Fermentative hydrogen production by a new alkaliphilic Clostridium sp. (strain PROH2) isolated from a shallow submarine hydrothermal chimney in Prony Bay,New Caledonia

The hydrogen-producing strain PROH2 pertaining to the genus Clostridium was successfully isolated from a shallow submarine hydrothermal chimney (Prony Bay, New Caledonia) driven by serpentinization processes. Cell biomass and hydrogen production performances during fermentation by strain PROH2 were studied in a series of batch experiments under various conditions of pH, temperature, NaCl and glucose concentrations. The highest hydrogen yield, 2.71 mol H₂/mol glucose, was observed at initial pH 9.5, 37 °C, and glucose concentration 2 g/L, and was comparable to that reported for neutrophilic clostridial species. Hydrogen production by strain PROH2 reached the maximum production rate (0.55 mM-H₂/h) at the late exponential phase. Yeast extract was required for growth of strain PROH2 and improved significantly its hydrogen production performances. The isolate could utilize various energy sources including cellobiose, galactose, glucose, maltose, sucrose and trehalose to produce hydrogen. The pattern of end-products of metabolism was also affected by the type of energy sources and culture conditions used. These results indicate that Clostridium sp. strain PROH2 is a good candidate for producing hydrogen under alkaline and mesothermic conditions.     

Enhanced hydrogen production from xylose and bamboo stalk hydrolysate by overexpression of xylulokinase and xylose isomerase in Klebsiella oxytoca HP1

Biofuels production from lignocellulose hydrolysates by microbe fermentation has merited attention because of the mild reaction conditions involved and the clean nature of the process. In this work, xylulokinase (XK) and xylose isomerase (XI) were overexpressed in Klebsiella oxytoca HP1 to enhance hydrogen production by the fermentation of xylose. The recombinant strains exhibited higher enzyme activity of XI or XK compared with the wild strain. Hydrogen production from pure xylose, xylose/glucose mixtures and bamboo stalk hydrolysate was significantly enhanced with the overexpression of XI and XK in K. oxytoca HP1 in terms of total hydrogen yield (THY), hydrogen yield per mole substrate (HYPM) and hydrogen production rate (HPR). The HYPM of K. oxytoca HP1/xylB and K. oxytoca HP1/xylA reached 1.93 ± 0.05 and 2.46 ± 0.05 mol H₂/mol xylose, respectively in pure xylose, while the value for the wild strain was 1.68 ± 0.04 mol H₂/mol xylose. The xylose consumption rate (XCR) for the recombinant strain was significantly higher than that for the wild strain, particularly in the early stage of fermentation. Relative to the wild type, hydrogen yield (HY) from 1 g of preprocessed bamboo powder of HP1/xylB and HP1/xylA increased by 33.04 and 41.31%, respectively. It was concluded that overexpression of XK or XI was able to promote hydrogen production from xylose and xylose/glucose mixtures by simultaneously increasing the utilization efficiency of xylose and weakening the inhibitory effect of glucose on xylose use. In addition, the results indicated that overexpression technology was an effective way to further increase hydrogen production from lignocellulosic hydrolysates.     

Feasibility study on the application of rhizosphere microflora of rice for the biohydrogen production from wasted bread

We performed an experiment of continuous anaerobic hydrogen fermentation as a pilot-plant-scale test, in which waste from a bread factory was fermented by microflora of rice rhizosphere origin. The community structure of microflora during anaerobic hydrogen fermentation was analyzed using PCR-DGGE, FISH, and quinone profiles. The relation of those results to hydrogen generation was discussed. Results show that a suitable condition was a reactor temperature of 35 °C, with HRT 12–36 h, volume load of 30–70 kg-COD_Cr/m³ day, and maximum hydrogen production rate of 1.30 mol-H₂/mol-hexose. Regarding characteristics of microflora during fermentation, PCR-DGGE results show specific 16S rDNA band patterns; Megasphaera elsdenii and Clostridium sp. of the hydrogen-producing bacteria were identified. M. elsdenii was detected throughout the fermentation period, while Clostridium sp. of hydrogen-producing bacteria was detected on the 46th day. Furthermore, FISH revealed large amounts of Clostridium spp. in the sample. The quinone profile showed that the dominant molecular species of quinone is MK-7. Because Clostridium spp. belong to MK-7, results suggest that the quinone profile result agrees with the results of PCR-DGGE and FISH. Microflora in the rhizosphere of rice plants can be a possible resource for effective bacteria of biohydrogen production.     

Statistical optimization of fermentative hydrogen production from xylose by newly isolated Enterobacter sp. CN1

Statistical experimental designs were applied for the optimization of medium constituents for hydrogen production from xylose by newly isolated Enterobacter sp. CN1. Using Plackett–Burman design, xylose, FeSO₄ and peptone were identified as significant variables which highly influenced hydrogen production. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. These variables were subsequently optimized using Box–Behnken design of response surface methodology (RSM). The optimum conditions were found to be xylose 16.15 g/L, FeSO₄ 250.17 mg/L, peptone 2.54 g/L. Hydrogen production at these optimum conditions was 1149.9 ± 65 ml H₂/L medium. Under different carbon sources condition, the cumulative hydrogen volume were 1217 ml H₂/L xylose medium, 1102 ml H₂/L glucose medium and 977 ml H₂/L sucrose medium; the maximum hydrogen yield were 2.0 ± 0.05 mol H₂/mol xylose, 0.64 mol H₂/mol glucose. Fermentative hydrogen production from xylose by Enterobacter sp. CN1 was superior to glucose and sucrose.     

Fermentative hydrogen production by a new mesophilic bacterium Clostridium sp. 6A-5 isolated from the sludge of a sugar mill

The fermentative hydrogen production capability of the newly isolated Clostridium sp. 6A-5 bacterium was studied in a batch cultivation experiment. Various culture conditions (temperature, initial pH, and glucose concentration) were evaluated for their effects on cell growth and hydrogen production (including the yield and rate) of Clostridium sp. 6A-5. Optimal cell growth was observed at 40 °C, initial pH 7.5–8, and glucose concentration 16–26 g/L. The optimal hydrogen yield was obtained at 43 °C, initial pH 8, and glucose concentration 10–16 g/L. Hydrogen began to evolve when cell growth entered the mid-exponential phase and reached the maximum production rate at the late exponential and stationary phases. The maximum hydrogen yield, and rate were 2727 mL/L, and 269.3 mL H₂/L h, respectively. These results indicate that Clostridium sp. 6A-5 is a good candidate for mesophilic fermentative hydrogen production.     

Microbial characterization of hydrogen-producing bacteria in fermented food waste at different pH values

An anaerobic fermentation of food waste was conducted in a 0.5 L bioreactor incubated at a thermophilic temperature of 55 °C to evaluate the effects of different controlled pH values (5.0, 5.5 and 6.0) on biohydrogen production. Effective biohydrogen production was found at controlled pH 5.5 and 6.0 corresponding to lower lactic acid production compared to pH 5.0. It was demonstrated that biohydrogen production from food waste was pH-dependent with hydrogen yields of 79, 76 and 23 mmol H₂/L-media/d for pH 5.5, 6.0 and 5.0, respectively. Specific microbial determination for Clostridium sp. and total bacteria quantification were carried out by the fluorescent in-situ hybridization (FISH) technique. The number of Clostridium sp. for acclimatized sludge, fermentation broth at pH 5.0, 5.5 and 6.0 were 2.9 × 10⁸, 3.6 × 10⁸, 7.8 × 10⁸ and 5.4 × 10⁸ cells/ml, respectively. The quantification analysis showed that 92% of the total bacteria belonged to Clostridium sp. from clusters I and XI from the sample at controlled pH 5.5. The denaturing gradient gel electrophoresis (DGGE) bands of the sample after heat-treatment, acclimatization and during fermentation indicated the presence of Bacteroidetes, Caloromator australicus sp. and Clostridium sp.     

Fermentative hydrogen production from different sugars by Citrobacter sp. CMC-1 in batch culture

In this study, production of hydrogen (H₂) from glucose, xylose, galactose, mannose, arabinose and rhamnose by a strain isolated from activated sludge was investigated. The strain, named as Citrobacter sp. CMC-1, was enriched in cellobiose amended minimal media. Based on 16S rRNA sequence, the CMC-1 strain is a close relative of Citrobacter amalonaticus strain SA01 (99%). Optimal cultivation parameters for H₂ production and growth such as pH and temperature were investigated. H₂ yields from glucose at optimal conditions (pH 6.0 and 34 °C) were 1.82 ± 0.02 mol-H₂/mol-glucose. Strain CMC-1 fermented galactose, mannose, xylose, arabinose and rhamnose. After 48 h incubation, the strain CMC-1 completely fermented all sugars tested, except arabinose. Increase in fermentation period lowered residual formate level in the media and improved H₂ production for galactose, mannose and xylose (1.68 ± 0.24, 1.93 ± 0.14 and 1.63 ± 0.07 mol-H₂/mol-substrate respectively).     

Biohydrogen production from cellobiose in phenol and cresol-containing medium using Clostridium sp. R1

Cellobiose fermentation in batch test using an isolated strain, Clostridium sp. R1, was investigated. The Clostridium sp. R1 achieved a maximum hydrogen yield of 3.5 mol H₂ mol⁻¹ cellobiose at pH 6 and 30 °C, higher than most yields reported in literature. This strain can generate hydrogen from a number of carbohydrates, including galactose, glucose, mannose, maltose, sucrose, and starch. This strain can also convert cellobiose to hydrogen in the presence of toxic phenol or cresol. The inhibition effects of phenolic compounds on strain R1 activity followed phenol > p-cresol > o-cresol > m-cresol. Co-culturing with another strain, Clostridium butyricum, can co-degrade some of the phenol as substrates. The new isolated strain can yield hydrogen from phenol-containing wastewaters.     

Effect of changing temperature on anaerobic hydrogen production and microbial community composition in an open-mixed culture bioreactor

The temperature effect (37–65 °C) on H₂ production from glucose in an open-mixed culture bioreactor using an enrichment culture from a hot spring was studied. The dynamics of microbial communities was investigated by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). At 45 and 60 °C the H₂ production was the highest i.e. 1.71 and 0.85 mol H₂/mol glucose, respectively. No H₂ was produced at temperatures 50 and 55 °C. At 37–45 °C, H₂ production was produced by butyrate type fermentation while fermentation mechanism changed to ethanol type at 60 °C. Clostridium species were dominant at 37–45 °C while at 50–55 °C and 60 °C the culture was dominated by Bacillus coagulans and Thermoanaerobacterium, respectively. In the presence of B. Coagulans the metabolism was directed to lactate production. The results show that the mixed culture had two optima for H₂ production and that the microbial communities and metabolic patterns promptly changed according to changing temperatures.     

Comparison of metabolic pathway for hydrogen production in wild-type and mutant Clostridium tyrobutyricum strain based on metabolic flux analysis

There has been a great interest in fermentative hydrogen production during recent decades. However, the low H₂ yield associated with fermentative hydrogen production process continues to hinder its industrial application. It is delectable that a maximum 3.9 mol H₂ per mol glucose was obtained in fed-batch fermentation mode with a butyric acid over-producing Clostridium tyrobutyricum mutant, which to our knowledge is the highest H₂ yield ever got in the fermentation process with Clostridium sp. This study aimed to better understand the change of flux profile within the whole metabolic network and to conduct the metabolic flux analysis of fermentative hydrogen production. For the first time, we constructed a metabolic flux model for the anaerobic glucose metabolism of C. tyrobutyricum ATCC 25755, and revealed the internal mechanism responsible for the redistribution of the carbon flux in the mutant strain in comparison with the wide-type. The MFA methodology was used to study the fractional flux response to variations in operational pH, and revealed that pH was a significant operational parameter effecting on the fermentative hydrogen production process. Furthermore, the presence of NADH-ferredoxin oxidoreductase activity in this anaerobe was demonstrated. By measuring the activities of related enzymes in the biosynthesis pathway of hydrogen, we thus concluded that the increased specific activities of both NFOR and hydrogen-catalyzing enzyme (hydrogenase) would be attributed to the hydrogen over-producing.     

Hydrogen production from xylose by moderate thermophilic mixed cultures using granules and biofilm up-flow anaerobic reactors

Continuous H₂ production from xylose by granules and biofilm up-flow anaerobic reactor using moderate thermophilic mixed cultures was investigated. The maximum H₂ yield of 251 mL H₂/g-xylose with H₂production rate of 15.1 L H₂/L⋅d was obtained from granules reactor operating at the organic loading rate (OLR) of 60 g-xylose/L⋅d and hydraulic retention time (HRT) of 4 h. Meanwhile the highest H₂ production rate of 13.3 L H₂/L⋅d with an H₂ yield of 221 mL H₂/g–xylose was achieved from the biofilm reactor. Both reactors were dominated by Thermoanaerobacterium species with acetate and butyrate as main fermentation products. The microbial community of the biofilm reactor was composed of Thermoanaerobacterium species, while granules reactor was composed of Clostridium sp., Thermoanaerobacterium sp. and Caloramator sp. The granular reactor was more microbial diversity and more balance between economic efficiency in term of the hydrogen production rate and technical efficiency in term of hydrogen yield.     

Hydrogen production performance by Enterobacter cloacae KBH3 isolated from termite guts

Two out of six bacterial isolates obtained from the guts of Globitermes sp. termites were identified as hydrogen-producing bacteria. One isolate, Enterobacter cloacae KBH3, was characterised using the BIOLOG identification system and 16S rRNA gene analysis. In a batch fermentation study to evaluate its growth in defined medium, E. cloacae KBH3 produced 154 ml H₂ per litre medium with approximately 50% hydrogen content. The carbon utilisation results suggest that E. cloacae KBH3 have the potential to be a good hydrogen producer. This strain is also able to produce hydrogen within a wide range of temperatures (28–40 °C) and pH (4.5–8). In several fermentation runs, the pH of the culture dropped from 6.5 to 5.36 within the first 3 h, which was mostly due to the biosynthesis of formate. An increase of cumulative hydrogen production was recorded as well as a decrease in the concentration of formate, indicating the importance of the formate pathway for hydrogen production. The highest rate of hydrogen production of 180.74 ml H₂/l/h was achieved when lactate and acetate were at their highest concentrations. Most of the hydrogen gas was produced during the exponential growth phase, and the biogas continued to be produced during the stationary phase. The specific growth rate was calculated to be 0.224 per hour while the hydrogen yield was 1.8 mol of hydrogen per mol of glucose. At the end of the batch study, the highest cumulative hydrogen production was 2404 ml H₂ per litre of fermentation medium.     

Anthrahydroquinone-2,6-disulfonate increases the rate of hydrogen production during Clostridium beijerinckii fermentation with glucose,xylose, and cellobiose

Fermentative hydrogen production is considered a reasonable alternative for generating H₂ as an energy carrier for electricity production using hydrogen fuel cells. The kinetics of hydrogen production from glucose, xylose and cellobiose were investigated using pure culture Clostridium beijerinckii NCIMB 8052. Adding anthrahydroquinone-2,6-disulfonate (AH₂QDS) at concentrations ranging from 100 μM to 500 μM increased the hydrogen production rates from 0.80 to 1.35 mmol/L-hr to 1.20–2.70 mmol/L-hr with glucose, xylose, or cellobiose as the primary substrates. AH₂QDS amendment also increased the substrate utilization rate and biomass growth rate by at least two times. These findings suggest that adding hydroquinone reducing equivalents influence cellular metabolism with hydrogen production rate, substrate utilization rate, and growth rate being simultaneously affected. Resting cell suspensions were conducted to investigate the influence of AH₂QDS on the hydrogen production rate from glyceraldehyde 3-phosphate, which is a shared intermediate in both glycolysis and pentose phosphate pathway. Data demonstrated that hydrogen production rate increased by 1.5 times when glyceraldehyde 3-phosphate was the sole carbon source, suggesting that the hydroquinone may alter reactions starting with or after glyceraldehyde 3-phosphate in central metabolism. These data demonstrate that adding hydroquinones increased overall metabolic activity of C. beijerinckii. This will eventually increase the efficiency of industrial scale production once appropriate hydroquinone equivalents are identified that work well in large-scale operations, since fermentation rate is one of the two critical factors (production rate and yield) influencing efficiency and cost.     

Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier

Biological mycelia pellets, which are formed spontaneously in the process of Aspergillus niger Y3 fermentation, were explored as carrier for immobilization of Clostridium sp. T2 to improve hydrogen production. Batch fermentation tests showed that optimal dosage and size of mycelia pellets for hydrogen production were 0.350 g 150 ml⁻¹ medium and 1.5 mm. Under these conditions, hydrogen production with immobilized cells on mycelia pellets was further investigated in continuous stirred-tank reactor (CSTR) with hydraulic retention time (HRT) ranging from 12 to 8 h. It obtained that the maximum hydrogen production rate reached 2.76 mmol H₂ L⁻¹ h⁻¹ at 10 h HRT, which was 40.8% higher than the carrier-free process, but slightly lower than the counterpart immobilized in sodium alginate with the value of 3.15 mmol H₂ L⁻¹ h⁻¹. SEM observation showed that abundant cells were closely adhered to mycelia pellets. The present results indicate the potential of using mycelia pellets as biological carrier for enhancing hydrogen production.     

Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well

Hydrogen producing novel bacterial strain was isolated from formation water from oil producing well. It was identified as Thermoanaerobacter mathranii A3N by 16S rRNA gene sequencing. Hydrogen production by novel strain was pH and substrate dependent and favored pH 8.0 for starch, pH 7.5 for xylose and sucrose, pH 8.0–9.0 for glucose fermentation at 70 °C. The highest H₂ yield was 2.64 ± 0.40 mol H₂ mol glucose at 10 g/L, 5.36 ± 0.41 mol H₂ mol – sucrose at 10 g/L, 17.91 ± 0.16 mmol H₂ g – starch at 5 g/L and 2.09 ± 0.21 mol H₂ mol xylose at 5 g/L. The maximum specific hydrogen production rates 6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type fermentation pathway (approximately 97%) was found to be dominant in strain A3N, whereas butyrate formation was found in sucrose and xylose fermentation. Lactate production increased with high xylose concentrations above 10 g/L.     

Isolation of a Clostridium acetobutylicum strain and characterization of its fermentation performance on agricultural wastes

A new solvent-producing Clostridium has been isolated from soil used in intensive rice cultivation. The 16S rRNA analysis of the isolate indicates that it is closely related to Clostridium acetobutylicum, with a sequence identity of 96%. The new isolate, named C. acetobutylicum YM1, produces biobutanol from multiple carbon sources, including glucose, fructose, xylose, arabinose, glycerol, lactose, cellobiose, mannitol, maltose, galactose, sucrose and mannose. This isolate can also utilize polysaccharides such as starch and carboxylmethyl cellulose (CMC) for the production of biobutanol. The ability of isolate YM1 to produce biobutanol from agro-industrial wastes was also evaluated for rice bran, de-oiled rice bran, palm oil mill effluent and palm kernel cake. The highest concentration of biobutanol (7.27 g/L) was obtained from the fermentation medium containing 2% (w/v) fructose, with a total acetone–butanol–ethanol (ABE) concentration of 10.23 g/L. The ability of isolate YM1 to produce biobutanol from various carbon sources and agro-wastes indicates the promise of the use of this isolate for the production of biobutanol, a renewable energy resource, from readily available renewable feedstocks.     

Fermentative hydrogen production by new marine Clostridium amygdalinum strain C9 isolated from offshore crude oil pipeline

The present study investigated hydrogen production potential of novel marine Clostridium amygdalinum strain C9 isolated from oil water mixtures. Batch fermentations were carried out to determine the optimal conditions for the maximum hydrogen production on xylan, xylose, arabinose and starch. Maximum hydrogen production was pH and substrate dependant. The strain C9 favored optimum pH 7.5 (40 mmol H₂/g xylan) from xylan, pH 7.5–8.5 from xylose (2.2–2.5 mol H₂/mol xylose), pH 8.5 from arabinose (1.78 mol H₂/mol arabinose) and pH 7.5 from starch (390 ml H₂/g starch). But the strain C9 exhibited mixed type fermentation was exhibited during xylose fermentation. NaCl is required for the growth and hydrogen production. Distribution of volatile fatty acids was initial pH dependant and substrate dependant. Optimum NaCl requirement for maximum hydrogen production is substrate dependant (10 g NaCl/L for xylose and arabinose, and 7.5 g NaCl/L for xylan and starch).     

Utilization of palm kernel cake as a renewable feedstock for fermentative hydrogen production

Fermentative hydrogen generation was studied using palm kernel cake (PKC) as sustainable cellulosic biomass. PKC was subjected to an acid hydrolysis approach using dilute H₂SO₄ (7% v/v). PKC hydrolysate obtained was then diluted (70%) and used as a substrate for hydrogen generation. Chemical analysis showed that the main fermentable sugars in diluted PKC hydrolysate were glucose, xylose and mannose with the concentrations of 2.75 g/L, 2.60 g/L and 27.75 g/L, respectively. Hydrogen production was carried out by the cultivation of Clostridium acetobutylicum YM1 on PKC hydrolysate. The effect of incubation temperature, the initial pH of culture medium and microbial inoculum size on hydrogen production was studied using a statistical model. The analysis of the model generated showed that the initial pH value of the culture medium and inoculum size had significant effects on the hydrogen production. The study showed that the optimum conditions for the biohydrogen production were 30.57 °C temperature, pH 5.5 and 20% inoculum size. A verification experiment was performed in the optimum conditions determined. Experimental results of the verification test showed that a cumulative hydrogen volume of 1575 ml/L was generated with consuming 2.75 g/L glucose, 2.20 g/L xylose and 16.31 g/L mannose.     

Exogenous anthrahydroquinone-2,6-disulfonate specifically increases xylose utilization during mixed sugar fermentation by Clostridium beijerinckii NCIMB 8052

The influence of a reduced extracellular electron shuttle (anthrahydroquinone-2,6-disulfonate, AH₂QDS) on substrate utilization and H₂ production was investigated at different glucose:xylose ratios using the fermentative culture Clostridium beijerinckii NCIMB 8052. Adding 250 μM AH₂QDS increased the total substrate utilization by 23–66%, and specifically xylose utilization by 20–54% at glucose:xylose ratios of 1:1, 1:3 and 1:9. Adding 250 μM AH₂QDS also increased the substrate utilization by 40–88% at all tested glucose:xylose ratios (which ranged from 1:9 to 9:1). Increasing AH₂QDS concentrations from 250 μM to 2 mM increased xylose utilization (>99% xylose consumed) as well as cumulative hydrogen production during mixed sugar fermentation. The extent of glucose utilization was consistent amongst all tests, which was expected. However, hydroquinone amendment specifically increased the extent of xylose utilized irrespective of the starting glucose:xylose ratio. To the best of our knowledge this is the first report of 100% xylose utilization by C. beijerinckii NCIMB 8052 grown under mixed sugar conditions. These data demonstrate that the substrate utilization, particularly xylose utilization, can be manipulated by amending extracellular redox active compounds. This will improve pentose utilization during fermentation of hydrolyzates that result from pre-treatment of lignocellulosic materials with wild type (non-GMO) cultures.     

Developing a thermophilic hydrogen-producing microbial consortia from geothermal spring for efficient utilization of xylose and glucose mixed substrates and oil palm trunk hydrolysate

Xylose and glucose are the major sugar components of lignocellulosic hydrolysate. This study aims to develop thermophilic hydrogen-producing consortia from eight sediments-rich samples of geothermal springs in Southern Thailand by repeated batch cultivation at 60 °C with glucose, xylose and xylose-glucose mixed substrates. Significant hydrogen production potentials were obtained from thermophilic enriched cultures encoded as PGR and YLT with the maximum hydrogen yields of 241.4 and 231.6 mL H₂/g sugar_consumed, respectively. After repeated batch cultivation the hydrogen yield from xylose-glucose mixed substrate of PGR increased to 375 mL H₂/g sugar_consumed which was 30% higher than that of YLT (287 mL H₂/g sugar_consumed). Soluble metabolites from xylose-glucose mixed substrates were composed mostly of butyric acid (20.6-21.8 mM), acetic acid (7.2-13.5 mM), lactic acid (8.2-11.7 mM) and butanol (4.4-13.0 mM). Denaturing gradient gel electrophoresis (DGGE) profiles illustrated small difference in microbial patterns of PGR enriched with glucose, xylose-glucose mixed substrate and xylose. The dominant populations were affiliated with low G + C content Gram-positive bacteria, Thermoanaerobacterium sp., Thermoanaerobacter sp., Caloramater sp. and Anoxybacillus sp. based on the 16S rRNA gene. Cultivation of the enriched culture PGR in oil palm trunk hydrolysate, the maximum hydrogen yield of 301 mL H₂/g sugar_consumed was achieved at hydrolysate concentration of 40% (v/v). At higher concentration to 80% (v/v), the hydrogen fermentation process was inhibited. Therefore, the efficient thermophilic hydrogen-producing consortia PGR has successfully developed and has great potential for production of biohydrogen from mixed sugars hydrolysate.