Volumetric Hydrogen Production Rate Research Articles

Dark fermentative biohydrogen production from synthetic cheese whey in an anaerobic structured-bed reactor: Performance evaluation and kinetic modeling

This study investigated the potential of dark fermentative hydrogen production from cheese whey in an acidogenic anaerobic structured-bed reactor (ASTBR). The reactor was operated at 25 °C with an organic loading rate (OLR) of 24 kg COD m−3 d−1 and a hydraulic retention time (HRT) of 24 h. The ASTBR had an average volumetric hydrogen production rate (VHPR) of 1.6 ± 0.7 L H2 L−1 d−1 and an average hydrogen yield (HY) of 1.4 ± 0.7 moL H2 mol−1 of consumed lactose. Batch experiments were performed with biomass from the ASTBR to characterize the main metabolic transformations involved in hydrogen production. An ADM1-based, unstructured kinetic model was developed and fitted to the obtained temporal profiles. The model allowed a satisfactory description of the process and indicated that hydrogen production from lactate and acetate could explain 74.5% of the total hydrogen volume produced. Because lactic-acid-producing bacteria are expected to ultimately colonize acidogenic reactors treating cheese whey, optimizing electron flow through this pathway may be a suitable strategy to enable long-term hydrogen production from cheese whey using mixed-culture fermentation.

The effect of hydraulic retention time on thermophilic dark fermentative biohydrogen production in the continuously operated packed bed bioreactor

The main objective of the study is to investigate the effect of hydraulic retention times on continuous dark fermentative biohydrogen production in an up-flow packed bed reactor (UPBR) containing a novel microorganism immobilization material namely polyester fiber beads. The hydrogen producing dark fermentative microorganisms were obtained by heat-pretreatment of anaerobic sludge from the acidogenic phase of an anaerobic wastewater treatment plant. Glucose was the sole carbon source and the initial concentration was 15 ± 1 g/L throughout the continuous feeding. UPBR was operated under the thermophilic condition at T = 48 ± 2 °C and at varying HRTs between 2 h and 6 h. The hydrogen productivity of continuously operated UPBR increased with increasing HRT. Hydrogen production volume varied between 4331 and 6624 ml/d, volumetric hydrogen production rates (VHPR) were obtained as 3.09–4.73 L H2/L day, and hydrogen production yields (HY) were 0.49 mol/mol glucose-0.89 mol/mol glucose depending on HRT. Maximum daily hydrogen volume (6624 ml/d), the yield (0.89 mol/mol glucose) and VHPR (4.73 L H2/L day) were obtained at HRT = 6 h. The production rate and the yield decreased with increasing organic loading rate due to substrate inhibition.

Coproduction of hydrogen and methane in a CSTR-IC two-stage anaerobic digestion system from molasses wastewater.

A continuous hydrogen and methane production system in a two-stage process has been investigated to increase energy recovery rate from molasses wastewater in this study. This system consisted of a continuous stirred-tank reactor for hydrogen production and an internal circulation (IC) reactor for methane production, and was studied under the influent organic loading rate (OLR) of 18, 24, 30 and 36kg COD/(m3·d) (COD: chemical oxygen demand). The maximum volumetric hydrogen production rate of 2.41 L/(L·d) was obtained at the OLR of 30kg COD/(m3·d) with a hydrogen content of 42%, and the maximum volumetric methane production rate of 2.4 L/(L·d) with a methane content of 74.45% was obtained at the OLR of 36kg COD/(m3·d) using the effluents of hydrogen fermentation as substrate. The maximum of 71.06% of the molasses wastewater energy was converted to biogas (hydrogen and methane) at the OLR of 30kg COD/(m3·d).

Enhancement of hydrogen production rate by high biomass concentrations of Thermotoga neapolitana

The objective of this study was to enhance the hydrogen production rate of dark fermentation in batch operation. For the first time, the hyperthermophilic pure culture of Thermotoga neapolitana cf. Capnolactica was applied at elevated biomass concentrations. The increase of the initial biomass concentration from 0.46 to 1.74 g cell dry weight/L led to a general acceleration of the fermentation process, reducing the fermentation time of 5 g glucose/L down to 3 h with a lag phase of 0.4 h. The volumetric hydrogen production rate increased from 323 (±11) to 654 (±30) mL/L/h with a concomitant enhancement of the biomass growth and glucose consumption rate. The hydrogen yield of 2.45 (±0.09) mol H2/mol glucose, the hydrogen concentration of 68% in the produced gas and the composition of the end products in the digestate, i.e. 62.3 (±2.5)% acetic acid, 23.5 (±2.9)% lactic acid and 2.3 (±0.1)% alanine, remained unaffected at increasing biomass concentrations.

The effect of HRT on biohydrogen production from acid hydrolyzed waste wheat in a continuously operated packed bed reactor

Biohydrogen production in a continuously operated up flow packed bed reactor was investigated at different hydraulic retention time (HRT) varying between 2 h and 13 h scouring sponge pad. The substrate was sugar solution obtained from hydrolysis of waste wheat at pH = 2 and 90 °C in an autoclave for 15 min. Experimental results indicated that hydrogen production volume and yield increases with decreasing HRT. The highest volumetric hydrogen production rate and yield were obtained as VHPR = 1.75 L H2/L d and YH2 = 1.6 moL H2/mol TS, respectively, at HRT = 2 h. Yields and rates at HRT = 2 h were almost two times of that obtained at HRT = 13 h. It can be concluded that metal mesh covered plastic scouring sponge pad is a suitable microorganism support particle to obtain high hydrogen yield and rate at short HRTs by dark fermentation.

Hydrogen metabolic patterns driven by Clostridium-Streptococcus community shifts in a continuous stirred tank reactor.

The hydrogen (H2) production efficiency in dark fermentation systems is strongly dependent on the occurrence of metabolic pathways derived from the selection of microbial species that either consume molecular H2 or outcompete hydrogenogenic bacteria for the organic substrate. In this study, the effect of organic loading rate (OLR) on the H2 production performance, the metabolic pathways, and the microbial community composition in a continuous system was evaluated. Two bacterial genera, Clostridium and Streptococcus, were dominant in the microbial community depending on the OLR applied. At low OLR (14.7-44.1gLactose/L-d), Clostridium sp. was dominant and directed the system towards the acetate-butyrate fermentation pathway, with a maximum H2 yield of 2.14molH2/molHexose obtained at 29.4gLactose/L-d. Under such conditions, the volumetric hydrogen production rate (VHPR) was between 3.2 and 11.6LH2/L-d. In contrast, relatively high OLR (58.8 and 88.2 gLactose/L-d) favored the dominance of Streptococcus sp. as co-dominant microorganism leading to lactate production. Under these conditions, the formate production was also stimulated serving as a strategy to dispose the surplus of reduced molecules (e.g., NADH2+), which theoretically consumed up to 5.72 LH2/L-d. In such scenario, the VHPR was enhanced (13.7-14.5 LH2/L-d) but the H2 yield dropped to a minimum of 0.74molH2/molHexose at OLR = 58.8gLactose/L-d. Overall, this research brings clear evidence of the intrinsic occurrence of metabolic pathways detrimental for biohydrogen production, i.e., lactic acid fermentation and formate production, suggesting the use of low OLR as a strategy to control them.

Impacts of nano-metal oxides on hydrogen production in anaerobic digestion of palm oil mill effluent – A novel approach

In the present study, hydrogen production from palm oil mill effluent (POME) was investigated with the incorporation of nanoparticles (NPs) comprising of nickel (NiO) and cobalt oxides (CoO). The NPs of NiO and CoO were prepared using hydrothermal method and were further applied to analyse, their effect on hydrogen production. The results demonstrated that, a maxima volumetric hydrogen production rate of 21 ml H2/L-POME/h with the hydrogen yield of 0.563 L H2/g-CODremoved was obtained with 1.5 mg/L concentration of NiO NPs. On the other hand, the addition of CoO NPs produced maximum volumetric hydrogen production rate of 18 ml H2/L-POME/h with a hydrogen yield of 0.487 L H2/g-CODremoved with 1.0 mg/L of CoO NPs. Results showed that addition of optimal concentration of NiO and CoO NPs to the POME enhances the hydrogen yield by 1.51 and 1.67 fold respectively. Besides, this addition of NiO and CoO enhanced the COD removal efficiency by 15 and 10% respectively as compared to an un-additive NPs POME. The toxicity of NPs was also tested using bacterial viability test, which revealed that application of 3.0 mg/L of NiO and CoO NPs to modified Luria-Bertani (LB) medium had 63% and 83% reduction in bacterial cell growth. The results concluded that supplementation of NiO and CoO NPs under an optimal range to the wastewater can improve the hydrogen productivity.

Amylolysis is predominated by cell-surface-bound hydrolase during anaerobic fermentation under mesophilic conditions

While knowing the amylolysis mechanism is important to effectively decompose corn starch fed into an anaerobic digestor, the objective of this study was to detect the activities and locations of α-amylase in a continuous reactor and batch cultures. In the continuous reactor operated at 35°C, the greatest cell-bound α-amylase activity was found to be 4.7 CU mL-1at hydraulic retention time (HRT)=9h, while the greatest volumetric hydrogen production rate (rH2) was observed at HRT=3h as 61mmolL-1 day-1. In the batch tests, the cell-bound α-amylase activities increased when the carbohydrate concentration decreased, and no significant reducing sugar accumulation was found in the serum bottles. By examining the specific hydrogen production rate (qH2) against different corn starch concentrations, the half-saturation constant (KSta) and the maximum qH2 were regressed to be 0.47gL-1 and 6mmol g-VSS-1d-1, respectively. The electronic microscopic images showed that the microbes could colonize on the starch granules without the disturbance of any floc-like materials. Conclusively, by excluding the methanogens and floc matrix, the secreted α-amylases are predominately bound on the cell surfaces and enabled the microbial cells favorably attach on large substrates for hydrolysis under the mesophilic condition.

Biohydrogen production at pH below 3.0: Is it possible?

Biological hydrogen production was investigated in continuous acidogenic reactors fed with sucrose at 30 °C without pH control. In the first experimental phase, three reactors were compared: a structured fixed-bed (FB), a granular UASB (UG) and a flocculent UASB (UF-1). They were run at 3.3 h HRT and 33 gCOD L−1d−1 OLR. Hydrogen production occurred throughout the experimental period with an average effluent pH of only 2.8. The FB, UG and UF-1 reactors presented volumetric hydrogen production rates (VHPR) of 95 ± 69, 45 ± 37 and 54 ± 32 mLH2 L−1h−1, respectively; and H2 yields (HY) of 1.5 ± 0.8, 0.8 ± 0.6 and 1.2 ± 0.7 molH2 mol−1 sucroseconsumed, respectively. The UF-1 reactor showed intermediate VHPR and HY, but no declining trend, contrary to what was observed in the FB reactor. Thus, aiming at continuous and long-term H2 production, a flocculent UASB was applied in the second experimental phase. In this phase, the HRT of the acidogenic reactor, which was named UF-2, was raised to 4.6 h, resulting in an OLR of 25 gCOD L−1d−1. The VHPR and the HY increased considerably to 175 ± 44 mLH2 L−1h−1 and 3.4 ± 0.7 molH2 mol−1 sucroseconsumed, respectively. These improvements were accompanied by greater sucrose removal, higher suspended biomass concentration, less production of lactate and more of acetate, and high ethanol concentration. Contradicting the current published literature data that reports strong inhibition of H2 production by dark fermentation at pH less than 4.0, the UF-2 reactor presented stable, long-term H2 production with satisfactory yields at pH 2.7 on average. 16 S rDNA sequencing revealed that two sequences assigned as Ethanoligenens and Clostridium accounted for over 70% of the microbiota in all the reactors. The non-necessity of adding alkalizing agents and the successful H2 production under very acid conditions, demonstrated in this study, open a new field of investigation in biological hydrogen production by dark fermentation towards a more sustainable and feasible technology.

Continuous hydrogen and methane production from Agave tequilana bagasse hydrolysate by sequential process to maximize energy recovery efficiency

Continuous H2 and CH4 production in a two-stage process to increase energy recovery from agave bagasse enzymatic-hydrolysate was studied. In the first stage, the effect of organic loading rate (OLR) and stirring speed on volumetric hydrogen production rate (VHPR) was evaluated in a continuous stirred tank reactor (CSTR); by controlling the homoacetogenesis with the agitation speed and maintaining an OLR of 44 g COD/L-d, it was possible to reach a VHPR of 6 L H2/L-d, equivalent to 1.34 kJ/g bagasse. In the second stage, the effluent from CSTR was used as substrate to feed a UASB reactor for CH4 production. Volumetric methane production rate (VMPR) of 6.4 L CH4/L-d was achieved with a high OLR (20 g COD/L-d) and short hydraulic retention time (HRT, 14 h), producing 225 mL CH4/g-bagasse equivalent to 7.88 kJ/g bagasse. The two-stage continuous process significantly increased energy conversion efficiency (56%) compared to one-stage hydrogen production (8.2%).

THE EFFECT OF MIXING MODES ON BIOHYDROGEN YIELD AND SPATIAL PH GRADIENT AT DARK FERMENTATION OF SOLID FOOD WASTE

Dark fermentation of multicomponent solid food waste by soil hydrogen-producing bacterial community was studied in horizontal batch reactor under different mechanical mixing modes; speed of mixing (rpm) and frequency of mixing (duration mixing and pause) were selected as target for optimization. Addition of pH indicator bromothymol blue to the culture liquid allowed to study patterns of formation of acidification zones in solid/liquid/gaseous fermentation system. It was detected that mixing speed can contribute to sustainable maintenance of optimal pH values and prevent formation of spatial pH gradient. Adjusting of mixing and pause duration resulted in significant decrease of digestion cycle and increase in volumetric hydrogen production rate. Established optimal mixing mode 24 rpm, 10 min of mixing/20 min of pause provided biohydrogen yield 123 LH2 kg-1 TSadded and reduction in the concentration of total suspended solids from 70 to 0.75 g/L in 4 days.

Dark-fermentative biohydrogen pathways and microbial networks in continuous stirred tank reactors: Novel insights on their control

In the present work, the influence of hydraulic retention time (HRT) on dark fermentation metabolism was evaluated through the operation and analysis of a series of four continuous stirred tank reactors (CSTR) at four HRT ranging from 6h to 24h. A maximum volumetric hydrogen production rate (VHPR) of 2000±149mL/L-d corresponding to an H2 yield of 0.86molH2/mollactose was observed at 6h HRT. In depth analysis of metabolite profiles and microbial communities showed that low values of HRT favored the emergence of a community dominated by Clostridiaceae-Lachnospiraceae-Enterobacteriaceae, which performed metabolic pathways co-producing hydrogen. In contrast, long HRT led to the establishment of Sporolactobacillaceae-Streptococcaceae microbial community that outcompeted hydrogen producing bacteria and was responsible of lactate production. Results suggested that these two communities mutually excluded themselves and HRT can act as an operational parameter to control the microbial communities and consequently the related metabolic pathways.

A novel tubular microbial electrolysis cell for high rate hydrogen production

Practical application of microbial electrolysis cells (MECs) for hydrogen production requires scalable reactors with low internal resistance, high current density, and high hydrogen recovery. This work reports a liter scale tubular MEC approaching these requirements. The tubular cell components (a platinum-coated titanium mesh cathode, an anion exchange membrane, and a pleated stainless steel felt anode) were arranged in a concentric configuration. The reactor had a low internal resistance (0.325 Ω, 19.5 mΩ m2) due to the high conductivity of the electrodes, a compact reactor configuration, and proper mixing. With acetate as electron donor, the MEC achieved a volumetric current density of 654 ± 22 mA L−1 (projected current density, 1.09 ± 0.04 mA cm−2) and a volumetric hydrogen production rate of 7.10 ± 0.01 L L−1 d−1 at an applied voltage of 1 V. The reactor also showed high hydrogen recovery (∼100%), high hydrogen purity (>98%), and excellent operational stability during the 3 weeks of operation. These results demonstrated that high hydrogen production rate could be achieved on larger scale MEC and this tubular MEC holds great potential for scaling up.

Continuous hydrogen production from enzymatic hydrolysate of Agave tequilana bagasse: Effect of the organic loading rate and reactor configuration

Lignocellulosic biomass is a promising alternative energy source, which after pretreatment can be efficiently used as feedstock for biological production of second generation biofuels. Hydrogen is considered an ideal biofuel and its production through dark fermentation has been recognized as a sustainable process. However, more studies with continuous systems are needed for its application at industrial scale. In this study, enzymatic hydrolysate of Agave tequilana bagasse was used for long-term continuous hydrogen production in both, a continuous stirred tank reactor (CSTR) and a trickling bed reactor (TBR), which were operated up to 87days under different organic loading rates (OLR) ranging from 17 to 60gCOD/L-d. Volumetric hydrogen production rate (VHPR) and hydrogen molar yield (HMY) in CSTR displayed an inverse correlation with maximum values of 2.53LH2/L-d and 1.35molH2/mol substrate, attained at OLR 52.2 and 40.2gCOD/L-d, respectively. In contrast, increasing OLR up to 52.9gCOD/L-d simultaneously enhanced VHPR and HMY in TBR, attaining values of 3.45LH2/L-d and 1.53molH2/molsubstrate, respectively. Acetate and butyrate were the main metabolites in both reactors, while lactate and propionate were detected in minor concentrations. Metabolites distribution, electron balances and hydrogen production trends obtained from both reactors suggest that CSTR may be more susceptible to inhibition by hydrogen accumulation than TBR. Apparent hydrogen consumption and susceptibility to high solids load were found to limit further OLR increments in CSTR and TBR, respectively.

Role of organic loading rate in bioenergy generation from palm oil mill effluent in a two-stage up-flow anaerobic sludge blanket continuous-stirred tank reactor

This contribution presents the technical possibilities for continuous hydrogen and methane production using an optimum organic loading rate of palm oil mill effluent in a two-stage reactor at a thermophilic temperature of 55 °C. The influence of four organic loading rates, namely, 30, 40, 50, and 60 kg COD/(m3 d) for hydrogen production and 8.3, 10.2, 13.1, 15.8 kg COD/(m3 d) for methane production, were investigated. Hydrogen production was controlled in an up-flow anaerobic sludge blanket reactor at a constant hydraulic retention time of 12 h. The maximum hydrogen content, volumetric hydrogen production rate and hydrogen yield were found to be 45%, 2.5 L H2/d and 33.48 mL H2/g COD, respectively, at the organic loading rate of 50 kg COD/(m3 d). The effluent from the hydrogenogenic reactor was further digested into methane in the continuous stirred tank reactor at a hydraulic retention time of 5 d. The maximum volumetric methane production rate and methane yield were 10.58 L CH4/d. and 0.11 m3 CH4/kg COD, respectively, at an organic loading rate of 13.1 kg COD/(m3 d). A total chemical oxygen demand removal of 91% was achieved in this two-stage process. The scientific contribution of this two-stage technology with an optimized organic loading rate may play a significant role in degrading palm oil mill effluent and developing an energy-efficient strategy for waste management.

Continuous two-staged co-digestion process for biohydrogen production from agro-industrial wastes

Mexico produces large amounts of organic residues. Twenty milliard tons of fruit and vegetable wastes (FVW) are produced yearly. On the other hand, the cheese processing industry produces crude cheese whey (CCW) with an annual production of 1 million metric tons. These types of residues are well characterized, and both constitute a potential feedstock for biohydrogen production, individually or as a mixture. Recently, we demonstrated the feasibility of utilizing CCW and FVW in a co-digestion process to produce biohydrogen. The possibility to design an effective process in a two stage configuration was evaluated based in our previous studies of the by-products and description of microbial ecology evolution of the co-digestion process. An initial assimilation of easily degradable carbohydrates and protein allowed cell growth and acetate and lactate as main byproducts. The co-digestion and two-stage processes increased several times the biohydrogen yield compared with those obtained from single substrate and in a co-digestion single stage process. Higher biohydrogen productivity was a result of reaction stability in comparison to single-substrate digestion and one-stage processes. The effect of hydraulic retention time (HRT) and organic loading rate (OLR) on the volumetric hydrogen production rate (VHPR) and yield (YH2) was evaluated in a two-staged co-digestion process of FVW and CCW. The system consisted of hydrolytic and hydrogenic bioreactors operated in batch mode and continuous regime (89 days), respectively. Eight conditions of HRTs and respective OLRs were evaluated. As HRT decreased, the VHPR increased proportionally, reaching the highest average values around 7 L L−1 d−1 at an HRT between 15 and 17.5 h. The highest YH2, (813.3 mL H2 g COD−1) was determined at 17.5 h (OLR, 80.02 g COD L−1 d−1). Based on the distribution of the final products, the processes occur sequentially and reach a steady state set by the OLR. Biohydrogen production is a result of a syntrophic microbial activity. In general, the two stages of the co-digestion process provided higher stability, reliability, and efficiency. Copyright © 2015 John Wiley & Sons, Ltd.

Hydrogen gas production with an electroformed Ni mesh cathode catalysts in a single-chamber microbial electrolysis cell (MEC)

Microbial electrolysis cells (MECs) are generally regarded as a promising future technology for manufacturing green hydrogen from organic material present in wastewaters and other renewable energy sources. However, the development of inexpensive and high-efficient cathode catalyst is the most critical challenge for MECs to become a commercialized H2 production technology. In this study, a non-noble metal electroformed Ni mesh cathode alternatives to typical cathode material (Pt/CC) was intensively examined in a single-chamber membrane-free MEC. To the best of our knowledge, the use of electroformed Ni mesh as the MEC cathode catalyst has not been reported so far. The MEC was operated in fed-batch mode and the performance of the Ni mesh cathode was compared with that of Pt/CC cathode in terms of columbic efficiencies (75 ± 4% vs. 72.7 ± 1%), overall hydrogen recovery (89.3 ± 4% vs. 90.9 ± 3%), overall energy efficiency (62.9 ± 5% vs. 69.1 ± 2%), the maximum volumetric hydrogen production rate (4.18 ± 1 m3 H2/m3 d vs. 4.25 ± 1 m3 H2/m3 d), volumetric current density (312 ± 9 A/m3 vs. 314 ± 5 A/m3). The obtained results in this study highlight the great potential of using the electroformed Ni mesh catalysts as a viable cathode material for hydrogen production in MECs.

The application of an innovative continuous multiple tube reactor as a strategy to control the specific organic loading rate for biohydrogen production by dark fermentation

Biohydrogen production in fixed-bed reactors often leads to unstable and decreasing patterns because the excessive accumulation of biomass in the bed negatively affects the specific organic loading rate (SOLR) applied to the reactor. In this context, an innovative reactor configuration, i.e., the continuous multiple tube reactor (CMTR), was assessed in an attempt to better control the SOLR for biohydrogen production. The CMTR provides a continuous discharge of biomass, preventing the accumulation of solids in the long-term. Sucrose was used as the carbon source and mesophilic temperature conditions (25°C) were applied in three continuous assays. The reactor showed better performance when support material was placed in the outlet chamber to enhance biomass retention within the reactor. Although the SOLR could not be effectively controlled, reaching values usually higher than 10gsucroseg−1VSSd−1, the volumetric hydrogen production and molar hydrogen production rates peaked, respectively, at 1470mLH2L−1d−1 and 45mmolH2d−1, indicating that the CMTR was a suitable configuration for biohydrogen production.

Improvement of biohydrogen production using a reduced pressure fermentation.

This study investigated the effect of reduced pressure on biohydrogen production in an upflow anaerobic sludge blanket (UASB) reactor from whey permeate. The results showed that the reduced pressure fermentation was more effective in enhancing biohydrogen production than dark fermentative hydrogen production at atmospheric pressure. Mesophilic fermentative biohydrogen production was investigated at a constant hydraulic retention time (HRT) of 24h and increasing organic loading rates (OLRs) of 20, 25, 30, 35kg COD/m(3)day. The reduced pressure fermentation was successfully operated at all OLRs tested. The maximum proportion of hydrogen in biogas of 47.7%, volumetric hydrogen production rate (VHPR) of 7.10LH2/day and hydrogen yield of 4.55molH2/kg COD removed occurred at the highest OLR. Increase in OLR affected the hydrogen production in UASB reactor exploited at atmospheric pressure. The reduced pressure process was able to remarkably improve the biohydrogen performance at high OLRs.

Inoculum pretreatment promotes differences in hydrogen production performance in EGSB reactors

Hydrogen production by dark fermentation is one of the most promising methods for obtaining clean energy. Inoculum pretreatments allow the selection of bacteria that have better performance in hydrogen production, because the selection of pretreatment limits the presence of some species while favoring others. In order to elucidate the inoculum pretreatment influence during the operation of two EGSB reactors, two pretreatments were assayed: heat shock and cell wash-out. Different organic loading rates (24–60 g glucose/L d) and hydraulic retention times (10–4 h) were applied to both reactors to determine population dynamics along 100 days of operation. Reactors exhibited differences in both volumetric hydrogen production rate and molar yield but with cell wash-out pretreatment showing better performance than heat shock pretreatment. Maximum molar yield (0.92 mol H2/mol hexose) and volumetric hydrogen production rate (4.23 L H2/L d) were obtained with organic loading rates of 36 g glucose/L d at HRT of 10 h in EGSB reactor inoculated with cell wash-out pretreated sludge. The microbial community of the reactors samples was analyzed by 16S rRNA genes profiles and the predominant bands were excised and their DNA sequence determined. Clostridium and representatives of Enterobacteriaceae were dominant, with a strong presence of Lactobacillus genus. The whole result indicates that the inoculum pretreatment has a strong initial effect during early stages of fermentation, after which the operating conditions have a greater impact on reactor performance.