The effect of heat pretreatment temperature on fermentative hydrogen production using mixed cultures

The effect of heat treatment at different temperatures on two types of inocula, activated sludge and anaerobically digested sludge, was investigated in batch cultures. Heat treatments were conducted at 65, 80 and 95 °C for 30 min. The untreated inocula produced less amount of hydrogen than the pretreated inocula, with lactic acid as the main metabolite. The maximum yields of 2.3 and 1.6 mol H₂/mol glucose were achieved for the 65 °C pretreated anaerobically digested and activated sludges, respectively. Approximately a 15% decrease in yield was observed with increasing pretreatment temperature from 65 to 95 °C concomitant with an increase in butyrate/acetate ratio from 1.5 to 2.4 for anaerobically digested sludge. The increase of pretreatment temperature of activated sludge to 95 °C suppressed the hydrogen production by lactic acid fermentation. DNA analysis of the microbial community showed that the elevated pretreatment temperatures reduced the species diversity.     

Evaluation of different pretreatment methods for preparing hydrogen-producing seed inocula from waste activated sludge

Waste activated sludge (WAS) is the most favorable inoculum for dark fermentative hydrogen-producing processes, because it can be collected economically. In order to accelerate the start-up process and develop the efficiency and stability of a hydrogen production system, pretreatment of the seed sludge has been examined to enrich hydrogen-producing bacteria. Six pretreatment methods including acid, base, heat-shock, aeration, chloroform and 2-bromoethanesulfonate (BES) were performed on WAS in batch cultures utilizing glucose as the substrate. The results showed that, at 35 °C and initial pH of 7.0, hydrogen yields of the pretreated sludge (except for BES) were higher than the control test. The pretreatment methods resulted in different distributions of soluble metabolites. Acid pretreatment at pH of 3 was the best among all six pretreatment methods, and the maximal hydrogen yield of 1.51 mol/mol-glucose-consumed and the maximal specific hydrogen production of 22.81 mmolH₂/g VSS were observed. The hydrogen yield of the acid treated sludge increased to 1.82 mol/mol-glucose-consumed after five repeated-batch cultivations. It was concluded that acid pretreatment is a simple, economic and effective method for enriching hydrogen-producing bacteria from WAS.     

Extreme-thermophilic biohydrogen production by an anaerobic heat treated digested sewage sludge culture

Biohydrogen production process from glucose using extreme-thermophilic H₂-producing bacteria enriched from digested sewage sludge was investigated for five cycles of repeated batch experiment at 70 °C. Heat shock pretreatment was used for preparation of hydrogen-producing bacteria comparing to an untreated anaerobic digested sludge for their hydrogen production performance and responsible microbial community structures. The results showed that the heat shock pretreatment completely repressed methanogenic activity and gave the maximum hydrogen production yield of 355-488 ml H₂/g COD in the second cycle of repeated batch cultivation with more stable gas production during the cultivation when compared with control. Hydrogen production was accompanied by production of acetic acid. The average specific hydrogen in five cycles experiment ranged from 150 to 200 ml H₂/g VSS. PCR-DGGE profiling showed that the extreme-thermophilic culture predominant species were closely affiliated to Thermoanaerobacter pseudethanolicus.     

Fermentative hydrogen production by diverse microflora

In this study, hydrogen production with activated sludge, a diverse bacterial source has been investigated and compared to microflora from anaerobic digester sludge, which is less diverse. Batch experiments were conducted at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The hydrogen production yields with activated sludge at 37 °C and 55 °C were 0.56 and 1.32 mol H₂/mol glucose consumed, respectively. While with anaerobically digested sludge hydrogen yield was 2.18 mol H₂/mol glucose consumed at 37 °C and 1.25 mol H₂/mol glucose consumed at 55 °C. The results of repeated batch experiments for 615 h resulted in average yields of 1.21 ± 0.62 and 1.40 ± 0.16 mol H₂/mol glucose consumed for activated sludge and anaerobic sludge, respectively. The hydrogen production with activated sludge was not stable during the repeated batches and the fluctuation in hydrogen production was attributed to formation of lactic acid as the predominant metabolite in some batches. The presence of lactic acid bacteria in microflora was confirmed by PCR-DGGE.     

Evaluation of pretreatment methods on harvesting hydrogen producing seeds from anaerobic digested organic fraction of municipal solid waste (OFMSW)

In order to harvest high-efficient hydrogen producing seeds, five pretreatment methods (including acid, heat, sonication, aeration and freeze/thawing) were performed on anaerobic digested sludge (AS) which was collected from a batch anaerobic reactor for treating organic fraction of municipal solid waste. The hydrogen production tests were conducted in serum bottles containing 20 gVS/L (24.8 g COD/L) mixture of rice and lettuce powder at 37 °C. The experimental results showed that the heat and acid pretreatment completely repressed the methanogenic activity of AS, but acid pretreatment also partially repressed hydrogen production. Sonication, freeze/thawing and aeration did not completely suppress the methanogen activity. The highest hydrogen yields were 119.7, 42.2, 26.0, 23.0, 22.7 and 22.1 mL/gVS for heated, acidified, freeze/thawed, aerated, sonicated and control AS respectively. A pH of about 4.9 was detected at the end of hydrogen producing fermentation for all tests. The selection of an initial pH can markedly affect the hydrogen producing ability for heated and acidified AS. The higher initial pH generated higher hydrogen yield and the highest hydrogen yield was obtained with initial pH 8.9 for heated AS.     

Effects of pretreatment methods on cassava wastewater for biohydrogen production optimization

Batch production of biohydrogen from cassava wastewater pretreated with (i) sonication, (ii) OPTIMASH BG^® (enzyme), and (iii) α-amylase (enzyme) were investigated using anaerobic seed sludge subjected to heat pretreatment at 105 °C for 90 min. Hydrogen yield at pH 7.0 for cassava wastewater pretreated with sonication for 45 min using anaerobic seed sludge was 0.913 mol H₂/g COD. Results from pretreatment with OPTIMASH BG^® at 0.20% and pH 7 showed a hydrogen yield of 4.24 mol H₂/g COD. Superior results were obtained when the wastewater was pretreated with α-amylase at 0.20% at pH 7 with a hydrogen yield of 5.02 mol H₂/g COD. In all cases, no methane production was observed when using heat-treated sludge as seed inoculum. Percentage COD removal was found to be highest (60%) using α-amylase as pretreatment followed by OPTIMASH BG^® at 54% and sonication (40% reduction rate). Results further suggested that cassava wastewater is one of the potential sources of renewable biomass to produce hydrogen.     

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 ± 0.07 mol H₂/mol glucose (mean ± S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 ± 0.03 mol H₂/mol glucose, 0.17 ± 0.01 mol H₂/mol glucose, 0.11 ± 0.01 mol H₂/mol glucose and 0.20 ± 0.04 mol H₂/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp.. However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community.     

Characteristics of fermentative hydrogen production with Klebsiella pneumoniae ECU-15 isolated from anaerobic sewage sludge

Klebsiella pneumoniae ECU-15 (EU360791), which was isolated from anaerobic sewage sludge, was investigated in this paper for its characteristics of fermentative hydrogen production. It was found that the anaerobic condition favored hydrogen production than that of the micro-aerobic condition. Culture temperature and pH of 37 °C and 6.0 were the most favorable for the hydrogen production. The strain could grow in several kinds of monosaccharide and disaccharide, as well as the complicated corn stalk hydrolysate, with the best results exhibited in glucose. The maximum hydrogen production rate and yield of 482 ml/l/h and 2.07 mol/mol glucose were obtained at initial glucose concentration of 30 g/L and 5 g/L, respectively. Fermentation results in the diluent corn stalk hydrolysate showed that cell growth was not inhibited. However, the hydrogen production of 0.65 V/V was relatively lower than that of the glucose (1.11 V/V), which was mainly due to the interaction between xylose and glucose.     

Effect of nickel loading on hydrogen production and chemical oxygen demand (COD) destruction from glucose oxidation and gasification in supercritical water

Gasification and partial oxidation of 0.25 molar glucose solution was conducted over different metallic nickel (Ni) loadings (7.5, 11, and 18 wt%) on different catalyst supports (θ-Al₂O₃ and γ-Al₂O₃) in supercritical water. Experiments were carried out at three different temperatures (T) of 400, 450, and 500 °C at constant pressure of 28 MPa and a 30 min reaction time (t). For comparison, some experiments were conducted using high loading commercial catalyst (65 wt% Ni on Silica–alumina). Hydrogen peroxide (H₂O₂) was used as a source of oxygen in the partial oxidation experiments. Oxygen to carbon molar ratios (MR) of 0.5–0.9 were examined to increase the hydrogen production via carbon monoxide (CO) production. Results showed that in the absence of the catalyst, the optimum molar ratio was 0.8 i.e. 80% of the amount of oxygen required for complete oxidation of glucose. At a molar ratio of 0.8, the hydrogen yield was 0.3 mol/mol, as compared to 0.2 mol/mol glucose at molar ratio of 0.5 and 0.9. This optimized oxygen dose was adopted as a base line for catalysts evaluation. The main gaseous products were carbon dioxide (CO₂), carbon monoxide (CO), hydrogen (H₂), and methane (CH₄). Results also showed that the presence of Ni increased the total gas yield increased in the 7.5–18 wt Ni/Al₂O₃ catalyst. An increase in MR from 0.55 to 0.8 increased the of carbon dioxide and hydrogen yields from 1.8 to 3.8 mol/mol glucose and from 0.9 to 1.1 mol/mol. The carbon monoxide and methane yields remain constant at 2 and 0.5 mol/mol glucose, respectively. The introduction of hydrogen peroxide (H₂O₂) prior to the feed injection inhibited the catalyst activity and did not increase the hydrogen yield whereas the introduction of H₂O₂ after 15 min of reaction time increased the hydrogen yield from 0.62 mol/mol to 1.5 mol/mol. This study showed that approximately the same hydrogen yield can be obtained from the synthesized low nickel alumina loading (18 wt%) catalyst as with the 65 wt% nickel on silica–alumina loading commercial catalyst. The highest H₂ yield of 1.5 mol/mol glucose was obtained with commercial Ni/silica–alumina with a BET surface area of 190 m²/g compared to 1.2 mol/mol with the synthesized Ni/θ alumina with a BET surface area of 46 m²/g.     

A pilot-scale high-rate biohydrogen production system with mixed microflora

A pilot-scale high-rate dark fermentative hydrogen production plant has been established in the campus of Feng Chia University to develop biohydrogen production pilot-plant technology. This pilot-plant system is composed of two feedstock storage tanks (0.75 m³ each), a nutrient storage tank (0.75 m³), a mixing tank (0.6 m³), an agitated granular sludge bed fermentor (working volume 0.4 m³), a gas-liquid-solid separator (0.4 m³) and a control panel. The seed mixed microflora was obtained from a lab-scale agitated granular sludge bed bioreactor. This pilot-scale fermentor was operated for 67 days at 35 °C, an organic loading rate (OLR) of 40-240 kg COD/m³/d, and the influent sucrose concentration of 20 and 40 kg COD/m³. Both biogas and hydrogen production rates increased with increasing OLR. However, the biomass concentration (volatile suspended solids, VSS) only increased with an increasing OLR at an OLR range of 40-120 kg COD/m³/d, whereas it decreased when OLR was too high (i.e., 240 kg COD/m³/d). The biogas consisted mainly of H₂ and CO₂ with a H₂ content range of 23.2-37.8%. At an OLR of 240 kg COD/m³/d, the hydrogen content in biogas reached its maximum value of 37% with a hydrogen production rate (HPR) of 15.59 m³/m³/d and a hydrogen yield of 1.04 mol H₂/mol sucrose. This HPR value is much higher than 5.26 m³/m³/d (fermented molasses substrate) and 1.56 m³/m³/d (glucose substrate) reported by other pilot-scale systems. Moreover, HPR was also greatly affected by pH. At an optimal pH of 5.5, the bacterial community became simple, while the efficient hydrogen producer Clostridium pasteurianum was dominant. The factors of energy output compared with the energy input (E_f) ranged from 13.65 to 28.68 on biohydrogen, which is higher than the E_f value on corn ethanol, biodiesel and sugarcane ethanol but in the similar range of cellulosic ethanol.     

Continuous biohydrogen production from liquid swine manure supplemented with glucose using an anaerobic sequencing batch reactor

Liquid swine manure supplemented with glucose (10 g/L) was used as substrate for hydrogen production using an anaerobic sequencing batch reactor at 37 ± 1 °C and pH 5.0 under different hydraulic retention times (HRTs). Decreasing HRT from 24 to 8 h caused an increasing hydrogen production rate from 0.05 to 0.15 L/h/L. Production rates of both total biogas and hydrogen were linearly correlated to HRT with R² being 0.993 and 0.997, respectively. The hydrogen yield ranged between 1.18 and 1.63 mol-H₂/mol glucose and the 12 h HRT was preferred for high production rate and efficient yield. For all the five HRTs examined, the glucose utilization efficiency was over 98%. The biogas mainly consisted of carbon dioxide and hydrogen (up to 43%) with no methane detected throughout the experiment. Ethanol and organic acids were the major aqueous metabolites produced during fermentation, with acetic acid accounting for 56–58%. The hydrogen yield was found to be related to the acetate/butyrate ratio.     

Enriching hydrogen-producing bacteria from digested sludge by different pretreatment methods

The pretreatment of digested sludge by different methods, including ionizing irradiation, heat-shock, acid and base, was performed for enriching hydrogen-producing bacteria. These methods were evaluated and compared based on their suitability in the enrichment of hydrogen-producing bacteria in dark fermentation with glucose as a substrate in batch tests. The experimental results showed that the seed sludge pretreated by ionizing irradiation achieved the best hydrogen production among the different pretreatment methods, and the maximum hydrogen production potential, maximum hydrogen production rate, hydrogen yield and substrate degradation rate were 525.6 mL, 37.2 mL/h, 267.7 mL/g glucose (2.15 mol/mol glucose) and 98.9%, respectively. Ionizing irradiation can be a good optional pretreatment method for enriching hydrogen-producing bacteria from digested sludge. The effect of ionizing irradiation on the microbial community structure dynamics of the pretreated sludge deserves further study, which will help us to understand the mechanisms leading to the effect of high bio-hydrogen production.     

Effect of organic loading on a novel hydrogen bioreactor

This study investigated the impact of six organic loading rates (OLR) ranging from 6.5 gCOD/L-d to 206 gCOD/L-d on the performance of a novel integrated biohydrogen reactor clarifier systems (IBRCSs) comprised a continuously stirred reactor (CSTR) for biological hydrogen production, followed by an uncovered gravity settler for decoupling of solids retention time (SRT) from hydraulic retention time (HRT). The system was able to maintain a high molar hydrogen yield of 2.8 mol H₂/mol glucose at OLR ranging from 6.5 to 103 gCOD/L-d, but dropped precipitously to approximately 1.2 and 1.1 mol H₂/mol glucose for the OLRs of 154 and 206 gCOD/L-d, respectively. The optimum OLR at HRT of 8 h for maximizing both hydrogen molar yield and volumetric hydrogen production was 103 gCOD/L-d. A positive statistical correlation was observed between the molar hydrogen production and the molar acetate-to-butyrate ratio. Biomass yield correlated negatively with hydrogen yield, although not linearly. Analyzing the food-to-microorganisms (F/M) data in this study and others revealed that, both molar hydrogen yields and biomass specific hydrogen rates peaked at 2.8 mol H₂/mol glucose and 2.3 L/gVSS-d at F/M ratios ranging from 4.4 to 6.4 gCOD/gVSS-d. Microbial community analysis for OLRs of 6.5 and 25.7 gCOD/L-d showed the predominance of hydrogen producers such as Clostridium acetobutyricum, Klebsiella pneumonia, Clostridium butyricum, Clostridium pasteurianum. While at extremely high OLRs of 154 and 206 gCOD/L-d, a microbial shift was clearly evident due to the coexistence of the non-hydrogen producers such as Lactococcus sp. and Pseudomonas sp.     

An integrated system for hydrogen and methane production during landfill leachate treatment

The patent-pending integrated waste-to-energy system comprises both a novel biohydrogen reactor with a gravity settler (Biohydrogenator), followed by a second stage conventional anaerobic digester for the production of methane gas. This chemical-free process has been tested with a synthetic wastewater/leachate solution, and was operated at 37 °C for 45 d. The biohydrogenator (system (A), stage 1) steadily produced hydrogen with no methane during the experimental period. The maximum hydrogen yield was 400 mL H₂/g glucose with an average of 345 mL H₂/g glucose, as compared to 141 and 118 mL H₂/g glucose for two consecutive runs done in parallel using a conventional continuously stirred tank reactor (CSTR, System (B)). Decoupling of the solids retention time (SRT) from the hydraulic retention time (HRT) using the gravity settler showed a marked improvement in performance, with the maximum and average hydrogen production rates in system (A) of 22 and 19 L H₂/d, as compared with 2–7 L H₂/d in the CSTR resulting in a maximum yield of 2.8 mol H₂/mol glucose much higher than the 1.1–1.3 mol H₂/mol glucose observed in the CSTR. Furthermore, while the CSTR collapsed in 10–15 d due to biomass washout, the biohydrogenator continued stable operation for the 45 d reported here and beyond. The methane yield for the second stage in system (A) approached a maximum value of 426 mL CH₄/gCOD removed, while an overall chemical oxygen demand (COD) removal efficiency of 94% was achieved in system (A).     

Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H₂/mol sugar and specific hydrogen production rate of 225 mmol of H₂/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.     

Gamma irradiation as a pretreatment method for enriching hydrogen-producing bacteria from digested sludge

Gamma irradiation was used as a pretreatment method for enriching hydrogen-producing bacteria from digested sludge. The experimental results demonstrated that 5.0 kGy was optimal dose among the different doses (0.5–10 kGy) applied in this study. The maximum cumulative hydrogen production, hydrogen yield, hydrogen production rate and substrate degradation efficiency of the sludge irradiated at such dose were 529.4 mL, 267.7 mL/g glucose, 37.25 mL/h and 98.9%, respectively when the fermentation conditions were as follows: at 36 °C, initial pH 7.0 and 10 g/L glucose as substrate. In comparison with the conventional pretreatment methods, such as heat-shock, acid, base, aeration and chloroform, gamma irradiation was more powerful pretreatment method for enriching hydrogen-producing bacteria. The effect of Gamma irradiation on the microbial community structure of the pretreated sludge needs further study.     

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.     

Fermentative bioenergy production from distillers grains using mixed microflora

The feasibility of hydrogen production from distillers grains substrate, an industrial cellulosic waste, was investigated. A substrate concentration of 80 g/L gave the maximum production at 50 °C and pH of 6.0 using sewage sludge. Four controllable factors with three levels: seed sludge (two sewage sludges and cow dung), temperature (40, 50, and 60 °C), pH (6, 7 and 8) and seed pretreatment (none, heat, and acid) were selected in Taguchi experimental design to optimize fermentation conditions. The peak hydrogen and ethanol productions were found with heat-treated cow dung seed, substrate concentration 80 g/L, 50 °C and pH 6. The peak hydrogen production rate and hydrogen yield were 7.9 mmol H₂/L/d and 0.40 mmol H₂/g-COD respectively whereas the peak ethanol production was 3050 mg COD/L and rate 0.22 g EtOH/L/d. A total bioenergy yield of 41 J/g substrate was obtained which was 21% and 79% from hydrogen and ethanol respectively.     

Feasibility of biohydrogen production from Gelidium amansii

The feasibility of hydrogen production from red algae was investigated. Galactose, the main sugar monomer of red algae, was readily converted to hydrogen by dark fermentation. The maximum hydrogen production rate and yield of galactose were 2.46 L H₂/g VSS/d and 2.03 mol H₂/mol galactose_added, respectively, which were higher than those for glucose (0.914 L H₂/g VSS/d and 1.48 mol H₂/mol galactose_added). The distribution of soluble byproducts showed that H₂ production was the main pathway of galactose uptake. 5-HMF, the main byproduct of acid hydrolysis of red algae causes noncompetitive inhibition of H₂ fermentation. 1.37 g/L of 5-HMF decreased hydrogen production rate by 50% compared to the control. When red algae was hydrolyzed at 150 °C for 15 min and detoxified by activated carbon, 53.5 mL of H₂ was produced from 1 g of dry algae with a hydrogen production rate of 0.518 L H₂/g VSS/d. Red algae, cultivable on vast tracts of sea by sunlight without any nitrogen-based fertilizer, could be a suitable substrate for biohydrogen production.     

Optimization of fermentative hydrogen production process by response surface methodology

The effect of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures was investigated in batch tests, and the optimization of fermentative hydrogen production process was conducted by response surface methodology with a central composite design. Experimental results showed that temperatures, initial pH and glucose concentrations had impact on fermentative hydrogen production individually and interactively. The maximum hydrogen yield of 289.8 mL/g glucose was estimated at the temperature of 38.6 °C, the initial pH of 7.2 and the glucose concentration of 23.9 g/L. The maximum hydrogen production rate of 28.2 mL/h was estimated at the temperature of 37.8 °C, the initial pH of 7.2 and the glucose concentration of 27.6 g/L. The maximum substrate degradation efficiency of 96.9% was estimated at the temperature of 39.3 °C, the initial pH of 7.0 and the glucose concentration of 26.8 g/L. Response surface methodology was a better method to optimize the fermentative hydrogen production process. Modified logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully.