Influence of pH on fermentative hydrogen production from sweet sorghum extract

The present study focused on the influence of pH on the fermentative hydrogen production from the sugars of sweet sorghum extract, in a continuous stirred tank bioreactor. The reactor was operated at a Hydraulic Retention Time of 12 h and a pH range of 3.5–6.5. The maximum hydrogen production rate and yield were obtained at pH 5.3 and were 1752 ± 54 mL H₂/d or 3.50 ± 0.07 L H₂/L reactor/d and 0.93 ± 0.03 mol H₂/mol glucose consumed or 10.51 L H₂/kg sweet sorghum, respectively. The main metabolic product at this pH value was butyric acid. The hydrogen productivity and yield were still at high levels for the pH range of 5.3–4.7, suggesting a pH value of 4.7 as optimum for hydrogen production from an economical point of view, since the energy demand for chemicals is lower at this pH. At this pH range, the dominant fermentation product was butyric acid but when the pH culture sharply decreased to 3.5, hydrogen evolution ceased and the dominant metabolic products were lactic acid and ethanol.     

Biohydrogen production from various feedstocks by Bacillus firmus NMBL-03

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

Phytosynthesized iron oxide nanoparticles and ferrous iron on fermentative hydrogen production using Enterobacter cloacae: Evaluation and comparison of the effects

The effects of FeSO₄ and synthesized iron oxide nanoparticles (0–250 mg/L) on fermentative hydrogen production from glucose and sucrose, using Enterobacter cloacae were investigated, to find out the enhancement of efficiency. The maximum hydrogen yields of 1.7 ± 0.017 mol H₂/mol glucose and 5.19 ± 0.12 mol H₂/mol sucrose were obtained with 25 mg/L of ferrous iron supplementation. In comparison, the maximum hydrogen yields of 2.07 ± 0.07 mol H₂/mol glucose and 5.44 ± 0.27 mol H₂/mol sucrose were achieved with 125 mg/L and 200 mg/L of iron oxide nanoparticles, respectively. These results indicate that the enhancement of hydrogen production on the supplementation of iron oxide nanoparticles was found to be considerably higher than that of ferrous iron supplementation. The activity of E. cloacae in a glucose and sucrose fed systems was increased by the addition of iron oxide nanoparticles, but the metabolic pathway was not changed. The results revealed that the glucose and sucrose fed systems conformed to the acetate/butyrate fermentation type.     

Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well

Hydrogen producing novel bacterial strain was isolated from formation water from oil producing well. It was identified as Thermoanaerobacter mathranii A3N by 16S rRNA gene sequencing. Hydrogen production by novel strain was pH and substrate dependent and favored pH 8.0 for starch, pH 7.5 for xylose and sucrose, pH 8.0–9.0 for glucose fermentation at 70 °C. The highest H₂ yield was 2.64 ± 0.40 mol H₂ mol glucose at 10 g/L, 5.36 ± 0.41 mol H₂ mol – sucrose at 10 g/L, 17.91 ± 0.16 mmol H₂ g – starch at 5 g/L and 2.09 ± 0.21 mol H₂ mol xylose at 5 g/L. The maximum specific hydrogen production rates 6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type fermentation pathway (approximately 97%) was found to be dominant in strain A3N, whereas butyrate formation was found in sucrose and xylose fermentation. Lactate production increased with high xylose concentrations above 10 g/L.     

Continuous hydrogen production from tofu processing waste using anaerobic mixed microflora under thermophilic conditions

We carried out continuous fermentative H₂ production from tofu (soybean curd)-processing waste (TPW) using anaerobic mixed microflora under thermophilic (60 °C) conditions and compared the rates and yields of H₂ production in a continuous stirred-tank reactor (CSTR) and a membrane bioreactor (MBR), wherein the membrane filtration unit was coupled to the CSTR. The TPW was diluted with tap water and then hydrolyzed by blending for 5 min in the presence of 0.5% HCl, and it was found that this protocol significantly increased the amount of soluble material in the mixture. The soluble chemical oxygen demand (SCOD)-to-total COD (TCOD) ratio jumped from 14% to 60%, and the soluble carbohydrate concentration was increased threefold, from 2.4 g/L to 7.2 g/L. Accordingly, H₂ production potential was increased 2.8-fold. In a CSTR operation using pretreated TPW as the substrate, a stable volumetric H₂ production rate (VHPR) of 8.17 ± 0.32 L H₂/L/d and a H₂ yield of 1.20 ± 0.05 mol H₂/mol hexose_added at 8-h HRT were achieved. Substantial increases in the VHPR and H₂ yield over those obtained with the CSTR were observed in the MBR operation. The role of the MBR was to increase the retention time of the solid substrate and the concentration of microorganisms, thereby enhancing the substrate utilization rate for H₂ production. Acetic and butyric acids were the main liquid-state metabolites produced during the fermentation process, thus indicating that the thermophilic operation provided favorable conditions for H₂ production from TPW. A maximum H₂ yield of 1.87 mol H₂/mol hexose_added was achieved at 8-h HRT and then gradually decreased to 1.00 mol H₂/mol hexose-equivalent at 2-h HRT. Meanwhile, the VHPR continuously increased to a maximum of 19.86 L H₂/L/d at 4-h HRT and then decreased with a high dilution rate as the HRT was lowered to 2 h (minimum). At 2-h HRT, the degradation of soluble carbohydrate was limited.     

Bioconversion of de-oiled Jatropha Waste (DJW) to hydrogen and methane gas by anaerobic fermentation: Influence of substrate concentration,temperature and pH

Cellulosic materials-based de-oiled Jatropha Waste (DJW) was fermented to H₂ and CH₄ using sewage sludge inoculum. Batch assays were performed at various substrate concentrations (40–240 g/L), temperatures (25–65 °C) and pHs (5.5–7.5). The peak hydrogen production rate (HPR) and hydrogen yield (HY) of 744.0 ± 11.3 mL H₂/L-d and 10.6 ± 0.2 mL H₂/g VS obtained when the optimal substrate concentration, pH, temperature were 200 g/L, 6.5, 55 °C, respectively. The peak methane production rate (MPR) of 178.4 ± 5.6 mL CH₄/L-d obtained while DJW concentration, pH, temperature were 200 g/L, 7.0, 45 °C, however, peak methane yield (MY) of 23.3 ± 0.1 mL CH₄/g VS obtained at 40 g/L, 7.0 and 55 °C, respectively. Effect of substrate concentration on HPR and MPR was elucidated using Monod model. Butyrate and acetate were the main soluble metabolic products. Maximal carbohydrate removal and COD reduction were achieved as 51.7 ± 0.7% and 68.3 ± 1.6%, respectively.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum TERI S7 from oil reservoir flow pipeline

Thermophilic dark fermentative hydrogen producing bacterial strain, TERI S7, isolated from an oil reservoir flow pipeline located in Mumbai, India, showed 98% identity with Thermoanaerobacterium thermosaccharolyticum by 16S rRNA gene analysis. It produced 1450–1900 ml/L hydrogen under both acidic and alkaline conditions; at a temperature range of 45–60 °C. The maximum hydrogen yield was 2.5 ± 0.2 mol H₂/mol glucose, 2.2 ± 0.2 mol H₂/mol xylose and 5.2 ± 0.2 mol H₂/mol sucrose, when the respective sugars were used as carbon source. The cumulative hydrogen production, hydrogen production rate and specific hydrogen production rate by the strain TERI S7 with sucrose as carbon source was found to be 1704 ± 105 ml/L, 71 ± 6 ml/L/h and 142 ± 13 ml/g/h respectively. Major soluble metabolites produced during fermentation were acetic acid and butyric acid. The strain TERI S7 was also observed to produce hydrogen continuously up to 48 h at pH 3.9.     

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

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

Biological hydrogen production from sweet sorghum syrup by mixed cultures using an anaerobic sequencing batch reactor (ASBR)

Continuous biological hydrogen production from sweet sorghum syrup by mixed cultures was investigated by using anaerobic sequencing batch reactor (ASBR). The ASBR was conducted based on the optimum condition obtained from batch experiment i.e. 25 g/L of total sugar concentration, 1.45 g/L of FeSO₄ and pH of 5.0. Feasibility of continuous hydrogen fermentation in ASBR operation at room temperature (30 ± 3 °C) with different hydraulic retention time (HRT) of 96, 48, 24 and 12 hr and cycle periods consisting of filling (20 min), settling (20 min), and decanting (20 min) phases was analyzed. Results showed that hydrogen content decreased with a reduction in HRT i.e. from 42.93% (96 hr HRT) to 21.06% (12 hr HRT). Decrease in HRT resulted in a decrease of solvents produced which was from 10.77 to 2.67 mg/L for acetone and 78.25 mg/L to zero for butanol at HRT of 96 hr-12 hr, respectively. HRT of 24 hr was the optimum condition for ASBR operation indicated by the maximum hydrogen yield of 0.68 mol H₂/mol hexose. The microbial determination in DGGE analysis indicated that the well-known hydrogen producers Clostridia species were dominant in the reacting step. The presence of Sporolactobacillus sp. which could excrete the bacteriocins causing the adverse effect on hydrogen-producing bacteria might responsible for the low hydrogen content obtained.     

Effects of temperature and substrate concentration on biological hydrogen production from starch

This study investigates the effects of temperature and substrate concentration on biological hydrogen production from starch using mixed cultures. In this work, although hydrogen was successfully produced under the thermophilic condition, stable hydrogen production was not observed under the mesophilic condition. In the thermophilic reactor, the maximum hydrogen yield was 2.8 mol H₂/mol glucose at 20 g/l-starch; however, hydrogen yield decreased drastically with the change of by-product distribution when substrate concentration was over 30 g/l-starch. A negative correlation was observed between the hydrogen yield and the total concentration of undissociated acids.     

Statistical optimization of fermentative hydrogen production from xylose by newly isolated Enterobacter sp. CN1

Statistical experimental designs were applied for the optimization of medium constituents for hydrogen production from xylose by newly isolated Enterobacter sp. CN1. Using Plackett–Burman design, xylose, FeSO₄ and peptone were identified as significant variables which highly influenced hydrogen production. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. These variables were subsequently optimized using Box–Behnken design of response surface methodology (RSM). The optimum conditions were found to be xylose 16.15 g/L, FeSO₄ 250.17 mg/L, peptone 2.54 g/L. Hydrogen production at these optimum conditions was 1149.9 ± 65 ml H₂/L medium. Under different carbon sources condition, the cumulative hydrogen volume were 1217 ml H₂/L xylose medium, 1102 ml H₂/L glucose medium and 977 ml H₂/L sucrose medium; the maximum hydrogen yield were 2.0 ± 0.05 mol H₂/mol xylose, 0.64 mol H₂/mol glucose. Fermentative hydrogen production from xylose by Enterobacter sp. CN1 was superior to glucose and sucrose.     

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.     

Impact of regulated pH on proto scale hydrogen production from xylose by an alkaline tolerant novel bacterial strain, Enterobacter cloacae DT-1

A hydrogen producing facultative anaerobic alkaline tolerant novel bacterial strain was isolated from crude oil contaminated soil and identified as Enterobacter cloacae DT-1 based on 16S rRNA gene sequence analysis. DT-1 strain could utilize various carbon sources; glycerol, CMCellulose, glucose and xylose, which demonstrates that DT-1 has potential for hydrogen generation from renewable wastes. Batch fermentative studies were carried out for optimization of pH and Fe²⁺ concentration. DT-1 could generate hydrogen at wide range of pH (5–10) at 37 °C. Optimum pH was; 8, at which maximum hydrogen was obtained from glucose (32 mmol/L), when used as substrate in BSH medium containing 5 mg/L Fe²⁺ ion. Decrease in hydrogen partial pressure by lowering the total pressure in the fermenter head space, enhanced the hydrogen production performance of DT-1 from 32 mmol H₂/L to 42 mmol H₂/L from glucose and from 19 mmol H₂/L to 33 mmol H₂/L from xylose. Hydrogen yield efficiency (HY) of DT-1 from glucose and xylose was 1.4 mol H₂/mol glucose and 2.2 mol H₂/mol xylose, respectively. Scale up of batch fermentative hydrogen production in proto scale (20 L working volume) at regulated pH, enhanced the HY efficiency of DT-1 from 2.2 to 2.8 mol H₂/mol xylose (1.27 fold increase in HY from laboratory scale). 84% of maximum theoretical possible HY efficiency from xylose was achieved by DT-1. Acetate and ethanol were the major metabolites generated during hydrogen production.     

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

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

Biohydrogen production from xylose by Thermoanaerobacterium thermosaccharolyticum KKU19 isolated from hot spring sediment

A thermophilic hydrogen producer was isolated from hot spring sediment and identified as Thermoanaerobacterium thermosaccharolyticum KKU19 by biochemical tests and 16S rRNA gene sequence analysis. The strain KKU19 showed the ability to utilize various kinds of carbon sources. Xylose was the preferred carbon source while peptone was the preferred organic nitrogen source. The optimum conditions for hydrogen production and cell growth on xylose were an initial pH of 6.50, temperature of 60 °C, a carbon to nitrogen ratio of 20:1, and a xylose concentration of 10.00 g/L. This resulted in a maximum cumulative hydrogen production, hydrogen production rate and hydrogen yield of 3020 ± 210 mL H₂/L, 3.95 ± 0.20 mmol H₂/L h and 2.09 ± 0.02 mol H₂/mol xylose consumed, respectively. Acetic and butyric acids were the main soluble metabolite products suggesting acetate and butyrate type fermentation.     

Biohydrogen production from pentose-rich oil palm empty fruit bunch molasses: A first trial

Oil palm empty fruit bunch (OPEFB) was pretreated by local plantation industry to increase the accessibility towards its fermentable sugars. This pretreatment process led to the formation of a dark sugar-rich molasses byproduct. The total carbohydrate content of the molasses was 9.7 g/L with 4.3 g/L xylose (C₅H₁₀O₅). This pentose-rich molasses was fed as substrate for biohydrogen production using locally isolated Clostridium butyricum KBH1. The effect of initial pH and substrate concentration on the yield and productivity of hydrogen production were investigated in this study. The best result for the fermentation performed in 70 mL working volume was obtained at the initial reaction condition of pH 9, 150 rpm, 37 °C and 5.9 g/L total carbohydrate. The maximum hydrogen yield was 1.24 mol H₂/mol pentose and the highest productivity rate achieved was 0.91 mmol H₂/L/h. The optimal pH at pH 9 was slightly unusual due to the presence of inhibitors, mainly furfural. The furfural content decreased proportionally as pH was increased. The optimal experiment condition was repeated and continued in fermentation volume of 200 mL. The maximum hydrogen yield found for this run was 1.21 mol H₂/mol pentose while the maximum productivity was 1.1 mmol H₂/L/h. The major soluble metabolites in the fermentation were n-butyric acid and acetic acid.     

Production of biohydrogen from waste wheat in continuously operated UPBR: The effect of influent substrate concentration

Utilization of waste materials is one of the most economical approaches to biohydrogen production. Continuous generation of biohydrogen in a bioreactor makes the process more economical with respect to the conventional physical and chemical method. The two main parameters that affect the biohydrogen production in a continuously operated bioreactor are hydraulic retention time (HRT) and influent substrate concentrations. The effect of influent substrate concentration on biohydrogen generation in an up-flow packed bed reactor (UPBR) at HRT = 3 h was investigated in this study. The substrate was waste wheat which was acid hydrolyzed in H₂SO₄ by adjusting the pH value to pH = 2, under high temperature as T = 90 °C in an autoclave to obtain fermentable sugar solution. A natural and porous support particle namely, aquarium biological sponge (ABS) was the microbial immobilization surface in the reactor. Total and hydrogen gas volumes, hydrogen percentage, influent and effluent substrate concentrations, VFA concentrations were monitored. The influent substrate concentration (TS_o) was varied between TS_o = 10 g/L and TS_o = 35 g/L. The process performance was evaluated as biohydrogen volume, percentages, rate and yield under varying operating conditions. The production volume (4275 ml/day) and the rate (3.05 L H₂/L day) were maximum at influent sugar concentration of TS_o = 25 g/L, but the yield reached to its maximum value as Y = 1.22 mol H₂/mol glucose at TS_o = 19 g/L. Substrate limitation and inhibitions were observed at influent concentrations of TS_o = 10 g/L and TS_o = 35 g/L, respectively. The results indicated that ABS could be suggested as a microbial support particle for hydrogen generation in immobilized systems.     

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.     

Thermophilic biohydrogen production from energy plants by Caldicellulosiruptor saccharolyticus and comparison with related studies

Air-dried samples of sweet sorghum, sugarcane bagasse, wheat straw, maize leaves and silphium were utilized without chemical pretreatment as sole energy and carbon sources for H₂ production by the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus. The specific H₂ production rates and yields were determined in the batch fermentation process. The best substrate was wheat straw, with H₂ production capacity of 44.7 L H₂ (kg dry biomass)^?1 and H₂ yield of 3.8 mol H₂ (mol glucose)^?1. Enzymatically pretreated maize leaves exhibited H₂ production of 38 L H₂ (kg dry biomass)^?1. Slightly less H₂ was obtained from homogenized whole plants of sweet sorghum. Sweet sorghum juice was an excellent H₂ source. Silphium trifoliatum was also fermented though with a moderate production. The results showed that drying is a good storage method and raw plant biomass can be utilized efficiently for thermophilic H₂ production. The data were critically compared with recently published observations.     

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.