Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710

Photofermentative hydrogen production is influenced by several parameters, including feed composition, pH levels, temperature and light intensity. In this study, experimental results obtained from batch cultures of Rhodobacter capsulatus DSM 1710 were analyzed to locate the maximum levels for the rate and yield of hydrogen production with respect to temperature and light intensity. For this purpose, a 3^k general full factorial design was employed, using temperatures of 20, 30 and 38 °C and light intensities of 100, 200 and 340 W/m². ANOVA results confirmed that these two parameters significantly affect hydrogen production. Surface and contour plots of the regression models revealed a maximum hydrogen production rate of 0.566 mmol H₂/L/h at 27.5 °C and 287 W/m² and a maximum hydrogen yield of 0.326 mol H₂/mol substrate at 26.8 °C and 285 W/m². Validation experiments at the calculated optima supported these findings.     

Fermentative hydrogen production from acetate using Rhodobacter sphaeroides RV

The study of photosynthetic hydrogen production by using Rhodobacter sphaeroides RV from acetate was described. We investigated the effects of light source (fluorescent, halogen and tungsten lamps), light intensity (1200–6000 lux), inoculum quantity (OD₆₆₀ 0.212–OD₆₆₀ 1.082) and initial pH (4.0–10.0) on biohydrogen production. The results indicated that the hydrogen production for halogen and tungsten lamps was better than it for fluorescent lamp as light source. The best light intensity of hydrogen production was 3600 lux for tungsten lamp as light source. Inoculum quantity experiments indicated that the higher hydrogen production volume and hydrogen conversion rate were obtained at initial OD₆₆₀ of 0.931. The effect of initial pH on hydrogen production indicated that the maximum hydrogen yield reached to 653.2 mmol H₂/mol acetate at initial pH 7.0.     

Hydrogen production by a marine photosynthetic bacterium, Rhodovulum sulfidophilum P5, isolated from a shrimp pond

A new hydrogen-producing photosynthetic bacterium, designated as Rhodovulum sulfidophilum P5, was enriched and isolated from the sludge of a marine shrimp cultivation farm. During fermentation, hydrogen was mainly produced in the late exponential and stationary phases. The optimum culture conditions of strain P5 for hydrogen production were NaCl concentration of 20 g/L, initial pH of 8, temperature of 30 °C, and light intensity of 100 μmol photons/m² s. The maximum hydrogen yield and rate were 2.56 ± 0.18 mol/mol acetate and 19.4 ± 1.6 mL/L h, respectively. Under optimum culture conditions, the hydrogen conversion efficiencies of P5 from acetate, propionate, and butyrate were (64.62 ± 5.05)%, (17.95 ± 0.72)%, and (41.83 ± 2.68)%, respectively. Taken together, these results suggest that this strain has a high salt tolerance and the potential to be used for biohydrogen production and biological treatment of marine organic wastewater.     

Hydrogen production from glucose by co-culture of Clostridium Butyricum and immobilized Rhodopseudomonas faecalis RLD-53

In this work, the effects of different parameters on co-culture hydrogen production using Clostridium Butyricum and immobilized Rhodopseudomonas faecalis RLD-53 were investigated. The maximum hydrogen yield of 4.134 mol H₂/mol glucose was obtained using 6 g/l glucose, 50 mmol/l phosphate buffer, initial pH of 7.5, the ratio of dark to photo bacteria of 1:10 and light intensity of 8000 lux. The maximum hydrogen production rate was 33.85 ml H₂/l/h. Phosphate buffer concentration was the most important parameter influencing hydrogen production in this co-culture. The ratio of acetate to butyrate increased from 0.74 to 1.82 in the soluble metabolites from C. butyricum with phosphate buffer concentration of 10–50 mmol/l. Experimental results could be of great significance for further pilot studies of co-culture hydrogen production.     

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 L bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H₂ mol⁻¹ glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol⁻¹ glucose and 0.6 mol butyrate mol⁻¹ glucose. For starch a maximum yield of 2.0 mol H₂ mol⁻¹ hexose, and a maximum production rate of 15 mol H₂ mol⁻¹ hexose h⁻¹ were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol⁻¹ hexose and 0.67 mol butyrate mol⁻¹ hexose.     

Hydrogen production from aqueous triethanolamine solution using Eosin Y-sensitized ZnO photocatalyst doped with platinum

Photocatalytic system for hydrogen production comprising ZnO as a photocatalyst, Eosin Y photo-sensitizer, triethanolamine electron donor and platinum co-catalyst is prepared and systematically tested under visible and simulated solar light irradiation. The results of laboratory experiments show that all studied parameters, such as a solution pH, Pt/ZnO dose, triethanolamine concentration and light intensity, notably influence the rate of photocatalytic hydrogen generation. The maximum hydrogen generation rate of 6.50 μmol min⁻¹ is achieved at pH 7.0 and 2 g L⁻¹ ZnO loaded with 0.75 wt% platinum, and 0.757 M triethanolamine concentration. The rate of hydrogen production as a function of triethanolamine concentration follows Langmuir-Hinshelwood kinetic model with reaction rate constant 4.50 μmol min⁻¹ and adsorption constant 14.84 M⁻¹ in solar light and 5.26 μmol min⁻¹ and 6.67 M⁻¹ in visible light, respectively. The reaction mechanism of hydrogen generation in the tested system is proposed and discussed.     

Fermentative production of hydrogen and soluble metabolites from crude glycerol of biodiesel plant by the newly isolated thermotolerant Klebsiella pneumoniae TR17

A thermotolerant fermentative hydrogen-producing strain was isolated from crude glycerol contaminated soil and identified as Klebsiella pneumoniae on the basis of the 16S rRNA gene analysis as well as physiological and biochemical characteristics. The selected strain, designated as K. pneumoniae TR17, gave good hydrogen production from crude glycerol. Culture conditions influencing the hydrogen production were investigated. The strain produced hydrogen within a wide range of temperature (30–50 °C), initial pH (4.0–9.0) and crude glycerol concentration (20–100 g/L) with yeast extract as a favorable nitrogen source. In batch cultivation, the optimal conditions for hydrogen production were: cultivation temperature at 40 °C, initial pH at 8.0, 20 g/L crude glycerol and 2 g/L yeast extract. This resulted in the maximum cumulative hydrogen production of 27.7 mmol H₂/L and hydrogen yield of 0.25 mol H₂/mol glycerol. In addition, the main soluble metabolites were 1,3-propanediol, 2,3-butanediol and ethanol corresponding to the production of 3.52, 2.06 and 3.95 g/L, respectively.     

Promoting hydrogen production by loading PdO and Pt on N–TiO2 under visible light

Nitrogen/titanium dioxide (N/TiO₂) visible light photocatalysts were prepared using the sol–gel method. The catalysts were characterized using transmission electron microscopy, reflective UV–visible spectroscopy, specific surface area measurements, and X-ray diffraction. The prepared catalysts were used to generate hydrogen gas through the water-splitting reaction under visible light (wavelengths greater than 400 nm). Various N/Ti addition ratios were tested, and the hydrogen generation rates were compared to determine the optimal ratio. The maximal hydrogen production rate (approximately 55 μmol h⁻¹ g⁻¹) was attained when the N/Ti ratio of N–TiO₂ was 10. When PdO and Pt were loaded onto the N–TiO₂ catalyst, the hydrogen generation rates increased to 544 and 772 μmol h⁻¹ g⁻¹, respectively. The highest hydrogen production rate (2460 μmol h⁻¹ g⁻¹) was obtained when bimetallic 0.05 wt% PdO-0.10 wt% Pt/N–TiO₂ was used. After three times use the hydrogen yield of the catalyst was maintained as 83%. A possible mechanism of water splitting catalyzed by this visible light photocatalyst is proposed.     

Hydrogen metabolism in the extreme thermophile Thermotoga neapolitana

Anaerobic growth of Thermotoga neapolitana led maximum to hydrogen yield of 3.85 ± 0.07 mol H₂/mol glucose and production rate of 51 ml/l/h. This productivity is strongly affected by stirring, pH buffering, N₂ sparging and culture/headspace volume ratio. Embden–Meyerhoff pathway is the only glycolytic route in T. neapolitana but, under the conditions used in this study, about 12–15% of the biogas requires consumption of protein source. Reduction of the hydrogen yields below the theoretical 4 mol H₂/mol glucose is mainly due to production of lactate and alanine that affect the availability of pyruvate/NADH for the hydrogenase, as well as to loss of part of glucose by conversion to fructose that is eventually released in the medium. Hydrogen productivity is modulated during the bacterial growth and major biogas synthesis is recorded in the stationary phase in concomitance with reduction of lactate synthesis. Apparently, this event is not consistent with an equal increase in acetate production. In agreement with the hydrogenase model recently proposed for the sister species Thermotoga maritima, this suggests that cellular NADH⁺ ratio has a crucial role on biogas synthesis.     

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.     

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.     

Continuous photosynthetic biohydrogen production from acetate-rich wastewater: Influence of light intensity

Photo-biohydrogen by microalgae is attractive sustainable energy caused by the utilization of solar energy and water. However, due to oxygen (O₂) sensitive hydrogenase (HydA) activity, effective control of O₂ and light intensity is critical for achieving sustainable photosynthetic hydrogen (H₂) production. Here we demonstrate continuous algal H₂ production using acetate-enriched fermenter effluent, achieving the complete O₂ cessation without sulfur depletion. Average H₂ production of 108 ± 4 μmol L^?1 for Chlamydomonas reinhardtii and 88 ± 7 μmol L^?1 for Chlorella sorokiniana at 100 μmol m^?2 s^?1 were observed for 15 days, respectively. The highest light energy to H₂ energy conversion efficiency (LHCE) of 1.61% for C. reinhardtii and 1.06% for C. Sorokiniana was obtained under low light intensity (50 μmol m^?2 s^?1) but the LHCE decreased with the increase of light intensity followed by photoinhibition, which led to a decrease of HydA activity and H₂ production. Low H₂ production was observed at 50 μmol m^?2 s^?1 under the highest LHCE, in which microalgae exhibited photoinhibition biomass growth kinetics to produce chlorophyll a (Chl a) for electron generation. These results demonstrate that light is a feasible strategy for producing electron for H₂ production under anoxygenic photosynthesis.     

Optimization of thermophilic fermentative hydrogen production by the newly isolated Caloranaerobacter azorensis H53214 from deep-sea hydrothermal vent environment

A unique thermophilic fermentative hydrogen-producing strain H53214 was isolated from a deep-sea hydrothermal vent environment, and identified as Caloranaerobacter azorensis based on bacterial 16S rRNA gene analysis. The optimum culture condition for hydrogen production by the bacterium, designated C. azorensis H53214, was investigated by the response surface methodology (RSM). Eight variables including the concentration of NaCl, glucose, yeast, tryptone, FeSO₄ and MgSO₄, initial pH and incubation temperature were screened based on the Plackett–Burman design. The results showed that initial pH, tryptone and yeast were significant variables, which were further optimized using the steepest ascent method and Box–Behnken design. The optimal culture conditions for hydrogen production were an initial pH of 7.7, 8.3 g L⁻¹ tryptone and 7.9 g L⁻¹ yeast. Under these conditions, the maximum cumulative hydrogen volume, hydrogen yield and maximum H₂ production rate were 1.58 L H₂ L⁻¹ medium, 1.46 mol H₂ mol⁻¹ glucose and 25.7 mmol H₂ g⁻¹ cell dry weight (CDW) h⁻¹, respectively. By comparison analysis, strain H53214 was superior to the most thermophilic hydrogen producers because of the high hydrogen production rate. In addition, the isolation of C. azorensis H53214 indicated the deep-sea hydrothermal environment might be a potential source for fermentative hydrogen-producing thermophiles.     

Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

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.     

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.     

Experimental design as a tool to study the reaction parameters in hydrogen production from photoinduced reforming of glycerol over CdS photocatalyst

The aim of this study was to set the reaction conditions of the photoinduced reforming of glycerol aqueous solution over Pt/hex-CdS under visible light irradiation for enhancement of hydrogen production by using a fractional factorial experimental design followed by a Box–Behnken design. The parameters assessed were irradiation time, mass of photocatalyst, concentration of glycerol, pH and electrolyte concentration (NaCl). The preliminary two-level fractional factorial design (2⁵⁻¹) showed that all of the investigated factors have significant effect in hydrogen production, being pH the most important parameter. The three factors Box–Behnken design showed maximum response for hydrogen production in pH 4.0, 55% glycerol and 1.5 mol L⁻¹ NaCl. The amount of hydrogen obtained under these conditions was 270% higher than our previous result, using the same photocatalyst and electron donor. In the ideal pH, >CdSH₂⁺and >CdOH species are predominant before irradiation, indicating that such species play an important role in the primary steps of the photoelectrochemical mechanism, which served as the basis for proposing a mechanism for hydrogen generation as well as glycerol photooxidation. Based on the surface response [NaCl] × [glycerol], a solution with salinity equivalent to approximately the natural seawater was tested and the result for hydrogen production was comparable to the best condition; besides, under this condition, the solubility of CdS in aqueous solution is reduced.     

Enhanced photo-hydrogen production of Rhodopseudomonas faecalis RLD-53 by EDTA addition

This study investigated the effect of EDTA concentration in medium on the growth, hydrogen production and nitrogenase activity of Rhodopseudomonas faecalis RLD-53 by batch cultures. Experimental results indicated that bacterial growth and hydrogen production were strongly inhibited with EDTA concentration increasing to 0.6–0.7 g/L. However, the lag time of hydrogen production and the trends of biomass at EDTA concentration of 0–0.5 g/L were similar. The maximum cumulative hydrogen volume of 3325 ml H₂/L culture, hydrogen production rate of 27.6 ml H₂/L/h and hydrogen yield of 2.97 mol H₂/mol acetate were obtained when EDTA concentration was at 0.3 g/L, and the maximum OD₆₆₀ attained 3.83. And the nitrogenase activity also reached a maximum value of 1331.9 μl-C₂H₄/h/mg dry weight in the medium containing Fe²⁺ and EDTA. These results showed that a proper concentration of EDTA can promote the availability of iron, thereby further enhancing the activity of nitrogenase and photo-hydrogen production.     

Fermentative hydrogen production by new marine Clostridium amygdalinum strain C9 isolated from offshore crude oil pipeline

The present study investigated hydrogen production potential of novel marine Clostridium amygdalinum strain C9 isolated from oil water mixtures. Batch fermentations were carried out to determine the optimal conditions for the maximum hydrogen production on xylan, xylose, arabinose and starch. Maximum hydrogen production was pH and substrate dependant. The strain C9 favored optimum pH 7.5 (40 mmol H₂/g xylan) from xylan, pH 7.5–8.5 from xylose (2.2–2.5 mol H₂/mol xylose), pH 8.5 from arabinose (1.78 mol H₂/mol arabinose) and pH 7.5 from starch (390 ml H₂/g starch). But the strain C9 exhibited mixed type fermentation was exhibited during xylose fermentation. NaCl is required for the growth and hydrogen production. Distribution of volatile fatty acids was initial pH dependant and substrate dependant. Optimum NaCl requirement for maximum hydrogen production is substrate dependant (10 g NaCl/L for xylose and arabinose, and 7.5 g NaCl/L for xylan and starch).     

Revivability of fermentative hydrogen producing bioreactors

In this study we investigated the revivability of a continuous biological hydrogen producing reactor after a period of feed interruption. Before the feed interruption, the hydrogen production yield was 1.36 mol H₂/mol glucose with butyric acid and acetic acid as the main metabolic products. However, after feed interruption, butyric acid formation completely stopped and the hydrogen yield decreased to 0.29 mol H₂/mol glucose. Lactic acid, ethanol and acetic acid became the main metabolites after re-start up. Reduction of organic loading rate together with increasing the pH after the feed interruption resulted in an increase in the hydrogen yield to 0.7 mol H₂/mol glucose. The microbial community dynamics showed complete elimination of Clostridium affiliated strains and predominance of Lactobacillus affiliated strains after the re-start up of the reactor.