Influence of Ni(2+) concentration on biohydrogen production

In this paper, the effect of Ni(2+) concentration ranging from 0 to 50mg/L on fermentative hydrogen production by mixed cultures was investigated in batch test. The results showed that at 35 degrees C and initial pH 7.0, Ni(2+) was able to enhance the hydrogen production rate with increasing Ni(2+) concentration from 0 to 0.2mg/L, and enhance the hydrogen production potential and hydrogen yield with increasing Ni(2+) concentration from 0 to 0.1mg/L. The maximum hydrogen production potential of 288.6mL and the maximum hydrogen yield of 296.1mL/g glucose were obtained at the Ni(2+) concentration of 0.1mg/L. In all tests, the major soluble metabolites produced by mixed cultures were ethanol, acetic acid and butyric acid, without propionic acid. Ni(2+) had little effect on the substrate degradation efficiency with increasing concentration from 0 to 50mg/L. Ni(2+) was able to enhance the biomass production yield with increasing Ni(2+) concentration from 0 to 0.1mg/L. The maximum biomass production yield of 232.5mg/g glucose was obtained at the Ni(2+) concentration of 0.1mg/L. In all tests, the final pH after fermentative hydrogen production was lower than the initial pH.     

Effect of some environmental parameters on fermentative hydrogen production by Enterobacter cloacae DM11

Fermentative hydrogen production was carried out by Enterobacter cloacae DM11, using glucose as the substrate. The effects of initial substrate concentration, initial medium pH, and temperature were investigated. Results showed that at an initial glucose concentration of 1.0% (m/v), the molar yield of hydrogen was 3.31 mol (mol glucose)(-1). However, at higher initial glucose concentration, both the rate and cumulative volume of hydrogen production decreased. The pH of 6.5 +/- 0.2 at a temperature of 37 degrees C was found most suitable with respect to maximum rate of production of hydrogen in batch fermentation. Activation enthalpies of fermentation and that of thermal deactivation of the present process were estimated following a modified Arrhenius equation. The values were 47.34 and 118.67 kJ mol(-1) K(-1), respectively. The effect of the addition of Fe(2+) on hydrogen production was also studied. It revealed that the presence of iron (Fe(2+)) in the media up to a concentration of 20 mg L(-1) had a marginal enhancing effect on total hydrogen production. A simple model developed from the modified Gompertz equation was applied to estimate the hydrogen production potential, production rate, and lag-phase time in a batch process, based on the cumulative hydrogen production curves, using the software program Curve Expert 1.3.     

Enhancement effect of hematite nanoparticles on fermentative hydrogen production

The effects of hematite nanoparticles concentration (0-1600 mg/L) and initial pH (4.0-10.0) on hydrogen production were investigated in batch assays using sucrose-fed anaerobic mixed bacteria at 35 °C. The optimum hematite nanoparticles concentration with an initial pH 8.48 was 200 mg/L, with the maximum hydrogen yield of 3.21 mol H₂/mol sucrose which was 32.64% higher than the blank test. At 200 mg/L hematite nanoparticles concentration, further initial pH optimization experiments indicated that at pH 6.0 the maximum hydrogen yield reached to 3.57 mol H₂/mol sucrose and hydrogen content was 66.1%. The slow release of hematite nanoparticles had been recorded by transmission electron microscopy (TEM). In addition, TEM analysis indicated that the hematite nanoparticles can affect the shape of bacteria, namely, its length increased from ca. 2.0-3.6 μm to ca. 2.6-5.6 μm, and width became narrower.     

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatumZ7产氢效率的因素,采用生理学方法对其进行了研究。结果表明:乙醇型发酵菌A.elongatumZ7的最适产氢温度为37℃,最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度>60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。     

Optimization of bio-hydrogen production from biodiesel wastes by Klebsiella pneumoniae

Biodiesel wastes containing glycerol were utilized by Klebsiella pneumoniae DSM 2026 to produce hydrogen. The optimization of medium components was performed using both Plackett-Burman and uniform design methods. Using the Plackett-Burman design, glycerol, yeast extract, NH(4)Cl, KCl and CaCl2 were found to be the most important components, which were further investigated by uniform design and second-order polynomial stepwise regression analysis. The optimized medium containing 20.4 g.L(-1) glycerol, 5.7 g.L(-1) KCl, 13.8 g.L(-1) NH(4)Cl, 1.5 g.L(-1) CaCl(2) and 3.0 g.L(-1) yeast extract resulted in 5.0-fold increased level of hydrogen (57.6 mL/50 mL medium) production compared to initial level (11.6 mL/50 mL medium) after 24 h of fermentation The optimization of fermentation condition (pH, temperature and inoculum) was also conducted. When the strain grew in the optimized medium under optimal fermentation condition in a 5-L stirred tank bioreactor for batch production, hydrogen yield and production reached 0.53 mol/mol and 117.8 mmol/L, respectively. The maximum hydrogen evolution rate was 17.8 mmol/(L.h). Furthermore, 1,3-propanediol (6.7 g.L(-1)) was also obtained from the liquid medium as a by-product.     

Kinetics of iron oxidation by Leptospirillum ferriphilum dominated culture at pH below one

The kinetics of ferrous iron oxidation by Leptospirillum ferriphilum (L. ferriphilum) dominated culture was studied in the concentration range of 0.1-20 g Fe(2+)/L and the effect of ferric iron (0-60 g Fe(3+)/L) on Fe(2+) oxidation was investigated at pH below one. Denaturing gradient gel electrophoresis of PCR amplified 16S rRNA genes followed by partial sequencing confirmed that the bacterial community was dominated by L. ferriphilum. In batch assays, Fe(2+) oxidation started without lag phase and the oxidation was completed within 1 to 60 h depending on the initial Fe(2+) concentration. The specific Fe(2+) oxidation rates increased up to around 4 g/L and started to decrease at above 4 g/L. This implies substrate inhibition of Fe(2+) oxidation at higher concentrations. Haldane equation fitted the experimental data reasonably well (R(2) = 0.90). The maximum specific oxidation rate (q(m)) was 2.4 mg/mg VS . h, and the values of the half saturation (K(s)) and self inhibition constants (K(i)) were 413 and 8,650 mg/L, respectively. Fe(2+) oxidation was competitively inhibited by Fe(3+) and the competitive inhibition constant (K(ii)) was 830 mg/L. The time required to reach threshold Fe(2+) concentration was around 1 day and 2.3 days with initial Fe(3+) concentration of 5 and 60 g/L, respectively. The threshold Fe(2+) concentration, below which no further Fe(2+) oxidation occurred, linearly increased with increasing initial Fe(2+) and Fe(3+) concentrations. Fe(2+) oxidation proceeds by L. ferriphilum dominated culture at pH below 1 even in the presence of 60 g Fe(3+)/L. This indicates potential of using and biologically regenerating concentrated Fe(3+) sulfate solutions required, for example, in indirect tank leaching of ore concentrates.     

Influence of metal ions on the formation of mycobactin and salicylic acid in Mycobacterium smegmatis grown in static culture

Mycobacterium smegmatis was grown on trace-metal-free medium in static culture. Throughout the growth phase, the concentration of mycobactin increased continuously, reaching a maximum of about 30 to 40 mug of mycobactin/mg of cell dry weight after 6 days; the concentration of salicylic acid remained approximately constant at 1 to 2 mug of salicylic acid/mug of cell dry weight. Fe(2+) (or Fe(3+)), Zn(2+), Mn(2+), and Mg(2+) were all essential to a maximum formation of mycobactin. Optimum concentrations required were: Fe(2+), about 1.8 mum; Mn(2+) and Zn(2+), about 0.5 mum; and Mg(2+), at least 0.17 mm. Higher levels of Fe(2+) (9 to 90 mum) and Zn(2+) (2 to 7 mum) repressed mycobactin to about half the maximum value. No other cation or anion apparently is required for mycobactin biosynthesis. Salicylic acid concentration increased about fourfold when iron was omitted from the medium, but this is not as great as the increase reported previously for this strain of M. smegmatis. Mycobactin formation in another strain of M. smegmatis, NCIB 8548, showed similar dependencies on Fe(2+), Zn(2+), and Mn(2+). Maximum accumulation of mycobactin with this strain was 85 mug of mycobactin/mg of dry cell weight, under iron-deficient (1.8 mum Fe(2+)) conditions.     

响应面法优化黑曲霉产异淀粉酶的培养条件

在液态发酵条件下,采用单因素实验确定了Aspergillus niger PZ331产异淀粉酶的最适碳源和氮源,分别为蔗糖和硝酸铵。在上述基础上利用Plackett-Burman设计对影响产异淀粉酶的因素进行评价,并筛选出硝酸铵、接种量、培养温度3个主要因素;继而利用响应面设计优化了最佳硝酸铵浓度、接种量和培养温度。最终确定了最优培养条件为：蔗糖10 g/L,硝酸铵10 g/L,磷酸氢二钾3 g/L,硫酸亚铁0.01 g/L,硫酸镁1 g/L,起始p H值4.2;接种量2%（孢子浓度为107cfu/m L）,30℃培养72 h,酶活达137.3μ/m L;比基础培养基的提高了1.71倍左右。     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

Evaluation of Arthrospira platensis extracellular polymeric substances production in photoautotrophic, heterotrophic and mixotrophic conditions

The kinetic study of Arthrospira platensis extracellular polymeric substances (EPS) production under different trophic modes??photoautotrophy (100???mol photons m^?2?s^?1), heterotrophy (1.5?g/L glucose), and mixotrophy (100???mol photons m^?2?s^?1 and 1.5?g/L glucose)??was investigated. Under photoautotrophic and heterotrophic conditions, the maximum EPS production 219.61?±?4.73 and 30.30?±?1.97?mg/L, respectively, occurred during the stationary phase. Under a mixotrophic condition, the maximum EPS production (290.50?±?2.21?mg/L) was observed during the early stationary phase. The highest specific EPS productivity (433.62?mg/g per day) was obtained under a photoautotrophic culture. The lowest specific EPS productivity (38.33?mg/g per day) was observed for the heterotrophic culture. The effects of glucose concentration, light intensity, and their interaction in mixotrophic culture on A. platensis EPS production were evaluated by means of 32 factorial design and response surface methodology. This design was carried out with a glucose concentration of 0.5, 1.5, and 2.5?g/L and at light levels of 50, 100, and 150???mol photons m^?2?s^?1. Statistical analysis of the model demonstrated that EPS concentration and EPS yield were mainly influenced by glucose concentration and that conditions optimizing EPS concentration were dissimilar from those optimizing EPS yield. The highest maximum predicted EPS concentration (369.3?mg/L) was found at 150???mol photons m^?2?s^?1 light intensity and 2.4?g/L glucose concentration, while the highest maximum predicted EPS yield (364.3?mg/g) was recorded at 115???mol photons m^?2?s^?1 light intensity and 1.8?g/L glucose concentration.     

Optimizing iron supplement strategies for enhanced surfactin production with Bacillus subtilis

Supplement of Fe(2+) into fermentation medium was utilized as a tool to optimize the iron-mediated enhancement of surfactin production from Bacillus subtilis ATCC 21332. Up to 3000 mg L(-)(1) of surfactin was produced using an iron-enriched minimal salt (MS) medium amended with an optimal Fe(2+) dosage of 4.0 mM, leading to 8-fold and 10-fold increase in cell concentration and surfactin yield, respectively, as compared to those without Fe(2+) supplement. In addition to resulting in an optimal production yield of surfactin, a supplement of 4.0 mM of Fe(2+) also propelled maximum overall surfactin production rate to a highest value of 24 mg L(-)(1) h(-)(1). Our results also show that production of surfactin followed a growth-associated kinetic model. The best yield coefficient estimated from the model was ca. 162 mg surfactin (g dry cell)(-)(1). The supernatant of the iron-enriched culture of B. subtilis ATCC 21332 exhibited the ability to emulsify kerosene and achieved a maximum emulsion index (E(24)) of 80% for culture supplemented with 4.0 mM of Fe(2+). Comparison of emulsion index and the corresponding surfactin production indicates that the emulsification activity was essentially contributed by surfactin.     

Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology

Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007, 23, 808-813). With this developed B. subtilis strain, the design of an optimum but cost-effective medium for high-level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett-Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box-Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake-flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 degrees C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost-effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high-level production of recombinant nattokinase in a fermenter.     

Optimization of a liquid medium for beauvericin production in <Emphasis Type="Italic">Fusarium redolens</Emphasis> Dzf2 mycelial culture

Beauvericin (BEA) is a proven and potent antibiotic compound useful for bio-control and a potential antifungal and anticancer agent for human. This study was to evaluate and optimize the nutrient medium for BEA production in mycelial liquid culture of a high BEA-producing fungus Fusarium redolens Dzf2 isolated from a medicinal plant. Among various organic and inorganic carbon and nitrogen sources, glucose and peptone were found the most favorable for the F. redolens Dzf2 mycelial growth and BEA production. Through a Plackett-Burman screening test on a basal medium, glucose, peptone, and medium pH were identified as the significant factors for mycelial growth and BEA production. These factors were optimized through central composite design of experiments and response surface methodology, as 49.0 g/L glucose, 13.0 g/L peptone and pH 6.6, yielding 198 mg/L BEA (versus 156 mg/L in the basal medium). The BEA yield was further increased to 234 mg/L by feeding 10 g/L glucose to the culture during exponential phase. The results show that F. redolens Dzf2 mycelial fermentation is a feasible and promising process for production of BEA.     

Effects of growth condition on the structure of glycogen produced in cyanobacterium Synechocystis sp. PCC6803

Growth and glycogen production were characterized for Synechocystis sp. strain PCC6803 grown under continuous fluorescent light in four variations of BG-11 medium: either with (G+) or without (G−) 5 mM glucose, and with a normal (N+, 1.5 g sodium nitrate/L) or a reduced (N−, 0.084 g sodium nitrate/L) nitrogen concentration. Glucose-supplemented BG-11 with a normal nitrogen concentration (N+G+) produced the highest growth rate and the greatest cell density. Although the maximum cell mass production was observed in the N+G+ medium, the highest glycogen yield (19.0 mg/g wet cell mass) was achieved under the glucose-supplemented, nitrogen-limiting condition (N−G+). The addition of glucose enhanced cell growth, while nitrogen limitation apparently directed carbon flux into glycogen accumulation rather than cell growth. Transmission electron microscopic analysis showed that, under nitrogen-limiting conditions (N−G+), glycogen particles accumulated in large amounts and filled the cytosol of the cells. Analysis by high-performance size-exclusion chromatography further revealed that the glycogen produced in N−G+ medium had the longest average branch chain-length (DP10.4) among the conditions tested. When the yield and structure of glycogen were examined in different growth phases, the greatest yield (36.6 mg/g wet cell mass) and the longest branch chain-length (DP10.7) were observed 2 days after the fully grown cells in the N+G+ medium were transferred to the growth restricting (N−G+) medium.     

Hydrogen production and metal-dye bioremoval by a Nostoc linckia strain isolated from textile mill oxidation pond

Biohydrogen production by Nostoc linckia HA-46, isolated from a textile-industry oxidation-pond was studied by varying light/dark period, pH, temperature and ratio of carbon-dioxide and argon in the gas-mixture. Hydrogen production rates were maximum under 18 h of light and 6 h of darkness, pH 8.0, 31°C, a CO(2):Ar ratio 2:10. Hydrogen production of the strain acclimatized to 20 mg/L of chromium/cobalt and 100 mg/L of Reactive red 198/crystal violet dye studied in N-supplemented/deficient medium was 6-10% higher in the presence of 1.5 g/L of NaNO(3). Rates of hydrogen production in the presence of dyes/metals by the strain (93-105 μmol/h/mg Chlorophyll) were significantly higher than in medium without metals/dyes serving as control (91.3 μmol/h/mg Chlorophyll). About 58-60% of the two metals and 35-73% of dyes were removed by cyanobacterium. Optimal conditions of temperature, pH and metals/dyes concentration for achieving high hydrogen production and wastewater treatment were found practically applicable as similar conditions are found in the effluent of regional textile-mills.     

响应面法优化荷叶离褶伞菌丝体产漆酶条件

为了对荷叶离褶伞产漆酶条件进行优化,在单因素实验基础上,通过最陡爬坡实验(PB)对培养基8因素进行筛选,获得影响产漆酶的3个显著性因素:葡萄糖,pH和KH₂PO₄;通过中心组合(CCD)设计及响应面分析确定了最优发酵条件:葡萄糖20.09g/L,酪蛋白1.5g/L,酵母提取物1.5g/L,MgSO₄ 3g/L,CuSO₄ 3.75mg/L,KH₂PO₄ 3.97g/L,pH 4.98,VB₁ 0.1g/L,愈创木酚12mg/L,该条件下,漆酶酶活为829.83U/mL,较未优化对照提高46.6%.     

玉米秸秆水解液发酵联产氢气与2,3-丁二醇的初步研究

选用实验室自行筛选的Klebsiella pneumoniae ECU-15,进行了玉米秸秆水解液发酵联产氢气和2,3-丁二醇的初步研究。结果表明:以葡萄糖为碳源时,两目标产物随培养条件的改变呈现相同的变化趋势,且最佳发酵温度为37℃,最佳pH为6.0,最佳初始糖浓度为30 g/L;不同比例葡萄糖/木糖为混合碳源时,均能实现氢气和2,3-丁二醇的联产过程,但随着木糖含量的增加,细胞产量、氢气产量和2,3-丁二醇的产量都有所下降,并且木糖的存在会降低葡萄糖的消耗速率;实验最后以玉米秸秆水解液和同比例模拟合成培养基为底物,初步探明了该菌株利用水解液发酵联产氢气和2,3-丁二醇的可行性,最终氢气产量为0.65 v/v,产氢得率为0.43 mol/mol sugar;2,3-丁二醇产量为5.05 g/L,得率为0.82 mol/mol sugar。     

Performance characteristics of a two-stage dark fermentative system producing hydrogen and methane continuously

The performance of a mesophilic two-stage system generating hydrogen and methane continuously from sucrose (10-30 g/L) was investigated. A hydrogen-generating CSTR followed by an upflow anaerobic filter were both inoculated with anaerobically digested sewage sludge, and ORP, pH, gas output, %H(2), %CH(4) and %CO(2) monitored. pH was controlled with NaOH, KOH or Ca(OH)(2). Using NaOH as alkali with 10 g/L sucrose, yields of 1.62 +/- 0.2 mol H(2)/mol hexose added and 323 mL CH(4)/gCOD added to the hydrogen and methane reactors respectively were achieved. The overall chemical oxygen demand (COD) reduction was 92.6% with 0.90 +/- 0.1 g/L sodium and 316 +/- 40 mg/L residual acetate in the methane reactor. Operation at 20 g/L sucrose and NaOH as alkali led to impaired volatile fatty acid (VFA) degradation in the methane reactor with 2.23 +/- 0.2 g/L sodium, 1,885 mg/L residual acetate, a hydrogen yield of 1.47 +/- 0.1 mol/mol hexose added, a methane yield of 294 mL/gCOD added and an overall COD reduction of 83%. Using Ca(OH)(2) as alkali with 20 g/L sucrose gave a hydrogen yield of 1.29 +/- 0.3 mol/mol hexose added, a methane yield of 337 mL/gCOD added and improved the overall COD reduction to 91% with residual acetate concentrations of 522 +/- 87 mg/L. Operation at 30 g/L sucrose with Ca(OH)(2) gave poorer overall COD reduction (68%), a hydrogen yield of 1.47 +/- 0.2 mol/mol hexose added, a methane yield of 138 mL/gCOD added and residual acetate 7,343 +/- 715 mg/L. It was shown that sodium toxicity and overloading are important issues for successful anaerobic digestion of effluent from biohydrogen reactors in high rate systems.     

Hydrogen production conditions from food waste by dark fermentation with Clostridium beijerinckii KCTC 1785

The optimum conditions for biological hydrogen production from food waste by Clostridium beijerinckii KCTC 1875 were investigated. The optimum initial pH and fermentation temperature were 7.0 and 40°C, respectively. When the pH of fermentation was controlled to 5.5, a maximum amount of hydrogen could be obtained. Under these conditions, about 2,737 mL of hydrogen was produced from 50 g COD/L of food waste for 24 h, and the hydrogen content in the biogas was 38%. Hydrogen production rate and yield were about 108 mL/L·h and 128 mL/g COD_degraded, respectively. High concentrations of acetic (< 5,000 mg/L) or butyric acid (< 3,000 mg/L) significantly inhibited hydrogen production.     

Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49

In order to increase the hydrogen yield from glucose, hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria Ethanoligenens harbinense B49 was investigated. The soluble metabolites from dark-fermentation mainly were ethanol and acetate, which could be further utilized for photo-hydrogen production. Hydrogen production by B49 was noticeably affected by the glucose and phosphate buffer concentration. The maximum hydrogen yield (1.83 mol H₂/mol glucose) was obtained at 9 g/l glucose. In addition, we found that the ratio of acetate/ethanol (A/E) increased with increasing phosphate buffer concentration, which is favorable to further photo-hydrogen production. The total hydrogen yield during dark- and photo-fermentation reached its maximum value (6.32 mol H₂/mol glucose) using 9 g/l glucose, 30 mmol/l phosphate buffers and immobilized R. faecalis RLD-53. Results demonstrated that the combination of dark- and photo- fermentation was an effective and efficient process to improve hydrogen yield from a single substrate.