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.     

Hydrogen and methane production via a two-stage processes (H2-SBR + CH4-UASB) using tequila vinasses

The feasibility of producing hydrogen and methane via a two-stage fermentation of tequila vinasses was evaluated in sequencing batch (SBR) and up-flow anaerobic sludge blanket (UASB) reactors. Different vinasses concentrations ranging from 500 mg COD/L to 16 g COD/L were studied in SBR by using thermally pre-treated anaerobic sludge as inoculum for hydrogen production. Peak volumetric hydrogen production rate and specific hydrogen production were attained as 57.4 ± 4.0 mL H₂/L-h and 918 ± 63 mL H₂/gVSS-d, at the substrate concentration of 16 g COD/L and 6 h of hydraulic retention time (HRT). Increasing substrate concentration has no effect on the specific hydrogen production rate. The fermentation effluent was used for methane production in an UASB reactor. The higher methane composition in the biogas was achieved as 68% at an influent concentration of 1636 mg COD/L. Peak methane volumetric, specific production rates and yield were attained as 11.7 ± 0.7 mL CH₄/L-h, 7.2 ± 0.4 mL CH₄/g COD-h and 257.9 ± 13.8 mL CH₄/g COD at 24 h-HRT and a substrate concentration of 1636 mg COD/L. An overall organic matter removal (SBR + UASB) in this two-stage process of 73–75% was achieved.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using xylan as the substrate

Thermophilic hydrogen production from xylan by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 isolated from elephant dung was investigated using batch fermentation. The optimum conditions for hydrogen production from xylan by the strain KKU-ED1 were an initial pH of 7.0, temperature of 55 °C and xylan concentration of 15 g/L. Under the optimum conditions, the hydrogen yield (HY), hydrogen production rate (HPR) and xylanase activity were 120.05 ± 15.07 mL H₂/g xylan, 11.53 ± 0.19 mL H₂/L h and 0.41 units/mL, respectively. The optimum conditions were then used to produce hydrogen from 62.5 g/L sugarcane bagasse (SCB) (equivalent to 15 g/L xylan) in which the HY and HPR of 1.39 ± 0.10 mL H₂/g SCB (5.77 ± 0.41 mL H₂/g xylan) and 0.66 ± 0.04 mL H₂/L h, respectively, were achieved. In comparison to the other strains, the HY of the strain KKU-ED1 (120.05 ± 15.07 mL H₂/g xylan) was close to that of Clostridium sp. strain X53 (125.40 mL H₂/g xylan) and Clostridium butyricum CGS5 (90.70 mL H₂/g xylan hydrolysate).     

Non-sterile bio-hydrogen fermentation from food waste in a continuous stirred tank reactor (CSTR): Performance and population analysis

Bio-hydrogen production from food waste by anaerobic mixed cultures was conducted in a continuous stirred tank reactor (CSTR). The hydraulic retention time (HRT) was optimized in order to maximize hydrogen yield (HY) and hydrogen production rate (HPR). The maximum hydrogen content (38.6%), HPR (379 mL H₂/L. d) and HY (261 mL H₂/g-VS_added) were achieved at the optimum HRT of 60 h. The major soluble metabolite products were butyric and acetic acids which indicated a butyrate-acetate type fermentation. Operation of CSTR at HRT 60 h could select hydrogen producing bacteria and eliminate lactic acid bacteria and acetogenic bacteria. The microbial community analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) revealed that the predominant hydrogen producer was Clostridium sp.     

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.     

Biohydrogen production from various feedstocks by Bacillus firmus NMBL-03

In present work our objective was to find out the potential substrates for fermentative hydrogen production using microbial culture of Bacillus firmus NMBL-03 isolated from municipal sludge. A wide variety of substrates (glucose, xylose, arabinose, lactose, sucrose, and starch) and carbohydrate rich waste products (bagasse hydrolysate, molasses, potato peel and cyanobacterial mass) have been used for dark fermentative hydrogen production. All studies were done at optimized physico-chemical conditions. Among all substrates glucose, arabinose, lactose, starch, molasses and bagasse hydrolysate were found to be the favorable substrates for hydrogen production. The highest VHPR i.e. 177.5 ± 7.07 ml/L.h (7.95 ± 0.29 mmol H₂/L.h) and maximum H₂ production (22.58 ± 2.65 mmol H₂/L) was achieved using starch as the substrate. The maximum yield 1.29 ± 0.11 mol H₂/mol reducing sugar was obtained from bagasse hydrolysate as substrate. Butyrate and acetate were detected as the end product in all the cases while lactate was also detected from glucose and cyanobacterial hydrolysate. Since considerable amount of H₂ also evolved when cyanobacterial mass was used, therefore this microbe can be exploited for hydrogen production through a three stage integrated system. Residual carbohydrate containing biomass left after cyanobacterial H₂ production can be utilized in dark fermentative H₂ production. Spent media obtained after dark fermentative H₂ production contain considerable amount of volatile fatty acids that can be potential substrate for photo fermentative H₂ 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.     

Evaluation of bio-hydrogen production using rice straw hydrolysate extracted by acid and alkali hydrolysis

This study was conducted to investigate the properties of hydrolysates obtained from acid and alkali hydrolysis and to evaluate the feasibility of employing them for bio-hydrogen production. High sugar concentrations of 16.8 g/L and 13.3 g/L were present in 0.5% and 1.0% H₂SO₄ hydrolysates, respectively. However, H₂SO₄ hydrolysis resulted in large amounts of short-chain fatty acids (SCFAs) and furan derivatives, which were removed by detoxification. In bio-hydrogen production, 1.0% H₂SO₄ hydrolysate showed a 55.6 mL of highest hydrogen production and 1.14 mol-H₂/mol-hexose equivalent_added of hydrogen yield. In control and 1.0% NaOH hydrolysate, 29.7 mL and 36.9 mL of hydrogen were produced, respectively. Interestingly, relatively high acetate and butyrate production resulted in lactate reduction. Also, NH₄OH hydrolysate produced less than 10 mL of hydrogen. Thus, these results indicate that hydrogen production and metabolite distribution can vary depending on the sugars and by-product composition in the hydrolysate.     

Production of biohydrogen from sugars and lignocellulosic biomass using Thermoanaerobacter GHL15

The effect of culture parameters on hydrogen production using strain GHL₁₅ in batch culture was investigated. The strain belongs to the genus Thermoanaerobacter with 98.9% similarity to Thermoanaerobacter yonseiensis and 98.5% to Thermoanaerobacter keratinophilus with a temperature optimum of 65–70 °C and a pH optimum of 6–7. The strain metabolizes various pentoses, hexoses, and disaccharides to acetate, ethanol, hydrogen, and carbon dioxide. However substrate inhibition was observed above 10 mM glucose concentration. Maximum hydrogen yields on glucose were 3.1 mol H₂ mol⁻¹ glucose at very low partial pressure of hydrogen. Hydrogen production from various lignocellulosic biomass hydrolysates was investigated in batch culture. Various pretreatment methods were examined including acid, base, and enzymatic (Celluclast^® and Novozyme 188) hydrolysis. Maximum hydrogen production (5.8–6.0 mmol H₂ g⁻¹ dw) was observed from Whatman paper (cellulose) hydrolysates although less hydrogen was produced by hydrolysates from other examined lignocellulosic materials (maximally 4.83 mmol H₂ g⁻¹ dw of grass hydrolysate). The hydrogen yields from all lignocellulosic hydrolysates were improved by acid and alkaline pretreatments, with maximum yields on grass, 7.6 mmol H₂ g⁻¹ dw.     

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.     

Effect of cornstalk hydrolysis on photo-fermentative hydrogen production by R. capsulatus

Cornstalk is a typical cellulose material, which can be used by photo-fermentative H₂ production after pretreatment. However, the pretreatment methods have different influence on photo fermentation. In this study, 25.0 g cornstalk was pretreated by HCl/NaOH/cellusase. The hydrolysis rates increased from 45.51% by ddH₂O-treatment to 60.79% by diluted HCl-treatment and 51.6% by NaOH-treatment. The corresponding reducing sugar yields were 0.13 g/g, 0.42 g/g and 0.01 g/g, respectively. Enzymatic treatment enhanced the corresponding cornstalk hydrolysis rates to 50.81%, 67.60% and 64.10% with reducing sugar yields of 0.22 g/g, 0.62 g/g and 0.26 g/g. The sorts and concentrations of carbon source for H₂ production vary among different hydrolysates. Photo-fermentative H₂ production of strain R. capsulatus JL1 and mutant JL1601 (cheR2^-) with hydrolysates were investigated. The maximum H₂ yield of 123.8 ± 14.2 mL/g by strain JL1 was obtained from alkali-enzyme pretreated cornstalk, while the H₂ yield of 224.9 ± 5.2 mL/g by mutant JL1601 (cheR2^-) was obtained with acid-enzyme hydrolysate as the substrates. Meanwhile, the alkali pretreated cornstalk was the worst for photo-fermentation of both strain JL1 and mutant JL1601 (cheR2^-). Nevertheless, the highest substrate conversion efficiencies for both strains were obtained from ddH₂O-pretreated hydrolysate. Two-step pretreated hydrolysates were more beneficial to H₂ production for mutant JL1601 (cheR2^-) but not for strain JL1.     

Evaluation of continuous biohydrogen production from enzymatically treated cornstalk hydrolysate

Enzymatically treated cornstalk hydrolysate was tested as substrate for H₂ production by Thermoanaerobacterium thermosaccharolyticum W16 in a continuous stirred tank reactor. The performance of strain W16 to ferment the main components of hydrolysate, mixture of glucose and xylose, in continuous culture was conducted at first, and then T. thermosaccharolyticum W16 was evaluated to ferment fully enzymatically hydrolysed cornstalk to produce H₂ in continuous operation mode. At the dilution rate of 0.020 h⁻¹, the H₂ yield and production rate reached a maximum of 1.9 mol H₂ mol⁻¹ sugars and 8.4 mmol H₂ L⁻¹ h⁻¹, respectively, accompanied with the maximum glucose and xylose utilizations of 86.3% and 77.6%. Continuous H₂ production from enzymatically treated cornstalk hydrolysate in this research provides a new direction for economic, efficient, and harmless H₂ production.     

Hydrogen and methane production potential of agave bagasse enzymatic hydrolysates and comparative technoeconomic feasibility implications

In the present work, agave bagasse enzymatic hydrolysates obtained with newly locally-available commercial enzymatic preparations were explored for their corresponding hydrogen and methane production potential in batch mode. The major levels in chemical oxygen demand and total carbohydrates were provided by enzymatic hydrolysates made with Zymapect and Stonezyme, respectively. Batch experiments demonstrated that Celluclast 1.5L achieves the maximum hydrogen productivity (1.88 L H₂/L), from 1.6 to 2.0-fold higher than other alternatives, whereas Zymapect attains the highest methane productivity (1.32 L CH₄/L), with high specific yield reached by both Stonezyme and Zymapect (162 and 163 L CH₄/kg bagasse), from 1.7 to 2.0-fold higher than other options. Finally, a preliminary techno-economic analysis allowed to elucidate that the cheapest alternatives for hydrogen and methane production at batch scale are Celluclast 1.5L and Stonezyme, respectively. Overall, the present analysis could serve as groundwork for the selection of the best enzymatic alternatives for hydrogen and methane production.     

Optimization of dilute acid and enzymatic hydrolysis for dark fermentative hydrogen production from the empty fruit bunch of oil palm

Pretreatment of the empty fruit brunch (EFB) from oil palm was investigated for H₂ fermentation. The EFB was hydrolyzed at various temperatures, H₂SO₄ concentrations, and reaction times. Subsequently, the acid-hydrolysate underwent enzymatic saccharification under various temperature, pH, and enzymatic loading conditions. Response surface methodology derived the optimum sugar concentration (SC), hydrogen production rate (HPR), and hydrogen yield (HY) as 28.30 g L⁻¹, 2601.24 mL H₂ L⁻¹d⁻¹, and 275.75 mL H₂ g⁻¹ total sugar (TS), respectively, at 120 °C, 60 min of reaction, and 6 vol% H₂SO₄, with the combined severity factor of 1.75. Enzymatic hydrolysis enhanced the SC, HY, and HPR to 34.52 g L⁻¹, 283.91 mL H₂ g⁻¹ TS, and 3266.86 mL H₂ L⁻¹d⁻¹, respectively, at 45 °C, pH 5.0, and 1.17 mg enzyme mL⁻¹. Dilute acid hydrolysis would be a viable pretreatment for biohydrogen production from EFB. Subsequent enzymatic hydrolysis can be performed if enhanced HPR is required.     

Direct fermentation of Laminaria japonica for biohydrogen production by anaerobic mixed cultures

A few studies have been made on fermentative hydrogen production from marine algae, despite of their advantages compared with other biomass substrates. In this study, fermentative hydrogen production from Laminaria japonica (one brown algae species) was investigated under mesophilic condition (35 ± 1 °C) without any pretreatment method. A feasibility test was first conducted through a series of batch cultivations, and 0.92 mol H₂/mol hexose_added, or 71.4 ml H₂/g TS of hydrogen yield was achieved at a substrate concentration of 20 g COD/L (based on carbohydrate), initial pH of 7.5, and cultivation pH of 5.5. Continuous operation for a period of 80 days was then carried out using anaerobic sequencing batch reactor (ASBR) with a hydraulic retention time (HRT) of 6 days. After operation for approximately 30 days, a stable hydrogen yield of 0.79 ± 0.03 mol H₂/mol hexose_added was obtained. To optimize bioenergy recovery from L. japonica, an up-flow anaerobic sludge blanket reactor (UASBr) was applied to treat hydrogen fermentation effluent (HFE) for methane production. A maximum methane yield of 309 ± 12 ml CH₄/g COD was achieved during the 90 days operation period, where the organic loading rate (OLR) was 3.5 g COD/L/d.     

Influence of linoleic acid,pH and HRT on anaerobic microbial populations and metabolic shifts in ASBRs during dark hydrogen fermentation of lignocellulosic sugars

In this study, controlling an anaerobic microbial community to increase the hydrogen (H₂) yield during the degradation of lignocelluosic sugars was accomplished by adding linoleic acid (LA) at low pH and reducing the hydraulic retention time (HRT) of an anaerobic sequencing batch reactor (ASBR). At pH 5.5 and a 1.7 d HRT, the maximum H₂ yield for LA treated cultures fed glucose or xylose reached 2.89 ± 0.18 mol mol⁻¹ and 1.94 ± 0.17 mol mol⁻¹, respectively. The major soluble metabolites at pH 5.5 with a 1.7 day HRT differed between the control and LA treated cultures. A metabolic shift toward H₂ production resulted in increased hydrogenase activity in both the xylose (13%) and glucose (34%) fed LA treated cultures relative to the controls. In addition, the Clostridia population and the H₂ yield were elevated in cultures treated with LA. A flux balance analysis for the LA treated cultures showed a reduction in homoacetogenic activity which was associated with reducing the Bacteriodes levels from 12% to 5% in the glucose fed cultures and 16% to 10% in the xylose fed cultures. Strategies for controlling the homoacetogens and optimal hydrogen production from glucose and xylose are proposed.     

Effect of hydraulic retention time on hydrogen production and chemical oxygen demand removal from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR)

This study evaluated hydrogen production and chemical oxygen demand removal (COD removal) from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR). The ABR was conducted based on the optimum condition obtained from the batch experiment, i.e. 2.25 g/L of FeSO₄ and initial pH of 9.0. The effects of the varying hydraulic retention times (HRT: 24, 18, 12, 6 and 3 h) on hydrogen production and COD removal in a continuous ABR were operated at room temperature (32.3 ± 1.5 °C). Hydrogen production rate (HPR) increased with a reduction in HRT i.e. from 164.45 ± 4.14 mL H₂/L.d (24 h HRT) to 883.19 ± 7.89 mL H₂/L.d (6 h HRT) then decreased to 748.54 ± 13.84 mL H₂/L.d (3 h HRT). COD removal increased with reduction in HRT i.e. from 14.02 ± 0.58% (24 h HRT) to 29.30 ± 0.84% (6 h HRT) then decreased to 21.97 ± 0.94% (3 h HRT). HRT of 6 h was the optimum condition for ABR operation as indicated.     

The production of biohydrogen by a novel strain Clostridium sp. YM1 in dark fermentation process

In view of increasing attempts for the production of renewable energy, the production of biohydrogen energy by a new mesophilic bacterium Clostridium sp. YM1 was performed for the first time in the dark fermentation. Experimental results showed that the fermentative hydrogen was successfully produced by Clostridium sp. YM1 with the highest cumulative hydrogen volume of 3821 ml/L with a hydrogen yield of 1.7 mol H₂/mol glucose consumed. Similar results revealed that optimum incubation temperature and pH value of culture medium were 37 °C and 6.5, respectively. The study of hydrogen production from glucose and xylose revealed that this strain was able to generate higher hydrogen from glucose compared to that from xylose. The profile of volatile fatty acids produced showed that hydrogen generation by Clostridium sp. YM1 was butyrate-type fermentation. Moreover, the findings of this study indicated that an increase in head space of fermentation culture positively enhanced hydrogen production.     

Comparison of the use of sucrose and glucose as a substrate for hydrogen production in an upflow anaerobic fixed-bed reactor

This study evaluated the use of two types of substrates, glucose and sucrose, feeding an anaerobic fixed-bed bioreactor. The biogas produced was composed of H₂ and CO₂, without methane. Maximum hydrogen yields were 3.22 mol H₂ mol⁻¹ sucrose_converted and 1.51 mol H₂ mol⁻¹ glucose_converted. The main intermediates were acetic acid, butyric acid, and ethanol. The greatest difference, however, was in the stability of the process. The operation of the reactor with sucrose exhibited a drop in biogas production, whereas operation with glucose was stable after a slight decrease in biogas production. This decrease may have been caused by the differential growth of microbial populations in each reactor, namely, the growth of organisms that use the Wood–Ljungdahl metabolic pathway.     

Sulfate-reducing bacteria as new microorganisms for biological hydrogen production

Sulfate-reducing bacteria (SRB) have an extremely high hydrogenase activity and in natural habitats where sulfate is limited, produce hydrogen fermentatively. However, the production of hydrogen by these microorganisms has been poorly explored. In this study we investigated the potential of SRB for H₂ production using the model organism Desulfovibrio vulgaris Hildenborough. Among the three substrates tested (lactate, formate and ethanol), the highest H₂ production was observed from formate, with 320 mL L⁻¹_medium of H₂ being produced, while 21 and 5 mL L⁻¹_medium were produced from lactate and ethanol, respectively. By optimizing reaction conditions such as initial pH, metal cofactors, substrate concentration and cell load, a production of 560 mL L⁻¹_medium of H₂ was obtained in an anaerobic stirred tank reactor (ASTR). In addition, a high specific hydrogen production rate (4.2 L g⁻¹_dcw d⁻¹; 7 mmol g⁻¹_dcw h⁻¹) and 100% efficiency of substrate conversion were achieved. These results demonstrate for the first time the potential of sulfate reducing bacteria for H₂ production from formate.