Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor

To achieve stable and efficient photo-fermentative hydrogen production, this work investigated photo-fermentative hydrogen production by forming biofilm on the surface of carrier in the biofilm reactor (BR). Results showed the hydrogen production performance was greatly improved by formed biofilm. The time of hydrogen production and efficiency of substrate utilization were enhanced obviously compared to the control reactor (CR). When the CR was used, hydrogen production stopped at 7th day and maximum cumulative hydrogen volume and hydrogen yield were 1730 ± 87 mL/L and 1.44 ± 0.07 mol H₂/mol acetate, respectively. However, in the BR hydrogen production volume of 3028 ± 150 mL/L and hydrogen yield of 2.52 ± 0.13 mol H₂/mol acetate were obtained, which were enhanced about 75% compared to that of the CR. The time of hydrogen production extended from 7 days of CR to 12 days of BR and the substrate conversion efficiency increased from 36% of CR to 63% of BR. It was worth noting at 8th day that substrate was almost utilized completely but hydrogen production still lasted for 4 days. This suggested that the formation of biofilm in BR was favorable to continuous hydrogen production and substrate utilization with high efficiency. Results demonstrated the BR can get a more stable and consistent operating process and it was a proper and potential way to produce hydrogen by photo-fermentative bacteria (PFB).     

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.     

Feasibility studies on continuous hydrogen production using photo-fermentative sequencing batch reactor

A novel photo-fermentative sequencing batch reactor (PFSBR) process assisted by activated carbon fibers (ACFs) was used to continuously produce hydrogen gas by Rhodopseudomonas faecalis RLD-53. Feasibility of continuous hydrogen production in PFSBR operation at different hydraulic retention times (HRTs) (48, 96, 144 and 192 h) and influent acetate concentrations (20, 40, 60 and 80 mmol/l) was investigated. The rate and yield of hydrogen production increased with HRTs from 48 to 144 h, and then decreased with the HRTs from 144 to 192 h. Regulation of the proper influent acetate concentration (60 mmol/l) not only increased hydrogen production by PFSBR, but also maintained quality of the effluent with high substrate removal efficiency (97.70%). Free R. faecalis RLD-53 was adsorbed on the surface of ACFs, initially isolated cells, then monolayer, and finally mature biofilm with three dimensional multilayers structures. The PFSBR reached a maximum hydrogen yield (3.12 mol H₂/mol acetate), and achieved a steady state when mature biofilm developed on ACFs. Therefore, photo-fermentative sequencing batch reactor was a promising process for continuous photo-fermentative hydrogen production.     

The effect of hydraulic retention time on thermophilic dark fermentative biohydrogen production in the continuously operated packed bed bioreactor

The main objective of the study is to investigate the effect of hydraulic retention times on continuous dark fermentative biohydrogen production in an up-flow packed bed reactor (UPBR) containing a novel microorganism immobilization material namely polyester fiber beads. The hydrogen producing dark fermentative microorganisms were obtained by heat-pretreatment of anaerobic sludge from the acidogenic phase of an anaerobic wastewater treatment plant. Glucose was the sole carbon source and the initial concentration was 15 ± 1 g/L throughout the continuous feeding. UPBR was operated under the thermophilic condition at T = 48 ± 2 °C and at varying HRTs between 2 h and 6 h. The hydrogen productivity of continuously operated UPBR increased with increasing HRT. Hydrogen production volume varied between 4331 and 6624 ml/d, volumetric hydrogen production rates (VHPR) were obtained as 3.09–4.73 L H₂/L day, and hydrogen production yields (HY) were 0.49 mol/mol glucose-0.89 mol/mol glucose depending on HRT. Maximum daily hydrogen volume (6624 ml/d), the yield (0.89 mol/mol glucose) and VHPR (4.73 L H₂/L day) were obtained at HRT = 6 h. The production rate and the yield decreased with increasing organic loading rate due to substrate inhibition.     

Thermophilic hydrogen production from starch wastewater using two-phase sequencing batch fermentation coupled with UASB methanogenic effluent recycling

The aim of this study was to evaluate the performance of thermophilic hydrogenesis coupled with mesophilic methanogenesis in which the effluent was recycled to the hydrogen reactor for starch wastewater treatment. With this system, the hydrogen production rate and yield were 3.45 ± 0.25 L H₂/(L·d) and 5.79 ± 0.41 mmol H₂/g COD_added respectively, and thus higher than the values of the control group without methanogenic effluent recycling. In addition, relatively higher contents of acetate and butyrate were obtained in the hydrogen reactor with recirculation. The methane reactors were operated with the effluent from the hydrogen reactor, and methane yield was stabilized at 0.21–0.23 L/g COD_removal in both. Analysis of the microbial communities further showed that methanogenic effluent recirculation enriched microbial communities in the hydrogen reactor. Two species of bacteria effective in hydrogenesis, Thermoanaerobacterium thermosaccharolyticum and Clostridium thermosaccharolyticum, dominated during hydrogen production, whereas archaea belonging to Euryarchaeota were detected and cultured in the methane reactor. The recycled effluent supplied alkaline substrates for the hydrogen producing bacteria. Alkali balance calculations showed that the amount of added alkali was reduced by 88%. This amount, required for hydrogen production from starch wastewater, was contributed by alkali in the methanogenic effluent, (2225 ± 140 mg CaCO₃/L), resulting in lower operational costs.     

Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy

The partial pressure of hydrogen is an extremely important factor for hydrogen generation. This study investigated the effect of reduced pressure (via vacuum) on hydrogen production in a CSTR reactor. The results show that the reduced pressure condition is more effective in enhancing H₂ production at lower HRT (e.g., 8–4 h) than at higher HRT (e.g., 12 h). The optimal hydrogen yield and overall hydrogen production efficiency occurred at a HRT of 6 h with a value of 4.50 mol H₂/mol sucrose and 56.2%, respectively. Meanwhile, at HRT 6 h the hydrogen production rate was 0.937 mol/L/d. In addition, the HPR could be further improved to 1.196 mol/L/d when the HRT was shortened to 4 h, obtaining a 37–271% increase in HPR when compared with that described in the relevant reports. For all experiments, butyrate and acetate were the two primary soluble metabolites, accounting for 85–99% of total soluble microbial products. Predominant production of acetate and butyrate demonstrates the efficient H₂ fermentation with reduced pressure processes.     

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.     

The kinetic characterization of photofermentative bacterium Rhodopseudomonas faecalis RLD-53 and its application for enhancing continuous hydrogen production

In this work, the kinetic characterization of hydrogen production by the photofermentative bacteria Rhodopseudomonas faecalis RLD-53 was investigated at different growth phase. During entire fermentation, 89.30% of total biomass was accumulated in exponential growth phase, while hydrogen yield was only 1.82 mol H₂/mol acetate at the expense of 51.25% substrate. In the stationary phase, biomass synthesis was minimal (7.51%), and 38.17% of the substrate was directly converted into hydrogen. As a result, hydrogen (59.19%) was mainly produced in stationary phase with highest hydrogen yield of 3.67 mol H₂/mol acetate. Consequently, bacteria in stationary phase were most effective for hydrogen production. Based on these findings, a novel membrane photobioreactor was developed to retain bacteria during stationary phase in reactor through membrane separation. Maximum rate (32.82 ml/l/h) and yield (3.27 mol H₂/mol acetate) of hydrogen production were achieved using membrane photobioreactor under the continuous operation. Therefore, using bacteria in stationary phase as hydrogen producer can offer considerable benefits for enhancing photo-hydrogen production.     

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.     

Psychrophilic hydrogen production from petrochemical wastewater via anaerobic sequencing batch reactor: techno-economic assessment and kinetic modelling

Independent hydrogen production from petrochemical wastewater containing mono-ethylene glycol (MEG) via anaerobic sequencing batch reactor (ASBR) was extensively assessed under psychrophilic conditions (15–25 °C). A lab-scale ASBR was operated at pH of 5.50, and different organic loading rates (OLR) of 1.00, 1.67, 2.67, and 4.00 gCOD/L/d. The hydrogen yield (HY) progressed from 134.32 ± 10.79 to 189.09 ± 22.35 mL/gMEG_initial at increasing OLR from 1.00 to 4.00 gCOD/L/d. The maximum hydrogen content of 47.44 ± 3.60% was achieved at OLR of 4.0 gCOD/L/d, while methane content remained low (17.76 ± 1.27% at OLR of 1.0 gCOD/L/d). Kinetic studies using four different mathematical models were conducted to describe the ASBR performance. Furthermore, two batch-mode experiments were performed to optimize the nitrogen supplementation as a nutrient (C/N ratio), and assess the impact of salinity (as gNaCl/L) on hydrogen production. HY substantially dropped from 62.77 ± 4.09 to 6.02 ± 0.39 mL/gMEG_initial when C/N ratio was increased from 28.5 to 114.0. Besides, the results revealed that salinity up to 10.0 gNaCl/L has a relatively low inhibitory impact on hydrogen production. Eventually, the cost/benefit analysis showed that environmental and energy recovery revenues from ASBR were optimized at OLR of 4.0 gCOD/L/d (payback period of 7.13 yrs).     

Methane free biohydrogen production from carbon monoxide using a continuously operated moving bed biofilm reactor

Biological water-gas shift (WGS) reaction is a green and sustainable alternative to thermochemical-catalytic WGS process for hydrogen production from carbon monoxide (CO). However, CO tolerant carboxydotrophic microbes for hydrogen production and scaling up the technology using a bioreactor system present challenges in successful application of this technology. This study demonstrated the capability of anaerobic microbial consortium for biohydrogen production from CO using a moving bed biofilm reactor (MBBR). The CO conversion pathway followed by the anaerobic biomass was first elucidated by inhibiting the methanogens present using 2-bromoethanesulfonate (BES) at an optimum concentration of 10 mmol/L. An increase in inlet CO concentration to the MBBR enhanced the H₂ production, but the CO conversion efficiency was low. More than 80% CO conversion efficiency was obtained only for a low inlet CO concentration. A maximum H₂ concentration of 19.5 mmol/L along with 2 mmol/L of acetate were obtained for 36 mmol/L of inlet CO concentration in the bioreactor. The carbon flux analysis showed that the CO was mainly utilized for methane free H₂ production, and only <10% of carbon flux was diverted towards acetate formation. Overall, this study demonstrated that MBBR system can be used for steady state biohydrogen production over a prolonged operation period.     

Influence of pretreatment and pH on the enhancement of hydrogen and volatile fatty acids production from food waste in the semi-continuously running reactor

In this study, FW effluent was used as a substrate for both hydrogen and volatile fatty acids (VFAs) production by a lab scale set up of the semi-continuously running reactor system with a mesophilic fermentation to examine the influence of pH and pretreatment. Repeated measurement analysis showed that the factors (pH and Pretreatment) significantly influenced H₂, VFAs concentration, VFA/soluble chemical oxygen demand (SCOD), and H₂/SCOD traits (P < 0.0001). Duncan comparisons showed that both concentration and yield of H₂ were the highest in the chemical treatment (CT) at pH 7, which were (280.82 ± 5.72) ml/L, and (4.44 ± 0.10) ml/g SCOD, respectively. While concentration and yield of the VFAs were the highest in the chemical treatment (CT) at pH 6, which were (55.44 ± 2.39) g/L, and (926.21 ± 42.27) mg/g SCOD, respectively. The butyrate and acetate for the optimal blend (pH 6, CT pretreatment) counted for 62.43% of the total VFAs.     

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.     

Hydrogen production using sono-biohydrogenator

Hydrogen production in a novel sonicated biological hydrogen reactor (SBHR) was investigated and compared with a continuous stirred tank reactor (CSTR). The two systems were operated at a hydraulic retention time (HRT) of 12 h and two organic loading rates (OLRs) of 21.4 and 32.1 g COD/L.d. The average hydrogen production rates per unit reactor volume for the conventional CSTR were 2.6 and 2.8 L/L.d, as compared with 4.8 and 5.6 L/L.d for SBHR, at the two OLRs, respectively. Hydrogen yields of 1.2 and 1.0 mol H₂/mol glucose were observed for the CSTR, respectively, while for the SBHR, the hydrogen yields were 2.1 and 1.9 mol H₂/mol glucose at the two OLRs, respectively. The hydrogen content in the SBHR’s headspace was higher than that in CSTR by 10% and 31% at OLRs of 21.4 and 32.1 g COD/L.d, respectively. Both glucose conversion efficiency and HAc/HBu ratio in the SBHR were higher than in the conventional CSTR at both OLRs. The biomass yield of about 0.32 g VSS/g COD observed in the CSTR and 0.23 g VSS/g COD in the SBHR substantiate the higher H₂ yield in the SBHR. DGGE analysis confirmed the specificity of the microbial hydrogen-producing culture in the SBHR, with two different hydrogen producers (Clostridium sp. and Citrobacter freundii) detected in the SBHR and not detected in the CSTR.     

Mesophilic biohydrogen production by Clostridium butyricum CWBI1009 in trickling biofilter reactor

This study investigates the mesophilic biohydrogen production from glucose using a strictly anaerobic strain, Clostridium butyricum CWBI1009, immobilized in a trickling bed sequenced batch reactor (TBSBR) packed with a Lantec HD Q-PAC^® packing material (132 ft²/ft³ specific surface). The reactor was operated for 62 days. The main parameters measured here were hydrogen composition, hydrogen production rate and soluble metabolic products. pH, temperature, recirculation flow rate and inlet glucose concentration at 10 g/L were the controlled parameters. The maximum specific hydrogen production rate and the hydrogen yield found from this study were 146 mmol H₂/L.d and 1.67 mol H₂/mol glucose. The maximum hydrogen composition was 83%. Following a thermal treatment, the culture was active without adding fresh inoculum in the subsequent feeding and both the hydrogen yield and the hydrogen production rate were improved. For all sequences, the soluble metabolites were dominated by the presence of butyric and acetic acids compared to other volatile fatty acids. The results from the standard biohydrogen production (BHP) test which was conducted using samples from TBSBR as inoculum confirmed that the culture generated more biogas and hydrogen compared to the pure strain of C. butyricum CWBI1009. The effect of biofilm activity was studied by completely removing (100%) the mixed liquid and by adding fresh medium with glucose. For three subsequent sequences, similar results were recorded as in the previous sequences with 40% removal of spent medium. The TBSBR biofilm density varied from top to bottom in the packing bed and the highest biofilm density was found at the bottom plates. Moreover, no clogging was evidenced in this packing material, which is characterized by a relatively high specific surface area. Following a PCA test, contaminants of the Bacillus genus were isolated and a standard BHP test was conducted, resulting in no hydrogen production.     

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.     

Hydrogen production by photo-fermentative bacteria immobilized on fluidized bio-carrier

In this work, a novel bio-carrier was used to immobilize photo-fermentative bacteria for hydrogen production. The results showed that the bacterial immobilization and hydrogen production were strongly affected by particle size, amount of bio-carrier and light intensity. Controlling the proper size of bio-carrier not only prevented light shading effect from each other, but also made solid carriers better fluidized during operation. The scanning electronic microscopy revealed that the biofilm formed by photo-fermentative bacteria on the surface of bio-carrier enhanced hydrogen production. Because of bio-carrier fluidization, each immobilized bacterium could receive light energy and produce hydrogen. With the optimal particle size (2 × 2 mm), amount (3% weight to volume ratio) and light intensity (6000 lux), the maximum hydrogen yield of 3.24 mol H₂/mol acetate and production rate of 36.06 ml/l/h were obtained in continuous operation stage. Bio-carrier was an effective solid carrier to immobilize photo-fermentative bacteria for improving hydrogen production.     

Effect of substrate concentration on fermentative hydrogen production from sweet sorghum extract

The aim of the present study was to assess the influence of substrate concentration on the fermentative hydrogen production from sweet sorghum extract, in a continuous stirred tank bioreactor. The reactor was operated at a Hydraulic Retention Time (HRT) of 12 h and carbohydrate concentrations ranging from 9.89 to 20.99 g/L, in glucose equivalents. The maximum hydrogen production rate and yield were obtained at the concentration of 17.50 g carbohydrates/L and were 2.93 ± 0.09 L H₂/L reactor/d and 0.74 ± 0.02 mol H₂/mol glucose consumed, corresponding to 8.81 ± 0.02 L H₂/kg sweet sorghum, respectively. The main metabolic product at all steady states was butyric acid, while ethanol production was high at high substrate concentrations. The experiments showed that hydrogen productivity depends significantly on the initial carbohydrate concentration, which also influences the distribution of the metabolic products.     

Fermentative hydrogen production with xylose by Clostridium and Klebsiella species in anaerobic batch reactors

Hydrogen production was obtained from low concentrations of xylose metabolized by heat treated inoculum obtained from the slaughterhouse wastewater treatment UASB reactor installed in Brazil. The molecular biological analysis Clostridium and Klebsiella species, recognized as H₂ and volatile acid producers, in addition to Burkholderia species and uncultivated bacteria. The assays were carried out in batch reactors: (1) 630.0 mg xylose/L, (2) 1341.0 mg xylose/L, (3) 1848.0 mg xylose/L and (4) 3588.0 mg xylose/L. The following yields were obtained: 3% (0.2 mol H₂/mol xylose), 8% (0.5 mol H₂/mol xylose), 10% (0.6 mol H₂/mol xylose) and 14% (0.8 mol H₂/mol xylose), respectively. The end products obtained were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors. The concentrations of xylose did not inhibit microbial growth and hydrogen production. This suggested that low concentrations of xylose should be added to wastewater to produce hydrogen.     

Bio-hydrogen production by a new isolated strain Rhodopseudomonas sp. WR-17 using main metabolites of three typical dark fermentation type

In this work, a new strain WR-17 was isolated for photo-fermentative hydrogen production and its hydrogen production capacity was investigated by utilizing main liquid byproducts of three dark fermentation types in batch culture. Experimental results indicated that strain WR-17 was identified as genus Rhodopseudomonas and maximum hydrogen yield of 2.42 mol H₂/mol acetate was obtained when the acetate was used as sole carbon source. Strain WR-17 had an excellent ability of using mixed short chain acids of three typical fermentations such as acetate and ethanol, acetate and butyrate, acetate and propionate. Result demonstrated that the metabolites of butyric acid-type fermentation as substrate is fitting to produce hydrogen and maximum cumulative hydrogen volume of 2156 ml/L-medium was obtained when acetate of 30 mmol/L and butyrate of 15 mmol/L were used. Therefore, butyric acid-type fermentation has great potential for further obtaining high hydrogen yield by the combining photo-fermentation.