Laurent Poughon

Céline Laroche

Catherine Creuly

Claude-Gilles Dussap

Christel Paille

Christophe Lasseur

Pieter Monsieurs

Wietse Heylen

Ilse Coninx

Felice Mastroleo

Natalie Leys

limnospira indica pcc8005 growth in photobioreactor: model and simulation of the iss and ground experiments

Keywords: MELiSSA, Limnospira indica PCC 8005, Photobioreactors, Model, ISS Flight experiment, Microgravity

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

Introduction

Long-term space missions (e.g., to Mars), including the establishment of a long-term, manned base, imply the development of a reliable life-support system including food supply and waste management. For far and long duration space missions, supplying all food, oxygen, and water from Earth will result in a tremendous weight and cost. Therefore, space life-support systems have to become increasingly regenerative. Food production can only be achieved by biological means, and the introduction of biological techniques opens a new set of solutions for other life-support requirements such as atmosphere, water, and waste management. For the last 30 years, the MELiSSA (Micro-Ecological Life-Support System Alternative) project, inspired by an aquatic ecosystem, has been set up to be a model for the study of regenerative life-support systems for long-term space missions [START_REF] Hendrickx | Microbial ecology of the closed artificial ecosystem MELiSSA (Micro-Ecological Life Support System Alternative): Reinventing and compartmentalizing the Earth's food and oxygen regeneration system for long-haul space exploration missions[END_REF][START_REF] Lasseur | MELiSSA: The European project of Closed Life Support System[END_REF][START_REF] Walker | MELiSSA the minimal biosphere: Human life, waste and refuge in deep space[END_REF]. MELiSSA has six major compartments (C1-C5) or subsystems, coupled together in a closed circulatory loop. Respectively, it involves anaerobic thermophilic bacteria (C1), photo-heterotrophic bacteria (C2) for waste degradation, nitrifying bacteria for ammonia and urine treatment (C3), photosynthetic cyanobacteria (C4a) and higher plants (C4b) for air revitalisation to supply the crew compartment (C5) with food. MELiSSA project is organized in five phases. Phase 1 is for basic research and development, phase 2 is focused on testing the processes in space conditions via space flight experiments, phase 3 concerns ground demonstrations of the MELiSSA loop and is mainly represented by the MELiSSA Pilot Plant [START_REF] Godia | MELiSSA Pilot Plant: A facility for ground demonstration of a closed life support system[END_REF], phase 4 is for technology transfer and phase 5 is for education and communication. Although the effects of space microgravity and its ground analogues on microorganisms have been studied for more than 50 years, conflicting and diverse results have been reported from different experiments, especially regarding microbial growth and secondary metabolism [START_REF] Huang | Effects of spaceflight and simulated microgravity on microbial growth and secondary metabolism[END_REF]. Microgravity has a significant impact on microorganisms in liquid culture [START_REF] Walther | Cultivation of Saccharomyces cerevisiae in a bioreactor in microgravity[END_REF][START_REF] Kacena | Bacterial growth in space flight: logistic growth curve parameters for Escherichia coli and Bacillus subtilis[END_REF]. Gravity affects a microbial (bioreactor) culture at two main scales: (i) it has a direct physical effect on the gas/liquid transfer, and more generally on fluid dynamics and mixing of bioreactor; and (ii) it affects the physiology and metabolism of microorganisms themselves. Several effects have been observed in microgravity [START_REF] Hendrickx | A Global Approach to Asses Stress Response of the Bioregenerative Life Support System Organism Rhodospirillum rubrum Under Space-Flight Related Environmental Conditions[END_REF][START_REF] Mastroleo | Low Earth orbit journey and ground simulations studies point out metabolic changes in the ESA life support organism Rhodospirillum rubrum[END_REF], 2009), but the responses are depending on the growth media and growth conditions used. Therefore, it is still difficult to know if the effects observed are a direct consequence of the microgravity on the cells or a cellular response to other environmental changes due to microgravity (such as partial pressure gradients of dissolved gas, inhomogeneous compounds distribution in fluids, etc.). Moreover, in space flight conditions, the radiation effects must be added to microgravity effects being even more important than the microgravity effects [START_REF] Mastroleo | Experimental design and environmental parameters affect Rhodospirillum rubrum S1H response to space flight[END_REF]. Niederwieser et al. (2018 b ) have reviewed the algal research in space, concluding that even though a large number of spaceflight experiments were conducted (mainly with Chlorella sp.), data are still quite limited, and no experiments have been successfully reported in-flight growth rate of algae. So, studies on the effects of microgravity in space on microbial growth and metabolism are still challenging both in terms of technology for bioreactor operation and control in microgravity and in terms of understanding specific cause-and-effect mechanisms of microbial responses to microgravity.

In the past decade several MELiSSA-phase 2 spaceflights microbial experiments were carried out: MESSAGE1 (2002), MESSAGE2 (2003), MOBILISATSIA (2004), BASE-A (2006), BASE-B/C/D (2008) [START_REF] Lasseur | MELiSSA: The European project of Closed Life Support System[END_REF]. These experiments were focused on gathering knowledge about the bacteria used in the MELiSSA loop or in closed life-support systems at the global level, including study of genomic, transcriptomic, and proteomic data to know more about their response to spaceflight conditions with special attention to cosmic radiation, and microgravity. MELiSSA-phase 2 is also focusing on demonstration of technology, concept and validation of mathematical models via projects such as BIORAT (a very simplified ecosystem reduced to gas exchange between a photo-bioreactor and a consumer compartment) or ArtEMISS (Arthrospira sp. gene Expression and mathematical Modelling on cultures grown in the International Space Station).

Arthrospira-B was the spaceflight experiment on ISS of the ArtEMISS project with the objective to determine the effect of space conditions, including reduced gravity and increased radiation, on Limnospira indica PCC8005 bacterial morphology, physiology, and metabolism, and how this influences the bioprocesses in the photobioreactor. The cyanobacteria Limnospira indica PCC8005 (also known as Arthrospira or spirulina) is a candidate for use in spacecraft biological life support systems, for carbon dioxide (CO2) and nitrate (NO3 -) removal, as well as oxygen (O2) and edible biomass production. The strain was selected for compartment C4a of the MELiSSA Biological Life Support System (BLSS). To ensure the reliability of such a BLSS it is necessary to characterize the response of Limnospira indica PCC8005 to in situ spaceflight conditions. The concept and demonstration of the space compatible membrane photobioreactor was first developed in early 2000 (Cogne et al, 2003 a, b ; Cogne et al., 2005). The ground demonstrator was designed in order to match two constraints: (i) to be adaptable to altered gravity conditions, ensuring gas / liquid exchanges through a membrane without any gas bubble exchange; (ii) to permit remote online monitoring of oxygen production rates by pressure increase measurement, enabling a direct assessment of both metabolic rates and biomass growth and allowing much more sensitive bioprocess analysis than classical end-of-point results. It must be outlined that Arthrospira-B is the first space flight experiment targeting a direct and on-line measurement of rates (oxygen production rate) offering a new and insightful investigation on time derivatives variables instead of integral variables such as biomass concentration permitting the assessment of microgravity effects onto the behaviour of cellular metabolism.

Arthrospira-B experiment was launched on December 15 th 2017 on board the SpaceX CRS-13 / Falcon 9 rocket for a 5 weeks experiment operated within the Biolab facility system of the Columbus module in ISS. This experiment was the first of its kind, having an active microbial bioreactor activated and running in space, with on-line remote monitoring of the bioprocess.

Although it is only a small scale and short duration pilot test, it is the first step for the development of future bio-based life support systems to enable sustainable human presence in space. Experimental device and samples collected during the spaceflight were recovered from the ISS for further analysis (genomic / transcriptomic proteomic / metabolomic analysis) four months after the launch (SpaceX CRS-14, May 5 th 2018).

The present paper presents the model that was used to monitor and predict the outcome of the bioprocess. The experimental total pressure data recorded online during the spaceflight experiment are also discussed, and their analysis versus (i) the same experiment performed in ground condition, and (ii) the simulation results using the predictive metabolic model of the behaviour of the cyanobacteria in the photobioreactor are presented. The analysis of biological, biochemical and biomolecular results collected during the experiment will be presented in a further paper.

Materials and methods

Strain and culture medium

The strain used is Limnospira indica PCC 8005, a gram-negative photosynthetic cyanobacterium, from the "Pasteur Culture Collection" (PCC), which was cultivated and maintained in axenic conditions. Recently, taxonomic classification of cyanobacteria has been revised and a new genus, Limnospira, and species indica has been established in place of the former Arthrospira sp. PCC 8005 [START_REF] Nowicka-Krawczyk | Detailed characterization of the Arthrospira type species separating commercially grown taxa into the new genus Limnospira (Cyanobacteria)[END_REF]. The culture medium is the Zarrouk medium [START_REF] Zarrouk | Contribution à l'étude d'une cyanophycée: influence de divers facteurs physiques et chimiques sur la croissance et la photosynthèse de Spirulina maxima[END_REF] modified by Cogne et al. (2003 c ), adjusted to pH 9.5 with a carbonate buffer: K2HPO4 0.5 g.L -1 ,MgSO4.7H2O 0.08 g.L -1 ,ZnSO4.7H2O 0.11 g.L -1 ,NaHCO3 10.5 g.L -1 ,NaNO3 2.5 g.L -1 ,CuSO4.5H2O 0.03 g.L -1 ,Na2CO3 7.6 g.L -1 ,EDTA 0.08 g.L -1 ,FeSO4.7H2O 0.01 g.L -1 ,CaCl2 0.03 g.L -1 ,K2SO4 1 g.L -1 ,MnCl2.4H2O 0.23 g.L -1 ,NaCl 1 g.L -1 . The bioreactors were sterilized and aseptically assembled before launch. All the inoculums (for space and ground experiments) came from the same preculture medium in order to prevent any physiological state difference. Cells (5mL at ~1.3g/L) were maintained in a separate compartment in the dark and at 4°C during launch. For inoculation, this small volume is injected in the culture chamber containing the growth medium and considering a 10-fold dilution. The culture was then run inside the reactor hermetically sealed from the outside atmosphere and all nutrients for growth were provided via the liquid Zarrouk medium, including dissolved CO2 and bicarbonate HCO3 -as CO2 source.

Photobioreactor and hardware

Four reactors on ground and four reactors on-board ISS were used in parallel to test the behaviour of four independent bacterial cultures (4 independent biological replicates). It was important to assess the reproducibility of the results in independent bacterial cultures, as cultures originating initially from the same inoculum, can nevertheless evolve differently over multiple generations. Each reactor was placed in a separate Integrated Experimental Container (IEC) (size 110x140x150mm, volume 2.2 L, mass 4.7 kg) designed and built by QinetiQ Space NV (Belgium). On-board ISS, the IEC was installed and operated within the Biolab facility in the European Columbus laboratory, providing IEC with power, atmosphere ventilation, as well temperature control via the Biolab thermal control unit. The two first reactors were installed on rotor A of Biolab on position A1 and A4 respectively while the two others were installed on rotor B on position B1 and B4. The two Biolab rotors had a separate atmosphere ventilation system but reactors on the same rotor share the same ventilation system. The IEC hardware (Figure 1) contained the following assemblies: the experiments container, the electronics assembly and the experimental unique equipment (EUE) (culture chamber with membrane and mixing unit, pump unit and liquid loop with 3-way valves, optical measurement unit, Zarrouk medium reservoir, sample reservoir and RNAlater® fixative reservoir for sample fixation).

The culture chamber (Figure 2) is the photobioreactor part of the EUE and contained: the liquid compartment for the growth of the cyanobacteria in a light transparent inflatable culture bag (51 mL); a gas permeable membrane (hydrophobic PTFE membrane of 57 ± 1 micron thickness; Sartorius-Sedim 11807 pore-size 0.2 micron); a magnetic stirrer sitting on top of the membrane and kept in place by the stirrer bar holder; a gas compartment (24 mL); a polycarbonate transparent windows and a LED PCB for illumination of the liquid compartment (36 LEDs delivering a continuous light intensity ranging between 20 and 50 µmol.m -2 .s -1 in the photosynthetic active range of 400-700nm; NICHIA NESL064AT). A cartridge solenoid valve (Staiger VA 204-715) vented the gas compartment inside the IEC, which was connected to the Biolab ventilation system (or ambient air for ground experiments). Venting occurred every 24h and/or if the pressure that is measured by the pressure sensor has risen above the threshold of 1.25 bar. During each venting cycle, air is flushed several seconds to ensure that air in gas chamber is replaced by ambient air. Temperature of the liquid chamber was recorded (44007RC Precision Epoxy NTC Thermistor) and was controlled with Biolab thermal control unit to obtain a temperature of 33°C ± 1°C in the bioreactor during the operational phase of the experiment.

The Optical Measurement Unit (OMU) is composed of a cell density measurement instrument (a spectrometer measuring light absorbance at 790 nm, 632 nm and 468 nm) and a photosynthetic activity measurement instrument (a miniaturized pulsed amplitude modulated (PAM) fluorometry measurement instrument) (Gademan Instrument/Heinz Walz GmbH). This OMU was placed on the external liquid loop (VLL=9mL), in which continuous biomass circulation was achieved by a peristaltic pump at an average flow rate of 1mL.min -1 . Each 12h, the circulation was stopped during 20 minutes to perform optical density and fluorimetry measurements on cells adapted to dark conditions.

Arthrospira-B experiment, timeline and data acquisition

The inoculum culture was dormant stored in the dark at 4°C in spent medium, in the liquid loop of the bioreactor in the IEC, during upload and on-board ISS storage; and reactivated by moving and mixing it with fresh Zarrouk medium in the culture chamber at the start of the bioreactor experiment in the Biolab facility. Cultures were initiated at a fixed cell (OD 790nm = 0.1) and medium composition (Modified Zarrouk at pH 9.5) and allowed to proliferate at a fixed light intensity (35 or 45 µmol.m -2 .s -1 ) and temperature (33.4 °C). During each batch, the liquid phase was circulating in the illuminated and stirred culture chamber (Vcc = 51mL) and in the non-illuminated liquid loop (VLL = 9mL) at an average flow rate of 1mL.min -1 allowing to assume a perfect mixing of the full liquid volume (60mL). The cyanobacterium was grown in a batch regime at conditions maintaining exponential cell proliferation during 1 week, then emptying the culture chamber and using the biomass remaining in the circulation loop (VLL = 9mL) to start the next batch. The manual replacement of the Zarrouk medium reservoir (90 mL) by the crew for 4 times, allowed for 4 successive batches to be run. This experiment allowed sufficiently large sample volumes (15 mL), to be taken at the end of each batch using RNAlater® to stabilize and protect cellular DNA, RNA and proteins of the sample for the further biomolecular analysis after the return of the experiment on Earth. The accumulative total radiation dose could be measured by means of dosimeters (luminescent detectors, type TLD-OSLD) and analysed also after the recovery of the experiment. Automatic measurements of temperature, gas pressure, optical density, fluorometry were online acquired during the experiment: every second for pressure and temperature, twice by day for optical measurements. The biomass concentration was directly correlated to the optical density. The fluorescence was recorded to calculate quantum yield. The gas pressure increase is the result of the oxygen production by Limnospira indica PCC 8005, so that online gas pressure measurement permits to calculate the instantaneous oxygen production rate rO2, and the total (or cumulated) oxygen produced (assuming no gas accumulation in the liquid). Cogne et al (2003 c ) have shown that the oxygen production measured by this way is consistent with the stoichiometry of the growth.

= . and = .

. where = .

In batch, the oxygen produced is linked to the biomass produced, by the YO2/X yield:

= /! . " and = /! #$ -$ 0 '
The ISS flight experiment (reactors A1, A4, B1, B4) was reproduced on ground at the same time with the same hardware (i.e. ground reactors GM1, GM2, GM3, GM4). Each of the reactors was run following the same timeline and the same incubation conditions. During the experiment, 4 batches were run and for each batch the timeline was decomposed in several stages: batch incubation (Batch1: 14d; Batch 2: 6d; Batch 3: 8d; Batch 4: 6d); automated sampling (30') followed by manual reservoir replacement by the crew (2h). and Algebraic Equations simultaneously using Matlab® R2015a ODE15s solver.

Radiative transfer (light) model

Considering the culture is heterogeneous in terms of light availability, it is treated as a system with uniformly distributed parameters along its depth (z). To determine the attenuation of light inside a photosynthetic culture, a radiative-transfer model designed for rectangular reactors lightened on one side, was adopted. This model will determine the value of irradiance, G, for any depth, z of the culture, following the two-flux model analytical solution of the dimensionless Eq. 1, [START_REF] Cornet | A Simple and reliable formula for assessment of maximum volumetric productivities in photobioreactors[END_REF] that has already been proved efficient in several studies [START_REF] Cogne | Design, Operation, and Modeling of a Membrane Photobioreactor to Study the Growth of the Cyanobacterium Arthrospira platensis in Space Conditions[END_REF][START_REF] Ifrim | Dynamic pH model for autotrophic growth of microalgae in photobioreactor: A tool for monitoring and control purposes[END_REF][START_REF] Pruvost | Chapter Five -Industrial Photobioreactors and Scale-Up Concepts[END_REF]:

( ) * + = 2 - ./
./0 1 0/2 3 4 56) 7 072 3 64 56) 0/2 8 3 45 7 072 8 3 645 experimentally by spectrophotometric tools using an integrating sphere. For the current study, collimated intensity is assumed, so that Eq.1 is used considering n = ∞.

The light availability is defined by the lightened liquid volume fraction:

B = CDE -1 , H + H 1 ,
where L0 is the depth for which light intensity is equal to the light compensation point; B represents the illuminated fraction of the reactor and depends on the incident light intensity and on the biomass concentration (Figure 5a).

Biological model

The biological model is composed of the growth kinetic model, accounting for the photosynthetic activity of the cells, and for the stoichiometry of the photosynthetic growth leading to the relationships between the compounds (nutrients and biomass) consumption and production rates.

Stoichiometry of the photosynthetic growth is summarized into a single mass and elemental (C,H,O,N,S,P) balanced equation (Eq. 2) where xEPS is the molar fraction of exopolysaccharide in the total biomass [START_REF] Dauchet | Chapter One -Photobioreactor Modeling and Radiative Transfer Analysis for Engineering Purposes[END_REF]: This overall equation was obtained from a structured approach of the metabolism of Limnospira indica PCC 8005 including anabolic reactions rates coupled to the rate of chemical energy carriers (JATP) and of reduced power cofactors (Jcov) driven by photosynthesis (Z scheme) [START_REF] Cornet | Kinetics and energetics of photosynthetic microorganisms in photobioreactors[END_REF]. It was demonstrated [START_REF] Dauchet | Chapter One -Photobioreactor Modeling and Radiative Transfer Analysis for Engineering Purposes[END_REF] that the energy transducing process can be described by the ratio (JATP)/(Jcov), which is termed the P/2e -ratio, and thus can be handled with the theory of the linear energy converter in terms of Linear Thermodynamics of Irreversible Processes (LTIP) as:

I + 0.
3 6 = 0/ .]0" .]0 .]0/"
x is obtained as the solution of the following second order equation: The mass fraction of exopolysaccharide that is predicted using the LTIP approach (Figure 5b) is consistent with experimental observations and metabolic analysis of Limnospira indica PCC 8005 growth for different light incident fluxes conditions (Cogne et al. 2003 a ). For P/2evalues greater than 1.5, the light energy absorbed by the microorganisms is above their metabolic capacities and can lead to physiological limitations. Therefore the working conditions for this micro PBR were fixed at a light incident flux q0 of 7.6 or 9.8 W.m -2 , which guaranties never to be in such light inhibiting conditions, even at start-up with a low biomass concentration (Figure 5b). In these conditions the mass fraction of exopolysaccharide in the total biomass was always maintained below 35%, reducing the problematic of cell aggregation and biofilm formation [START_REF] Cornet | Kinetics and energetics of photosynthetic microorganisms in photobioreactors[END_REF][START_REF] Cornet | A Simple and reliable formula for assessment of maximum volumetric productivities in photobioreactors[END_REF].

The respiration of Limnospira indica PCC 8005 in the dark was assumed to be negligible for a perfectly mixed illuminated bioreactor with a low residence time of the liquid medium in the dark zone considering light inhibition of respiration in the illuminated zone. The kinetic of growth is equal to the average photosynthetic growth rate associated to Eq. 2, <rx>, calculated for the full liquid volume of the reactor by integrating the local light flux Gz along the culture depth [START_REF] Cornet | Kinetics and energetics of photosynthetic microorganisms in photobioreactors[END_REF][START_REF] Cornet | A Simple and reliable formula for assessment of maximum volumetric productivities in photobioreactors[END_REF]:

〈 " 〉 = 1 -h i j Φ l mn $ 1 o p q c l + q c e H
h is the design dark fraction of the reactor, i j the maximum energetic yield for photon conversion, Φ the mass quantum yield for the Z-scheme of photosynthesis, l the half saturation constant for photosynthesis. The kinetic rate of a compound (i) involved in the mass balanced growth equation Eq. 2 is calculated:

〈 r 〉 = r/" 〈 " 〉 r/" is the mass yield g(i). gX -1 calculated accordingly to the stoichiometric coefficients of Eq.

2.

The carbon source for the growth in Eq. 2 is CO2, but in practice both dissolved CO2 and HCO3 -support photosynthesis. On ground, in a pH-controlled reactor, the carbon source for cyanobacteria growing at pH 9 +/-1 remains mainly in the bicarbonate HCO3 -form.

Therefore classical models neglect the issue of carbon limitation; carbon is either considered as Total Inorganic Carbon (TIC) or HCO3 -. For the current study, pH was not controlled leading to pH increase above 10 in the culture medium and to a potential C limitation. A Monod limiting term limC was introduced in the biomass growth kinetic equation for the carbon source considered (HCO3 -): limC = HCO3 -/(KC+HCO3 -), with KC the half saturation constant for HCO3 -. Because of intracellular bicarbonate accumulation mechanism [START_REF] Giordano | CO2 CONCENTRATING MECHANISMS IN ALGAE: Mechanisms, Environmental Modulation, and Evolution[END_REF]) and the influence of both light limitation and pH, it was difficult to get reliable values for Kc from literature data. Most of values for cyanobacteria are ranging between 10 -2 -10 -4 mol C.L -1 . For the current study a value of 5 10 -3 mol HCO3 -.L -1 was used.

Liquid model and pH model

The evolution of compounds concentration in the liquid phase for the micro PBR operated in batch was expressed in term of mass balance equation for a perfectly mixed reactor:

D = 〈 r 〉 -(H r
(H r is the gas-liquid transfer rate for the compound (i) through the membrane expressed in same unit as 〈 r 〉. Considering the PTFE hydrophobic membrane, this term was relevant only for molecular CO2 and O2 and was equal to zero for other compounds.

The CO2 required for the growth (fixed by RubisCo) was obtained via dehydration of the bicarbonate inside the cell (HCO3 -<-> CO2aq + OH -), a reaction that releases hydroxyl ions (OH -) causing an increase in pH for the pH-uncontrolled micro PBR. pH affects both the growth and the relative distribution of ionic species. A model predicting pH evolution as a function of net CO2 uptake was used. The model was first described by [START_REF] Achard | Prediction of pH in complex aqueous mixtures using a group-contribution method[END_REF] and used by Cogne et al (2003 a ) and for the prediction of pH in a toric PBR with pH control by periodic gas CO2 pulses [START_REF] Ifrim | Dynamic pH model for autotrophic growth of microalgae in photobioreactor: A tool for monitoring and control purposes[END_REF]. The pH model results in a multi-solute system of 10 distinct species: H3PO4, H2PO4 -, HPO4 2-, PO4 3-, CO2, HCO3 -CO3 2-, H2O, OH -and H + , linked by chemical equilibrium relations. The chemical equilibrium relations were expressed as the one for water: l s = n t/ + n t7 /n s , where n r = B r u r is the activity for a compound (i) ,

B r being the molar activity coefficient based on concentration and u r the compound concentration. Activities were calculated by the model of Pitzer [START_REF] Edwards | Vapor-liquid equilibria in multicomponent aqueous solutions of volatile weak electrolytes[END_REF].

Finally, the thermodynamic model for calculating the chemical equilibrium of species in the liquid phase was composed of 19 unknown (concentrations and activities) and solved using 19 relations (10 Pitzer relations for activities, 6 chemical equilibrium relations, 2 compounds species mass balances, 1 electroneutrality relation). The pH itself was calculated as vP = -wxy P /

Gas pressure model and gas/liquid equilibrium

The evolution of partial pressure of CO2 and O2 in the micro PBR gas compartment is expressed as:

r = z { | (} 10 7S | H (H r
(H r is in units of mol(i).L -1 .h -1 ; the volume is expressed in L and pressures in Pa. Total pressure was obtained as sum of partial pressures.

The gas transfer rate through the membrane was calculated as:

(H r = ~ • r | H P r u r -r
With ~ the membrane surface (m 2 ), • r the membrane permeability for (i) in mol.Pa -1 .m -2 .h -1 , P r the Henry constant for (i) in Pa.L.mol -1 .

Results and discussion

4.1 Experimental results: Ground and ISS Figure 6 presents the results for the 4 ISS reactors namely A1, B1, A4, B4. The pressure of the gas compartment was recorded online (Figure 6). The experiment was designed in order that all four reactors were operated in same conditions, so that the same results were expected.

Due to experimental and technical issues, this was not always the case. For the first batch (culture start up) only the reactor B4 gave the expected biomass production and pressure profile with a pressure increase up to the relieve set point of 1.25 bar. Reactor A1 had a lower pressure increase than expected in batch 1, 2 and 3, but suddenly recovered the expected pressure profile at the end of batch 3 and for the entire batch 4. Reactor B1 had never start (no biomass growth and no pressure increase) and was stopped after 21 days.

Reactor A4 showed no measurable biomass production for the start up first batch. The growth started at the end of batch 2 with a low-pressure increase profile in the last 2 batches (number 3 and 4), which looks like the pressure profile of reactor A1-batch1 but with a crushed shape. Inspection of the reactor after its return on Earth indicated a pressure leak from 1.15 bar. The biomass profiles of reactor A4 -batches3, 4-were similar to the A1-batch1. For reactor B4, the growth stopped after 290h, and biomass decay was then observed. Medium renewal for the batch 2 did not permit to restore the culture. No gas production was recorded potentially due to a malfunctioning pressure sensor or of the pressure release valve.

The online monitoring data of the 4 ground reactors, namely GM1, GM2, GM3 and GM4 are presented in Figure 7. Only pressure results are presented; fluorescence measurements require about 20 min, so that on ground biomass sedimentation occurs leading to a nonreliable value for OD. Several issues when running occurred also for ground experiments.

Batch 1 for GM1 and GM2 had to be stopped due to a liquid leak that required repair. The 2 reactors were restarted for batch2. After their restart, reactors GM1 and GM2 had a similar pressure profile (batches 2 and 3). GM3 showed a low gas production few days after its start-up. A stirring failure was identified when visually inspecting the reactor that has been confirmed after dismantling the reactor after the experiment. GM3 reactor was then ran with mixing of the liquid compartment only by the way of the external liquid loop (1 mL.min - 1 ), leading to heterogeneity of the liquid phase and gas transfer limitation in liquid boundary layer surrounding the membrane surface. GM3 reactor was kept in this degraded mode and is further used as a reference to characterize a reactor with stirring difficulties. GM4 reactor had presented no technical failures, but like for the B4 ISS reactor at the end of the batch 1, the biomass and oxygen production suddenly decreased, and could only be recovered in batch 4. As the growth decrease was not observed when restarting GM1 and GM2 (batch 2,3,4), the interruption of GM4 and B4 were interpreted as linked to the long batch duration (14 days for batch 1, versus 7 days for other batches), which has led to toxic high pH and/or carbon substrate limitation.

For each reactor, analysis of pressure and biomass (Figure 8) allowed to calculate the oxygen production rate and yield /! (Table 1). Theoretically /! is constant for a constant light flux and is predicted equal to 1.9 gO2.gX -1 by the stoichiometric equation. For A1-batch4 and B4-batch1 the calculated oxygen production yield /! was close to the theoretical one, taking into account the experimental gross errors in calculations due to the OD to dry mass conversions and the small volumes of the reactors. The values in the order of 0.5 gO2.gX -1 for A1-batches 1-3 were too low when compared to stoichiometric theoretical value, suggesting either a pressure problem (such as gas leakage or pressure sensor malfunction) or a biomass overestimation. Considering the results of ISS A1 batch 1-3 until day 27 (~650h) are similar to the results obtained for on-ground GM3 reactor that has been identified as a reactor that has encountered a stirrer issue, it is concluded that the same difficulty has occurred on the ISS reactor. A normal stirring has been recovered for ISS A1 batch 4 indicating that the stirrer has recovered a normal functioning, showing the reversibility of the malfunction as it has been often observed on ground for this case of dysfunction.

The experiment was designed as a 4 reactors -4 batches/reactors for both ISS and ground.

Except the initial biomass concentration, which could not be strictly controlled, impacting the early growth phase (lag phase and adaptation phase), all reactors were operated in same conditions (temperature, light, reactor design). So ideally, the 16 batches were planned to be 16 replications that can be compared between ISS and ground. In a worst case, knowing that at least for the ISS it was not possible to correct a technical issue, it was expected that relevant results could be obtained for some batches. Over the 32 batches, 3 gave the expected oxygen production rate of 0.33 mmol O2.L -1 .h -1 and 7 a production rate of 0.23 +-0.03 mmol O2.L -1 .h -1 (Table 1). For 6 batches the oxygen production rate calculated was 0.1 +/-0.03 mmol O2.L -1 .h -1 . 16 batches have failed (either technical or inoculation defaults) and no pressure build-up could be used. The cumulative pressure was a good way to compare the different batches (Figure 9) and to discriminate reactors with low oxygen production.

For batches with 0.1 +/-0.03 mmol O2.L -1 .h -1 (A1-b1-b2-b3, GM1-b4, GM3-b2-b3), the calculated growth rate is lower by a factor of 3 than the normal growth rate with the applied light energy transfer conditions. It has been observed on-ground (GM1-b4, GM3-b2-b3) that such a functioning was characteristic of a dysfunction of the magnetic stirrer leading to degraded O2 transfer from the liquid phase to the gas phase leading to a high O2 gas partial pressure into the liquid phase. A low production rate of 0.1 mmol O2.L -1 .h -1 is characteristic of limiting O2 transfer between liquid and gas coming from a stirring issue. This behaviour has been observed both on ground and on ISS, leading to higher oxygen dissolved concentrations into the liquid phase, therefore inhibiting biomass growth. Moreover, the sudden re-increase in O2 production rate in ISS reactor A1-b3-b4 confirms this explanation and was interpreted as an unblocking of the stirrer.

One ISS batch (A1-b4) can be compared to the ground batches GM4-b1 and GM4-b4 for the maximal production rates obtained of 0.33 mmol O2.L -1 .h -1 . Even considering that the number of replications is not sufficient to draw firm conclusions, it can be claimed that beyond technical issues it is possible to achieve similar productivities and rates of production biomass and oxygen on ground and in microgravity.

The last group of batches with an intermediate calculated productivity of 0.23 +-0.03 mmol O2.L -1 .h -1 lower than the nominal productivity (B4-b1, GM1-b2-b3, GM2-b2-b3-b4, GM3-b1)

was mainly observed on ground experiments. 9a). Reactors GM4-b1 and GM3-b1 are taken as reference experiments. As can be seen in Figure10, the model fits quite satisfactorily the experimental results for GM4-batch1, without any fitting or adjustment of the model parameters. This confirms the predictability of the model, which was already applied for various ground photobioreactors (Cogne et al., 2003 b , Cornet et al., 2009, Ifrim et al., 2014, Dauchet et al., 2016). The decrease of the growth is linked to a carbon limitation (bicarbonate in the model) due to the high pH value reached after 250h of culture. It has been calculated that the growth stop near 300h happens for a pH of 11.5, what was consistent with [START_REF] Pandey | Standardization of pH and Light Intensity for the Biomass Production of Spirulina platensis[END_REF] who has observed a 50% decrease in pigments at pH 10 and 11, and no growth at pH 12 in flask cultures. Decrease of pigments content associated to metabolic and cell damage for pH above 11-11.5 was probably the reason why even after medium replacement for the batch 2, the culture could not be rescued.

The experimental results of GM3-b1 do not fit satisfactorily with the model without any parameter adjustment (Figure 11). The calculated O2 productivity is lower than for GM4-b1

(Table 1). This behaviour could be explained either by a non-perfectly mixed liquid phase (GM3 had indeed issues with the magnetic stirrer) and/or by the presence of a gas bubble into the liquid, leading to an increased gas buffer volume and consequently to cushion the pressure increase. Knowing that the model parameters were not adapted for a non-perfectly mixed liquid volume, the model was further tuned (Figure 11) by considering a 5 mL gas bubble into the liquid phase resulting into a good fit of the experimental cumulative pressure with the model. This confirms that the presence of gas bubbles in the liquid compartment can lead to small changes in the gas / liquid volume ratio, which in turn can that have a significant effect onto the gas pressure evolution.

Reactor B4-b1 and A1-b4 are the only 2 ISS reactors with a behaviour suggesting no technical problems related to mixing or to pressure measurement. A1-b4 showed exactly the same trend as GM4-b1. The theoretical model represents quite satisfactorily the behaviour of the last batch of this reactor. B4-b1 showed the same trend as the GM3-b11 reactor and again, the tuned model can be used to represent the ISS reactor (Figure 12). The pressure profile was better predicted than for ground experiment because environmental parameters (pressure, humidity, composition) on-board ISS and of Biolab facility were better controlled than for the ground experiments. Again, the model failed to correctly represent the experimental results when pH has reached high values. After a threshold pH predicted at 11.3, the growth rate presents a sharp decrease. For ISS reactor, the limiting effect of bicarbonate availability at high pH was a bit lower than on ground, so that the culture was maintained only in light-limited conditions until the pH threshold was observed. This is explained by the fact the carbon limitation happens a bit later due to the higher CO2 fraction in the gas flushing the reactor (ground air is 0.04% while ISS air is 0.4%). A better fit is obtained by reducing the half saturation constant for bicarbonate in the model. The mechanistic interpretation shows that gas-liquid transfer of CO2 through the membrane involves a non-negligible transfer resistance of molecular CO2 from gas to liquid (at pH higher than 9.5). Considering that for ISS reactors pCO2 is 10 times higher than on ground conditions, the appearance of the gas transfer resistance limitation effect is therefore significantly hindered. The model of carbon uptake considers that bicarbonate is the main carbon source. However at the higher pH values (higher than 11) molecular CO2 that is the chemical species undertaken by RUBISCO complex must be considered as the main carbon source. Therefore higher pCO2 on ISS has a tendency to reduce the limitation in ISS conditions. Niederwieser et al. (2018 a ) has discussed the influence of increased CO2 partial pressure on algae growth in spacecraft conditions and they have given similar conclusions for the growth rates of algae.

Conclusion

The Arthrospira-B experiment was successfully run; relevant results have been obtained from online measurement of the oxygen production rate by Limnospira Indica PCC8005 in a micro Photobioreactor operated in microgravity. Considering the 51 green algae space experiments reviewed by Niederwieser et al. (2018 b ), Arthrospira-B is the first successful dynamic culture experiment in space, allowing direct measurement and calculation of reliable growth and oxygen production rates. However, three main issues for the ISS experiment have to be reported. 1) The remotely activated automated inoculation and reactor start-up failed for one reactor in space, but the procedure was successful for the 3 other space reactors, and all ground reactors. 2) There were some technical difficulties with the stirrer and gas pressure measurement, leading to a non-perfectly mixed liquid phase, for which the current model was not completely applicable. It was still interesting to note that the continuous circulation of the liquid in the liquid loop was sufficient to maintain a suitable liquid phase mixing for operating the PBR in ground and microgravity conditions with about 1/3 of the maximum oxygen productivity. The lower oxygen productivity in non-perfectly mixed liquid phase may be due to presence of gas bubbles into the liquid phase and limiting gas / liquid transfer across the liquid boundary layer at the surface of the membrane. It has also been observed that when a perfectly mixed behaviour was recovered, the oxygen production profile was recovered too.

3) The space bioreactor compatible with Biolab facility on ISS could not account for a pH sensor or regulation, so that pH of the process was uncontrolled, leading to a steep pH increase when bicarbonate was consumed. A complete model of pH reconstruction using element and electrolytes balances was derived and used to reconstruct post-fight the pH profiles of the cultures. The model predicted a max pH threshold value of 11.3-11.4 for biomass and oxygen production both on ISS and on ground, to avoid an unrecoverable biomass death. Except for reactors showing stirring difficulties, oxygen productivity and therefore biomass growth were comparable on ground and in microgravity experiments. The model also fits both on ground and microgravity experiments except when high pH conditions (above 11) were reached. In microgravity the growth in such bicarbonate-pH limiting condition was less affected than in ground conditions, maybe due to higher CO2 fraction in the gas used to flush the IEC on ISS.

The experiment had been planned with 16 batches in order to obtain enough data for comparison of the growth and oxygen production on-board ISS and on ground. But the technical issues encountered have reduced the number of results and a definitive conclusion on the influence or not of the microgravity on the growth of Limnospira indica PCC8005 remains difficult to assess. Nevertheless, it was observed that for on-board ISS and on ground reactors, the oxygen production rates measurements were comparable.

Consequently, the terrestrial bioreactor growth model is completely applicable for representing 0-g conditions, provided the modifications of physical environmental variables have been correctly assessed by a mechanistic approach in the model. The present model can still be improved if in the future for predicting the growth under degraded conditions such as high pH or imperfectly mixed reactor.

  structure and assumptions Cogne et al. (2003 b ) have proposed, developed and implemented the principle of a dynamic model for a similar membrane photobioreactor. The photosynthetic growth of microalgae under light limiting conditions has been modelled associating four main sub-models (Figure 4), namely a radiative-transfer model, a biological model, a liquid phase mass balance model and a gas phase mass balance model. The four sub-models are coupled and interdependent from each other. Biological phenomena (biomass growth, oxygen production rate) are ratelimited by the light availability inside the culture. Therefore, a radiative-transfer model is required to express light attenuation which itself is function of the biomass concentration.Similarly, gas and liquid phases are linked by gas/liquid equilibrium and gas transfer rate through the membrane. The magnetic stirrer is supposed to provide an efficient agitation in the liquid phase ensuring perfectly mixed conditions in the liquid phase of the reactor within the time scale of the accumulation of substrates and products and of characteristic time of biomass growth. The location of the stirrer at the membrane wall permits to minimize the thickness of the liquid boundary layer, reducing the mass transfer resistance between the liquid bulk and the membrane. The nutrients (N, S, P sources) were in excess, but below inhibition levels. Temperature and gas pressure are controlled, respectively by the Biolab thermal control unit and by the pressure relief valve of the reactor; pH is uncontrolled and increases progressively due to CO2 fixation for the biomass growth. Consequently as pH affects the ionic equilibrium of the solution and can affect the growth, a pH prediction model is also included in the liquid sub-model.The state variables are calculated by solving the model Ordinary Differential Equations (ODE)

  photobioreactor and L is the depth of the photobioreactor. The optical and radiative

  from P/2e-: xEPS = 1.33 (P/2e --1.23).

  673 + 0.137 N < O P + 1.192 1 -N < O PR S + 0.005 +

	0.01N < O P U V + 0.06 1 -N < O P S V →
	IP 0.XYY/ . ZV" [\? .V X/ .XVX" [\? R .0] 07" [\? U . X/ . 0" [\? . Y 07" [\? + 1.444 -
	0.484N < O	(Eq. 2)

  These ground reactors have often shown some difficulties with irregular adherence of the stirring barrel leading to a similar interpretation as previously. It was also often observed in previous ground experiments that this intermediate productivity could be explained by the presence of a gas bubble in the liquid compartment, reducing the liquid volume and increasing the gas volume. If it still difficult to conclude whether this group of batches with intermediate productivity would have to be related to a stirrer dysfunction (reduced liquid gas transfer rate) or to a bubble appearance in the liquid. It might also be explained by a pressure problem (such as gas leakage or pressure sensor malfunction), the reason being most probably identical for all reactors.4.2 Model versus experimentsOne of the objectives of the experiments was to validate and to use the theoretical model in microgravity conditions. For multiple experiments together, it was easier to use the cumulative pressure than the pressure profiles for comparing experimental data and model data. First the model must be validated with the ground experiments. As the current model

	assumes perfectly mixed and perfectly tight and isolated reactor, only the batches showing
	reasonable oxygen production profiles without indications of severe technical issues were
	used (Figure

Acknowledgement

This work is co-financed by the European Space Agency and the Belgian Science Policy (Belspo) and is part of the MELiSSA project (http://www.melissafoundation.org). The Arthrospira-B flight experiment was part of the ArtEMISS project of ESA (ESA-AO2004-085) lead by SCK•CEN (Belgian Nuclear Research Centre). The ArtEMISS scientific team thanks the ESA and NASA astronauts, and all the teams of QinetiQ, ESA, MUSC, and BIOTESC who made the space flight experiment possible.

Batch 1 Batch 2

Batch 3 Batch 4 A1 0.42 g.g -1 0.10 mmolO2.L -1 .h -1 b) 0.32 g.g -1 0.10 mmolO2.L


Djalma Antônio

Ferraz Nunes

Clémence Júnior

Eric Pages

Nicolas Latrille

Marcelo Bernet

Eric Zaiat
email: zaiat@sc.usp.br

Trably

Antônio Djalma

Nunes Ferraz Júnior

Clémence Pages
email: clemence.pages@inra.fr

Eric Latrille
email: eric.latrille@inra.fr

Nicolas Bernet
email: nicolas.bernet@inra.fr

Marcelo Zaiat

Eric Trably
email: eric.trably@inra.fr

Biogas sequestration from the headspace of a fermentative system enhances hydrogen production rate and yield

Keywords: biohydrogen, dark fermentation, biogas sequestration, ppH2, sugarcane molasses

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

INTRODUCTION

The production of biohydrogen (BioH2) from organic waste is a promising biotechnological process with gains at energetic, societal and environmental levels [START_REF] Toledo-Alarcón | Basics of bio-hydrogen production by dark fermentation[END_REF].

However, BioH2 production by dark fermentation (DF) is still a technological challenge for being a very sensitive process, requiring careful balancing of the following parameters: pH [START_REF] Koyama | Kinetics of thermophilic acidogenesis of typical Brazilian sugarcane vinasse[END_REF] [3], temperature [START_REF] Júnior | Mesophilic hydrogen production in acidogenic packed-bed reactors (apbr) using raw sugarcane vinasse as substrate: Influence of support materials[END_REF], organic loading rate (OLR) [START_REF] Júnior | Effect of organic loading rate on hydrogen production from sugarcane vinasse in thermophilic acidogenic packed bed reactors[END_REF] and specific organic loading rate (sOLR) [START_REF] Júnior | High organic loading rate on thermophilic hydrogen production and metagenomic study at an anaerobic packed-bed reactor treating a residual liquid stream of a Brazilian biorefinery[END_REF].

In fermentative systems using non-sterile mixed cultures, high H2 yields are associated with a mixture of acetate and butyrate fermentation pathways end-products, while low H2 yields are associated with other reduced end-products such as lactate, solvents (ethanol, butanol and acetone) and alanine. To date, hydrogen yields in fermentative systems are mostly ranging between 1.2 -2.3 molH2.mol -1 hexose which represent only 30 -50% of the theoretical maximum hydrogen yield (4 molH2.mol - 1 hexose, glucose) [START_REF] Hallenbeck | Fermentative hydrogen production: Principles, progress, and prognosis[END_REF] [8] [9] [START_REF] Júnior | Applied Metagenomics for Biofuel Development and Environmental Sustainability[END_REF].

Multiple reasons have been associated to low hydrogen yields such as (i) anabolic consumption of the substrate for biomass synthesis [START_REF] Júnior | Mesophilic hydrogen production in acidogenic packed-bed reactors (apbr) using raw sugarcane vinasse as substrate: Influence of support materials[END_REF] (ii) inappropriate fermentative conditions [START_REF] Koyama | Kinetics of thermophilic acidogenesis of typical Brazilian sugarcane vinasse[END_REF] [11] (iii) hydrogenotrophic activity [START_REF] Fm Braga | Thermophilic biohydrogen production using a UASB reactor: performance during long-term operation[END_REF] (iv) homoacetogenic activity [START_REF] Carrillo-Reyes | Strategies to cope with methanogens in hydrogen producing UASB reactors: Community dynamics[END_REF] and (v) inhibition by partial pressure of hydrogen [START_REF] Kraemer | Measurement of H 2 consumption and its role in continuous fermentative hydrogen production[END_REF].

The partial pressure of hydrogen ( ) is an extremely important factor especially for continuous BioH2 production [START_REF] Doremus | Agitation and pressure effects on acetone-butanol fermentation[END_REF] [16] [17] [18]. This factor is explained by Le Chatelier's Principle that says "all chemical equilibrium responds to an increase in the pressure, causing the reaction to move in the opposite sense to that, which rises the pressure." In biological multipahses systems, this event is associated to the limitation of the liquid-to-gas mass transfer. The liquid-to-gas mass transfer limitation arises because the gas production rate is higher than the transfer rate to the gas phase [START_REF] Barca | Fermentative hydrogen production in an up-flow anaerobic biofilm reactor inoculated with a co-culture of Clostridium acetobutylicum and Desulfovibrio vulgaris[END_REF] [START_REF] Pauss | Liquid-to-Gas mass transfer in anaerobic processes: Inevitable transfer limitations of methane and hydrogen in the biomethanation process[END_REF]. Such a limitation have caused H2 supersaturation in the liquid with concentrations of H2 between 5-and 71/fold higher than the equilibrium value [START_REF] Pauss | Liquid-to-Gas mass transfer in anaerobic processes: Inevitable transfer limitations of methane and hydrogen in the biomethanation process[END_REF] [21]. Thereby, during the fermentation process, as the in bioreactors increases, H2

synthesis decreases [START_REF] Kisielewska | Improvement of biohydrogen production using a reduced pressure fermentation[END_REF]. This also can be explained through Metabolic pathways shifts are also observed in function of the . According to Hallenbeck [START_REF] Hallenbeck | Fermentative hydrogen production: Principles, progress, and prognosis[END_REF] in Clostridial-type hydrogen producing fermentation at low , the NADH generated during glycolysis can be reoxidized, probably by a NADH-dependent

[FeFe] hydrogenase. At moderate to high , this reaction is unfavorable, and NADH is reoxidized by the formation of reduced organic compounds (previously mentioned).

As a consequence, low hydrogen yields are achieved.

Few methods to control the have been investigated: sparging (i.e., gas flushing to remove other dissolved gas, in this case H2), removing H2 from the system or reactor operation at low pressure. Mizuno et al [START_REF] Mizuno | Enhancement of hydrogen production from glucose by nitrogen gas sparging[END_REF] evaluated the influence of sparging in a continuous stirred tank (CSTR) fed with a mineral salts-glucose medium (10.7 gCOD.L -1 ). Nitrogen gas was sparged at a flow rate of 15 times the specific hydrogen production rate (sHPR) observed in a control CSTR (i.e., without sparging) that was 1.446 mL H2. min -1 . g -1 biomass. An increase of 68% of hydrogen yield was achieved with sparging (1.43 molH2.mol -1 glucose).

Besides nitrogen, other gases such as internal biogas and only carbon dioxide with different flow rates (100 -400 mL.min -1 ) were investigated in a CSTR fed with a mineral salts-sucrose medium (20 gCOD.L -1 ) (Kim and co-authors, [START_REF] Kim | Effect of gas sparging on continuous fermentative hydrogen production[END_REF]). The best performances were obtained by CO2 sparging at 300 ml.min -1 , resulting in the highest H2 yield of 1.68 molH2.mol -1 hexose converted. Concomitant to the increase of hydrogen production and yield, too much sparging produces dilute gas stream, creating a serious problem with respect to the H2 separation from the sparging gas [START_REF] Levin | Biohydrogen production : prospects and limitations to practical application[END_REF].

Fast collection of biogas was also studied as the control method. Liang et al.

[26] investigated the biogas removal using a vacuum pump (31.4 kPa) and membrane purification of H2 from a fermentation system (Batch reactor; 2.5 g glucose added). The authors reported that silicone rubber was effective in reducing the , improving the hydrogen production by 10% (2.6 -3 mmol H2.g -1 VSS. h -1 ) and the hydrogen yield by 15% (0.84 -0.92 molH2. mol -1 glucose). Lee et al. [START_REF] Lee | Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy[END_REF] investigated the effect of working with reduced pressure in a CSTR, they worked with pressures similar to the ones in Liu

and Wang [START_REF] Liu | Directional enhancement of fermentative coproduction of hydrogen and acetic acid from glucose via control of headspace pressure[END_REF], between 0.2 and 0.9 atm, and concluded that H2 production can be improved in fermentative systems with reduced pressure.

Interestingly, no difference regarding H2 production was observed in pure culture system (Clostridium butyricum strain SC-E1) under vacuum (28 kPa) and nonvacuum. Glucose-polypeptone at 0.5 and 1.0% concentration were used as substrate, resulting in maximum hydrogen yields of 1.8 -2.2 and molH2.mol -1 glucose for all condition evaluated [START_REF] Kataoka | STUDIES ON HYDROGEN PRODUCTION HYDROGEN-PRODUCING ANAEROBIC BACTERIA[END_REF].

Recently, another strategy to remove BioH2 from the fermentative systems has been tested. Massanet-Nicolau et al. [START_REF] Massanet-Nicolau | Maximising biohydrogen yields via continuous electrochemical hydrogen removal and carbon dioxide scrubbing[END_REF] reported a system with electrochemical H2 removal and carbon dioxide absorption as an effective strategy to increase H2 yields and avoid its consumption. Also, membrane systems are suggested as a way to separate and purify H2 [START_REF] Ramírez-Morales | Simultaneous production and separation of biohydrogen in mixed culture systems by continuous dark fermentation[END_REF].

Despite these mentioned studies on this subject, more detailed research on this topic is necessary to enable the production of BioH2 at larger scale and with continuous operation of the fermentation process. In this study, regular collection of biogas from headspace of a fermentative continuous system was carried out aiming to control the in the process and thus, attempt to maintain a high hydrogen productivity. The dynamics of the microbial community was also studied based on the sequencing of the V4 region of 16S rRNA gene for Bacteria using High-Throughput Sequencing (MiSeq Sequencing System -Illumina).

MATERIALS AND METHODS

Seed sludge

The seed sludge was taken from an industry of commercialization of sugarcane and sugar beet plant (UASB-type reactor). The total volatile solids (TVS) concentration of the sludge was 53.7 g/L. Heat-treatment was applied to the sludge at 90°C for 1 h to inactivate hydrogen consumers and to harvest spore-forming anaerobic bacteria such as Clostridium sp. [START_REF] Júnior | Impact of organic loading rate on biohydrogen production in an up-flow anaerobic packed bed reactor (UAnPBR)[END_REF].

Feeding solution

A mineral salts-sugarcane molasses solution of 21 g COD L -1 was used as carbon source in a feeding medium composed by the following macro-and micronutrients (mg L -1 ): NiSO4.6H2O, 0.5; FeSO4.7H2O, 2.5; FeCl3.6H2O, 0.25; CoCl2.2H2O, 0.04; CaCl2.2H2O, 2.06; SeO2, 0.036; HCl, 0.25, according to Del Nery [START_REF] Nery | Utilização de lodo anaeróbio imobilizado em gel no estudo de partida de reatores de fluxo ascendente com manta de lodoNo Title[END_REF]. The C/N ratio of molasses was 52.7.

Reactor design and operational conditions

Experiments were carried out in four continuous stirred reactors of 4 L with a working volume of 2 L (Figure 1). Each reactor was equipped with a stirring system made of a Rushton turbine and a marine propeller to ensure a homogeneous mixture. A revolution counter was connected to access to the measurement of the stirring velocity which was 250 rpm. The gas flow rate was measured with a peristaltic pump calibrated at each different levels of pressure. Pressure was regulated with a control device combining a pressure sensor and a peristaltic pump following a two-band control law. A combined sensor was connected to the reactor for measuring the redox potential and pH (4010/120/Pt100, Mettler Toledo). The pH and redox meter (M300 -Mettler Toledo)

was connected to a computer for on-line data acquisition (home-made software Odin in collaboration with INRIA teams). The pH was set and controlled at 5.5 by adding NaOH (2 M) with a peristaltic pump. Temperature in the reactor was also controlled using a platinum probe Pt100 and a heating electric resistance. The temperature was maintained constant at 37 ± 0.5 °C. The hydraulic retention time (HRT) was 6 h, resulting in an organic loading rate (OLR) of 84.2 gCOD.L -1 . d -1 , as suggested in [START_REF] Júnior | Effect of organic loading rate on hydrogen production from sugarcane vinasse in thermophilic acidogenic packed bed reactors[END_REF].

The total pressure tested is presented in Table 1.

[Figure 1 -Please here]

[Table 1 -Please here]

The experimental setup was based on the following assumptions: (i) the second column of Table 1 presents the initial TP of the four independent conditions (R1 to R4), to evaluate the real influence of total pressure on hydrogen producing-system. This also represents the condition where the microbial community had the same operating history;

(ii) other conditions (the third and fourth columns) were performed to evaluate how the microbial community responds to a variation of total pressure to evaluate whether hydrogen production was inhibited or if such inhibition is irreversible or reversible; (iii) the steady-state for each operating condition was considered when the coefficient of variance of the hydrogen production rate (HPR) was less than 10% based on its mean value from the ten last HRT of each operating phase; (iv) considering the steady-state of hydrogen-producing systems, the of the headspace and the concentration of dissolved hydrogen in the liquid medium ([H2] Liq.) were estimated by Dalton's and Henry's Law, respectively; (v) Experiments without pressure control was not carried out. Thereby, the condition at 100 kPa was set as control both to compare data between conditions and reactors, and microbial community response to the total pressure variation (also controlled). (vi) The inspected pressure range was chosen for be near atmospheric pressure and to evaluate the sensibility of the process.

Chemical analysis

Biogas composition was analyzed as previously described in [START_REF] Motte | Total solids content: a key parameter of metabolic pathways in dry anaerobic digestion[END_REF]. Reduced sugars and fermentation end-products were quantified using high performance liquid chromatography (HPLC; HPX 87 column -Biorad) coupled to a refractometer (Waters R410). The eluent used was a H2SO4 solution (0.222 μl L -1 ). The operating conditions were: elution flow, 0.4 mL min -1 ; temperature of column, 35 °C; temperature of refractometer, 40 °C. Microbial cells (biomass) concentration was determined as volatile suspended solids (VSS) by filtration at 1.2 μm, according to [START_REF]APHA. Standard methods for the examination of water and wastewater[END_REF].

DNA extraction, PCR amplification and High-Throughput Sequencing of hydrogen-producing systems samples

At the end of each operating condition, microbial cells were collected after centrifugation (12000 × g; 15 min) of 2 mL of culture. Genomic DNA was extracted using the Wizard Genomic DNA Purification kit (Promega). The V3-4 region of the Kozich et al. [START_REF] Kozich | Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform[END_REF] in Mothur version 1.33.0. Alignment and taxonomic outline was made using release information: SILVA 102, as provided by Schloss et al. [START_REF] Schloss | Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities[END_REF]. The software PAUP* (version 4.0b10) was used to infer a phylogeny -criterion of maximum parsimony [START_REF] Yang | Phylogenetic analysis using parsimony and likelihood methods[END_REF]. Bootstrap support was calculated using 1500 repetitions. SumTrees (version 3.3.1) of the DendroPy package (version 3.12.0) was used to map bootstrap values to the best phylogeny [START_REF] Sukumaran | DendroPy: a Python library for phylogenetic computing[END_REF]. Sequences of most abundant operational taxonomic unit (OTU) found in the biofilm were deposited in the NCBI Genbank database under the following accession name SUB5433515 (MK765997 -MK766231).

Calculations

Hydrogen Production Rate (HPR, mL-H2 h -1 ) and Hydrogen Yield (HY, mol-H2 mol -1 suc eq.) were calculated using Equations 1 and 2 -4, respectively.

HPR = Qg . %H2 (1)
HY = ((Qg. nH2)/V) / ((Q.(CS0 -CSF))/MMS) (2) nH2 = %H2 . n (3) n = (P.V) / (R.T) (4)
where, Qg is the biogas flow, %H2 is the hydrogen content in biogas, nH2 is the number of mol of hydrogen, V is the volume of gas of the sample, Q is the liquid flow in the reactor, CS0 is the influent substrate concentration, CSF is the effluent substrate concentration, and MMS is the sucrose molar mass, n value corresponds to the total number of moles of sample (i.e., %H2, %CO2 and %CH4), P is the gas pressure, R is the universal ideal gas constant, and T is the absolute temperature.

The theoretical expected hydrogen production and the acetate produced from homoacetogenesis were calculated using Equations ( 5) and ( 6) as proposed by Luo et al. [START_REF] Mota | Biohydrogen production at pH below 3.0: Is it possible?[END_REF] and Ferraz Júnior et al [START_REF] Levin | Biohydrogen production : prospects and limitations to practical application[END_REF]:

H2 theoretical = 2[A] + 2[B] -[P] (5)
Acetate homoacetogenesis = (2[A] + 2[B] -[P] -[H2])/6 (6)
where [A], [B], [P] and [H2] are the measured acetic, butyric and propionic acids; and the hydrogen concentrations in mM, respectively.

The COD balance expressed as COD recovery (Equation 7) of the fermentative process was calculated as follows:

COD recovery % = (COD final/COD0) * 100 [START_REF] Hallenbeck | Fermentative hydrogen production: Principles, progress, and prognosis[END_REF] where COD0 is the COD of molasses fed and COD final is the sum of the mass, expressed as g-COD, of every outlet component of the fermentative system, as proposed by Ferraz Júnior et al [START_REF] Júnior | Effect of organic loading rate on hydrogen production from sugarcane vinasse in thermophilic acidogenic packed bed reactors[END_REF].

Principal component analysis (PCA) was performed using STATISCA 10.

Primarily, a factor analysis was performed to identify the number of independent factors [START_REF] Pauss | Liquid-to-Gas mass transfer in anaerobic processes: Inevitable transfer limitations of methane and hydrogen in the biomethanation process[END_REF]. The Kaiser criterion was used to decide the factors that could be retained for interpretation [START_REF] Kaiser | The Application of Electronic Computers to Factor Analysis[END_REF]. The factors cut off was identified through the point of wherein the eigenvalue level drop off continuously based on Catell [START_REF] Cattell | The Scree Test For The Number Of Factors[END_REF].

RESULTS AND DISCUSSION

Hydrogen production (HPR) and yield (HY)

Four similar stirred reactors were operated in parallel with the same conditions of pH, temperature, stirring, initial concentration of substrate and HRT. However, different initial total pressures (TP) were applied (R1 -80 kPa; R2 -100 kPa; R3 -120 kPa; and R4 -140 kPa). The steady-state was reached after approximately 60 HRT from the time when the TP of R1, R2 and R3 was increased to 100, 120 and 140 kPa, respectively (Phase II). The R4 was disassembled, according to the experimental design.

A second steady-state was achieved within the same period as Phase I for the remaining reactors. Then, R1 and R2 had their total pressure increased to 120 and 140 kPa, respectively, and R3 decreased to 100 kPa (Phase III). The steady-state Phase III was also achieved after 15 days.

Hydrogen content of biogas was around 47 -54% for all reactors and conditions. Methane was not observed in the biogas suggesting that the inoculum heattreated, and the operating conditions inhibited the methanogenesis and favored the hydrogen-producing process. The partial pressure of hydrogen ( ) was determined by Dalton's law and the values ranged between 41-70 kPa. These values are slightly higher than [START_REF] Liang | Behavioral study on hydrogen fermentation reactor installed with silicone rubber membrane[END_REF] and [START_REF] Kataoka | STUDIES ON HYDROGEN PRODUCTION HYDROGEN-PRODUCING ANAEROBIC BACTERIA[END_REF].

The different total pressure (TP) showed a strong influence on hydrogen production rate (HRP, mL-H2 h -1 ) (Figure 1). The highest HPR (406.1±36.8 mL-H2 h -1 )

was achieved in R1 with the lowest TP of 80 kPa. At atmospheric pressure (100 kPa), the HRP decreased 25% in relation to Phase I. The HPR decreased even more (52%)

with the increase of TP to 120 kPa.

The same behavior was observed in R2 and R3. The increase of the TP from 100 to 140 kPa; and from 120 to 140 kPa, was reflected in HPR decrease of 70% (R2) and 17% (R3), respectively (Phase I). When the TP was alleviated to 100 kPa in R3, the HPR increased by 90% in Phase I. In addition, the highest TP (140 kPa) as applied to R4 resulted in the lowest value of HRP (61.6±5.8 mL-H2 h -1 ) (Table 2). These findings show that gas removal had a positive effect on HPR. The increase of the TP with a consequent increase of the influenced negatively the Bio-H2 production.

Hydrogen yield (HY) followed the same trend as HPR, being the maximum and the minimum values achieved of 4.51 and 0.56 mol-H2 mol -1 suc eq. when the TP was 80 and 140 kPa, respectively (Table 2). The HY at initial TP of 80 KPa represented an increase of 61.6% and 705% comparing to the controlled atmospheric pressure (100 kPa) and to the highest TP evaluated (140 kPa), respectively, reaffirming the high influence of TP on biological hydrogen production process.

[Figure 2 -Please here]

[Table 2 -Please here]

Based on the of the headspace, the concentration of dissolved hydrogen in the liquid medium ([H2] Liq.) was estimated by Henry's Law. The correlation between

[H2] Liq. and; HPR and HY indicated a linear coefficient of 0.979 and 0.968, respectively (Figure 3).

[Figure 3 -Please here] By applying linear regression analysis on the experimental results, the equations

Intermediates products from molasses fermentation

The conversion of sucrose, the main carbon source presented in the mineral salts-sugarcane molasses, was higher than 99% for all reactors and conditions.

However, reducing sugars such as glucose and fructose remained in the acidogenic reactors liquid outlet in percentage between 29% and 37.4% (Figure S1). These findings are similar to the ones reported by [START_REF] Mota | Biohydrogen production at pH below 3.0: Is it possible?[END_REF] who evaluated different configurations of reactor to produce hydrogen from sucrose.

In addition to the molasses fermentation products, the organic acids were quantified to investigate the main metabolic pathways in the hydrogen-producing systems. Table 3 shows that the main intermediates products were acetate (49.1 -22 mM) followed by lactate (16.7 -27.8 mM), ethanol (10.9 -33.2 mM) and butyrate (16.1 -24.1 mM). Traces of propionate (0.1 mM) were detected in conditions with TP higher than 100 kPa (Table 3).

At steady state, the metabolite yields were 0.4 -0.9 mol acetate. mol -1 suc. eq.; 0.3 -0.5 mol lactate mol -1 suc. eq.; 0.2 -0.6 mol ethanol mol -1 suc. eq.; and 0.3 -0.4 mol butyrate mol -1 suc. eq. Similar values of organic acids yields have been reported by Palomo-Briones et al.

[43] who studied the influence of OLR on hydrogen production using a cheese wheyfed CSTR. Despite the carbon source being different from this study, heat pretreatment of the sludge and operating conditions of pH, temperature, stirring, OLR and HRT were analogous.

The theoretical hydrogen production was also estimated for each TP evaluated according to the organic acids concentrations detected, mainly acetate, butyrate and propionate. The measured hydrogen ranged between 9.9% and 44.2% of the theoretical hydrogen estimated (Table 3), suggesting the homoacetogenesis pathway especially at a TP of 140 kPa. Ferraz Júnior et al. [START_REF] Júnior | High organic loading rate on thermophilic hydrogen production and metagenomic study at an anaerobic packed-bed reactor treating a residual liquid stream of a Brazilian biorefinery[END_REF] and Corona & Razo-Flores [START_REF] Corona | Continuous hydrogen and methane production from Agave tequilana bagasse hydrolysate by sequential process to maximize energy recovery efficiency[END_REF] reported similar values for the measured H2 and theoretical H2 ratio. Finally, it is worth mentioning that biomass, residual sugars, organic acids and H2 represented between and 87.4 and 105.2% of the COD fed to the fermentation systems (Figure S1). In this study, the COD fed drive to hydrogen production increased from 2.31% to 18% as the TP decreased from 140 kPa to 80 kPa.

Microbial community analysis

16S ribosomal DNA gene sequences at steady states were analyzed by Illumina MiSeq technology to characterize the microbial community structure and reveal the total pressure-associated changes. Microbial composition of all TP evaluated is depicted in Figure 4A.

More than 400 thousand partial 16S ribosomal DNA gene sequences were obtained out of which 94 -98% were assigned to the domain Bacteria more specifically phylum Firmicutes. No sequence was assigned to the domain Archaea (9.6% of inoculum) by the end of reactors operation, indicating that the sludge pretreatment added to the operating conditions inhibited successfully methanogenesis.

More precisely, the most abundant microorganism harbored in all reactor and conditions were Sporolactobacillus (57 -82%) followed by species of genus Clostridium (14 -31%) and Ethanoligenens (1.2 -4.6%) (Figure 4A). This low microbial diversity is considered as a common characteristic in Bio-H2 producing systems [START_REF] Fuess | Temporal dynamics and metabolic correlation between lactate-producing and hydrogen-producing bacteria in sugarcane vinasse dark fermentation: The key role of lactate[END_REF] and apparently, the strong pressure of selection becomes accentuated in reactor with suspended biomass [START_REF] Etchebehere | Microbial communities from 20 different hydrogen-producing reactors studied by 454 pyrosequencing[END_REF]. Remarkably, no drastic change in the microbial community was observed at different TP, suggesting that both HPR and HY were directly affected by the mass transfer process (Liquid-Gas) or even by inhibition of synthesis/consumption of hydrogen at the cellular level rather than microbial composition (Figure 4B). However, specific studies must be carried out to validate such a statement.

Both Sporolactobacillus and Clostridium have been reported as obligate anaerobes capable of producing endospores [START_REF] Júnior | Impact of organic loading rate on biohydrogen production in an up-flow anaerobic packed bed reactor (UAnPBR)[END_REF]. Therefore, these two genera are strongly associated to the heat-treatment of seed sludge that is able to inactivate hydrogen consumers, primely methanogenic archaea, and induce the formation of sporeforming anaerobic bacteria [START_REF] Toledo-Alarcón | Basics of bio-hydrogen production by dark fermentation[END_REF]. However, Sporolactobacillus is described as homofermentative, lactic acid-producing organisms [START_REF] Kitahara K | SPOROLACTOBACILLUS NOV. SUBGEN[END_REF] [47] while species within the Clostridium genus have been well proved to possess a high ability to produce hydrogen independently of the reactor configuration [START_REF] Mota | Biohydrogen production at pH below 3.0: Is it possible?[END_REF], organic loading rate [START_REF] Júnior | Impact of organic loading rate on biohydrogen production in an up-flow anaerobic packed bed reactor (UAnPBR)[END_REF],

immobilization [START_REF] Júnior | Mesophilic hydrogen production in acidogenic packed-bed reactors (apbr) using raw sugarcane vinasse as substrate: Influence of support materials[END_REF] or in suspension [12] [48]. With less dominance, in this study, but not less important, the genus Ethanoligenens harbors the most promising hydrogenproducing organisms due to their capability to generate hydrogen at high rates and efficiency [START_REF] Ren | Cloning, Expression, and Characterization of an Acetate Kinase from a High Rate of Biohydrogen Bacterial Strain Ethanoligenens sp. hit B49[END_REF].

[Table 3 -Please here]

Aiming to better understand the interaction among the indicators of reactor performances, a principal component analysis (PCA) was performed (Figure 5). Two principal components accounted for nearly 74% of the dataset variance. The results showed two well-defined axes or principal components (PC): PC 1 which represents the main effect of HPR, HY, acetate yield, TP 80 kPA and TP 100 kPa opposing TP 140 kPa; and PC II which represents TP 120 kPa, lactate, butyrate and ethanol yields opposing TP 140 kPa. PC I reaffirms that the higher values of HPR and HY were archived at the lowest TP evaluated while PC II indicates a direct effect of high pressures on byturate, lactate and ethanol yields. The results also showed an inverse relationship between ethanol yield and TP 140 kPa. It should be noticed that the microbial community was not computed in the PCA analysis due to its quite low variability (Figure 4B).

[Figure 4 -Please here]

[Figure 5 -Please here]

Highly efficient Bio-H2 condition with regard to the literature

The operation of the dark fermentative high-rate CSTR fed with a mineral saltssugarcane molasses solution at low total pressure (i.e., TP and of 80 kPa and 41 kPa, respectively) was found to favor successfully the Bio-H2 production. The observed Bio-H2 yields were even slightly one of the highest value when compared to other reports (Table 4).

[Table 4 -Please here]

References linked to (3)

For acetate-type fermentation (glucose-model), the breakdown of pyruvate yields (2 moles of H2 per mole of glucose), and an additional 2 moles of H2 per mole of glucose is derived through Reaction 4 [START_REF] Baghchehsaraee | Effect of extrinsic lactic acid on fermentative hydrogen production[END_REF]. The reduction of hydrogenase by NADH is energetically unfavorable under standard conditions unless at extremely low (< 0.1 kPa) [START_REF] Hallenbeck | Fundamentals of the fermentative production of hydrogen[END_REF]. Based on the Gibb's free energy change, butyrate-type fermentation is more energetically favorable and thus NAD is often used in butyrate-type fermentation. In this sense, the combination of acetate and butyrate-type fermentation might occur simultaneously during H2 production using mixed cultures; and therefore, the maximum hydrogen yield may never exceed 2.5 moles of H2 per mole of glucose (i.e., 62.5% of its maximum theoretical yield) (Reaction 5) [START_REF] Hawkes F | Continuous dark fermentative hydrogen production by mesophilic microflora: Principles and progress[END_REF] [57] [START_REF] Noblecourt | High hydrogen production rate in a submerged membrane anaerobic bioreactor[END_REF]. In the case of sucrose as carbon source, this value is equivalent to 5 moles of H2. Based on this assumption, the low pressure applied in this study achieved a HY of 4.51 mol-H2 mol -1 suc eq., which represents 95% of the maximum hydrogen yield through mixed biological path (Reaction 5).

NADH:ferrodoxin oxireductase activity

NADH + H + → H2 + NAD + ∆Gº' +18.1 kJ.mol -1 (4)
Acetate and butyrate-type fermentation using mixed culture

C6H12O6 + 0.5H2O → 0.75CH3(CH2)2COOH + 0.5CH3COOH + 2CO2 + 2.5H2 (5)
In addition, Procentese et al. [START_REF] Procentese | Continuous lactose fermentation by Clostridium acetobutylicum--assessment of solventogenic kinetics[END_REF] reported that species of Clostridium acetobutylicum could be inhibited by the accumulation of acetate (26 Mm) and butyrate (34 Mm). In the present study, acetate and butyrate were in the range of the inhibitory concentrations (Table 3). In fermentative systems, these acids normally accumulate in the growth medium as dead-end metabolites, since the conversion of these acids into additional H2 is thermodynamically unfavorable. Consequently, a redirection of the cellular metabolic pathways towards solvent production is often taken. As an illustration, Clostridium beijerinckii strains have been reported to reconsume the produced acids at low pH, converting them into ethanol, isopropanol and butanol [START_REF] Andersch | Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum[END_REF] [60]. Considering the low abundance of Ethanoligenens (1.2 -4.6%), ethanol concentrations detected in the acidogenic reactors liquid outlet was attributed to solventogenesis rather than the ethanol-type fermentation thus, not being accounted in the theoretical hydrogen production (Equation 1).

Homoacetogenesis still occurred at low pressure

In the anaerobic digestion process, hydrogenotrophic methanogenesis (Reaction 6) is thermodynamically more favorable than homoacetogenesis (Reaction 7) in standard conditions [START_REF] Zhang | Stable acetate production in extreme-thermophilic (70°C) mixed culture fermentation by selective enrichment of hydrogenotrophic methanogens[END_REF]. Acetate evolution as sole metabolite in the liquid phase can only occur at below 0.06 kPa. Homoacetogenesis is also a possible pathway that consumes hydrogen and generates acetate in anaerobic digestion, but the threshold for acetate production through this pathway is 0.25 kPa at 35°C, which is high when compared to the thresholds of 0.06 kPa (hydrogenotrophic methanogenesisis pathway)

[14] [START_REF] Poehlein | An Ancient Pathway Combining Carbon Dioxide Fixation with the Generation and Utilization of a Sodium Ion Gradient for ATP Synthesis[END_REF].

In this study, the value at low-pressure was still 160 times higher than the threshold for acetate production by homoacetogenesis, indicating that even if methanogenesis was prevented by heat-pretreatment of sludge and operating condition, homoacetogenesis could have occured. The steady-state operation, low pH (5.5) and HRT of 6 h might also favor such reaction 7.

Hydrogenotrophic methanogenesis

4H2 + HCO3 -+ H + → CH4 + 3H2O ∆Gº' -135.5 kJ.mol -1 (6) Homoacetogenesis 4H2 + 2HCO3 -+ H + → CH3COO -+ 4H2O
∆Gº' -104.5 kJ.mol -1 (7) In an experiment at atmospheric pressure performed by Corona and Razo-Flores [START_REF] Corona | Continuous hydrogen and methane production from Agave tequilana bagasse hydrolysate by sequential process to maximize energy recovery efficiency[END_REF], the increase in the agitation speed from 150 to 300 rpm was implemented as a strategy to collect the hydrogen gas from the liquid phase and avoid its consumption by homoacetogens. The authors reported that values between 30% and 38% of the measured acetate came from homoacetogenesis, being the lower value of the acetate estimated from homoacetogenesis was achieved at the highest stirring condition. This finding is in accordance with the values obtained in this study at TP condition of 80 kPa and 100 kPa (i.e., of 41 kPa and 49 kPa, respectively). Consistently, when TP of 140 kPa ( of 70 kPa) was applied, acetate issued from homoacetogenesis reached values up to 56.6% due to a higher availability of hydrogen in the liquid medium, resulting in the worst condition for Bio-H2 production even with agitation speed set at 250 rpm (operating conditionsubhead 2.2.).

Lactate-type fermentation might comprise an additional pathway to produce

Bio-H2

Lactic acid bacteria (LAB) are often detected in mesophilic hydrogen producing consortia as bacteria that accompany hydrogen producers [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: useful insights for driving the ecosystem function[END_REF]. However, the real role of LAB in hydrogen-producing systems and their influence on hydrogen producers are still unclear.

Noike et al. [START_REF] Noike | Inhibition of hydrogen fermentation of organic wastes by lactic acid bacteria[END_REF]; Ren et al. [START_REF] Ren | Cloning, Expression, and Characterization of an Acetate Kinase from a High Rate of Biohydrogen Bacterial Strain Ethanoligenens sp. hit B49[END_REF] and Gomes et al. [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] reported inhibition of hydrogen producers by LAB due to substrate competition (replacement of hydrogen fermentation by lactic acid fermentation) and excretion of bacteriocins. In contrast, a positive role of LAB in dark fermentation process has also been reported [START_REF] Rafrafi | Sub-dominant bacteria as keystone species in microbial communities producing bio-hydrogen[END_REF].

Fluorescence In Situ Hybridization (FISH) images from a high-rate fermentative hydrogen system suggested that Streptococcus cells acted as seeds for granule formation [START_REF] Hung | Quantitative analysis of a high-rate hydrogen-producing microbial community in anaerobic agitated granular sludge bed bioreactors using glucose as substrate[END_REF]. It is particularly important in CSTR, since this may help increasing biomass concentration into the reactor leading it to higher Bio-H2 production [START_REF] Rafrafi | Sub-dominant bacteria as keystone species in microbial communities producing bio-hydrogen[END_REF]. Yang et al.

[67] even declared the isolation of Lactobacillus bacteria capable of hydrogen production during lactose fermentation.

Corroborating to the positive role of LAB in dark fermentation process, several clostridia have also demonstrated the ability to ferment lactate. Clostridium propionicurn uses the acrylate pathway to metabolize lactate, as a sole carbon and energy source [START_REF] Cato | Genus Clostridium Prazmowski 1880[END_REF]. Clostridium acetobutylicum cultures metabolize lactate in corn steep liquor [START_REF] Datta | Modulation of acetone-butanol-ethanol fermentation by carbon monoxide and organic acids[END_REF]. Clostridium beijerinckii [START_REF] Bhat | Clostridium lacto-acetophilum Nov. Spec. and the Role of Acetic Acid in the Butyric Acid Fermentation of Lactate[END_REF] and Clostridium tyrobutryicum [START_REF] Woolford | The silage fermentation[END_REF] require acetate as co-substrate to utilize lactate but the role of acetate and the pathway of lactate metabolism have not been defined.

More recently, hydrogen and butyrate were produced from a mixture of acetate (50.8 mM) and lactate (33.3 mM) using Clostridium diolis JPCC H-3. A molar ratio of consumption of acetate to lactate was 1:2 and the very favorable Gibbs free energy of the reaction (Reaction 10) strongly suggests that this reaction would have proceeded [START_REF] Matsumoto | Hydrogen production by fermentation using acetic acid and lactic acid[END_REF]. Interestingly, in this study, Sporolactobacillus species were the most abundant microorganisms in all reactors and conditions while lactate was the second most abundant organic acids detected in the acidogenic reactor liquid outlet. As previously mentioned, a moderate relationship was found between butyrate and lactate yields indicating a direct interaction within these two metabolic intermediates (Figure 5).

Considering the actual concentrations of intermediates, butyrate and H2 synthesis from lactate and acetate is favorable (Table 5). Therefore, the consumption of lactate using acetate as co-substrate was suggested to be an additional pathway to produce H2 under the evaluated conditions.

[Table 5 -Please here]

References linked to Table 5: [START_REF] Kleerebezem R | A Generalized Method for Thermodynamic State Analysis of Environmental Systems[END_REF]

Conclusions

In this study, it was shown that the sequestration of biogas from bioreactor headspace enhanced the hydrogen production rate and yield. The higher hydrogen yield (4.51 mol-H2 . mol -1 suc eq.) achieved was obtained under a low total pressure of 80 kPa.

Interestingly, the composition of the microbial community did not change with the increase and /or decrease of the total pressure. Acetate from homoacetogenesis was accounted even at low pressure conditions. In addition, observations suggest that lactate-type fermentation might play a key role in dark fermentation and might be more considered as additional pathway to produce hydrogen. f. Value obtained from the ration of hydrogen measured and the estimated hydrogen produced via acetic and butyric pathways. The authors do not express HY in mol.mol -1 probably due to the lignocellulose hydrolysates be composed by glucose, xylose, arabinose, cellobiose, lignin fragments, among others. ---140 / -79.8 ΔG° were calculated at 25 °C and standard concentrations. ΔG were calculated at pH 5.5, 37 °C and the intermediates concentrations as shown in Table 3. Gibbs' energy values were computed in accordance with Kleerebezem and Van Loosdrecht [START_REF] Yang | Biohydrogen production from cheese processing wastewater by anaerobic fermentation using mixed microbial communities[END_REF].

  plus the respective linkers over 30 amplification cycles at 65° C (annealing temperature). An index sequence was added using the primers AATGA-TACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC and CAAGCAGAAGACGGCATACGAGAT-index-TGACTGGAGTTCAGACGTGT (PCR -12 cycles). The PCR products were purified and loaded onto the Illumina MiSeq cartridge according to the manufacturer's instructions for sequencing (pairedend; 250 bp reads) which was performed at the GeT PlaGe sequencing center of the genotoul life science network in Toulouse, France (get.genotoul.fr). Quality checking was made using a slightly modified version of the Standard Operation Procedure by

( 4 )

 4 and (5) were obtained to describe the influence of [H2] Liq. on HPR and HY, respectively. The [H2] Liq. of 0.57 mg. L -1 resulted in maximum values of HPR and HY while the [H2] Liq. of 0.99 mg. L -1 resulted in the lower values of the respective variables indicating that during fermentation process, as the in bioreactors decreases, BioH2 synthesis increases (vice versa). HPR = -821.15*[H2] Liq. + 890.1 R² = 0.979 (4) HY = -9.2954*[H2] Liq. + 9.6264 R² = 0.968 (5)

Figure captions Figure 1 .Figure 2 .

 12 Figure captions Figure 1. Schematic of experimental apparatus to control the total pressure of headspace of the hydrogen-producing systems fed with a mineral salts-sugarcane molasses solution. CSTR reactor image taken from

Figure 3 .

 3 Figure 3. Linear fit of the experimental data obtained from monitoring of fermentative systems fed with a mineral salts-sugarcane molasses solution with different total pressure (TP, kPa). A. Hydrogen production rate (HPR, mL-H2 h -1 ) and concentration of hydrogen dissolved in the liquid medium ([H2] Liq.) ratio. B. Hydrogen yield (HY), mol-H2 mol -1 suc eq.) and concentration of hydrogen dissolved in the liquid medium([H2] Liq.) ratio.

Figure 4 .

 4 Figure 4. A. Composition of microbial community of hydrogen-producing systems at different total pressure (TP, kPa) fed with a mineral salts-sugarcane molasses solution. B. Correlation between Clostridium, Ethanoligenens and Sporolactobacillus species within TP (kPa).

Figure 5 .

 5 Figure 5. Principal components analysis of hydrogen-producing systems at different total pressure (TP, kPa) fed with a mineral salts-sugarcane molasses solution.

Table 4 :

 4 [If butyrate or ethanol are the fermentation products, 4 moles of H2 per mole of sucrose are rather obtained (Reaction 2 and 3) [51] [52] [53].

	24] [23] [26] [29] [50] [10] [44]

independently of the TP imposed (Figure

4A

). Theoretically, 8 moles of H2 per mole of sucrose (4 moles of H2 from glucose and other 4 moles of H2 from fructose) can be produced if acetate is obtained as the only fermentation product (Reaction 1).

  One of the first reports of lactate conversion to butyrate and hydrogen was the study made by Thauer et al.[START_REF] Thauer | Energy conservation in chemotrophic anaerobic bacteria[END_REF] (Reaction 8). However, this reaction does not include acetate reduction. Later, in experiments made with Clostridium acetobutylicum strain P262, acetate was included in the equation and the Gibbs free energy was estimated at

	approximately -53.8 kJ.mol -1 (Reaction 9) [73]. These last authors added that lactate
	utilization was catabolized by an inducible NAD-independent lactate dehydrogenase
	(iLDH) with the Michaelis constant of enzyme reaction (Km) of 3.2 mM for D-lactate.
	Lactate conversion to butyrate and hydrogen
	2CH3CH(OH)COOH → CH3(CH2)2COOH + 2CO2 + 2H2 + H2O
	∆Gº' -56.5kJ.mol -1	(8)
	Lactate and acetate conversion to butyrate and hydrogen via NAD-iLDH pathway
	1.4CH3CH(OH)COOH + 0.6CH3COOH → CH3(CH2)2COOH + 1.4CO2 + 0.8H2 + H2O
	∆Gº' -53.8 kJ.mol -1	(9)
	Lactate and acetate conversion to butyrate and hydrogen
	CH3COOH + 2CH3CH(OH)COOH → 3/2CH3(CH2)2COOH + H2 + 2CO2 + H2O
	∆Gº' -156.6 kJ.mol -1

Total pressure (kPa) Clostridium sp.

		kPa
		kPa
		kPa
		kPa
		kPa
		kPa
		kPa
		kPa
		kPa
		kPa
		0%	20%	40%	60%	80%	100%
				Relative abundance
		Sporolactobacillus	Clostridium sp.	Ethanoligenens
	A	Lactobacillus		Lactococcus		unclassified
					Ethanoligenens
		Sporolactobacillus		Linear (Clostridium sp.)
	B	Linear (Ethanoligenens)	Linear (Sporolactobacillus)

Table 1 .

 1 Experimental design and different total pressure (kPa) applied to the hydrogen-producing systems fed with a mineral salts-sugarcane molasses solution. . The steady-state of the reactor was adopted as criterion of condition change.

			Total pressure (kPa)
	Reactor	Initial TP and independent	Condition of TP Increment/Decrement a
		condition a
	R1	80	100	120
	R2	100	120	140
	R3	120	140	100
	R4	140	-	-

a

Table 2 .

 2 Hydrogen production rate (HPR, mL-H2 h -1 ) and hydrogen yield ((HY), mol-H2 mol -1 suc eq.) of fermentative systems fed with a mineral salts-sugarcane molasses solution.

	Reactor	Total pressure (TP; kPa) / Partial pressure of hydrogen ( 80 / 41 100 / 49 120 / 61 140 / 70 ; kPa)
	R1	406 (4.51)	302 (3.02)	194 (2.15)	-
	R2	-	332 (2.79)	210 (1.88)	102 (0.64)
	R3	-	338(3.35)	210 (1.52)	102 (0.63)
	R4	-	-	-	62 (0.56)

Table 3 .

 3 Intermediates of hydrogen-producing systems at different total pressure (TP, kPa) and fed with a mineral salts-sugarcane molasses solution. Mean value ± standard deviation; n=6. b. Theoretical hydrogen production and yield are based on the acetate, butyrate and propionate produced according to Ferraz Júnior et al. (2014b). c. Acetate from homoacetogenesis was calculated according to Luo et al.[START_REF] Lee | Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy[END_REF]. d. Calculated according to Ferraz Júnior et al.[START_REF] Noblecourt | High hydrogen production rate in a submerged membrane anaerobic bioreactor[END_REF].

	Reactor	Conditions	Acetate a (mM)	Butyrate a (mM)	Propionate a (mM)	Ethanol a (mM)	Lactate a (mM)	Experimental H2 (mM)	Theoretical H2 b (mM)	Exp. H2/ Theoretical H2 (%)	Acetate from homoacetogenesis/ Total acetate c (%)	CODrec d (%)
		80 kPa	37.3 ± 1.7	24.1 ± 1.1	0.0 ± 0.0	19.5 ± 2.2 18.9 ± 0.0	54.2 ± 4.9	122.7	44.2	30.7	99.1
	R1	100 kPa	39 ± 1.4	18.4 ± 2.3	0.1 ± 0.0	30.4 ± 4.3 21.1 ± 2.2	40.3 ± 3.4	114.5	35.2	31.8	98.2
		120 kPa	35.6 ± 1.2	24.1 ± 1.2	0.1 ± 0.0	19.5 ± 2.1 21.1 ± 1.1	25.8 ± 2.6	119.2	21.7	43.8	100.2
		100 kPa	33.2 ± 0.3	21.8 ± 0.9	0.0 ± 0.0	26.2 ± 3.5 27.8 ± 1.7	44.3 ± 3.1	110	40.2	33	100.6
	R2	120 kPa	35.2 ± 3.1	21.8 ± 1.1	0.1 ± 0.0	33.2 ± 0.5 25.7 ± 1.4	28 ± 2.5	114	24.6	40.7	104.8
		140 kPa	29.5 ± 1.6	19.5 ± 1.0	0.1 ± 0.0	13 ± 2.2	26.1 ± 1.9	13.6 ± 1.0	97.8	13.9	47.6	87.4
		120 kPa	44 ± 1.7	20.7 ± 1.1	0.1 ± 0.0	26.1 ± 6.5 26.1 ± 3.3	28 ± 2.8	129.3	21.7	38.3	105
	R3	140 kPa	28.8 ± 1.0	21.8 ± 0.7	0.1 ± 0.0	17.4 ± 3.2 22.2 ± 1.1	13.6 ± 2.2	101.1	13.5	50.6	89.6
		100 kPa	49.1 ± 3.4	16.1 ± 0.6	0.0 ± 0.0	10.9 ± 2.2 16.7 ± 1.2	45.1 ± 4.4	130.4	34.6	28.9	96.7
	R4	140 kPa	22 ± 0.2	19.5 ± 1.2	0.0 ± 0.0	15.2 ± 4.3	20 ± 6.7	8.2 ± 0.9	83.1	9.9	56.6	90.8
	a.

Table 4 .

 4 Maximum hydrogen yield reported from different methods of controlling the partial pressure of hydrogen (

	).

Table 5 .

 5 Gibb's energy of lactate and acetate conversion into butyrate and hydrogen reaction.

	Reactor	Total pressure (TP; kPa) / Gibb's energy (∆Gº; kJ.mol -1 )
	R1	80 / -80.2	100 / -79.5	120 / -79.5	-
	R2	-	100 / -77.8	120 / -78.3	140 / -78.2
	R3	-	100 / -81.0	120 / -78.2	140 / -78.2
	R4

Acknowledgement

The authors gratefully acknowledge the financial support from FAPESP (Projects 2013/15665-8 and 2015/21650-9). Special thanks to Etienne Consoni, William Level, Dr. Afifi Akiar, Dr. Antonella Marone, Dr. Jordan Seira, Dr. Diane Plouchart, Dr. Julie Jimenez, Dr. Gabriel Capson-Tojo, Dr. Wendy Jabberwocky, Dr. Silvio Riggio, Dr. Martha Minale, Dr. Javiera Belen, Felipe Guilayn, Lucia Braga, Aurelie Bichot, Helene Thomas, Virginie Rossard, Alice Danel, Diane Ruiz, Pacôme Prompsy, Albane Lomenede, Clément Van Vlierberghe and Fernanda Peiter. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical statement The authors confirm that the article does not contain any studies with human participants or animals.