Dark fermentative hydrogen production from simple sugars and various wastewaters by a newly isolated Thermoanaerobacterium thermosaccharolyticum SP-H2

The hydrogen-producing bacterium SP-H2 was isolated from a thermophilic acidogenic reactor inoculated with municipal sewage sludge and processing a carbohydrate-rich simulated food waste. Based on the 16S rRNA gene sequence, the bacterium was identified as Thermoanaerobacterium thermosaccharolyticum. The maximum growth rate was observed at 55–60 °C and pH 7.5. The H₂-producing activity of the bacterium was studied using mono-, di- and tri-saccharides related to both hexoses (maltose, glucose, mannose, fructose, lactose, galactose, sucrose, raffinose, cellobiose) and pentoses (xylose and arabinose), as well as using real wastewaters (cheese whey, confectionery wastewater, sugar-beet processing wastewater). The highest H₂ yield was observed during dark fermentation (DF) of maltose (1.91 mol H₂/mol hexose or 77.8 mmol H₂/L). The maximum H₂ production rate was observed during DF of xylose (13.3 ml H₂/g COD/h) and cellobiose (2.47 mmol H₂/L/h). The main soluble metabolite products were acetate, ethanol and butyrate. The acetate concentration had a statistically significant positive correlation with the H₂ content in biogas and the specific H₂ yield. Based on the results of the correlation analysis, it was tentatively assumed that in the formic acid (mixed-acid) type fermentation, the rate of H₂ production was higher than in the butyric acid type fermentation. With regard to real wastewater, cheese whey and confectionery wastewater were distinguished by a higher H₂ yield (152 ml H₂/g COD) and H₂ production rate (0.57 mmol H₂/L/h), respectively. The highest concentrations of confectionery wastewater and cheese whey, at which the DF process took place, were 5915 and 7311 mg COD/L, respectively. At the same time, SP-H2 dominated in the microbial community, despite the presence of indigenous microorganisms in wastewater. Thus, T. thermosaccharolyticum SP-H2 is a promising strain for DF of carbohydrate-rich unsterile wastewater under thermophilic conditions.     

Sustainable fermentation approach for biogenic hydrogen productivity from delignified sugarcane bagasse

Improper lignocellulosic wastes management causes severe environmental pollution and health damage. Conversion of such wastes particularly sugarcane bagasse (SCB) onto bioenergy is a sustainable approach due to a continuous depletion of conventional biofuels. The delignification of SCB is necessary to proceed for bio-genic H₂ productivity by anaerobic bacteria. The effect of autoclaving, pre-acidification/autoclaving and pre-alkalization/autoclaving of SCB on glucose recovery and subsequently H₂ productivity by dark fermentation was comprehensively investigated. Pre-acidified SCB with 1% H₂SO₄ (v/v) provided H₂ productivity of 8.5 ± 0.14 L/kg SCB and maximum H₂ production rate (R_m) of 105.9 ± 8.3 mL/h. Those values were dropped to 2.7 ± 0.13 L/kg SCB and 58.3 ± 12.9 mL/h for fermentation of delignified SCB with 2% H₂SO₄. This was linked to high levels of total phenolic compounds (1775.3 ± 212 mg/L) in the feedstock. Better H₂ productivity of 13.9 ± 0.58 L/kg SCB and R_m of 133.9 ± 3.6 mL/h was achieved from fermentation of pre-alkalized SCB with 1%KOH (v/v). 256.8 ± 9.8 U/100 mL of α-amylase, 165.7 ± 7.6 U/100 mL of xylanase, 232.8 ± 6.1 U/100 mL of CM-Cellulase, 176.5 ± 5.0 U/100 mL of polyglacturanase and 0.702 ± 0.013 mg M B. reduced/min. of hydrogenase enzyme was accounted for the batches supplied with delignified SCB by KOH. The Clostridium and Bacillus spp. was dominance and prevalence resulting a higher H₂ productivity and yield. A novel strain of Archea and alpha proteobacterium were also identified and detected.     

H2-rich biogas recirculation prevents hydrogen supersaturation and enhances hydrogen production by Thermotoga neapolitana cf. capnolactica

This study focused on the supersaturation of hydrogen in the liquid phase (H_2aq) and its inhibitory effect on dark fermentation by Thermotoga neapolitana cf. capnolactica by increasing the agitation (from 100 to 500 rpm) and recirculating H₂-rich biogas (GaR). At low cell concentrations, both 500 rpm and GaR reduced the H_2aq from 30.1 (±4.4) mL/L to the lowest values of 7.4 (±0.7) mL/L and 7.2 (±1.2) mL/L, respectively. However, at high cell concentrations (0.79 g CDW/L), the addition of GaR at 300 rpm was more efficient and increased the hydrogen production rate by 271%, compared to a 136% increase when raising the agitation to 500 rpm instead. While H_2aq primarily affected the dark fermentation rate, GaR concomitantly increased the hydrogen yield up to 3.5 mol H₂/mol glucose. Hence, H_2aq supersaturation highly depends on the systems gas-liquid mass transfer and strongly inhibits dark fermentation.     

Simultaneous biohydrogen (H2) and bioplastic (poly-β-hydroxybutyrate-PHB) productions under dark,photo, and subsequent dark and photo fermentation utilizing various wastes

The present study is focused on bio hydrogen (H₂) and bioplastic (i.e., poly-β-hydroxybutyrate; PHB) productions utilizing various wastes under dark fermentation, photo fermentation and subsequent dark-photo fermentation. Potential bio H₂ and PHB producing microbes were enriched and isolated. The effects of substrate (rice husk hydrolysate, rice straw hydrolysate, dairy industry wastewater, and rice mill wastewater) concentration (10–100%) and pH (5.5–8.0) were examined in the batch mode under the dark and photo fermentation conditions. Using 100% rice straw hydrolysate at pH 7, the maximum bio H₂ (1.53 ± 0.04 mol H₂/mol glucose) and PHB (9.8 ± 0.14 g/L) were produced under dark fermentation condition by Bacillus cereus. In the subsequent dark-photo fermentation, the highest amounts of bio H₂ and PHB were recorded utilizing 100% rice straw hydrolysate (1.82 ± 0.01 mol H₂/mol glucose and 19.15 ± 0.25 g/L PHB) at a pH of 7.0 using Bacillus cereus (KR809374) and Rhodopseudomonas rutila. The subsequent dark-photo fermentative bio H₂ and PHB productions obtained using renewable biomass (i.e., rice husk hydrolysate and rice straw hydrolysate) can be considered with respect to the sustainable management of global energy sources and environmental issues.     

Hydrogen production from sludge of poultry slaughterhouse wastewater treatment plant pretreated with microwave

This study aims to produce hydrogen from sludge of poultry slaughterhouse wastewater treatment plant (5% total solid) by anaerobic batch fermentation with Enterobactor aerogenes or mixed cultures from hot spring sediment as the inoculums. Sludge was heated in microwave at 850 W for 3 min. Results indicated that a soluble chemical oxygen demand (sCOD) of pretreated sludge was higher than that of raw sludge. Pretreated sludge inoculated with E. aerogenes and supplemented with the Endo nutrient had a higher hydrogen yield (12.77 mL H₂/g tCOD) than the raw sludge (0.18 mL H₂/g tCOD). When considered the hydrogen yield, the optimum initial pH for hydrogen production from microwave pretreated sludge was 5.5 giving the maximum value of 12.77 mL H₂/g tCOD. However, when considered the hydrogen production rate (R_m), the optimum pH for hydrogen production would be 9.0 with the maximum R_m of 22.80 mL H₂/L sludge·h.     

Parametric optimization of the dark fermentation process for enhanced biohydrogen production from the organic fraction of municipal solid waste using Taguchi method

In this work, the Taguchi method was used to optimize the dark fermentative H₂ production from the organic fraction of municipal solid waste (OFMSW). The experiments were planned using the L16 orthogonal array design with each trial conducted at different levels of substrate concentration, inoculum-to-substrate ratio (ISR), and temperature. Based on the results, the optimal setting of the process parameters was the substrate concentration of 6 g-VS/L, ISR 0.5, and temperature of 55 °C. Furthermore, substrate concentration was the most important parameter affecting bio-H₂ production among the three process parameters considered. Finally, a confirmation experiment under optimal conditions yielded 62.5 mL H₂/g-VS_added, which was higher than all the bio-H₂ yield values obtained in the other conditions tested in this study. The measured and predicted bio-H₂ yields in the verification test were also very close to each other, confirming the reliability of the Taguchi method in optimizing the bio-H₂ production process.     

Enhanced H2 production from corn stalk by integrating dark fermentation and single chamber microbial electrolysis cells with double anode arrangement

The low conversion efficiency of substrate is one of the main bottlenecks in dark fermentation for bio-H₂ production. Herein, an enhanced H₂ yield from corn stalk was achieved by integrating dark fermentation and single chamber microbial electrolysis cells (MECs). In the dark fermentation stage, a H₂ yield of 129.8 mL H₂/g-corn stalk and an average H₂ production rate of 1.73 m³/m³ d were recorded at 20 g/L of corn stalk and initial pH 7.0. The effluent from dark fermentation was diluted and further employed as feedstock to generate H₂ by MECs. A H₂ yield of 257.3 mL H₂/g-corn stalk, an HPR of 3.43 ± 0.12 m³/m³ d and an energy efficiency of 166 ± 10% were obtained with the effluent COD of 3995.5 mg/L under 0.8 V applied voltage. During MECs operation stage, about 90 ± 2% of acetate was converted to H₂ and the corresponding COD removal reached 44 ± 2% in MECs. Overall, the H₂ yield can reach 387.1 mL H₂/g-corn stalk by integrating dark fermentation and MECs, which had nearly tripled as against that of dark fermentation.     

Synergistic strategy for the enhancement of biohydrogen production from molasses through coculture of Lactobacillus brevis and Clostridium saccharobutylicum

This study aimed to evaluate the capacity of different inoculum sources and their bacterial diversity to generate hydrogen (H₂). The highest Simpson (0.7901) and Shannon (1.581) diversity indexes for H₂-producing bacterial isolates were estimated for sewage inocula. The maximum cumulative H₂ production (H_max) was 639.6 ± 5.49 mL/L recorded for the sewage inoculum (SS30) after 72 h. The highest H₂-producing isolates were recovered from SS30 and identified as Clostridium saccharobutylicum MH206 and Lactobacillus brevis MH223. The H_max of C. saccharobutylicum, L. brevis, and synergistic coculture was 415.00 ± 24.68, 491.67 ± 15.90, and 617.67 ± 3.93 mL/L, respectively. The optimization process showed that the H_max (1571.66 ± 33.71 mL/L) with a production rate of 58.02 mL/L/h and lag phase of 19.33 h was achieved by the synergistic coculture grown on 3% molasses at 40 °C, pH 7, and an inoculum size of 25% (v/v). This study revealed the economic feasibility of the synergistic effects of coculture on waste management and biohydrogen production technology.     

Comparative evaluation of the mesophilic and thermophilic biohydrogen production at optimized conditions using tequila vinasses as substrate

The objective of this work was to comparatively evaluate the production of biohydrogen (bio-H₂) from tequila vinasses at optimized mesophilic and thermophilic conditions and to elucidate the main metabolic routes involved. Optimal temperatures of 35 °C and 55 °C, and pH of 5.5 maximized the bio-H₂ production rates, 25.5 ± 0.01 NmL h⁻¹ and 169.9 ± 8.9 NmL h⁻¹ in the mesophilic and thermophilic regimens, respectively. During the operation of anaerobic sequencing batch reactors, the thermophilic process allowed a volumetric bio-H₂ production rate of 519 ± 13 NmL-H₂ L⁻¹ d⁻¹ equivalent to 750 ± 19 NmL-H₂ L_vinasse⁻¹, while the mesophilic one 448 ± 23 NmL-H₂ L⁻¹ d⁻¹ and 647 ± 33 NmL-H₂ L_vinasse⁻¹, respectively. Furthermore, the gas produced under thermophilic conditions showed high hydrogen content (86.5%). Finally, formate degradation and glucose fermentation to acetic and butyric acids were the main metabolic routes involved in bio-H₂ production under thermophilic conditions, while at mesophilic conditions, the lactate and formate degradation pathways governed.     

Enhancement of biohydrogen production in industrial wastewaters with vinasse pond consortium using lignin-mediated iron nanoparticles

The aim of this work was the enhancement of biohydrogen production by an anaerobic bacterial consortium with incorporation of lignin-mediated iron nanoparticles in the fermentation medium. Lignin magnetic nanoparticles (LMNP), identified as magnetite by XRD, exhibited spherical shape and average particle size of 8.6 nm, while lignin non-magnetic nanoparticles (LNMNP) exhibited high agglomeration and an amorphous nature in TEM and XRD, respectively. The fermentation medium (pH 7) was composed of 88% soft drink wastewater (SDW) and 12% corn steep liquor (CSL) and supplemented with NaHCO₃ (1.0 g/L) and cysteine-HCl (0.5 g/L). Under optimal conditions, BioH₂ production was 17.67 ± 0.54 mL, after 48 h of fermentation at 37 °C. Addition of LMNP and LNMNP increased BioH₂ production in 91.0 and 74.3%, respectively. Additionally, 200 mg/L of LMNP and LNMNP in the fermentation medium improved the BioH₂ yields (mL H₂/g COD_removed) in 2.8- and 2.3-fold, respectively.     

Evaluation of sludge liquors from acidogenic fermentation and thermal hydrolysis process as feedstock for microbial electrolysis cells

In this study, mesophilic acidogenic fermentation, thermophilic acidogenic fermentation, and thermal hydrolysis process (THP) were compared to generate sludge liquors for bioenergy recovery with microbial electrolysis cells (MECs). The results showed that THP at 170 °C was the most effective for hydrolysis of particulate organics in sewage sludge, while fermentation under thermophilic temperature led to the highest accumulation of volatile fatty acids (VFAs) in sludge liquor. However, THP yielded the highest percentage of acetate in VFAs, which resulted in superior MEC performance compared to fermented sludge liquors in terms of current density (2.7 vs. ~1.3 A/m²), coulombic efficiency (50% vs. 31–34%), bio-H₂ potential (1114 vs. 839–881 mL), and H₂ production rate (50.3 mL/d vs. 28–32 mL/d). The utilization sequence of the VFAs was found to be acetate > butyrate > propionate. Overall, our results show that generating sludge liquors through THP could provide a feasible solution to produce bio-H₂ from sewage sludge; however, coulombic efficiencies should be further improved before practical application.     

Bio-hydrogen production from cornstalk wastes by orthogonal design method

One-factor-at-a-time design and orthogonal design were used in the experimental design methods to optimize bio-hydrogen (bio-H₂) production from cornstalk wastes by anaerobic fermentation. Three series of experiments were designed to investigate the effects of substrate concentration, initial pH and orthogonal design on the bio-H₂ production by using the natural sludge as inoculant. Experimental results indicate that substrate concentration was the most significant condition for optimal hydrogen production. The optimum orthogonal design method was proposed to be at an enzymatic temperature of 50 °C, an enzymatic time of 72 h, an initial pH of 7.0 and a substrate concentration of 10 g/L. The proposed method facilitated the optimization of optimum design parameters, only with a few well-defined experimental sets. Under the proposed condition, the maximum cumulative H₂ yield was 141.29 ml g^?1-CS (cornstalk, or 164.48 ml g^?1-TS, total solid, TS = 0.859 W_{dried cornstalk}), with an average H₂ production rate of 12.31 ml g^?1-CS h^?1. The hydrogen content reached 57.85% and methane was not detected in the biogas.     

Fermentative production of hydrogen from cassava processing wastewater by Clostridium acetobutylicum

This work reports on the effect of initial substrate concentration on COD consumption, pH, and H₂ production during cassava processing wastewater fermentation by Clostridium acetobutylicum ATCC 824. Five initial COD wastewater concentrations, namely 5.0, 7.5, 10.7, 15.0, and 30.0 g/L, were used. The results showed that higher substrate concentrations (30.0 and 15.0 COD/L) led to lower H₂ yield as well as less efficient substrate conversion into H₂. On the other hand, initial COD concentrations of 10.7, 7.5 and 5 g/L furnished 1.34, 1.2 and 2.41 mol H₂/mol glucose, with efficiency of glucose conversion into H₂ of 34, 30, and 60% (mol/mol), respectively. These results demonstrate that cassava processing wastewater, a highly polluting effluent, can be successfully employed as substrate for H₂ production by C. acetobutylicum at lower COD concentrations.     

Comparative study on the production of biohydrogen from rice mill wastewater

This study presents the production of biohydrogen from rice mill wastewater. The acid hydrolysis and enzymatic hydrolysis operating conditions were optimized, for better reducing sugar production. The effect of pH and fermentation time on biohydrogen production from acid and enzymatic hydrolyzed rice mill wastewater was investigated, using Enterobacter aerogenes and Citrobacter ferundii. The enzymatic hydrolysis produced the maximum reducing sugar (15.8 g/L) compared to acid hydrolysis (14.2 g/L). The growth data obtained for E. aerogenes and C. ferundii, fitted well with the Logistic equation. The hydrogen yields of 1.74 mol H₂/mol reducing sugar, and 1.40 mol H₂/mol reducing sugar, were obtained from the hydrolyzate obtained from enzymatic and acid hydrolysis, respectively. The maximum hydrogen yield was obtained from E. aerogenes compared to C. ferundii, and the optimum pH for better hydrogen production was found to be in the range from 6.5 to 7.0. The chemical oxygen demand (COD) reduction obtained was around 71.8% after 60 h of fermentation.     

Newly-isolated hydrogen-producing bacteria and biohydrogen production by Bacillus coagulans MO11 and Clostridium beijerinckii CN on molasses and agricultural wastewater

Biohydrogen is one of the most potential alternative fuels for the future. Among various substances from agricultural factory waste, molasses and wastewater from ethanol distillation plants are the main focus of this study. To overcome the limitation of bio-H₂ production, the direct screening of waste substrates for an efficient producer using co-culture free technique is an interesting choice. Competent species with H₂-producing ability and valuable volatile acid such as acetic acid were selected. Several bacteria were isolated and had their species identified by microbial 16SrDNA sequence analysis and phylogenetic analysis by Maximum Likelihood method. Bacillus coaglulans, B. circulans, Sporolactobacillus inulinus, Lactobacillus spp., Pediococcus acidilactici, Paenibacillus thermophilus, Chronobacter sakazaki and Clostridium beijerinckii were found in both reducing-sugar rich waste and wastewater. For bio-H₂ application in this criteria, potent natural hydrogen producer B. coagulans MO11 and C. beijerinckii CN were picked to test the growth ability on different substrates (carbon-dioxide, l-arabinose, d-xylose, d-glucose, d-fructose, sucrose, maltose, cellobiose, starch and others). Wastewater from sugarcane factories and other agricultural factories in Thailand were tested for physical and biochemical characteristics which would make them suitable renewable substrates. Among all, Bacillus coagulans MO11 could produce hydrogen gas using molasses and ethanol refinery wastewater effectively (1.634 mol_H2/mol hexose) as detected by Drager tube, which was the maximum yield in this study. In comparison, the experiment performed by using B. coagulans MO11 and C. beijerinckii CN fed with 40X diluted molasses wastewater showed better production of H₂ gas in Clostridium beijerinckii CN during the final 72-hr gas collection (79 mL/L and 125 mL/L respectively). The results pointed in the same direction when replacing 40X diluted molasses with 5X diluted refinery wastewater supplemented with trace elements.     

Whey waste as potential feedstock for biohydrogen production

In-house isolate Clostridium sp. IODB-O3 was exploited for biohydrogen production using cheese whey waste in batch fermentation. Analysis of cheese whey shows, it is enriched with lactose, lactic acid and protein components which were observed most favourable for biohydrogen production. Biohydrogen yield by IODB-O3 was compared with the cultures naturally occurring in waste solely or in combinations, and found that Clostridium sp. IODB-O3 was the best producer. The maximum biohydrogen yield obtained was 6.35 ± 0.2 mol-H₂/mol-lactose. The cumulative H₂ production (ml/L), 3330 ± 50, H₂ production rate (ml/L/h), 139 ± 5, and specific H₂ production (ml/g/h), 694 ± 10 were obtained. Clostridium sp. IODB-O3 exhibited better H₂ yield from cheese whey than the reported values in literature. Importantly, the enhancement of biohydrogen yield was observed possibly due to absence of inhibitory compounds, presence of essential nutrients, protein and lactic acid fractions which supported better cell growth than that of the lactose and glucose media. Carbon balance was carried out for the process which provided more insights in IODB-O3 metabolic pathway for biohydrogen production. This study may help for effective utilization of whey wastes for economic large scale biohydrogen production.     

Preparation of active hydrogen-producing cultures from palm oil mill sludge for biohydrogen production system

Methods are investigated to prepare active hydrogen (H₂)-producing cultures originating from palm oil mill sludge using dark fermentation. The first successful method that produces potent H₂-producing cultures and avoids growing H₂-consuming methanogens involves heat pretreatment of the sludge at 100 °C for 2 h and then the sludge sample is shocked in an ice bath for 15 min. Subsequently, a glucose solution rich in nutrients (glucose-based substrate) of 14.80 g chemical oxygen demand (COD)/L is fed in to enrich the H₂-producing cultures. The H₂ production reaches 78.63% on day 31. The second method involves acid pretreatment of sludge with 10 M hydrochloric acid at pH 3 for 48 h. Glucose-based substrate of 25.47 g COD/L is fed into the system. The H₂ production is 69.41% on day 27. For both methods, the H₂ production is stable after the H₂ content reached its maximum. The operation is performed semi-continuously using a hydraulic retention time of 1 day and at 30 °C. The optimum bacterial cells-to-COD level of substrate is approximately 0.60 in the start-up medium. The fermentation medium has an optimum initial pH of 5 and a final pH of 5.2–5.3. These two methods are recommended to produce active H₂-producing cultures for plant start-up in bio-H₂ production.     

Enhanced hydrogen gas production from mixture of beer spent grains (BSG) and distiller's grains (DG) with glycerol by Escherichia coli

Escherichia coli perform mixed acid fermentation and produce hydrogen gas (H₂) as one of the fermentation end products. E. coli can ferment sugars like glucose, xylose and alcohols like glycerol. It has been shown that E. coli has the ability to utilize pretreated organic waste (BSG or DG) or mixtures of it with glycerol and H₂ can be produced. H₂ evolution was maximum when the concentration of BSG was 4% and DG - 10% yielding 1.4 mmol L⁻¹ H₂. H₂ evolution was prolonged to ~24–120 h when mixtures of glycerol and DG or BSG wastes were applied. Moreover, in hycE (lacking large subunit of Hyd-3) or hyfG (lacking large subunit of Hyd-4) single mutants H₂ production was absent compared to wild type suggesting that Hyd-3 and Hyd-4 are responsible for H₂ generation. In addition, multiple mutant enhanced cumulative H₂ production ~3–4 fold. Taken together it can be proposed that BSG or DG wastes either together or in mixture with glycerol can be applied to obtain E. coli biomass and produce bio-H₂. The novel data can be used to further control effectively the application of organic waste resources as a feedstock for developing bio-H₂ production technology.     

Effect of iron and molybdenum addition on photofermentative hydrogen production from olive mill wastewater

Photofermentative hydrogen production from olive mill wastewater (OMW) by Rhodobacter sphaeroides O.U.001 was assessed under iron and molybdenum supplementation. Control cultures were only grown with 2% OMW containing media. The analysis included measurements of biomass accumulation, hydrogen production, pH variations of the medium, and changes in the chemical oxygen demand (COD) of the wastewater. Growth under control and Mo-supplemented experiments yielded about the same amount of biomass (∼0.4 g dry cell weight per L culture). On the other hand, Mo addition slightly enhanced the total volume of H₂ gas production (62 mL H₂), in comparison with the control reactor (40 mL H₂). Fe-supplemented cultures showed a significant increase on H₂ production (125 mL H₂), tough having a longer lag time for the observation of the first H₂ bubbles (24 h), compared to the control (15 h) and Mo-supplemented ones (15 h). Fe-added cultures also yielded better wastewater treatment by achieving 48.1% degradation of the initial chemical oxygen demand (COD) value compared to the control reactor having 30.2% COD removal efficiency. Advances described in this work have the potential to find applications in hydrogen industry while attempting an effective management of cheap feedstock utilization.     

Evaluation of process performance on biohydrogen production in continuous fixed bed reactor (C-FBR) using acid algae hydrolysate (AAH) as feedstock

In this study, a novel inoculation method to mitigate the inhibition of 5-hydroxymethylfurfural (5-HMF) is proposed. Acid algae hydrolysate containing 1.5 g 5-HMF/L and 15 g hexose/L hexose was fed to a continuous fixed bed reactor (C-FBR) partially packed with hybrid-immobilized beads. The inoculation method enabled a high rate of H₂ production, due to the reduction of 5-HMF inhibition and enhanced biofilm formation. Maximum hydrogen production was achieved at a hydraulic retention time of 6 h with a hydrogen production rate (HPR) of 20.0 ± 3.3 L H₂/L-d and a hydrogen yield (HY) of 2.3 ± 0.4 mol H₂/mol hexose _added. Butyrate and acetate were the major soluble metabolic products released during fermentation. Quantitative real-time polymerase chain reaction analysis revealed that Clostridium butyricum comprised 94.3% of the total bacteria, which was attributed to the high rate of biohydrogen production.