Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 in a UASB reactor and bacterial quantification under non-sterile conditions

Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 was investigated in an up-flow anaerobic sludge blanket (UASB) reactor. The reactor was operated under non-sterile conditions at 40^○C and initial pH 8.0 at different hydraulic retention times (HRTs) (2–12 h) and glycerol concentrations (10–30 g/L). Decreasing the HRT led to an increase in hydrogen production rate (HPR) and hydrogen yield (HY). The highest HPR of 242.15 mmol H₂/L/d and HY of 44.27 mmol H₂/g glycerol consumed were achieved at 4 h HRT and glycerol concentrations of 30 and 10 g/L, respectively. The main soluble metabolite was 1,3-propanediol, which implies that Klebsiella sp. was dominant among other microorganisms. Fluorescence in situ hybridization (FISH) revealed that the microbial community was dominated by Klebsiella sp. with 56.96, 59.45, and 63.47% of total DAPI binding cells, at glycerol concentrations of 10, 20, and 30 g/L, respectively.     

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.     

Influence of Cu concentration on the biohydrogen production of continuous stirred tank reactor

In this paper, the effect of hydraulic retention time (HRT, 16 h–4 h) on fermentative hydrogen production by mixed cultures was firstly investigated in a sucrose-fed anaerobic continuous stirred tank reactor (CSTR) at 35 °C and initial pH 8.79. After stable operations at HRT of 16–6 h, the bioreactor became unstable when the HRT was lowered to 4 h. The maximum hydrogen yield reached 3.28 mol H₂/mol-Sucrose at HRT 4 h. Supplementation of Cu²⁺ at HRT 4 h improved the operation stability through enhancement of substrate degradation efficiency. The effect of Cu²⁺ concentration ranging from 1.28 to 102.4 mg/L on fermentative hydrogen production was studied. The results showed that Cu²⁺ was able to enhance the hydrogen production yield with increasing Cu²⁺ concentration from 1.28 to 6.4 mg/L. The maximum hydrogen yield of 3.31 mol H₂/mol-Sucrose and the maximum hydrogen production rate of 14.44 L H₂/Day/L-Reactor were obtained at 6.4 mg/L Cu²⁺ and HRT 4 h Cu²⁺ at much higher concentration could inhibit the hydrogen production, but it could increase substrate degradation efficiency (12.8 and 25.6 mg/L Cu²⁺). The concentration of Cu²⁺ had effect on the distribution of soluble metabolite.     

Continuous production of hydrogen from oat straw hydrolysate in a biotrickling filter

Hydrogen was produced in a biotrickling filter (BF) packed with perlite and fed with oat straw acid hydrolysate at 30 °C. Inlet chemical oxygen demand (COD) from 1.2 to 35 g/L and hydraulic retention time (HRT) between 24 h and 6 h were assayed. With increasing inlet COD or decreasing HRT, H₂ production rate (HPR) increased but H₂ production yield (HY) decreased. Maximum HPR of 81.4 mL H₂/L_reactor h (3.3 mmol H₂/L_reactor h) and HY of 2.9 mol H₂/mol_{hexose consumed} were found at an inlet COD of 0.05 g_COD/L h (HRT 24 h) and 2.9 g_COD/L h (HRT 12 h), respectively. Maximum hydrogen composition in gas was 45 ± 4% (v/v) with CO₂ as balance. Methane was not detected. Maximum HPR and inlet COD used in this work were higher than others reported for reactors with suspended or fixed biomass. However, implementation of strategies for biomass control to avoid reactor clogging is needed.     

Converting waste molasses liquor into biohydrogen via dark fermentation using a continuous bioreactor

This study investigated the effects of substrate concentration, HRT (hydraulic retention time), and pre-treatment of the substrate molasses on biohydrogen production from waste molasses (condensed molasses fermentation solubles, CMS) with a CSTR (continuously-stirred tank reactor). First, the hydrogen production was performed with various CMS concentrations (40–90 g COD/L, total sugar 8.7–22.6 g/L) with 6 h HRT. The results show that the maximal hydrogen production rate (HPR) occurred at 80 g COD/L substrate (19.8 g ToSu/L, ToSu: Total Sugar), obtaining an HPR of 0.417 mol/L/d. However, maximum hydrogen yield (HY) of 1.44 mol H₂/mol hexose and overall hydrogen production efficiency (HPE) of 25.6% were achieved with a CMS concentration of 70 g COD/L (17.3 g ToSu/L). The substrate inhibition occurred when CMS concentration was increased to 90 g COD/L (22.6 g ToSu/L). Furthermore, it was observed that the optimal HPR, HY, and HPE all occurred at HRT 6 h. Operating at a lower HRT of 4 h decreased the hydrogen production performance because of lower substrate utilization efficiency. The employment of pre-heating treatment (60 °C for 1 h) of the substrate could markedly enhance the fermentation performance. With 6 h HRT and substrate pre-heating treatment, the HPE raised to 29.9%, which is 18% higher than that obtained without thermal pretreatment.     

Effects of substrate concentration and hydraulic retention time on hydrogen production from common reed by enriched mixed culture in continuous anaerobic bioreactor

This study aims to investigate the effect of substrate concentration and hydraulic retention time (HRT) on hydrogen production in a continuous anaerobic bioreactor from unhydrolyzed common reed (Phragmites australis) an invasive wetland and perennial grass. The bioreactor has capacity of 1 L and working volume of 600 mL. It was operated at pH 5.5, temperature at 37 °C, hydraulic retention time (HRT) 12 h, and variation of substrate concentration from 40, 50, and 60 g COD/L, respectively. Afterward, the HRT was then varied from 12, 8, to 4 h for checking the optimal biohydrogen production. Each condition was run until reach steady state on hydrogen production rate (HPR) which based on hydrogen percentage and daily volume. The results were obtained the peak of substrate concentration was at the 50 g COD/L with HRT 12 h, average HPR and H₂ concentration were 28.71 mL/L/h and 36.29%, respectively. The hydrogen yield was achieved at 106.23 mL H₂/g CODre. The substrate concentration was controlled at 50 g COD/L for the optimal HRT experiments. It was found that the maximum of average HPR and H₂ concentration were 43.28 mL/L/h and 36.96%, respectively peak at HRT 8 h with the corresponding hydrogen yield of 144.35 mL H₂/g CODre. Finally, this study successful produce hydrogen from unhydrolyzed common reed by enriched mixed culture in continuous anaerobic bioreactor.     

Exploring optimal conditions for thermophilic fermentative hydrogen production from cassava stillage

This study investigated the effects of seed sludges, alkalinity and HRT on the thermophilic fermentative hydrogen production from cassava stillage. Five different kinds of sludges were used as inocula without any pretreatment. Though batch experiments showed that mesophilic anaerobic sludge was the best inoculum, the hydrogen yields with different seed sludges were quite similar in continuous experiments in the range of 82.9–92.3 ml H₂/gVS without significant differences which could be attributed to the establishment of Uncultured Thermoanaerobacteriaceae bacterium-dominant microbial communities in all reactors. It is indicated that results obtained from batch experiments are not consistent with those from continuous experiments and all the tested seed sludges are good sources for continuous thermophilic hydrogen production from cassava stillage. The influent alkalinity of 6 g NaHCO₃/L and HRT 24 h were optimal for hydrogen production with hydrogen yield of 76 ml H₂/gVS and hydrogen production rate of 3215 ml H₂/L/d. Butyrate was the predominant metabolite in all experiments. With the increase in alkalinity of more than 6 g/L, the concentration of VFA/ethanol increased while hydrogen yield decreased due to the higher concentration of acetate and propionate. The decrease in HRT resulted in the higher hydrogen production rate but lower hydrogen yield. Variation of hydrogen yields were quite correlated with butyrate/acetate (B/A) ratio with different influent alkalinities, however, butyrate was important parameter to justify the hydrogen yields with various HRTs.     

Biohydrogen from molasses with ethanol-type fermentation: Effect of hydraulic retention time

Though ethanol-type fermentation has many advantages for improving hydrogen production rate (HPR) in continuously mode hydrogen producing system, information on this fermentation is very deficient. The effect of hydraulic retention time (HRT) on biohydrogen production and operational stability of ethanol-type fermentation was investigated in a continuous stirred tank reactor (CSTR) using molasses as substrate. Five HRTs were examined, ranging from 4 to 10 h. At HRT 5 h, the highest HPR of 12.27 mmol L⁻¹ h⁻¹ was obtained from ethanol-type fermentation in the pH range of 4.3–4.4. During the whole operation process, ethanol, butyrate and acetate were the predominant metabolites. A total COD concentration of ethanol and acetate accounted for above 73.3% of total soluble microbial products. Linear regression showed that HPR and ethanol production rate were proportionately correlated at all HRTs which could be expressed as y = 0.9821x − 3.5151 (r² = 0.9498). It is meaningful that the proposed recovery of both hydrogen and ethanol from fermentation process can improve energy production rate and economic profit. Results demonstrated that the best energy production rate was 15.50 kJ L⁻¹ h⁻¹, occurred at HRT = 5 h.     

Aspect ratio effect of bioreactor on fermentative hydrogen production with immobilized sludge

The phenomenon of bacterial wash-out frequently occurs in the traditional continuous stirred tank reactor (CSTR) systems at low hydraulic retention time (HRT). In this study, the effect of different aspect ratios, height (H) to diameter (D) of 1:1, 3:1 and 5:1, of a CSTR with immobilized anaerobic sludge on hydrogen (H₂) production were investigated. The pH, volatile suspended solids (VSS) and total solids (TS) concentrations of the seed sludge were 6.8, 33.3 and 65.1 g/L, respectively. Thermally treated sludge was immobilized by silicone gel entrapment approach. The entrapped-sludge system operated stably at a low HRT without suffering from cell wash-out. Hence, the hydrogen production rate (HPR) was enhanced by increasing organic loading rates. The immobilized sludge CSTRs were operated at 40 °C with sucrose (10, 20, 30 and 40 g COD/L) and Endo nutrient medium at different HRTs (4, 2, 1 and 0.5 h). It was found that the granule formation enhanced HPR. The maximum HPR and the H₂ yield were found to be 15.36H₂ L/h/L and 3.16 mol H₂/mol sucrose, respectively, with the H₂ content in the biogas above 44% for all tests runs.     

Improving hydrogen production from cassava starch by combination of dark and photo fermentation

The combination of dark and photo fermentation was studied with cassava starch as the substrate to increase the hydrogen yield and alleviate the environmental pollution. The different raw cassava starch concentrations of 10–25 g/l give different hydrogen yields in the dark fermentation inoculated with the mixed hydrogen-producing bacteria derived from the preheated activated sludge. The maximum hydrogen yield (HY) of 240.4 ml H₂/g starch is obtained at the starch concentration of 10 g/l and the maximum hydrogen production rate (HPR) of 84.4 ml H₂/l/h is obtained at the starch concentration of 25 g/l. When the cassava starch, which is gelatinized by heating or hydrolyzed with α-amylase and glucoamylase, is used as the substrate to produce hydrogen, the maximum HY respectively increases to 258.5 and 276.1 ml H₂/g starch, and the maximum HPR respectively increases to 172 and 262.4 ml H₂/l/h. Meanwhile, the lag time (λ) for hydrogen production decreases from 11 h to 8 h and 5 h respectively, and the fermentation duration decreases from 75–110 h to 44–68 h. The metabolite byproducts in the dark fermentation, which are mainly acetate and butyrate, are reused as the substrates in the photo fermentation inoculated with the Rhodopseudomonas palustris bacteria. The maximum HY and HPR are respectively 131.9 ml H₂/g starch and 16.4 ml H₂/l/h in the photo fermentation, and the highest utilization ratios of acetate and butyrate are respectively 89.3% and 98.5%. The maximum HY dramatically increases from 240.4 ml H₂/g starch only in the dark fermentation to 402.3 ml H₂/g starch in the combined dark and photo fermentation, while the energy conversion efficiency increases from 17.5–18.6% to 26.4–27.1% if only the heat value of cassava starch is considered as the input energy. When the input light energy in the photo fermentation is also taken into account, the whole energy conversion efficiency is 4.46–6.04%.     

Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse

Immobilized Clostridium butyricum TISTR 1032 on sugarcane bagasse improved hydrogen production rate (HPR) approximately 1.2 times in comparison to free cells. The optimum conditions for hydrogen production by immobilized C. butyricum were initial pH 6.5 and initial sucrose concentration of 25 g COD/L. The maximum HPR and hydrogen yield (HY) of 3.11 L H₂/L substrate·d and 1.34 mol H₂/mol hexose consumed, respectively, were obtained. Results from repeated batch fermentation indicated that the highest HPR of 3.5 L H₂/L substrate·d and the highest HY of 1.52 mol H₂/mol hexose consumed were obtained at the medium replacement ratio of 75% and 50% respectively. The major soluble metabolites in both batch and repeated batch fermentation were butyric and acetic 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.     

Hydrogen production from butyrate by a marine mixed phototrophic bacterial consort

A newly enriched marine phototrophic bacterial consort was studied for its capability of hydrogen production in batch cultivations using butyrate as the sole carbon source. Analyses of denaturing gradient gel electrophoresis (DGGE) profiles showed that the mixed bacterial consort consisted mainly of Ectothiorhodospira, Sporolactobacillus, and Rhodovulum. Important parameters investigated include temperature, light intensity, initial pH, and butyrate concentration. The pH of the culture medium significantly increased as fermentation proceeded. Optimal cell growth was observed at temperature of 25–35 °C, light intensity of 80–120 μmol photons/m² s, initial pH of 8, butyrate concentration of 20–40 mmol/l. Optimal conditions for hydrogen production were 30 °C, light intensity of 80 μmol photons/m² s, initial pH 8. The increase of butyrate concentration (10–50 mmol/l) resulted in higher hydrogen production, but the yield of hydrogen production (mol H₂/mol butyrate) gradually decreased with increasing butyrate concentration. The maximal hydrogen yield and hydrogen production rate were estimated to be 2.52 ± 0.12 mol H₂/mol butyrate and 19.40 ± 2.32 ml/l h, respectively. These results indicate that optimization of the culture conditions resulted in a simultaneous increase in biohydrogen production and cell growth.     

Continuous biogenic hydrogen production from dilute acid pretreated algal hydrolysate using hybrid immobilized mixed consortia

This study investigated the bioconversion of dilute acid (2% H₂SO₄) pretreated red algae (Gelidium amansii) hydrolysate into H₂ by anaerobic fermentation in a continuous stirred tank reactor under mesophilic conditions using hybrid immobilized cells as microbial catalyst. Two different hydraulic retention times (HRT) of 24 h and 16 h with a feed concentration of 15 g/L hexose equivalent have been investigated over 85 days of operation to evaluate H₂ production performance and stability of the continuous system. The highest hydrogen production rate (HPR) and hydrogen yield (HY) of 2.7 L/L/d and 1.3 mol/mol substrate hexose_added was achieved at 24 h HRT, while further operation at 16 h HRT led to a significant drop in the hydrogen production with a HPR and HY values of 1.8 L/L/d and 0.7 mol/mol substrate hexose_added, respectively. The bacterial community analysis characterized by 454 pyrosequencing revealed that the changes in HRT significantly influence the composition of the dominant microflora. At longer HRT (24 h), the phyla Firmicutes was abundant over 98%, whereas at shorter HRT (16 h), Proteobacteria being the dominant populations with 84%. These outcomes suggested that controlling appropriate HRT is prerequisite for efficient hydrogen production.     

Photo fermentative hydrogen production using dominant components (acetate, lactate, and butyrate) in dark fermentation effluents

Engineering strategies were applied to promote the phototrophic H₂ production of an indigenous purple nonsulfur bacterium Rhodopseudomonas palustris WP3-5 using major components (i.e., acetate, butyrate, and lactate) of dark fermentation effluents as carbon sources. First, performance of cell growth and photo-H₂ production on each carbon source was examined individually. It appeared that acetate was the most effective carbon source for photo-H₂ production, giving an overall H₂ production rate and H₂ yield of 12.68 ml/h/l and 67.1%, respectively. Next, the effect of substrate concentration of each carbon source on photo-hydrogen production was investigated. Kinetic models were developed to describe the correlation between maximum specific growth rate/specific H₂ production rate and the substrate concentration. The results show that using acetate and lactate as the carbon source, the kinetics for the cell growth and photo-hydrogen production can be described by Monod-type and Michaelis–Menten models, respectively, whereas substrate inhibition occurred when using butyrate as the carbon source. The continuous cultures were also conducted at a hydraulic retention time of 96 h using synthetic dark fermentation soluble metabolites (with a 5 and 10 fold dilution) as the influent. The phototrophic H₂ production efficiency was stably maintained for over 30 days with an overall H₂ yield 10.30 and 11.97 mol H₂/mol sucrose, when using 5-fold and 10-fold diluted dark fermentation effluent, respectively, as the substrate for dark fermentation. This demonstrates the feasibility of using the sequential dark and photo fermentation for high-yield biohydrogen production.     

Hydrogen fermentation of different galactose–glucose compositions during various hydraulic retention times (HRTs)

This study investigated the effects of sugar composition and hydraulic retention time (HRT) on continuous hydrogen fermentation. Continuously-stirred tank reactors (CSTRs) were inoculated with heat-treated digester sludge and fed with 15 g/L of glucose, galactose and galactose: glucose mixture (8:2 ratio-simulating the hydrolysate composition of macroalgae) at HRTs of 6–24 h. Peak hydrogen production rate (HPR) and hydrogen yield (HY) of 4.49 L/L/d and 1.62 mol/mol glucose_added were attained while using glucose as feedstock at HRTs of 6 and 18 h, respectively. Meanwhile, galactose provided a peak HPR and HY of 2.35 L/L/d and 1.00 mol/mol galactose_added during the HRTs of 12 and 24 h, respectively. In case of mixed sugars (galactose 8: glucose 2) the production performances were inferior to the sole sugar conditions due to the low substrate utilization of less than 65%, which showed a maximal HPR and HY of 2.75 L/L/d and 0.48 mol/mol carbohydrate_added at the HRTs of 6 and 18 h, respectively.     

Biohydrogen production from palm oil mill effluent (POME) by two stage anaerobic sequencing batch reactor (ASBR) system for better utilization of carbon sources in POME

The production of biohydrogen through dark fermentation of palm oil mill effluent (POME) was evaluated in two-stages of biohydrogen in an anaerobic sequencing batch reactor (ASBR) system using enriched mixed culture for the first time. This study attempts to examine the effect of HRT and its interaction behavior with the solid retention time (SRT), and the sugar consumption. The effluent after discharged from the thermophilic reactor contained 7.61 g/L TC and 22.87 g/L TSS was fed to the secondary mesophilic reactor system. Results indicated that the overall sugar consumption reached 88.62% at the optimum HRT of 12 h with the SRT set to 20 h. The optimum hydrogen yield and HPR in the thermophilic stage were 2.99 mol H₂/mol-sugar and 8.54 mmol H₂/L·h respectively, while for the mesophilic stage were 1.19 mol H₂/mol-sugar and 1.47 mmolH₂/L·h respectively. The overall HPR showed an improvement and increase from 8.54 mmol H₂/L·h to 10.34 mmol H₂/L.h. Microbial community analysis of mixed culture in the two-stage thermophilic (55.0 °C) and mesophilic (37.0 °C) ASBR reactor was dominated by Thermoanaerobacterium sp. based on the PCR-DGGE technique.     

Combination of dark- and photo-fermentation to enhance hydrogen production and energy conversion efficiency

In this study, we investigated a two-phase process of combining the dark- and photo-fermentation methods to reutilize the residual solution derived from dark fermentation and increase the hydrogen yield (HY) from glucose. In dark fermentation, an orthogonal experimental design was used to optimize the culture medium for Clostridium butyricum (C. butyricum). The optimal culture medium composition was determined as glucose 20 g/l, NaCl 3 g/l, MgCl₂ 0.1 g/l, FeCl₂ 0.1 g/l, K₂HPO₄ 2.5 g/l, l-cysteine 0.5 g/l, vitamin solution 10 ml/l, and trace element solution 10 ml/l. In this method, the maximum HY increased from 1.59 to 1.72 mol H₂/mol glucose and hydrogen production rate (HPR) from 86.8 to 100 ml H₂/l/h. The metabolite byproducts from dark fermentation, mostly containing acetate and butyrate, were inoculated with Rhodopseudomonas palustris (R. palustris) and reutilized to produce hydrogen in photo-fermentation. In photo-fermentation, the maximum HY was 4.16 mol H₂/mol glucose, and the maximum removal ratios of acetate and butyrate were 92.3% and 99.8%, respectively. Combining dark fermentation and photo-fermentation caused a dramatic increase of HY from 1.59 to 5.48 mol H₂/mol glucose. The conversion efficiency of heat value in dark fermentation surged from 13.3% to 46.0% in the two-phase system.     

Strategy for enhancing photo-hydrogen production yield by repeated fed-batch cultures

In order to obtain the high H₂ yield, photo-hydrogen production by Rhodopseudomonas faecalis strain RLD-53 in fed-batch culture using acetate as the sole carbon was studied. In repeated fed-batch culture, biomass increased rapidly from 0.05 to 0.65 g/l within 120 h, with the specific maximal growth rate estimated 0.68 × 10⁻³ g/h. After 120 h, biomass increased slowly and after each time feeding biomass increased slightly. The specific cumulative H₂ volumes in each phase were 2791.3, 1161.7, 1445.5 and 840.6 ml H₂/l-culture, respectively. The average H₂ yield was 3.17 mol H₂/mol acetate based on the whole process while the average substrate conversion efficiency reached 79.3%. Specific maximum H₂ production rate and H₂ content was 37.2 ml H₂/l/h and 95.5%, respectively. The results demonstrated the repeated fed-batch mode obtained higher efficiency for hydrogen production, feeding acetate concentration and control of pH were important to fed-batch culture hydrogen production.