Vegetable waste as substrate and source of suitable microflora for bio-hydrogen production

Self-fermentation of cellulosic substrates to produce biohydrogen without inoculum addition nor pretreatments was investigated. Dark fermentation of two different substrates made of leaf-shaped vegetable refuses (V) and leaf-shaped vegetable refuses plus potato peels (VP), was taken in consideration. Batch experiments were carried out, under two mesophilic anaerobic conditions (28 and 37 °C), in order to isolate and to identify potential H₂-producing bacterial strains contained in the vegetable extracts. The effect of initial glucose concentration (at 1, 5 and 10 g/L) on fermentative H₂ production by the isolates was also evaluated.H₂ production from self-fermentation of both biomasses was found to be feasible, without methane evolution, showing the highest yield for V biomass at 28 °C (24 L/kg VS). The pH control of the culture medium proved to be a critical parameter. The isolates had sequence similarities ≥98% with already known strains, belonging to the family Enterobacteriaceae (γ-proteobacteria) and Streptococcaceae (Firmicutes). Four genera found in the samples, namely Pectobacterium, Raoultella, Rahnella and Lactococcus have not been previously described for H₂ production from glucose. The isolates showed higher yield (1.6–2.2 mol H₂/mol glucose_added) at low glucose concentration (1 g/L), while the maximum H₂ production ranged from 410 to 1016 mL/L and was obtained at a substrate concentration of 10 g/L. The results suggested that vegetable waste can be effectively used as both, substrate and source of suitable microflora for bio-hydrogen production.     

Dark fermentative biohydrogen production by Acinetobacter junii-AH4 utilizing various industry wastewaters

Hydrogen (H₂) is one of the most promising renewable energy sources, anaerobic bacterial H₂ fermentation is considered as one of the most environmentally sustainable alternatives to meet the potential fossil fuel demand. Bio-H₂ is the cleanest and most effective source of energy provided by the dark fermentation utilizing organic substrates and different wastewaters. In this study, the bio-H₂ production was achieved by using the bacteria Acinetobacter junii-AH4. Further, optimization was carried out at different pH (5.0–8.0) in the presence of wastewaters as substrates (Rice mill wastewater (RMWW), Food wastewater (FWW) and Sugar wastewater (SWW). In this way, the optimized experiments excelled with the maximum cumulative H₂ production of 566.44 ± 3.5 mL/L (100% FWW at pH 7.5) in the presence of Acinetobacter junii-AH4. To achieve this, a bioreactor (3 L) was employed for the effective production of H₂ and Acinetobacter junii-AH4 has shown the highest cumulative H₂ of 613.2 ± 3.0 mL/L, HPR of 8.5 ± 0.4 mL/L/h, HY of 1.8 ± 0.09 mol H₂/mol glucose. Altogether, the present study showed a COD removal efficiency of 79.9 ± 3.5% by utilizing 100% food wastewater at pH 7.5. The modeled data established a batch fermentation system for sustainable H₂ production. This study has aided to achieve an ecofriendly approach using specific wastewaters for the production of bio-H₂.     

Enhanced hydrogen production from sewage sludge by cofermentation with wine vinasse

The cofermentation of sewage sludge and wine vinasse at different mixing ratios to enhance hydrogen production was investigated. Batch experiments were carried out under thermophilic conditions with thermophilic sludge inoculum obtained from an acidogenic anaerobic reactor. The results showed that the addition of wine vinasse enhances the hydrogen production of sewage sludge fermentation. The highest hydrogen yields, 41.16 ± 3.57 and 43.25 ± 1.52 mL H₂/g VS_added, were obtained at sludge:vinasse ratios of 50:50 and 25:75, respectively. These yields were 13 and 14 times higher than that obtained in the monofermentation of sludge (3.17 ± 1.28 mL H₂/g VS_added). The highest VS removal (37%) was obtained at a mixing ratio of 25:75. Cofermentation had a synergistic effect the hydrogen yield obtained at a sludge:vinasse ratio of 50:50 was 40% higher, comparing to the sum of each waste. Furthermore, kinetic analysis showed that Cone and first-order kinetic models fitted hydrogen production better than the modified Gompertz model.     

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.     

Optimization of culture conditions for biohydrogen production from sago wastewater by Enterobacter aerogenes using Response Surface Methodology

Sago wastewater (SWW) causes pollution to the environment due to its high organic content. Annually, about 2.5 million tons of SWW is produced in Malaysia. In this study, the potential of SWW as a substrate for biohydrogen production by Enterobacter aerogenes (E. aerogenes) was evaluated. Response Surface Methodology (RSM) was employed to find the optimum conditions. From preliminary optimization, it was found that the most significant factors were yeast extract, temperature, and inoculum size. According to Face Centered Central Composite Design (FCCCD), the maximum hydrogen concentration and yield were 630.67 μmol/L and 7.42 mmol H₂/mol glucose, respectively, which is obtained from the sample supplemented with 4.8 g/L yeast extract concentration, 5% inoculum, and incubated at the temperature of 31 °C. Cumulative hydrogen production curve fitted by the modified Gompertz equation suggested that H_max, R_max, and λ from this study were 15.10 mL, 2.18 mL/h, and 9.84 h, respectively.     

Bacterial community analyses by pyrosequencing in dark fermentative H2-producing reactor using organic wastes as a feedstock

Hydrogen(H₂)-producing bacterial community structures of the dark fermentation system in a batch reactor were investigated during 48 h by analyzing 16S rRNA gene sequences obtained from pyrosequencing. Organic wastes composed of food waste and sewage sludge were used as a feedstock. After heat treatment (90 °C for 20 min) of the feedstock, H₂ was naturally evolved under anaerobic mesophilic conditions, showing a H₂ yield of 2.26 mol H₂/mol hexose_added. The bacterial community structure of the initial inoculum (microbial community at the starting point (0 h)) combined with heat treated food waste and sewage sludge was mainly comprised of Proteobacteria and Bacteroidetes. After 6 h operation, the sequences that belong to other groups except Firmicutes decreased dramatically and were not observed at all in the latter samples. Clostridium spp., which were negligible in the inoculum, took over the main bacterial community by taking charge of H₂ production. Among the phylum Firmicutes, the sequences closely related with Clostridium sordellii ATCC 9714^T, Clostridium perfringens ATCC 13124^T, and Clostridium butyricum ATCC 19398^T became predominant in the time series within 48 h. Overall, the results showed how fast the Clostridium spp. overwhelmed the bacterial community in dark fermentative H₂ production conditions, where they were at a negligible amount at the start.     

Hydrogen production by sequential dark and photofermentation using wet biomass hydrolysate of Spirulina platensis: Response surface methodological approach

Microalgae and cyanobacteria can be used as a potential biomass to produce hydrogen from stored glycogen and starch through fermentation and photofermentation. In this study, the potential of algal biomass i.e. Spirulina platensis hydrolysate as a substrate for sequential fermentative (I-stage) and photo-fermentative (II-stage) biohydrogen production was evaluated. Response Surface Methodology (RSM) was employed to find the optimum photofermentation conditions. From the preliminary optimization experiments, it was found that the significantly affecting factors for H₂ production were pH, dilution fold (D.F.) of fermentate and Fe(II) sulfate concentration during photofermentation (second stage). In the present study, 1% (w/v) Spirulina platensis hydrolyzate produced 23.06 ± 3.63 mmol of H₂ with yield of 1.92 ± 0.20 mmol H₂/g COD reduced. In the second stage experiment 1510 ± 35 mL/l hydrogen was produced using inoculum volume-20.0% (v/v) and inoculum age-48 h of co-culture of Rhodobacter sphaeroides NMBL-01 and Bacillus firmus NMBL-03 under conditions pH-5.95, D.F. of dark fermentate-20.30 folds, Fe(II) sulfate concentration-0.412 μM, temperature-32±2 °C and light intensity-2.5 klux.     

Bioaugmentation on hydrogen production from food waste

The aim of this work was to evaluate the effect of two hydrolytic (Paenibacillus polymyxa and Bacillus subtilis) and two fermentative (Clostridium saccharobutylicum and Clostridium beijerinckii) strains on hydrogen (H₂) production in dark fermentation by batch testing. Food waste was used as a substrate, pretreated anaerobic sludge was used as the inoculum, and different concentrations of the evaluated microorganisms were used. Bioaugmentation with 3.5 × 10⁹ CFU/mL/L_reactor B. subtilis showed the best performance, obtaining a production of 84.5 mL H₂/g SV and a reduction in the lag phase (from 7.9 h in control to 3.5 h). Bioaugmentation with B. subtilis in an anaerobic sequencing batch reactor exhibited a significant effect on volumetric productivity, reaching a maximal increase of 344% of H₂ production in comparison with that obtained without the addition of the strain. The increase in H₂ was observed in a short period of time (4 cycles), after which H₂ production returned to the original H₂ production baseline. During all reactor operations, the main volatile fatty acids produced were acetic acid and butyric acid. Microbial community analysis when bioaugmentation was applied showed an importance of lactic acid bacteria abundance, such as that of Bifidobacterium and Lactobacillus, whose metabolic activity was crucial in reactor performance. The added concentration of microorganisms is a critical parameter for the bioaugmentation process.     

Microbial hydrogen production from sewage sludge bioaugmented with a constructed microbial consortium

A constructed microbial consortium was formulated from three facultative H₂-producing anaerobic bacteria, Enterobacter cloacae IIT-BT 08, Citrobacter freundii IIT-BT L139 and Bacillus coagulans IIT-BT S1. This consortium was tested as the seed culture for H₂ production. In the initial studies with defined medium (MYG), E. cloacae produced more H₂ than the other two strains and it also was found to be the dominant member when consortium was used. On the other hand, B. coagulans as a pure culture gave better H₂ yield (37.16 ml H₂/g COD_consumed) than the other two strains using sewage sludge as substrate. The pretreatment of sludge included sterilization (15% v/v), dilution and supplementation with 0.5% w/v glucose, which was found to be essential to screen out the H₂ consuming bacteria and ameliorate the H₂ production. Considering (1:1:1) defined consortium as inoculum, COD reduction was higher and yield of H₂ was recorded to be 41.23 ml H₂/g COD_reduced. Microbial profiling of the spent sludge showed that B. coagulans was the dominant member in the constructed consortium contributing towards H₂ production. Increase in H₂ yield indicated that in consortium, the substrate utilization was significantly higher. The H₂ yield from pretreated sludge (35.54 ml H₂/g sludge) was comparatively higher than that reported in literature (8.1–16.9 ml H₂/g sludge). Employing formulated microbial consortium for biohydrogen production is a successful attempt to augment the H₂ yield from sewage sludge.     

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.     

A robust and efficient bioprocess of hydrogen production from crude glycerol by Clostridium beijerinckii G117

Clostridia can produce hydrogen (a renewable-biofuel) from crude glycerol (CG). Reportedly, the indigenous CG-impurities (eg.methanol, soap and salt) interfere with clostridial-process, and reduces hydrogen production. Thus, it is important to develop a robust bioprocess to directly utilize native-CG for hydrogen production. In this study, among four Clostridia tested, Clostridium beijerinckii G117 is selected based on its high hydrogen (>290 mL/g) production from CG. Furthermore, optimization of process-parameters enhances hydrogen production from strain G117 by ～5 fold from 1195 ± 45 to 5893 ± 25 mL/L with 2.16 mol/mol yield. The order of impact of process-parameters (optimum value in parenthesis) on hydrogen production is: CG concentration (12.5 g/L)>inoculum size (10%v/v)>inoculum age (12 h)>temperature (39 °C)>initial medium-pH (6.4)>L-cysteine (1 g/L)>operational volume (30 mL)>agitation rate (150 rpm). Notably, this optimized bioprocess records >90% efficiency by determining total mass and electron balances. Interestingly, this optimized bioprocess retains adequate robustness to yield 1.94–2.02 mol/mol hydrogen with elevated CG-impurities including alcohols/salts/surfactants.     

Biohydrogen production from sugarcane bagasse hydrolysate by elephant dung: Effects of initial pH and substrate concentration

Pre-heated elephant dung was used as inoculum to produce hydrogen from sugarcane bagasse (SCB) hydrolysate. SCB was hydrolyzed by H₂SO₄ or NaOH at various concentrations (0.25-5% volume) and reaction time of 60 min at 121 °C, 1.5 kg/cm² in the autoclave. The optimal condition for the pretreatment was obtained when SCB was hydrolyzed by H₂SO₄ at 1% volume which yielded 11.28 g/L of total sugar (1.46 g glucose/L; 9.10 g xylose/L; 0.72 g arabinose/L). The maximum hydrogen yield of 0.84 mol H₂/mol total sugar and the hydrogen production rate of 109.55 mL H₂/L day were obtained at the initial pH 6.5 and initial total sugar concentration 10 g/L. Hydrogen-producing bacterium (Clostridium pasteurianum) and non hydrogen-producing bacterium (Flavobacterium sp.) were dominating species in the elephant dung and in hydrogen fermentation broth. Sporolactobacillus sp. was found to be responsible for a low hydrogen yield obtained.     

Optimization of molecular hydrogen production by Rhodobacter sphaeroides O.U.001 in the annular photobioreactor using response surface methodology

Photofermentative H₂ production at higher rate is desired to make H₂ viable as cheap energy carrier. The process is influenced by C/N composition, pH levels, temperature, light intensity etc. In this study, Rhodobacter sphaeroides strain O.U 001 was used in the annular photobioreactor with working volume 1 L, initial pH of 6.7 ± 0.2, inoculum age 36 h, inoculum volume 10% (v/v), 250 rpm stirring and light intensity of 15 ± 1.1 W m⁻². The effect of parameters, i.e. variation in concentration of DL malic acid, L glutamic acid and temperature on the H₂ production was noted using three factor three level full factorial designs. Surface and contour plots of the regression models revealed optimum H₂ production rate of 7.97 mL H₂ L⁻¹ h⁻¹ at 32 °C with 2.012 g L⁻¹ DL malic acid and 0.297 g L⁻¹ L glutamic acid, which showed an excellent correlation (99.36%) with experimental H₂ production rate of 7.92 mL H₂ L⁻¹ h⁻¹.     

Comparison of suspended and granular cell anaerobic bioreactors for hydrogen production from acid agave bagasse hydrolyzates

Acid agave bagasse hydrolyzates have been used as a substrate for hydrogen production, however, bioreactors are unstable and with poor performance. Granular biomass could be more successful in producing hydrogen from acid agave bagasse hydrolyzates in comparison with suspended biomass. Thus, this study aimed to evaluate the effect of increasing concentrations of acid agave hydrolyzates on hydrogen production, to compare the hydrogen productivity and stability of granular biomass in an expanded granular sludge bed (EGSB) reactor and suspended biomass in an anaerobic sequencing batch reactor (AnSBR) fed with acid hydrolyzates, and finally to determine the variation of microbial communities established in both bioreactor configurations. In batch tests, the heat-treated inoculum produced hydrogen from acid agave hydrolyzates without observing inhibition at 6.3 g/L of carbohydrates (_CHO). This hydrolyzate concentration was used to start up the AnBSR, which reached a productivity of 226 ± 53 mL H₂/L⋅d at organic loading rates (OLR) from 3.2 to 4.5 g_CHO/L⋅d. The hydrogen production stability index decreased from 0.8 to 0.6 at increasing OLR, and the AnSBR failed at the highest OLR of 5.7 g/L⋅d. The EGSB reactor reached the highest productivity of 361 ± 130 mL H₂/L⋅d at an OLR of 7.4 g_CHO/L⋅d, but with a low stability index of 0.6. Independently of the bioreactor configuration, microbial communities associated with the production of acetate/lactate were successfully established in both configurations with the prevalence of Lactobacillus spp. A low abundance of typical H₂ producers like Clostridium was always observed over the whole period of operation (<10% of the total abundance). In sum, the hydrogen productivity from acid agave hydrolyzates was higher for the EGSB reactor than for the AnSBR, but with much lower stability. The evidence provided by this study suggests the establishment of metabolic pathways for hydrogen production from organic acids.     

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.     

Fermentative hydrogen production by Clostridium butyricum and Escherichia coli in pure and cocultures

The effect of coculture of Clostridium butyricum and Escherichia coli on hydrogen production was investigated. C. butyricum and E. coli were grown separately and together as batch cultures. Gas production, growth, volatile fatty acid production and glucose degradation were monitored. Whilst C. butyricum alone produced 2.09 mol-H₂/mol-glucose the coculture produced 1.65 mol-H₂/mol-glucose. However, the coculture utilized glucose more efficiently in the batch culture, i.e., it was able to produce more H₂ (5.85 mmol H₂) in the same cultivation setting than C. butyricum (4.62 mmol H₂), before the growth limiting pH was reached.     

Enhanced methane fermentation of municipal sewage sludge by microbial electrochemical systems integrated with anaerobic digestion

Sewage sludge from a municipal wastewater treatment plant was fed into a microbial electrochemical system, combined with an anaerobic digester (MES-AD), for enhanced methane production and sludge stabilization. The effect of thermally pretreating the sewage sludge on MES-AD performance was investigated. These results were compared to those obtained from control operations, in which the sludge was not pretreated or MES integration was absent. The soluble chemical oxygen demand (SCOD) in the raw sewage sludge after pretreatment was 31% higher than the SCOD in untreated sludge (5804.85 mg/L vs. 4441.46 mg/mL). The methane yield and proportion of methane in biogas generated by the MES-AD were higher than those of the control systems, regardless of the pretreatment process. The maximum methane yield (0.28 L CH₄/g COD) and methane production (1139 mL) were obtained with the MES inoculated with pretreated sewage sludge. Methane yield and production with this system using pretreated sewage were 47% and 56% higher, respectively, than those of the control (0.19 L CH₄/g COD, 730 mL). Additionally, the maximum SCOD removal (89%) and current generation were obtained with the MES inoculated with a pretreated substrate. These results suggested that sewage sludge could be efficiently stabilized with enhanced methane production by synergistic combination of MES-AD system with pretreatment process.     

Bioconversion of de-oiled Jatropha Waste (DJW) to hydrogen and methane gas by anaerobic fermentation: Influence of substrate concentration,temperature and pH

Cellulosic materials-based de-oiled Jatropha Waste (DJW) was fermented to H₂ and CH₄ using sewage sludge inoculum. Batch assays were performed at various substrate concentrations (40–240 g/L), temperatures (25–65 °C) and pHs (5.5–7.5). The peak hydrogen production rate (HPR) and hydrogen yield (HY) of 744.0 ± 11.3 mL H₂/L-d and 10.6 ± 0.2 mL H₂/g VS obtained when the optimal substrate concentration, pH, temperature were 200 g/L, 6.5, 55 °C, respectively. The peak methane production rate (MPR) of 178.4 ± 5.6 mL CH₄/L-d obtained while DJW concentration, pH, temperature were 200 g/L, 7.0, 45 °C, however, peak methane yield (MY) of 23.3 ± 0.1 mL CH₄/g VS obtained at 40 g/L, 7.0 and 55 °C, respectively. Effect of substrate concentration on HPR and MPR was elucidated using Monod model. Butyrate and acetate were the main soluble metabolic products. Maximal carbohydrate removal and COD reduction were achieved as 51.7 ± 0.7% and 68.3 ± 1.6%, respectively.     

Statistical optimization of biohydrogen production from sucrose by a co-culture of Clostridium acidisoli and Rhodobacter sphaeroides

Statistically based experimental designs were applied to optimize the fermentation process parameters for hydrogen (H₂) production by co-culture of Clostridium acidisoli and Rhodobacter sphaeroides with sucrose as substrate. An initial screening using the Plackett–Burman design identified three factors that significantly influenced H₂ yield: sucrose concentration, initial pH, and inoculum ratio. These factors were considered to have simultaneous and interdependent effects. A central composite design and response surface analysis were adopted to further investigate the mutual interactions among the factors and to identify the values that maximized H₂ production. The optimal substrate concentration, initial pH, and inoculum ratio of C. acidisoli to R. sphaeroides were 11.43 g/L sucrose, 7.13, and 0.83, respectively. Using these optimal culture conditions, substrate conversion efficiency was determined as 10.16 mol H₂/mol sucrose (5.08 mol H₂/mol hexose), which was near the expected value of 10.70 mol H₂/mol sucrose (5.35 mol H₂/mol hexose).     

Extreme-thermophilic biohydrogen production by an anaerobic heat treated digested sewage sludge culture

Biohydrogen production process from glucose using extreme-thermophilic H₂-producing bacteria enriched from digested sewage sludge was investigated for five cycles of repeated batch experiment at 70 °C. Heat shock pretreatment was used for preparation of hydrogen-producing bacteria comparing to an untreated anaerobic digested sludge for their hydrogen production performance and responsible microbial community structures. The results showed that the heat shock pretreatment completely repressed methanogenic activity and gave the maximum hydrogen production yield of 355-488 ml H₂/g COD in the second cycle of repeated batch cultivation with more stable gas production during the cultivation when compared with control. Hydrogen production was accompanied by production of acetic acid. The average specific hydrogen in five cycles experiment ranged from 150 to 200 ml H₂/g VSS. PCR-DGGE profiling showed that the extreme-thermophilic culture predominant species were closely affiliated to Thermoanaerobacter pseudethanolicus.