoQuant M200, Tecan). DNA were stored at -20°C prior sequencing. The procedure for 16S rRNA gene sequencing and raw data processing was described elsewhere by Carmona-Martinez (2015). The V3over 30 amplification cycles at an annealing temperature of 65.0°C. In a second PCR reactor of 12 cycles, an index sequence was added using the primers AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC and CAAGCAGAAGACGGCATACGAGAT-index-GTGACTGGAGTTCAGACGTGT. The PCR products were then sent for sequencing on a Illumina MySeq cartridge in GeT PlaGe sequencing center of the genotoul life science network in Toulouse, France (get.genotoul.fr) according to the procedure described in (Carmona-Martínez et al., 2015). P a g e 13 | 32 2.3.2 Data processing and statistical analysis A slightly modified version of the Standard Operation Procedure was used to process the raw data for alignment and assignation of the sequences in MOTHUR version 1.33.0. SILVA release 102. This version is more detailed by Carmona-Martinez et al. (2015). R CRAN software (version 3.3.3) was used to examine 16S abundance file. Considering the dispersion in the total number of reads identified in each sample, bacterial OTUs abundances were normalized. Only OTUs that exceeded 0.5% in at least one sample have been taken into account for the histogram representation. P a g e 14 | 32 3 Results and discussion

S4.

  It was divided into two bar charts corresponding to two H2 production phases. High variability observed within the triplicate of bioreactors required to distinguish the results for each replicate. Contrary to what could be expected, a great reproducibility was revealed in the metabolic pathways used by the mixed culture during the first H2 production phase. All replicates produced four types of metabolite (acetate, butyrate, formate and lactate) in relatively unchanged proportions. Acetate and butyrate production were the major fermentation products (3.22±0.05 mmol and 3.58±0.11 mmol, respectively) suggesting that Clostridium spp could be predominant within the mixed culture during the first phase.

Figure caption Figure 1 :

 caption1 Figure caption

Figure 2 :

 2 Figure 2: Ultimate metabolites concentration (mmol) of the different reactors inoculated with

Figure 3 :

 3 Figure 3: Relative abundances of the bacterial genera generated by 16S rRNA gene

Figure S1 :

 S1 Figure S1: Cumulated H2 production (mL) over time (number of hours) of a pure culture of

Figure S4 :

 S4 Figure S4: Metabolites concentration (mmol) of the different reactors inoculated with the

Figure S5 :

 S5 Figure S5: Ultimate metabolites concentration (mmol) of the different reactors inoculated

  E. coli synthesized 1.64±0.15 mmol of acetate, 1.65±0.05 mmol of propionate and 0.73±0.06 mmol of lactate while no butyrate was detected during these incubations.[START_REF] Hosseini | Hydrogen production from renewable and sustainable energy resources: Promising green energy carrier for clean development[END_REF] demonstrated that Escherichia coli was able to use acetate and formate pathways to synthesize H2. However, formate is hardly detectable. The presence of lactate suggested that E. coli partly shunted theoretical hydrogen potential towards solventogenesis.

	This could be induced by acetate inhibition which could orientate E. coli metabolic pathways
	towards non-acetate producing pathways.
	L. bulgaris and E. casseliflavus produced respectively between 8.7±0.1 mmol and
	6.5±0.5 mmol of lactate from glucose while D. vulgaris and C. necator did not produce any
	metabolite confirming their incapacity to produce H2.
	acetobutylicum produced 3.37±0.50 mmol of acetate, 5.03±0.21 mmol of butyrate, 1.20±0.17
	mmol of propionate and 2.17±0.06 mmol of formate while C. pasteurianum produced
	3.90±0.20 mmol of acetate, 6.17±0.11 mmol of butyrate, 0.30±0.01 mmol of propionate and
	1.76±0.06 mmol of formate. It could be noted that C. acetobutylicum produced more
	propionate than C. pasteurianum. Propionic acid pathway is a hydrogen consuming pathway
	that leads to limited hydrogen production. Here, it seems that in these conditions C.
	acetobutylicum metabolic pathways were less efficient for hydrogen production than C.
	pasteurianum which is underlined by a lower butyrate level and consequently to lower H2
	yield.
	P a g e 19 | 32

Both Clostridies used similar metabolic pathways and produced four types of metabolites (acetate, butyrate, propionate and formate), in relatively unchanged proportions. C.

3.2.3 Confrontation of mixed culture with pure strains

Results presented in Figure

2

showed that C. acetobutylicum, C. pasteurianum, E. coli; and C. necator additions strongly directed ecosystem metabolic pathways towards acetate and butyrate, although small amounts of other metabolites (lactate, formate and ethanol) were still produced. Interestingly, metabolites synthesized by these four triplicates as well as by the triplicate consisting of the mixed culture confronted with L. bulgaris were more reproducible within each other than for the mixed culture alone. Lactobacillus bulgaris, promoted the synthesis of lactate, repressed acetate and butyrate pathways thus inducing a decrease in H2

  D. vulgaris was not detected in the final ecosystem. But similarly to the bioreactors within which no second H2 production phase occurred, Sporolactobacillus Nevertheless, it confirms that the mixed culture instability could lead to situations where strong spore forming competitors such as lactic acid bacteria could emerge and greatly affect H2 production. Interestingly, this instability always appeared after a first H2 production phase during which rearrangements occurred within the ecosystem, probably because of a shift in substrate composition from glucose to metabolites such as VFA. It can be hypothesized that acetate production could create inhibitory conditions that were less favorable to C. butyricum. During this shift, new interactions were implemented and sporeforming bacteria could grow. Results prove that this instability could be avoided in batch reactors by two strategies: either by bioaugmenting with specific H2 producers which were able to resist to acetate production and pH drop, or by using other strains (eg., E. coli or C. necator) which seemed to be able to prevent the growth of competitors such as lactic acid

	4 Conclusions
	The distinct addition of E. coli and C. necator was proved to reduce metabolic and H2
	production instability of a mixed culture. Bioaugmentations with C. acetobutylicum, C.
	pasteurianum and L. bulgaris also mitigated this variability but induced stronger trophic
	competition that greatly limited final H2 production. Divergence in performances were
	associated to populations shifts in the mixed culture but also to the appearance of spore
	forming, lactate producing genera such as Sporolactobacillus. This outcome highlighted
	potentialities to use pure strains to enhance process stability and limit the stochastic
	emergence of substrate competitors within mixed cultures.
	bacteria.
	and C. algifaecis gained importance (34% and 8%, respectively) at the expense of C.
	acetobutylicum (28%) and C. butyricum (30%). Thus, it can be hypothesized that either these
	strains were not metabolically active or that they did not influence the interactions within the
	mixed culture.
	P a g e 25 | 32 P a g e 26 | 32

producers such as C. acetobutylicum and C. butyricum were less represented in the ecosystem (40% and 22 %, respectively). Interestingly, C. algifaecis was significantly present with a relative abundance of 8%. E-supplementary data of this work can be found in online version of the paper. P a g e 27 | 32

  -1 )Hmax (mL H 2 ) λ (hour) Carbon conversion yield (mol H 2. mol glucose -1 )

		Production phase n°1	126.8 ± 52.6	224.2 ± 49.2	8.4 ± 0.3	1.95 ± 0.10
	Mixed culture	Production phase n°2	49.5 ± 23.8	176.3 ± 151.5	-	1.50 ± 1.28
		Total	-	431.9 ± 159.3	-	1.68 ± 0.62
	Clostridium acetobutylicum		303.0 ± 40.4	463.1 ± 26.6	19.4 ± 0.3	1.65 ± 0.10
	Clostridium pasteurianum		77.5 ± 7.0	514.4 ± 32.5	14.2 ± 0.5	1.79 ± 0.12
	Escherichia coli		18.3 ± 0.7	54.0 ± 7.7	19.2 ± 0.6	0.18 ± 0.03
	Mixed culture + Clostridium acetobutylicum		50.1 ± 3.5	274.8 ± 42.4	9.7 ± 0.2	1.17 ± 0.21
	Mixed culture + Clostridium pasteurianum		93.9 ± 23.1	349.1 ± 14.8	8.3 ± 0.1	1.45 ± 0.06
	Mixed culture + Lactobacillus bulgaris		142.7 ± 8.3	169.5 ± 36.1	8.7 ± 0.4	1.21 ± 0.23
		Production phase n°1	112.0 ± 40.1	224.5 ± 23.9	7.9 ± 0.1	1.61 ± 0.27
	Mixed culture + Escherichia coli	Production phase n°2		207.2 ± 33.6	-	2.04 ± 0.09
		Total	-	436.0 ± 49.5	-	1.77 ± 0.28
		Production phase n°1	164.0 ± 26.4	197.7 ± 15.4	9.7 ± 0.1	1.47 ± 0.16
	Mixed culture + Cupriavidus necator	Production phase n°2		247.3 ± 68.3	-	2.08 ± 0.55
		Total	-	445.6 ± 79.9	-	1.75 ± 0.31
		Production phase n°1	106.3 ± 36.5	189.6 ± 26.7	8.3 ± 0.2	1.46 ± 0.20
	Mixed culture + Enterococcus casseliflavus	Production phase n°2		120.9 ± 81.7	-	1.00 ± 0.65
		Total	-	310.5 ± 107.4	-	1.24 ± 0.42
		Production phase n°1	169.5 ± 36.4	212.2 ± 21.3	9.4 ± 0.1	1.47 ± 0.18
	Mixed culture + Desulfovibrio vulgaris	Production phase n°2		138.4 ± 126.5	-	1.21 ± 1.05
		Total	-	350.6 ± 138.5	-	1.35 ± 0.51

Acknowledgments

We gratefully acknowledge the financial support of the French National Research Agency (ANR) through the InGEcoH Project entitled "Ecological engineering of microbial ecosystems producing biohydrogen by dark fermentation" (contract number 2008-BIOE-005-01). We also thank Yan Rafrafi and Anaïs Bonnafous for having participated to this work. P a g e 28 | 32


Ndeye Safietou Fall

Nafissatou Diagne
email: nafissatou.diagne@ird.fr

Oleg Mediannikov
email: oleg.mediannikov@ird.fr

Florence Fenollar
email: florence.fenollar@univ-amu.fr

Philippe Parola
email: philippe.parola@univ-amu.fr

Cheikh Sokhna
email: cheikh.sokhna@ird.fr

Didier Raoult
email: didier.raoult@gmail.com

J.-C Lagier
email: jclagier@yahoo.fr

Jean-Christophe Lagier

Prof Jean-Christophe Lagier

Ndeye Safietou

Jean-Christophe Lagier

Detection of Borrelia crocidurae in a vaginal swab after miscarriage, rural Senegal, Western Africa

Keywords: Borrelia crocidurae, self vaginal sampling, spontaneous miscarriage

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Overdiagnosis of malaria in endemic areas overshadows the presence of other pathogens, such as bacteria that induce fever [START_REF] Gwer | Over-diagnosis and co-morbidity of severe malaria in African children: a guide for clinicians[END_REF]. In Senegal, several studies have already reported the presence of DNA from pathogenic bacteria in the blood of patients with non-malarial fever, including tick-borne relapsing fever (TBRF) borrelia [START_REF] Mediannikov | Borrelia crocidurae infection in acutely febrile patients, Senegal[END_REF][START_REF] Parola | Tick-borne relapsing fever borreliosis, rural senegal[END_REF]Talagrand-Reboulet al., 2018). TBRF borrelias are risk factors for pregnant women and can cause miscarriages (Giakomelou et al., 2016). Borrelia are known to readily penetrate different biological barriers. This is explained by their helical shape and their movement ability and is facilitated by the use of the host protease.

In recent years, reliable and effective vaginal self-sampling has been used in the diagnosis of several infections, including HPV and bacteria causing sexually transmitted infections (STI) [START_REF] Lagier | Vaginal selfsampling as a diagnosis tool in low-income countries and potential applications for exploring the infectious causes of miscarriage[END_REF]. First, we carried out a project to assess the feasibility of taking vaginal samples in rural areas of Senegal associated with the detection of the carriage of some bacteria or Human papillomavirus, as previously reported [START_REF] Fall | Feasibility, Acceptability, and Accuracy of Vaginal Self-Sampling for Screening Human Papillomavirus Types in Women from Rural Areas in Senegal[END_REF]. Our study on miscarriages, which began in March 2016, consists of self-sampling women who have had a miscarriage or other pregnancy problems and determining whether pathogens are responsible for abortions or other pregnancy problems in rural areas of Senegal. To the best of our knowledge, we report here the first detection of Borrelia crocidurae in self vaginal sampling in such clinical involvement.

The study was performed in health posts in Dielmo, Ndiop and Niakhar (Diohine, Toucar, Ngayohéme) in Senegal, West Africa [START_REF] Fall | Feasibility, Acceptability, and Accuracy of Vaginal Self-Sampling for Screening Human Papillomavirus Types in Women from Rural Areas in Senegal[END_REF]. Women who had miscarriage performed a vaginal self-sampling (Transwab M40 COMPLIANT for bacterial detection). An informed consent was obtained from patients. The ethical authorization for our study was granted by the national ethics committee of Senegal (n°83/MSAS/DPRS/CNFRS). The microorganisms, including those associated with STI and those known to cause abortion, were tested by qPCR and will be the subject of a study published separately. When a sample was positive for one gene, a second qPCR targeting another gene was performed (Table 1). To check the quality DNA extracts, we tested the human albumin gene as previously described [START_REF] Fall | Feasibility, Acceptability, and Accuracy of Vaginal Self-Sampling for Screening Human Papillomavirus Types in Women from Rural Areas in Senegal[END_REF].

From March 2016 to July 2017 we tested 132 vaginal samples from controls (women living in the same geographic areas and with no history of complicated pregnancy) and 26 vaginal samples from patients. All samples are of great quality (100% positive for human albumin). Using qPCR, we obtained 3 vaginal samples positive for Borrelia sp. 16S in patients and none in controls. However, only 1 was confirmed by a qPCR targeting the ITS4 (intergenic spacer) locus. A standard PCR targeting the ITS locus confirmed our diagnosis (Table 1). The sequencing of the ITS locus amplicons (539 base pairs, Genbank accession number: LT984797) exhibited a 100% sequence similarity with B. crocidurae (KF176340.1) isolated from Ornithodoros sonrai, the vector of borreliosis, collected in the same geographic areas. This sample was collected from a woman who underwent an abortion after 12 weeks of pregnancy. The difference between patients (1/26) and controls (0/132) was significant for B. crocidurae. The patient lives in Niakhar area and had a history of 3 spontaneous abortions.

The 25 patients who tested negative for B. crocidurae had had an abortion from 6 to 24 weeks of pregnancy (mean of 20 weaks). The patient with a positive sample for Borrelia lives in an environment with horse, calf, goat, donkey and sheep and has seen many rats in her house but never ticks. Her house was built in bricks and the soil was non-cemented dirt. Floor and walls included some crevices (Figure 1 A andB). The 25 patients who had miscarriage but with a negative sample for Borrelia lives in same geographical area and no difference regarding the repartition of animals was observed.

Our study suggests that vaginal self-sampling could be a valuable technique to detect B. crocidurae. The use of a consequent number of controls living in the same geographical area and the use of stringent protocols confirm the accuracy of our results. The fact that women were taking samples from the brick and cement dispensary and not at homes, and the fact that all controls were negative, excluded an environmental contamination of the sample. Tick-borne relapsing fever (TBRF) is transmitted to humans through the bites of soft ticks of the genus Ornithodoros. Wild rodents and insectivores are common reservoir hosts. TBRF is responsible for recurring fever associated with spirochetemia and constitutes the main cause of non-malarial fever in Senegal (Talagrand-Reboulet al., 2018). Previous studies performed in Rwanda and in Tanzania demonstrated a 30% risk for pregnancy loss and a perinatal mortality rate of 15% in case of tick-borne relapsing fever (Talagrand-Reboulet al., 2018). In addition of Borrelia crocidurae infections, our study suggests that vaginal self-sampling is a method adapted to the detection of infectious causes of spontaneous miscarriage in lowincome countries [START_REF] Lagier | Vaginal selfsampling as a diagnosis tool in low-income countries and potential applications for exploring the infectious causes of miscarriage[END_REF]. Certainty of a link between the vaginal detection of Borrelia crocidurae and the causality of abortion will require further more in-depth studies.

Nevertheless, this technique of sampling could be coupled with Point-of-Care laboratories, which have previously demonstrated their efficiency in exploring the non-malarial causes of fever [START_REF] Drancourt | The Point-of-Care Laboratory in Clinical Microbiology[END_REF] and could be used to elucidate the infectious causes of miscarriage in low-income countries.

Figure 1 :A:

 1 Figure 1:

Table 1 :

 1 Molecular biology tools used for the identification of Borrelia crocidurae

	Gene	Targeted sequence
	Borrelia sp. 16S	Bor_16S_3 F : AGCCTTTAAAGCTTCGCTTGTAG,
		Bor_16S_3R : GCCTCCCGTAGGAGTCTGG,
		Bor_16S_3P : 6FAM-CCGGCCTGAGAGGGTGAACGG
	qPCR targeting the	Bor_ITS4_F: GGCTTCGGGTCTACCACATCTA,
	ITS4	Bor_ITS4_R : CCGGGAGGGGAGTGAAATAG,
		Bor_ITS4_P : 6FAM-TGCAAAAGGCACGCCATCACC
	Standard PCR	ITS F : AGGGGGGTGAAGTCGTAACAAG
	targeting the ITS	ITS R : GTCTGATAAACCTGAGGTCGGA
	locus

FUNDING SOURCE This work has benefited from the French State support, managed by 'Agence Nationale pour la Recherche' including the 'Programme d'Investissements d'avenir' under the reference Méditerranée Infection 10-IAHU-03. This work was supported by the Région Provence-Alpes-Côte d'Azur and European funding FEDER PRIMI. CONTRIBUTION Study design: JCL, DR, CS; Data collection: NSF, ND, JCL; Data analysis: NSF, OM, FF, PP, JCL, CS, DR; Writing: NSF, PP, DR, JCL

CONFLICT OF INTEREST STATEMENT

None

ETHICAL APPROVAL:

The ethical authorization for our study was granted by the national ethics committee of Senegal (n°83/MSAS/DPRS/CNFRS).


Marina Al Daccache

Mohamed Koubaa
email: m.koubaa@escom.fr

Dominique Salameh

Richard G Maroun

Nicolas Louka

Eugène Vorobiev

Marina Al Daccache

Ultrasound-assisted fermentation for cider production from Lebanese apples

Keywords: Hanseniaspora sp., ultrasound-assisted fermentation, stimulation, cider, low alcohol content, Lebanese apples

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Ultrasound (US) is defined as acoustic waves whose frequency is equal or greater than 20 kHz. It has been widely applied in medical diagnosis, sonochemical treatment, ultrasonic cleaning of surfaces, and in submarines [START_REF] Chisti | Sonobioreactors: using ultrasound for enhanced microbial productivity[END_REF][START_REF] Zhao | Applications of ultrasound to enhance mycophenolic acid production[END_REF]. Most applications for microbial inactivation in food processing use low frequency US, with a frequency ranging from 20 to 100 kHz [START_REF] Chisti | Sonobioreactors: using ultrasound for enhanced microbial productivity[END_REF][START_REF] Zinoviadou | Fruit juice sonication: Implications on food safety and physicochemical and nutritional properties[END_REF]. This is mainly related to the several benefits of US, which is considered as "green" technology, compared to conventional processes. The benefits of US include shortening the processing time (seconds to minutes) with high reproducibility and accuracy, reducing the processing cost, generating highly pure product, eliminating some of the downstream purification steps, as well as reducing the post-treatment of wastewater [START_REF] Chemat | Review of green food processing techniques. Preservation, transformation, and extraction[END_REF][START_REF] Chemat | Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review[END_REF].

While high-intensity US (10-1000 W.cm -2 ) is usually effective in disrupting microbial cells, low-intensity US (<10 W.cm -2 ) can result in an intensification of several biotechnological processes without damaging the cells [START_REF] Chisti | Sonobioreactors: using ultrasound for enhanced microbial productivity[END_REF][START_REF] Gogate | A review of applications of cavitation in biochemical engineering/biotechnology[END_REF][START_REF] Pagnossa | Ultrasound: beneficial biotechnological aspects on microorganisms-mediated processes[END_REF]. The mechanisms underlying productivity improvements have not been so far identified, although factors such as improved gas-liquid mass transfer (liquid phase gas absorption) and solid-liquid mass transfer (dissolution of a solid in the liquid phase) play an important role [START_REF] Sainz Herrán | Effects of ultrasound on culture of Aspergillus terreus[END_REF]. Furthermore, US induces cavitation that generates microbubbles at different sites in the liquid. The formed microbubbles grow during the rarefaction phase and then implode and collapse during the compression phase, releasing thereby a wave shock in the medium [START_REF] Chemat | Review of green food processing techniques. Preservation, transformation, and extraction[END_REF]. Besides, pressure fluctuation during US treatments can induce stress on the cells, possibly promoting cell growth and proliferation, as well as changes in the metabolic processes [START_REF] Sainz Herrán | Effects of ultrasound on culture of Aspergillus terreus[END_REF][START_REF] Hu | Influences of ultrasound and Ohmic heating on growth of Sake yeast[END_REF][START_REF] Dai | Effects of low-intensity ultrasound on the growth, cell membrane permeability and ethanol tolerance of Saccharomyces cerevisiae[END_REF].

Several applications of low-intensity US have been described in food processing, and particularly in food fermentation to improve the performance of microorganisms, and the quality of the end product [START_REF] Awad | Applications of ultrasound in analysis, processing and quality control of food: A review[END_REF][START_REF] Kwiatkowska | Stimulation of bioprocesses by ultrasound[END_REF][START_REF] Ojha | Ultrasound technology for food fermentation applications[END_REF]. It has been reported that the application of US in the dairy fermentation can induce some modifications in macromolecules such as enzymes [START_REF] Huang | Effects of ultrasound on microbial growth and enzyme activity[END_REF][START_REF] Ma | Effect of energy-gathered ultrasound on Alcalase[END_REF], reduce the yogurt fermentation time [START_REF] Feifei | Research progress of applications of ultrasonic technology in food industry[END_REF][START_REF] Polonorum | Applications of ultrasound in food technology[END_REF], and improve the yogurt's rheological properties [START_REF] Vercet | Rheological properties of yoghurt made with milk submitted to manothermosonication[END_REF]. Besides, US was used for the proliferation of probiotics (e.g. Bifidobacterium sp.), which caused an increase in the lactose hydrolysis, and transgalactosylation in milk [START_REF] Nguyen | Stimulating fermentative activities of bifidobacteria in milk by highintensity ultrasound[END_REF][START_REF] Yeo | Effect of ultrasound on the growth of probiotics and bioconversion of isoflavones in prebiotic-supplemented soymilk[END_REF].

One of the promising fields described in the literature for the application of US-assisted fermentation is the production of alcoholic beverages. For instance, Matsuura and co-workers reported a significant reduction of wine and beer fermentation time (by 50%) when applying US intensity of 30 mW.cm -2 with a frequency of 43 kHz. An acceleration of ethanol production and a decrease in the dissolved CO2 were also noted [START_REF] Matsuura | Acceleration of cell growth and ester formation by ultrasonic wave irradiation[END_REF]. A similar observation was reported by Lanchun and co-workers by applying US with a frequency of 24 kHz, a power of 2 W, and a stimulation time of 1 s after intermitting for 15 s during 30 min of the exponential phase [START_REF] Lanchun | The research into the influence of low-intensity ultrasonic on the growth of S. cerevisiaes[END_REF]. Results showed growth acceleration by 33% of the Saccharomyces cerevisiae cells. The impact of US depends as well on the period of growth phase (e.g. adaptation, exponential, etc). For example, the effect of the energy applied during the Lag phase of S. cerevisiae yeast culture was studied [START_REF] Jomdecha | Effects of pulse ultrasonic irradiation on the lag phase of Saccharomyces cerevisiae growth[END_REF]. Applying US energy between 330 and 360 W.s.m -3 decreased the Lag phase duration by 1 h compared to the untreated suspension, whereas applying higher energy than 850 W.s.m -3 led to a longer Lag phase and a growth reduction. There is a great variability in the sensitivity of different microorganisms to US, which strongly depends on the properties of the medium and the parameters of the treatment (intermittent or continuous application of US). For example, it has been shown that the intermittent sonication (25 kHz, 300 W.m -3 ) of S. cerevisiae yeasts throughout the fermentation more than doubled the ethanol yield, whereas the same effect did not occur for the continuously-treated cultures [START_REF] Schläfer | Improvement of biological activity by low energy ultrasound assisted bioreactors[END_REF]. A 4-fold increase in ethanol productivity was observed when applying US (35 kHz, 1.48 W.cm -2 ) to a 10% cycle (equivalent to sonication for 1 min followed by a rest period of 9 min) on S. cerevisiae MTCC 170 [START_REF] Singh | Mechanistic insight into ultrasound induced enhancement of simultaneous saccharification and fermentation of Parthenium hysterophorus for ethanol production[END_REF]. By contrast, a recent study has reported that the application of either direct US (at different intensities ranging from 23 to 32 W.L -1 ) or indirect US (1.4 W.L -1 intensity) had negative effects on yeast performances [START_REF] Huezo | Effects of ultrasound on fermentation of glucose to ethanol by Saccharomyces cerevisiae[END_REF]. Both reductions in the rates of glucose consumption and ethanol production were also observed. Thus, high variability was observed for the impact of US on fermentation, which depends on the type of microorganism, the US parameters, the growth phase, etc. This makes understanding the mechanisms underlying the process more difficult, and therefore more studies are required. In this line, the current work is devoted to study the impact of USassisted fermentation on Hanseniaspora sp. growth and glucose uptake during cider production. The genus Hanseniaspora was the major yeast developed during spontaneous apple juice fermentation [START_REF] Valles | Yeast species associated with the spontaneous fermentation of cider[END_REF][START_REF] Bedriñana | Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias[END_REF]. Besides, Capozzi and co-workers demonstrated the positive impact of Hanseniaspora sp. in alcoholic fermentation and in cider fermentation [START_REF] Capozzi | Impact of coinoculation of Saccharomyces cerevisiae, Hanseniaspora uvarum and Oenococcus oeni autochthonous strains in controlled multi starter grape must fermentations[END_REF], which can positively contribute to the aroma profile of the fermented apple juice [START_REF] De Arruda Moura Pietrowski | Influence of fermentation with Hanseniaspora sp. yeast on the volatile profile of fermented apple[END_REF].

Materials and methods

Chemicals, yeast, and apples

Peptone and ethanol (C2H6O) were bought from Fisher Scientific (Illkirch, France).

Yeast extract was supplied by Sigma-Aldrich (Saint-Quentin-Fallavier, France). Glucose and fructose were provided by Merck Millipore (Guyancourt, France).

Hanseniaspora sp. strain used in this work belongs to the authors' laboratory collection.

The strain was isolated previously from spontaneous fermentation of a Lebanese apple juice obtained from the "Ace spur" variety [START_REF] Al Daccache | New indigenous yeast strains "Hanseniaspora Meyeri-Libani" for the elaboration of cider[END_REF].

The apples ("Ace spur" variety) used to conduct fermentation experiments were supplied from Bqaatouta village (Keserwan region, Lebanon).

Fermentation processes 2.2.1. Hanseniaspora sp. pre-culture

The pre-culture of the Hanseniaspora sp. strain was performed by taking one colony from YPD agar (1 % yeast extract, 2 % peptone, 2 % dextrose, and 2 % agar) plate into 250 mL of YPD (1 % yeast extract, 2 % peptone, and 2 % dextrose) medium under sterile conditions (20 min at 121 °C in an autoclave HMC HV-110L (HMC Europe GmbH, Germany)). The pre-culture was incubated during 45 h at 30 °C temperature and 250 rpm agitation speed in a shaker (Thermo Scientific, France).

Fermentations in synthetic medium

Control fermentations

A volume of 1.5 L of YPD medium (1 % yeast extract, 2 % peptone, and 2 % dextrose) was introduced into a 2-L fermenter (LSL Biolafitte S.A.). After sterilization as abovementioned and cooling to 30 °C, the medium was inoculated by a pre-culture to get an initial concentration of 4.5*10 6 cells.mL -1 . The control fermentation in batch mode without medium circulation (Figure 1.A) was performed at 250 rpm agitation speed, and the temperature was maintained at 30 °C. After recording the kinetics of growth and glucose consumption in batch mode without medium circulation, a treatment chamber was designed to allow the circulation of the medium and the yeast treatment by US during the fermentation (Figure 1.B). For this purpose, two parallel stainless-steel plates (3 mm width, 6 cm diameter) were fixed between two Teflon rings creating a channel for the flowing liquid. The volume of the treatment chamber was around 40 mL (corresponding to a ratio of 1:50 between the volume of the fermenter and the volume of the chamber). Two pipe fitting connectors were adjoined to make it run as a continuous chamber. A transducer (100 W, 40 kHz) fixed to the chamber and connected to the power supply (220 V) converted the available electrical voltage line into high-frequency waves. A peristaltic pump allowed the medium circulation from the reactor to the treatment chamber and back into the reactor. Various circulation flow rates were tested (25, 50, and 75 mL.min -1 ) without US application to get the same growth and glucose consumption kinetics obtained in batch mode without circulation (Figure 1.A). The total length of the tubes used was equal to 90 cm. The treatment chamber and the tubes were sterilized as above-mentioned.

US-assisted fermentations

The designed treatment chamber (Figure 1.B) was used for the stimulation of Hanseniaspora sp. yeast. The stimulation conditions were first optimized using the synthetic medium. The US treatment was done in a cyclic mode with variable periods of US pulses of Δtp = 0.5, 1, and 2 s, followed by pauses of Δtw = 6 s (Figure 2). The variation of US application period allowed the optimization of the treatment. US-assisted fermentation was initially performed during the first 6 h of fermentation (associating both the Lag and the Log phases). US-assisted fermentation was then performed during either Lag phase (3 h) or Log phase (3 h) using each time optimal US treatment.

Fermentations in apple juice

Control fermentations

The apples were first washed and then used to extract the juice using a fruit juice centrifuge separator (Moulinex, France). A volume of 1.5 L of apple juice was introduced into a 2-L sterile fermenter and inoculated by the pre-culture to get an initial concentration of 3.0*10 6 cells.mL -1 . Cider production was conducted for 100 h in batch mode, with medium circulation at 30 °C temperature and 250 rpm agitation speed. The flow rate used for the circulation was fixed to allow obtaining the same growth kinetics compared to the control without medium circulation. The kinetics of growth, substrates consumption, and ethanol production were recorded during the fermentation. The content of soluble compounds was followed by measuring the °Brix using a digital refractometer (Anggur DR401, France).

US-assisted fermentations

The kinetics of growth, substrate consumption, and ethanol production of US-assisted fermentation were compared to that of control fermentation (section 2.2.3.1). US-assisted fermentation of apple juice was recorded during the first 12 h of the fermentation (including the Lag phase). The application of US was also performed on the pre-culture for 6 h (including the Lag and Log phases), followed by apple juice fermentation without US treatment inoculated with the treated pre-culture.

US treatment was done in a pulsed mode with sequential application of n US pulses. The total time of US cycles was 6 h (21600 s) and can be defined as follows: tt=n(Δtp+Δtw), where Δtp corresponds to the US pulse duration (Δtp = 0.5, 1, and 2 s), and Δtw is the pause duration (Δtp = 6 s). The number of pulses was n = 21600 /(Δtp+Δtw) = 2700, 3086 and 3324, while the effective time of US treatment was tUS = n . Δtp corresponding to 1662 s, 3086 s, and 5400 s.

The US generator allowed recording the energy E (Wh) during the treatment. The specific energy consumption W (J.mL -1 ) was estimated according to equation (1):

J. = * Equation (1)
where E is the energy (Wh) provided by the generator, and V is the volume of the culture medium in the bioreactor (mL).

Kinetics of growth, substrates consumption, and ethanol production

The kinetics of growth, substrates consumption, and ethanol production were recorded for all the fermentation processes (control and assisted by US).

Determination of the growth kinetics

The growth kinetics were followed by both measuring the optical density (absorbance) at 600 nm, and counting the cell number (cells.mL -1 ). Samples were taken periodically from the bioreactor and their absorbance values were determined using a Thermo Spectronic BioMate 3 UV-visible spectrophotometer (Thermo Scientific, France). The samples were diluted several times in water, and the cells were counted using a Thoma cell counting chamber (Preciss, France). To detect the viable cells, the samples were mixed with a 0.01 % v/v solution of methylene blue. The optical density was followed only for the fermentation in the synthetic medium, while for the apple juice fermentations the biomass concentration was just recorded by cell counting. The biomass concentration X (cells.mL -1 ) was calculated according to equation ( 2): . = . .

Equation (2)

where a is the number of cells counted, d is the dilution factor, b is the number of squares counted in the chamber, and 250000 corresponds to a constant included in the formula of the Thoma cell.

The maximal specific growth rate µmax (h -1 ) was determined by plotting Ln X (cells.mL -1 )

versus time (h). The µmax (h -1 ) values corresponded to the slopes of the linear curves obtained during the exponential phase.

Carbon substrates quantification

The carbon substrates were quantified enzymatically using a glucose and fructose assay kit (LTA, Milano, Italy).

Ethanol quantification 10

The samples taken at different times of the fermentation processes were centrifuged at 6000 rpm for 10 min to eliminate the yeast suspensions and apple particles. The ethanol content was determined by density measurements using a 5-mL glass pycnometer (Thermo Fisher, France). Calibration curve for ethanol quantification was performed using reconstituted solutions of apple juice/ethanol (1-10%, v/v).

The ethanol production rate γ (gethanol.gbiomass -1 .h -1 ), the biomass yield YX/S (gbiomass.gsubstrate - 1 ), and the product yield (YP/S (gethanol.gsubstrate -1 ) were calculated according to equations ( 3), (4), and (5), respectively.

/ !"# $$ . $% $&' &( = ) * * )
Equation ( 3)

+/ (&, -". . $% $&' &( = + ) + * * )
Equation ( 4)

/ (&, -". . !"# $$ . ℎ = + ) + * & ) & * Equation (5)
where X0 and Xf are respectively the initial and the final biomass concentrations, P0 and Pf are respectively the initial and the final ethanol concentrations, and S0 and Sf are respectively the initial and the final substrate concentrations.

Statistical analyses

Each experiment was repeated three times. One-way ANOVA was used for the statistical analysis of the data with the help of Statgraphics Plus software (version 5.1, Statpoint Technologies Inc., Warrenton, VA, USA). A significance level of 5% was taken for each analysis. The error bars presented in the figures correspond to the standard deviations.

Results and discussion

Optimization of the experimental parameters

Treatment during the 6 first hours of fermentation

A medium circulation flow rate of 75 mL.min -1 allowed obtaining the similar growth kinetics than that of the control fermentation without circulation. It was thereby used for USassisted fermentations. The US parameters were optimized on the synthetic medium by applying US, first during both Lag (adaptation) and Log (exponential) growth phases (6 h), and then separately during either the Lag phase (3 h) or the Log phase (3 h). Results in figure 3 show an increase of the biomass (yeast) concentration with US treatment during both Lag and Log phases (6 h). The biomass concentration increased more rapidly under the US treatment than for the control samples, especially for the pulses duration Δtp = 0.5 and 1 s, which corresponded to respectively 1662 and 3086 s of effective US treatment. The stimulation effect was more pronounced when applying US pulses for 0.5 s compared to that for pulses of 1 s. Furthermore, increasing the pulse time up to 2 s (effective US treatment of 5400 s) seemed to decrease the yeast growth (Figure 3). The results of the US treatment of Hanseniaspora sp. cells concur with those reported in the literature showing that cell stimulation is energy-dependent, and the application of high energy can lead to a cell growth reduction and yeast inactivation [START_REF] Jomdecha | Effects of pulse ultrasonic irradiation on the lag phase of Saccharomyces cerevisiae growth[END_REF].

Treating the cell suspension for 6 h (pulses of 0.5 s followed each by a pause of 6 s) allowed obtaining a significant increase in the final biomass concentration (after 8 h of fermentation) by 63 %, which corresponded to an increase in the number of cells from 9.0*10 7 (for the control (untreated) medium) to 1.5*10 8 cells.mL -1 (for the US-treated medium). These results concur with the measurements of the optical densities (absorbance values at 600 nm), which showed a significant increase after 8 h of fermentation (from 2.43

for the control experiment to 2.97 for the US-assisted fermentation) (Figure 3.B). Moreover, the highest specific growth rates µmax of 0.54 and 0.56 h -1 were observed for the US-assisted fermentations with pulses of 0.5 s and 1 s, respectively, with no significant differences between both growth rates (Table 1). These results concur with those of Barukčić and coworkers, who reported an intensification of the cell growth by ≈1 log cycle following US treatment, which led to a decrease in the fermentation time [START_REF] Barukčić | Influence of high intensity ultrasound on microbial reduction, physico-chemical characteristics and fermentation of sweet whey[END_REF]. Besides, stimulation of probiotics in milk was reported for the cultures exposed to low-frequency US [START_REF] Nguyen | Stimulating fermentative activities of bifidobacteria in milk by highintensity ultrasound[END_REF][START_REF] Nguyen | Effect of high intensity ultrasound on carbohydrate metabolism of bifidobacteria in milk fermentation[END_REF]. Ewe and co-workers noted as well an increase up to 9% of the US-treated cells compared to that of the control during the fermentation of biotin-soymilk [START_REF] Ewe | Effects of ultrasound on growth, bioconversion of isoflavones and probiotic properties of parent and subsequent passages of Lactobacillus fermentum BT 8633 in biotin-supplemented soymilk[END_REF]. achieved with the US pulse duration of Δtp =0.5 s. Likewise, after 8 h of fermentation, the °Brix decreased from 5.9% (g.100 mL -1 ) for the untreated culture to 4.8% (g.100 mL -1 ) for the US-assisted fermentation (with Δtp =0.5 s). Accordingly, the biomass yields per gram of glucose consumed were significantly higher for the US-treated cultures (pulses of 0.5 and 1 s), compared to that of the control fermentation (Table 1). It has been reported in the literature that US may increase the permeability of the cell membrane through the microbubbles activity, creating temporary pores on the cell membrane [START_REF] Bekeredjian | Impact of microbubbles on shock wave-mediated DNA uptake in cells in vitro[END_REF]. Furthermore, the formation of reversible pores could improve the transmembrane flow and thereby decreasing the membrane resistance [START_REF] Deng | Ultrasound-induced cell membrane porosity[END_REF] and accelerating the transfer of substrates, which leads to stimulating the cell growth [START_REF] Huang | Effects of ultrasound on microbial growth and enzyme activity[END_REF][START_REF] Lanchun | The research into the influence of low-intensity ultrasonic on the growth of S. cerevisiaes[END_REF].

Treatment during either Lag or Log phase

For the suspensions treated during the first 3 h (Lag phase), an increase in the yeast biomass concentration by 43% was observed after 8 h of fermentation (Figure 4.A).

However, only an increase of 17% of yeast stimulation was recorded for the cells treated during the Log phase (from 3 to 6 h of fermentation). Additionally, faster sugar depletion was noticed during the Lag phase, compared to the Log phase (Figure 4.C). It was also confirmed by a faster reduction of the °Brix for the treatment during the Lag phase (Figure 4.D). This observation was previously reported by Lanchun and co-workers, who mentioned that the effects of US on the growth of S. cerevisiae were significant during the Lag and the Log phases, but not during the stationary phase [START_REF] Lanchun | The research into the influence of low-intensity ultrasonic on the growth of S. cerevisiaes[END_REF]. They also noted that the yeast cells were less resistant to environmental stress during the Lag phase, preparing for cell growth and reproduction [START_REF] Lanchun | The research into the influence of low-intensity ultrasonic on the growth of S. cerevisiaes[END_REF].

Energy consumption

Regarding the energy consumption, the US treatment performed during 6 h with pulse duration of 2 s consumed the highest energy (269 J.mL -1 ), and led to a growth kinetics decrease compared to the US treatments with pulses of 0.5 s and 1 s (Table 1), which consumed less amount of energy (respectively 155 and 197 J.mL -1 ). The same amount of energy (77 J.mL -1 ) was consumed for the US treatments during one growth phase (either Lag or Log); however, different impacts on the growth kinetics were observed.

Thus, the most effective parameters for Hanseniaspora sp. cells stimulation, among those tested during this study, could be the application of 0.5 s US pulses followed by 6 s rest period during either both Lag and Log phases or the Lag phase alone.

US-assisted fermentation of apple juice

The impact of US on ethanol accumulation during cider production was evaluated using Hanseniaspora sp. yeast. An US treatment with pulse duration of 0.5 s followed by a pause of 6 s was applied to either the pre-culture (YPD medium) or the apple juice yeast medium.

The growth phase durations of Hanseniaspora sp. on apple juice were determined during the conventional fermentation of the apple juice (without US treatment, with or without medium circulation).

Biomass growth kinetics

Results in figure 5.A show an increase in the biomass concentration by ≈52% when the apple juice yeast suspension was treated by US during the first 12 h of fermentation (without treating the pre-culture). On the other hand, the biomass concentration increased by 42% when the pre-culture was treated by US, followed by apple juice fermentation without any treatment (Figure 5.A). The raise in the biomass concentration could be assigned to the stimulation of the yeast cells' division during the US treatment. Different impacts of US have been reported at the cellular and molecular level. US could promote microbial growth by separating the cell groups, increasing thereby the membrane permeability and impacting the cellular functions and components [START_REF] Huang | Effects of ultrasound on microbial growth and enzyme activity[END_REF]. The values of the maximal growth rate, µmax (Table 2) showed accelerated growth kinetics by 10 folds for the US-assisted fermentation. As a result, the stationary phase was reached faster. These results concur with that of the literature, where higher µmax values were reported for Lactobacillus sakei cells treated with US [START_REF] Ojha | Integrated phenotypicgenotypic approach to understand the influence of ultrasound on metabolic response of Lactobacillus sakei[END_REF].

Moreover, the treatment of the pre-culture showed a significant effect on the fermentation kinetics. A higher µmax value was obtained compared to the control, which indicates that the stress of the yeasts during the pre-culture may influence later the fermentation kinetics of apple juice. These results concur with the results of Matsuura and coworkers, who observed a reduction of the fermentation time by 50% when using US (30 mW.cm -2 , 43 kHz) [START_REF] Matsuura | Acceleration of cell growth and ester formation by ultrasonic wave irradiation[END_REF]. Besides, an increase in the biomass production was reported for Kluyveromyces marxianus fermentation treated by 20 kHz sonication at an intensity of 11.8

W.cm -2 for a duty cycle less than 20% (a 10% duty cycle: 1 s of sonication followed by a pause of 10 s) [START_REF] Sulaiman | Ultrasound-assisted fermentation enhances bioethanol productivity[END_REF]. Furthermore, when the fermentation was treated during the Lag phase, a 1 h reduction of the Lag phase was observed by Jomdecha & Prateepasen [START_REF] Jomdecha | Effects of pulse ultrasonic irradiation on the lag phase of Saccharomyces cerevisiae growth[END_REF]. Finally, an ultrasonic treatment of 1 s after intermitting for 15 s during 30 min can stimulate the growth of S. cerevisiae by around 33.3%, and then the stationary phase was reached 4 h earlier compared to that of the control [START_REF] Lanchun | The research into the influence of low-intensity ultrasonic on the growth of S. cerevisiaes[END_REF][START_REF] Lanchun | The influence of low-intensity ultrasonic on some physiological characteristics of Saccharomyces cerevisiae[END_REF]. Besides, Yang et al. have reported that US treatment enhanced the growth of Brevibacterium sp. [START_REF] Yang | The ultrasonic effect on the mechanism of cholesterol oxidase production by Brevibacterium sp[END_REF].

Ethanol production and sugar consumption kinetics

In terms of ethanol production, results in figure 5.B show a decrease in the ethanol concentration by 0.41 and 0.55%, respectively for the treated pre-culture for 6 h, and the treated juice during the first 12 h, compared to the control. Besides, a decrease in the specific ethanol production rate γmax was observed in comparison with the control (Table 2). Figures

5.C and 5.D show an increase in glucose consumption while the same quantity of fructose

was used for the control and US-assisted fermentations. This result is also reflected by the yield values, showing that the ethanol yield production per gram of fructose was constant for all the fermentation processes, whereas the ethanol yield per gram of glucose decreased for the US-assisted fermentations (Table 2).

These results could be explained by the fact that the fastest biomass kinetic (juice treated by US during the first 12 h) could be associated with the lowest ethanol content (reduction of 0.55% (v,v) and ethanol production rate, and with the highest glucose consumption. The yeast cells seem to consume more glucose to produce more biomass and CO2 rather than producing ethanol. In fact, it was reported that one of the probable impacts of the US is improving the substrate consumption and oxygen uptake leading to a higher cell biomass concentration [START_REF] Huang | Effects of ultrasound on microbial growth and enzyme activity[END_REF][START_REF] Avhad | Ultrasound assisted production of a fibrinolytic enzyme in a bioreactor[END_REF][START_REF] Bochu | The influence of Ca 2+ on the proliferation of S. cerevisiae and low ultrasonic on the concentration of Ca 2+ in the S. cerevisiae cells[END_REF]. Some studies have also reported that US may enhance the enzyme activity [START_REF] Yeo | Effect of ultrasound on the growth of probiotics and bioconversion of isoflavones in prebiotic-supplemented soymilk[END_REF][START_REF] Nguyen | Effect of high intensity ultrasound on carbohydrate metabolism of bifidobacteria in milk fermentation[END_REF][START_REF] Avhad | Ultrasound assisted production of a fibrinolytic enzyme in a bioreactor[END_REF][START_REF] Guiseppi-Elie | Ultrasonic processing of enzymes: Effect on enzymatic activity of glucose oxidase[END_REF]. In fact, applying US causes a strong micro-convection in the medium, which may increase the mass transfer of substrates through the cell membrane, and alters the enzyme conformation and the enzyme-substrate affinity [START_REF] Pagnossa | Ultrasound: beneficial biotechnological aspects on microorganisms-mediated processes[END_REF]. Since higher concentration of substrate is accessible by cells, associated enzyme activities may be promoted, leading to higher glucose consumption and thereby higher biomass production. A thorough study of enzymes' kinetics could be of great interest for a better understanding of microbial stimulation by ultrasound.

It is important to note that different impacts of US on fermentation have been reported in the literature depending on the intensity, the duration of the treatment, and the type of microorganism being treated [START_REF] Galván-D'alessandro | Fermentation assisted by pulsed electric field and ultrasound: A review[END_REF][START_REF] Moncada | Effect of mild sonication conditions on the attributes of Lactobacillus delbrueckii ssp. bulgaricus LB-12[END_REF]. A recent study demonstrated a decrease in ethanol production for the fermentations assisted by US [START_REF] Huezo | Effects of ultrasound on fermentation of glucose to ethanol by Saccharomyces cerevisiae[END_REF]. A direct US treatment at 23 and 32

W.L -1 , or an indirect US (1.4 W.L -1 ) treatment had undesirable effects on S. cerevisiae activity and viability and induced a reduction in glucose consumption rates and ethanol production [START_REF] Huezo | Effects of ultrasound on fermentation of glucose to ethanol by Saccharomyces cerevisiae[END_REF]. Ojha and co-workers studied the impact of US on L. sakei, and revealed that several metabolic pathways were affected by US [START_REF] Ojha | Integrated phenotypicgenotypic approach to understand the influence of ultrasound on metabolic response of Lactobacillus sakei[END_REF]. Results also showed that the UStreated L. sakei had significant variations in nutrient consumption. The ultrasonicated cells preferred to consume other carbon sources in comparison with the untreated cells. These results may explain the enhancement in the glucose uptake for the US-treated cells in the current study (Figure 5.C). The metabolic pathway of Hanseniaspora sp. treated cells may be redirected to produce more biomass than to produce ethanol. Thus, the results obtained in the current study may highlight that US treatment of Hanseniaspora sp. cells could be a promising way to reduce the ethanol content in alcoholic beverages and to control the sugar/ethanol conversion rate. Particularly, US is classified as a technology that preserves the qualitative characteristics of the treated products, such as flavor, odor, and visual appearance.

However, different disadvantages were reported in some studies affecting the final product's texture [START_REF] Arvanitoyannis | Use of ultrasounds in the food industry-Methods and effects on quality, safety, and organoleptic characteristics of foods: A review[END_REF]. In the current study, organoleptic analyses are required in order to evaluate the volatile organic compounds and the aromatic profile of the untreated and US-treated ciders.

Conclusions

In this work, the effects of US treatment on Hanseniaspora sp. yeast cells were first studied and optimized using synthetic medium. For an effective treatment time of 1662 s, applied during both the Lag and Log phases (6 h) for pulse duration of 0.5 s followed by 6 s pause, the yeast concentration and the biomass yield increased significantly. The depletion of glucose was also significantly faster. For this short period of US exposure, the cells were more sensitive to the treatment during the Lag phase rather than during the Log phase. The yeast concentration and the biomass yield increased significantly during the Lag phase.

Afterward, the impact of US treatment on Hanseniaspora sp. cells was studied during the apple juice fermentation. The yeast concentration and the biomass yield increased significantly when the cells were treated during the first 12 h of the fermentation. Besides, the ethanol content was reduced by 0.55% (v/v) and the biomass production rate was 10 folds higher than the control. Furthermore, the yeast concentration and the biomass yield increased significantly for the US-treated cells.

The obtained results demonstrate the potential for the practical implementation of the US fermentation technology to reduce the ethanol content during the fermentation processes and especially using Hanseniaspora sp. yeast cells. Nevertheless, further investigations are required to understand the mechanisms behind microbial stimulation and ethanol reduction. Tables Table 1. Energy consumptions, and fermentation process performances (Y (g biomass. g glucose -1 ), µmax) during US treatment. 0.5 s pulse / 6 s pause denotes applying US pulses of 0.5 s followed by a pause period of 6 s.

W (J.mL -

Figures 3 .

 3 Figures 3.C and 3.D present respectively the evolution of glucose concentrations and

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Experimental set-up for the optimization of US-assisted fermentation of

Figure 3 .

 3 Figure 3. A. Growth kinetics, B. Optical densities at 600 nm, C. Glucose consumption

Figure 4 .

 4 Figure 4. A. Growth kinetics, B. Optical density measurements at 600 nm, C. Glucose

Figure 5 .

 5 Figure 5. A. Growth kinetics, B. Ethanol production kinetics, C. Glucose consumption

Figures Figure 1 .

 1 Figures

Figure 2

 2 Figure 2.

Table 2 .

 2 Fermentation process performances, and energy consumptions for the control and US-assisted fermentations.

		1 )	µmax (h -1 )	Y (g biomass . g
				glucose	-1 )
	Untreated	-	0.39 ±0.004	0.08 ± 0.003
		US treatment during both Lag and Log phases (6 h)
	0.5 s pulse / 6 s	155 ± 3	0.54 ± 0.01	0.15 ± 0.002
	pause
	1 s pulse / 6 s	197 ± 5	0.56 ± 0.003	0.14 ± 0.001
	pause
	2 s pulse / 6 s	269 ± 7	0.43 ± 0.002	0.12 ± 0.003
	pause
		US treatment (0.5 pulse/6 s pause) during either Lag
			or Log phases (3 h)
	Lag phase	79 ± 1.5	0.50 ± 0.005	0.12 ± 0.002
	treated
	Log phase	77± 1.4	0.48 ± 0.01	0.12 ± 0.004
	treated

Acknowledgments

The authors wish to thank the Research council of Saint-Joseph University of Beirut, Project FS103 and the Lebanese National Council for Scientific research (CNRS-FS129).

Conflict of interest

The authors declare no conflict of interest