Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

Fermentative hydrogen production from hydrolyzed cellulosic feedstock prepared with a thermophilic anaerobic bacterial isolate

Hydrogen gas was produced via dark fermentation from natural cellulosic materials and α-cellulose via a two-step process, in which the cellulosic substrates were first hydrolyzed by an isolated cellulolytic bacterium Clostridium strain TCW1, and the resulting hydrolysates were then used as substrate for fermentative H₂ production. The TCW1 strain was able to hydrolyze all the cellulosic materials examined to produce reducing sugars (RS), attaining the best reducing sugar production yield of 0.65 g reducing sugar/g substrate from hydrolysis of α-cellulose. The hydrolysates of those cellulosic materials were successfully converted to H₂ via dark fermentation using seven H₂-producing bacterial isolates. The bioH₂ production performance was highly dependent on the type of cellulosic feedstock used, the initial reducing sugar concentration (C_RS,o) (ranging from 0.7 to 4.5 mg/l), as well as the composition of sugar and soluble metabolites present in the cellulosic hydrolysates. It was found that Clostridium butyricum CGS5 displayed the highest H₂-producing efficiency with a cumulative H₂ production of 270 ml/l from α-cellulose hydrolysate (C_RS,o = 4.52 mg/l) and a H₂ yield of 7.40 mmol/g RS (or 6.66 mmol/g substrate) from napier grass hydrolysate (C_RS,o = 1.22 g/l).     

Advances in biohydrogen production processes: An approach towards commercialization

Biological H₂ production has an edge over its chemical counterpart mainly because it is environmentally benign. Despite having simpler technology, higher evolution rate of H₂ and the wide spectrum of substrate utilization, the major deterrent of anaerobic dark fermentation process stems from its lower achievable yields. Theoretically, the maximum H₂ yield is 4 mol H₂/mol glucose when glucose is completely metabolized to acetate or acetone in the anaerobic process. But it is somewhat difficult to achieve the complete degradation of glucose to carbon dioxide and H₂ through anaerobic dark fermentation. Moreover, this yield appears too low to be economically viable as an alternative to the existing chemical or electrochemical processes of hydrogen generation. Intensive research studies have already been carried out on the advancement of these processes, such as the development of genetically modified microorganism, improvement of the reactor designs, use of different solid matrices for the immobilization of whole cells, development of two-stage processes, and higher H₂ production rates. Maximum H₂ yield is found to be 5.1 mol H₂/mol glucose. However, major bottlenecks for the commercialization of these processes are lower H₂ yield and rate of H₂ production. Competent microbial cultures are required to handle waste materials efficiently, which are usually complex in nature. This will serve dual purposes: clean energy generation and bioremediation. Scale-up studies on fermentative H₂ production processes have been done successfully. Pilot plant trials of the photo-fermentation processes require more attention. Use of cheaper raw materials and efficient biological H₂ production processes will surely make them more competitive with the conventional H₂ generation processes in near future.     

Biohydrogen production by Ethanoligenens harbinense B49: Nutrient optimization

A new hydrogen-producing bacterial strain Ethanoligenens harbinense B49 was examined for its capability of H₂ production with glucose as sole carbon source. The H₂ production was significantly affected by the concentration of the yeast powder and phosphate in the synthetic medium. The optimized concentration of yeast powder was 0.3–0.5 g/L and the maximum hydrogen yield was obtained at the concentration of phosphate about 100–150 mmol/L. The dynamics of hydrogen production showed that rapid evolution of hydrogen appeared to start after the middle-phase of exponential growth (about 8 h). The maximum H₂ yield and specific hydrogen production rate were estimated to be 2.26 mol H₂/mol glucose and 27.74 mmol H₂/g cell, respectively, when 10 g/L of glucose was present in the medium. The possible pathway of hydrogen production by Ethanoligenens sp. B49 during glucose fermentation was oxidative decarboxylation of pyruvate and the NADH pathway.     

The effect of dilution and l-malic acid addition on bio-hydrogen production with Rhodopseudomonas palustris from effluent of an acidogenic anaerobic reactor

In this study, H₂ was produced from cheese whey wastewater in a two-stage biological process: i) first stage; thermophilic dark fermentation ii) second stage; the photo fermentation using Rhodopseudomonas palustris strain DSM 127 (R. palustris). The effect of both dilution and addition of l-malic acid on the hydrogen production was investigated. Among the dilution rates used, 1/5 dilution ratio was found to produce the best hydrogen production (349 ml H₂/g COD_fed). On the other hand, It was seen that the mixing the effluent with l-malic acid at increasing ratios had further positive effect and improved the hydrogen production significantly. It was concluded that dilution of the feeding helps to reduce the nitrogen content and the volatile fatty acid content that might be otherwise harmful to the photo-heterotrophic organisms. Overall hydrogen production yield (for dark + photo fermentation) was found to vary 2 and 10 mol H₂/mol lactose. Second conclusion is that cheese whey effluent should be mixed with a co-substrate containing l-malic acid such as apple juice processing effluents before fed into the photo fermentation reactor.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.     

Effective hydrogen production using waste sludge and its filtrate

Waste activated sludge from a wastewater treatment plant is rich in polysaccharides and proteins and thus is a potential substrate for producing hydrogen. In this study, the hydrogen yield could be largely enhanced by using filtrates of waste sludge. The hydrogen yield was effectively increased from 1.34 mg H₂/gTCOD (waste sludge) to 4.44 mg H₂/gTCOD (filtrate). The changes of nutrients such as SCOD, protein and carbohydrate in sludge and its filtrate during fermentation have obviously diversity. It implied that the nutrients could be further released from the solid phase of the sludge during fermentation. In addition, the fermentation of the sludge was advantageous for releasing nutrients, but the H₂ production might be lower at high substrate concentrations as a result of the inhibition products formed during hydrogen production. Therefore, the solid phase of waste sludge could not be utilized by the anaerobes as nutrient and it might absorb certain products, release toxic metals or deliver toxic substances during fermentation. The changes of pH indicated that conditions were favorable for hydrogen production from the filtrate. The 16S rRNA gene sequence, phylogenetic and biochemical character analyses demonstrated that strain GZ1 was a new strain of Pseudomonas and suitable for hydrogen production.     

Anaerobic fluidized bed reactor with expanded clay as support for hydrogen production through dark fermentation of glucose

This study evaluated hydrogen production in an anaerobic fluidized bed reactor (AFBR) fed with glucose-based synthetic wastewater. Particles of expanded clay (2.8–3.35 mm) were used as a support material for biomass immobilization. The reactor was operated with hydraulic retention times (HRT) ranging from 8 to 1 h. The hydrogen yield production increased from 1.41 to 2.49 mol H₂ mol⁻¹ glucose as HRT decreased from 8 to 2 h. However, when HRT was 1 h, there was a slight decrease to 2.41 mol H₂ mol⁻¹ glucose. The biogas produced was composed of H₂ and CO₂, and the H₂ content increased from 8% to 35% as HRT decreased. The major soluble metabolites during H₂ fermentation were acetic acid (HAc) and butyric acid (HBu), accounting for 36.1–53.3% and 37.7–44.9% of total soluble metabolites, respectively. Overall, the results demonstrate the potential of using expanded clay as support material for hydrogen production in AFBRs.     

Microbial diversity of hydrogen-producing bacteria in batch reactors fed with cellulose using leachate as inoculum

Hydrogen production using cellulosic residues offers the possibility of waste minimization with renewable energy recovery. In the present study, heat-treated biomass purified from leachate was used as inoculum in batch reactors for hydrogen production fed with different concentrations of cellulose (2.5, 5.0 and 10 g/L), in the presence and absence of exogenous cellulase. The heat-treated biomass did not degrade cellulose and hydrogen production was not detected in the absence of cellulase. In reactors with cellulase, the hydrogen yields were 1.2, 0.6 and 2.3 mol H₂/mol of hydrolyzed cellulose with substrate degradation of 41.4, 28.4 and 44.7% for 2.5, 5.0 and 10 g/L cellulose, respectively. Hydrogen production potentials (P) varied from 19.9 to 125.9 mmol H₂ and maximum hydrogen production rates (R_m) were among 0.8–2.3 mmol H₂/h. The reactor containing 10 g/L of cellulose presented the highest P and R_m among the conditions tested. The main acid produced in reactors were butyric acid, followed by acetic, isobutyric and propionic acids. Bacteria similar to Clostridium sp. (98–99%) were identified in the reactors with cellulase. The heat-treated leachate can be used as an inoculum source for hydrogen production from hydrolyzed cellulose.     

Hydrogen production and end-product synthesis patterns by Clostridium termitidis strain CT1112 in batch fermentation cultures with cellobiose or α-cellulose

Hydrogen (H₂) production and end-product synthesis were characterized in a novel, mesophilic, cellulolytic, anaerobic bacterium, Clostridium termitidis strain CT1112, isolated from the gut of the termite, Nasutitermes lujae. Growth curves, pH patterns, protein content, organic acid synthesis, and H₂ production were determined. When grown on 2 g l⁻¹ cellobiose and 2 g l⁻¹ α-cellulose, C. termitidis displayed a cell generation time of 6.5 h and 18.9 h, respectively. The major end-products synthesized on cellobiose included acetate, hydrogen, CO₂, lactate, formate and ethanol, where as on cellulose, the major end-products included hydrogen, acetate, CO₂ and ethanol. The concentrations of acetate were greater than ethanol, formate and lactate on both cellobiose and α-cellulose throughout the entire growth phase. Maximum yields of acetate, ethanol, hydrogen and formate on cellobiose were 5.9, 3.7, 4.6 and 4.2 mmol l⁻¹ culture, respectively, where as on cellulose, the yields were 7.2, 3.1, 7.7 and 2.9 mmol l⁻¹ culture, respectively. Hydrogen and ethanol production rates were slightly higher in C. termitidis cultured on cellobiose when compared to α-cellulose. Although, the generation time on α-cellulose was longer than on cellobiose, H₂ production was favored corresponding to acetate synthesis, thereby restricting the carbon flowing to ethanol. During log phase, H₂, CO₂ and ethanol were produced at specific rates of 4.28, 5.32, and 2.99 mmol h⁻¹ g dry weight⁻¹ of cells on cellobiose and 2.79, 2.59, and 1.1 mmol h⁻¹ g dry weight⁻¹ of cells on α-cellulose, respectively.     

A bench scale study of fermentative hydrogen and methane production from food waste in integrated two-stage process

A two-stage fermentation process combining hydrogen and methane production for the treatment of food waste was investigated in this paper. In hydrogen fermentation reactor, the indigenous mixed microbial cultures contained in food waste were used for hydrogen production. No foreign inoculum was used in the hydrogen fermentation stage, the traditional heat treatment of inoculum was not applied either in this bench scale experiment. The effects of the stepwise increased organic loading rate (OLR) and solid retention time (SRT) on integrated two-stage process were investigated. At steady state, the optimal OLR and SRT for the integrated two-stage process were found to be 22.65 kg VS/m³ d (160 h) for hydrogen fermentation reactor and 4.61 (26.67 d) for methane fermentation reactor, respectively. Under the optimum conditions, the maximum yields of hydrogen (0.065 m³ H₂/kg VS) and methane (0.546 m³ CH₄/kg VS) were achieved with the hydrogen and methane contents ranging from 29.42 to 30.86%, 64.33 to 71.48%, respectively. Biodegradability analysis showed that 5.78% of the influent COD was converted to the hydrogen in H₂-SCRD and 82.18% of the influent COD was converted to the methane in CH₄-SCSTR under the optimum conditions.     

Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent

A hydrogen producer was successfully isolated from anaerobic digested palm oil mill effluent (POME) sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced hydrogen concurrently with cell growth. A controlled study was done on a synthetic medium at an initial pH value of 6.0 with 10 g/L glucose with the maximum hydrogen production at 948 mL H₂/L-medium and the volumetric hydrogen production rate at 172 mL H₂/L-medium/h. The supplementation of yeast extract was shown to have a significant effect with a maximum hydrogen production of 992 mL H₂/L-medium at 4 g/L of yeast extract added. The effect of pH on hydrogen production from POME was investigated. Experimental results showed that the optimum hydrogen production ability occurred at pH 5.5. The maximum hydrogen production and maximum volumetric hydrogen production rate were at 3195 mL H₂/L-medium and 1034 mL H₂/L-medium/h, respectively. The hydrogen content in the biogas produced was in the range of 60–70%.     

Sequencing batch reactor enhances bacterial hydrolysis of starch promoting continuous bio-hydrogen production from starch feedstock

Bio-hydrogen production from starch was carried out using a two-stage process combining thermophillic starch hydrolysis and dark H₂ fermentation. In the first stage, starch was hydrolyzed by Caldimonas taiwanensis On1 using sequencing batch reactor (SBR). In the second stage, Clostridium butyricum CGS2 was used to produce H₂ from hydrolyzed starch via continuous dark hydrogen fermentation. Starch hydrolysis with C. taiwanensis On1 was operated in SBR under pH 7.0 and 55 °C. With a 90% discharge volume, the reducing sugar (RS) production from SBR reactor reached 13.94 g RS/L, while the reducing sugar production rate and starch hydrolysis rate was 0.92 g RS/h/L and 1.86 g starch/h/L, respectively, which are higher than using other discharge volumes. For continuous H₂ production with the starch hydrolysate, the highest H₂ production rate and yield was 0.52 L/h/L and 13.2 mmol H₂/g total sugar, respectively, under a hydraulic retention time (HRT) of 12 h. The best feeding nitrogen source (NH₄HCO₃) concentration was 2.62 g/L, attaining a good H₂ production efficiency along with a low residual ammonia concentration (0.14 g/L), which would be favorable to follow-up photo H₂ fermentation while using dark fermentation effluents as the substrate.     

Fermentative biohydrogen production by mixed anaerobic consortia: Impact of glucose to xylose ratio

Glucose and xylose are the dominant monomeric carbohydrates present in agricultural materials which can be used as potential building blocks for various biotechnological products including biofuels production. Hence, the imperative role of glucose to xylose ratio on fermentative biohydrogen production by mixed anaerobic consortia was investigated. Microbial catabolic H₂ and VFA production studies revealed that xylose is a preferred carbon source compared to glucose when used individually. A maximum of 1550 and 1650 ml of cumulative H₂ production was observed with supplementation of glucose and xylose at a concentration of 5.5 and 5.0 g L⁻¹, respectively. A triphasic pattern of H₂ production was observed only with studied xylose concentration range. pH impact data revealed effective H₂ production at pH 6.0 and 6.5 with xylose and glucose as carbon sources, respectively. Co-substrate related biohydrogen fermentation studies indicated that glucose to xylose ratio influence H₂ and as well as VFA production. An optimum cumulative H₂ production of 1900 ml for 5 g L⁻¹ substrate was noticed with fermentation medium supplemented with glucose to xylose ratio of 2:3 at pH 6. Overall, biohydrogen producing microbial consortia developed from buffalo dung could be more effective for H₂ production from lignocellulosic hydrolysates however; maintenance of glucose to xylose ratio, inoculum concentration and medium pH would be essential requirements.     

Hydrogen production by combined dark and light fermentation of ground wheat solution

Ground waste wheat was subjected to combined dark and light batch fermentation for hydrogen production. The dark to light biomass ratio (D/L) was changed between 1/2 and 1/10 in order to determine the optimum D/L ratio yielding the highest hydrogen formation rate and the yield. Hydrogen production by only dark and light fermentation bacteria was also realized along with the combined fermentations. The highest cumulative hydrogen formation (CHF = 76 ml), hydrogen yield (176 ml H₂ g⁻¹ starch) and formation rate (12.2 ml H₂ g⁻¹ biomass h⁻¹) were obtained with the D/L ratio of 1/7 while the lowest CHF was obtained with the D/L ratio of 1/2. Dark–light combined fermentation with D/L ratio of 1/7 was faster as compared to the dark and light fermentations alone yielding high hydrogen productivity and reduced fermentation time. Dark and light fermentations alone also yielded considerable cumulative hydrogen, but slower than the combined fermentation.     

Biological hydrogen production from corn stover by moderately thermophile Thermoanaerobacterium thermosaccharolyticum W16

This study addressed the utilization of an agro-waste, corn stover, as a renewable lignocellulosic feedstock for the fermentative H₂ production by the moderate thermophile Thermoanaerobacterium thermosaccharolyticum W16. The corn stover was first hydrolyzed by cellulase with supplementation of xylanase after delignification with 2% NaOH. It produced reducing sugar at a yield of 11.2 g L⁻¹ glucose, 3.4 g L⁻¹ xylose and 0.5 g L⁻¹ arabinose under the optimum condition of cellulase dosage 25 U g⁻¹ substrate with supplement xylanase 30 U g⁻¹ substrate. The hydrolyzed corn stover was sequentially introduced to fermentation by strain W16, where, the cell density and the maximum H₂ production rate was comparable to that on simulated medium, which has the same concentration of reducing sugars with hydrolysate. The present results suggest a promising combined hydrogen production process from corn stover with enzymatic hydrolysis stage and fermentation stage using W16.     

Different ratios of carbon sources in the fermentation of cheese whey and glucose as substrates for hydrogen and ethanol production in continuous reactors

The fermentation of glucose, cheese whey and the mixture of glucose and cheese whey were evaluated in this study from two inocula sources (sludge from a UASB reactor for swine wastewater treatment and poultry slaughterhouse) for hydrogen production in continuous anaerobic fluidized bed reactors (AFBR). For all fermentations, a hydraulic retention time (HRT) of 6 h and a substrate concentration of 5 g COD L⁻¹ were used. In glucose fermentation, the maximum hydrogen yield (HY) was 1.37 mmol H₂ g⁻¹ COD. The co-fermentation of the cheese whey and glucose mixture was favorable for the concomitant production of hydrogen and ethanol, with yields of up to 1.7 mmol H₂ g⁻¹ COD and 3.45 mol EtOH g⁻¹ COD in AFBR2. The utilization of cheese whey as a sole substrate resulted in an HY of 1.9 mmol H₂ g⁻¹ COD. Throughout the study, ethanol fermentation was evident.     

Control strategies for hydrogen production through co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53

This study evaluated hydrogen production by co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53 with different control strategies. To enhance cooperation of dark and photo-fermentation bacteria during hydrogen production process, the glucose concentration, phosphate buffer concentration and initial pH were controlled at 6 g/l, 50 mmol/l and 7.5, respectively. The maximum yield and rate of hydrogen production were 3.10 mol H₂/mol glucose and 17.2 mmol H₂/l/h, respectively. Ethanol from E. harbinense B49 in acetate medium can enhance hydrogen production by R. faecalis RLD-53 except the ratio of ethanol to acetate (R_E/A) among 0.8 to 1.0. Control of the proper phosphate buffer concentration (50 mmol/l) not only increased acetic acid production by E. harbinense B49, but also maintained stable pH of co-culture system. Therefore, the results showed that co-culture of E. harbinense B49 and immobilized R. faecalis RLD-53 was a promising way of converting glucose into hydrogen.     

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.     

The effect of pH on the production of biohydrogen by clostridia: Thermodynamic and metabolic considerations

This study evaluates the effect of pH (4-7) on fermentative biohydrogen production by utilizing three isolated Clostridium species. Fermentative batch experiments show that the maximum hydrogen yield for Clostridium butyricum CGS2 (1.77 mmol/mmol glucose) is achieved at pH 6, whereas a high hydrogen production with Clostridium beijerinckii L9 (1.72 mmol/mmol glucose) and Clostridium tyrobutyricum FYa102 (1.83 mmol/mmol glucose) could be achieved under uncontrolled pH conditions (initial pH of 6.4-6.6 and final pH of 4-4.2). Low hydrogen yields (0-0.6 mmol/mmol glucose) observed at pH 4 are due likely to inhibitory effects on the microbial growth, although a low pH can be thermodynamically favorable for hydrogen production. The low hydrogen yields (0.12-0.64 mmol/mmol glucose) observed at pH 7 are attributed not only to thermodynamically unfavorable, but also metabolically unfavorable for hydrogen production. The relatively high levels of lactate, propionate, or formate observed at pH 7 reflect presumably the high enzymatic activities responsible for their production, together with the low hydrogenase activity, resulting in a low hydrogen production. A correlation analysis of the data from present and previous studies on biohydrogen production with pure Clostridium cultures and mixed microflora indicates a close relation between the hydrogen yield (YH₂) and the (YH₂)/(2(YHAc+YHBu)) ratio, with the observed correlation coefficient (0.787) higher than that (0.175) between YH₂ and the molar ratio of butyrate to acetate (B/A). Based on the (YH₂)/(2(YHAc+YHBu)) ratios observed at different pHs, a control of pH at 5.5-6.8 would seem to be an effective means to enhance the fermentative biohydrogen production.