Bio-hydrogen production enhancement by co-cultivating Rhodopseudomonas palustris WP3-5 and Anabaena sp. CH3

Photobiological H₂ production is a promising method for renewable energy development. An innovative system that co-cultivating Rhodopseudomonas palustris WP3-5 and Anabaena sp. CH3 was carried out to estimate the effect of co-cultivation on H₂ production enhancement. H₂ production prolongation and enhancement were observed due to the light and metabolic compatibility of these two strains. Co-culture system served by acetate and fructose as carbon source can accumulate H₂ in 140.8 mL, almost double than the sum of individuals. Moreover, the enhancement of H₂ production was significantly affected by the mixed ratio of two strains. The mixed ratio (WP3-5:CH3) of 1:2 showed a highest H₂ production rate in 44.8 mL-H₂/L-culture/h, and both 2:1 and 1:2 exhibited a relatively high substrate conversion efficiency during the latest period of cultivation, whereas the mixed ratio of 1:1 and 3:1 only revealed a prolongation in H₂ production due to metabolic compatibility of two strains.     

Sustained outdoor H2 production with Rhodopseudomonas palustris cultures in a 50 L tubular photobioreactor

Sunlight represents an important choice for low cost photosynthetic cultures irradiation, especially in up-scaled processes. The aim of this study was the assessment of the hydrogen production performances of Rhodopseudomonas palustris strain 42OL cultivated outdoors, under sunlight, in a 50 L tubular photobioreactor. Moreover, bacteriochlorophyll a fluorescence was used for the first time as a tool for monitoring the physiological status of Rp. palustris mass cultures.     

The effect of irradiance growing on hydrogen photoevolution and on the kinetic growth in Rhodopseudomonas palustris, strain 42OL

The relationship between hydrogen generation and the age of culture was investigated under fed-batch growth conditions. The specific growth rate (μ_e) was determined during the log phase of the growth curve and the μ_eMax was 0.02643 h⁻¹. Boltzmann's sigmoidal regression model was used to determine the specific rate of hydrogen evolution (μH): the maximum was 0.04440 h⁻¹. At low irradiance (36–75 W m⁻²), an inverse relationship was found between μH and I; after increasing the irradiance further, μH reached a plateau (0.00916 h⁻¹). The maximum reactor yield of cumulative hydrogen (4.5 l) was obtained at an irradiance of 320 W m⁻², but the highest hydrogen evolution rate (17.217 ml h⁻¹) was achieved at 500 W m⁻². The light conversion efficiency reached its maximum (6.91%) at the lowest irradiance investigated (36 W m⁻²); when the irradiance increased further, it decreased progressively down to 0.36%.     

Optimization of phototrophic hydrogen production by Rhodopseudomonas palustris PBUM001 via statistical experimental design

Phototrophic hydrogen production by indigenous purple non-sulfur bacteria, Rhodopseudomonas palustris PBUM001 from palm oil mill effluent (POME) was optimized using response surface methodology (RSM). The process parameters studied include inoculum sizes (% v/v), POME concentration (% v/v), light intensity (klux), agitation (rpm) and pH. The experimental data on cumulative hydrogen production and COD reduction were fitted into a quadratic polynomial model using response surface regression analysis. The path to optimal process conditions was determined by analyzing response surface three-dimensional surface plot and contour plot. Statistical analysis on experimental data collected following Box-Behnken design showed that 100% (v/v) POME concentration, 10% (v/v) inoculum size, light intensity at 4.0 klux, agitation rate at 250 rpm and pH of 6 were the best conditions. The maximum predicted cumulative hydrogen production and COD reduction obtained under these conditions was 1.05 ml H₂/ml POME and 31.71% respectively. Subsequent verification experiments at optimal process values gave the maximum yield of cumulative hydrogen at 0.66 ± 0.07 ml H₂/ml POME and COD reduction at 30.54 ± 9.85%.     

Optimization of H2 photo-fermentation from benzoate by Rhodopseudomonas palustris using a desirability function approach

The operational conditions of H₂ production from benzoate by Rhodopseudomonas palustris was optimized in batch experiments by using the response surface methodology (RSM) and a desirability function approach. The estimated best H₂ production performance, i.e., maximum substrate-to-hydrogen conversion efficiency of 0.61 and light conversion efficiency of 3.20%, was achieved at: benzoate concentration 9.97 mM, NH₄Cl concentration 0.21 g/L and pH 6.76. These parameters were employed for running a continuous H₂ production bioreactor, for which the optimal hydraulic retention time was found to be 6 d. Under a light–dark cycle, R. palustris survived and maintained their H₂ production activity in the continuous operating system. The results demonstrate that it is possible to operate an outdoor photo-bioreacror for continuous H₂ production by utilizing aromatic compound as substrate. This works offer implications for guiding the design and operation of more energy–productive processes for treatment of aromatic compound-containing wastewater treatment.     

Fermentative hydrogen production from macro-algae Laminaria japonica using anaerobic mixed bacteria

In this study, one macro-alga (Laminaria japonica) was used for fermentative hydrogen production by anaerobic mixed bacteria. The saccharification efficiency and hydrogen production by L. japonica with four different pretreatment methods, including heat, acid, alkaline and ultrasonic treatment, were investigated. The results showed that the saccharification efficiency from L. japonica that was pretreated with acid was the highest among the four methods. The saccharification efficiency for the total reducing sugars in the acid-pretreated L. japonica was 350.54 ± 19.89 mg/g (mean ± S.E.). The cumulative hydrogen production was 66.68 ± 5.68 mL/g from the heat-pretreated L. japonica, whereas that of L. japonica that was subjected to acid, alkaline, and ultrasonic pretreatment and the control was 43.65 ± 6.87 mL/g, 15.00 ± 3.89 mL/g, 23.56 ± 4.56 mL/g and 10.00 ± 1.21 mL/g, respectively. In addition, the effects of substrate concentration and initial pH on hydrogen production from heat-pretreated L. japonica were also analyzed. The results showed that the maximum hydrogen production was 83.45 ± 6.96 mL/g with a hydrogen concentration of approximately 28.4% from heat-pretreated L. japonica when the initial pH and substrate concentration were determined to be 6.0 and 2%, respectively. Heat pretreatment was the most effective method for increasing fermentative hydrogen production when L. japonica was used as the only substrate.     

The effect of Ni,Fe and Mg concentration on photo-hydrogen production by Rhodopseudomonas faecalis RLD-53

In the present study, the effect of Ni²⁺ (0–10 μmol/l), Fe²⁺ (0–200 μmol/l) and Mg²⁺ (0–15 mmol/l) concentration on photo-hydrogen production from acetate was investigated by batch culture. Results showed that under a proper concentration range, Ni²⁺ was able to enhance the hydrogen production rate and the hydrogen yield; Fe²⁺ was able to increase the hydrogen yield, and hydrogen production rate was enhanced only when the culturing time was 24–72 h. Ni²⁺ and Fe²⁺ at a higher concentration inhibited cell growth. When Ni²⁺ and Fe²⁺ concentrations were 4 μmol/l and 80 μmol/l, respectively, maximal hydrogen yield of 2.87 and 2.78 mol H₂/mol acetate was obtained when batch culturing at 35 °C with initial pH 7.0. Mg²⁺ did not significantly affect hydrogen production and hydrogen yield which maintained at about 2.45 mol H₂/mol acetate, but it was favorable to cell growth.     

Light fermentation of dark fermentation effluent for bio-hydrogen production by different Rhodobacter species at different initial volatile fatty acid (VFA) concentrations

Three different Rhodobacter sphaeroides (RS) strains (RS–NRRL, RS–DSMZ and RS–RV) and their combinations were used for light fermentation of dark fermentation effluent of ground wheat containing volatile fatty acids (VFA). In terms of cumulative hydrogen formation, RS–NRRL performed better than the other two strains producing 48 ml H₂ in 180 h. However, RS–RV resulted in the highest hydrogen yield of 250 ml H₂ g⁻¹ TVFA. Specific hydrogen production rate (SHPR) with the RS–NRRL was also better in comparison to the others (13.8 ml H₂ g⁻¹ biomass h⁻¹). When combinations of those three strains were used, RS–RV + RS–DSMZ resulted in the highest cumulative hydrogen formation (90 ml H₂ in 330 h). However, hydrogen yield (693 ml H₂ g⁻¹ TVFA) and SHPR (12.1 ml H₂ g⁻¹ biomass h⁻¹) were higher with the combination of the three different strains. On the basis of Gompertz equation coefficients mixed culture of the three different strains gave the highest cumulative hydrogen and formation rate probably due to synergistic interaction among the strains. The effects of initial TVFA and NH₄–N concentrations on hydrogen formation were investigated for the mixed culture of the three strains. The optimum TVFA and NH₄–N concentrations maximizing the hydrogen formation were determined as 2350 and 47 mg L⁻¹, respectively.     

A pilot-scale study of biohydrogen production from distillery effluent using defined bacterial co-culture

We evaluated the feasibility of improving the scale of hydrogen (H₂) production from sugar cane distillery effluent using co-cultures of Citrobacter freundii 01, Enterobacter aerogenes E10 and Rhodopseudomonas palustris P2 at 100 m³ scale. The culture conditions at 100 ml and 2 L scales were optimized in minimal medium and we observed that the co-culture of the above three strains enhanced H₂ productivity significantly. Results at the 100 m³ scale revealed a maximum of 21.38 kg of H₂, corresponding to 10692.6 mol, which was obtained through batch method at 40 h from reducing sugar (3862.3 mol) as glucose. The average yield of H₂ was 2.76 mol mol⁻¹ glucose, and the rate of H₂ production was estimated as 0.53 kg/100 m³/h. Our results demonstrate the utility of distillery effluent as a source of clean alternative energy and provide insights into treatment for industrial exploitation.     

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.     

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.     

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.     

The kinetic characterization of photofermentative bacterium Rhodopseudomonas faecalis RLD-53 and its application for enhancing continuous hydrogen production

In this work, the kinetic characterization of hydrogen production by the photofermentative bacteria Rhodopseudomonas faecalis RLD-53 was investigated at different growth phase. During entire fermentation, 89.30% of total biomass was accumulated in exponential growth phase, while hydrogen yield was only 1.82 mol H₂/mol acetate at the expense of 51.25% substrate. In the stationary phase, biomass synthesis was minimal (7.51%), and 38.17% of the substrate was directly converted into hydrogen. As a result, hydrogen (59.19%) was mainly produced in stationary phase with highest hydrogen yield of 3.67 mol H₂/mol acetate. Consequently, bacteria in stationary phase were most effective for hydrogen production. Based on these findings, a novel membrane photobioreactor was developed to retain bacteria during stationary phase in reactor through membrane separation. Maximum rate (32.82 ml/l/h) and yield (3.27 mol H₂/mol acetate) of hydrogen production were achieved using membrane photobioreactor under the continuous operation. Therefore, using bacteria in stationary phase as hydrogen producer can offer considerable benefits for enhancing photo-hydrogen production.     

Introducing pyruvate oxidase into the chloroplast of Chlamydomonas reinhardtii increases oxygen consumption and promotes hydrogen production

In an anaerobic environment, the unicellular green algae Chlamydomonas reinhardtii can produce hydrogen (H₂) using hydrogenase. The activity of hydrogenase is inhibited at the presence of molecular oxygen, forming a major barrier for large scale production of hydrogen in autotrophic organisms. In this study, we engineered a novel pathway to consume oxygen and correspondingly promote hydrogen production in Chlamydomonas reinhardtii. The pyruvate oxidase from Escherichia coli and catalase from Synechococcus elongatus PCC 7942 were cloned and integrated into the chloroplast of Chlamydomonas reinhardtii. These two foreign genes are driven by a HSP70A/RBCS2 promoter, a heat shock inducing promoter. After continuous heat shock treatments, the foreign genes showed high expression levels, while the growth rate of transgenic algal cells was slightly inhibited compared to the wild type. Under low light, transgenic algal cells consumed more oxygen than wild type. This resulted in lower oxygen content in sealed culture conditions, especially under low light condition, and dramatically increased hydrogen production. These results demonstrate that pyruvate oxidase expressed in Chlamydomonas reinhardtii increases oxygen consumption and has potential for improving photosynthetic hydrogen production in Chlamydomonas reinhardtii.     

Expression of aldehyde dehydrogenase gene increases hydrogen production from low concentration of acetate by Rhodobacter sphaeroides

Rhodobacter sphaeroides RV (RV) is a hydrogen-producing bacterium exhibiting the highest yield of hydrogen production from organic acids such as lactate and acetate, which are the byproducts of hydrogen fermentation by hydrogen-producing anaerobic bacteria. Co-fermentation of the RV strain with anaerobic bacteria is an efficient method of hydrogen production. However, less than 21 mM acetate is produced by the anaerobic bacteria, which is too low for efficient hydrogen production by the RV strain; it requires approximately 75 mM acetate. In this study, 2 distinct isozymes of aldehyde dehydrogenase from Rhodospirillum rubrum were separately overexpressed in the RV strain. The recombinant RV strains that were designated as RVAD1 and RVAD2, exhibited 13-fold higher ALDH activities than the wild-type RV strain. Hydrogen yields of both of the recombinant strains were 1.4-fold higher than that of the RV strain in 21 mM acetate. In 43 mM acetate, the RVAD1 strain showed higher yield, though the RVAD2 strain showed lower yield as compared to that of the RV strain. In 64 mM acetate and all concentrations of lactate tested (21, 43 and 64 mM), the yields of the recombinant strains were lower than those of the RV strain. The intact (empty) expression plasmid increased the ALDH activity and had little effect on the hydrogen production in acetate, however, it decreased the production in lactate. At the beginning of the fermentation process, when very little hydrogen had been produced, the recombinant strains expressing the ALDH gene consumed smaller amounts of acetate compared to the wild-type strain. We have discussed the effects of ALDH on hydrogen production in this report.     

Effect of ferrous ion on photo heterotrophic hydrogen production by Rhodobacter sphaeroides

The effect of ferrous ion (0–3.2 mg/l) on photo heterotrophic hydrogen production was studied in batch culture using sodium lactate as substrate. The results showed that hydrogen production by Rhodobacter sphaeroides   was significantly suppressed when Fe²⁺${\mathrm{Fe}}^{2+}$ was limited. Hydrogen production increased linearly with an increase in Fe²⁺${\mathrm{Fe}}^{2+}$ concentration in the range of 0–1.6 mg/l; reaching a maximum at 2.4 mg/l. When hydrogen production was suppressed in the above medium, a pH increase to 8.9 was observed, and the ratio of lactate utilized to total organic carbon removal was found to be increased, indicating that more soluble organic products were produced. Under the Fe²⁺${\mathrm{Fe}}^{2+}$ limited conditions, ferrous iron was shown to have a greater effect on hydrogen production by Rb. sphaeroides than that by the anaerobic heterotrophic bacterium Clostridium butyricum.     

Study of hydrogen production by three strains of Chlorella isolated from the soil in the Algerian Sahara

In this study, the photosynthetic hydrogen production rates by some strains of green microalgae were investigated. Three strains of Chlorella isolated from arid soil and foggaras's water in the Algerian Sahara were used. Chlorella sorokiniana strain Ce, Chlorella salina strain Mt and Chlorella sp strain Pt6 produced hydrogen gas under sulphur-deprived conditions, but its rate was dependent on strain type and oxygen partial pressure in medium. In C. sorokiniana strain Ce, the maximum value of hydrogen accumulated was 147 ml at 222 h at 2% of O₂ pressure. Compared to C. sorokiniana strain Ce, C. salina strain Mt and Chlorella sp strain Pt6 produced less amount of hydrogen, but they were able to sustain with an O₂ partial pressure of up to 11–15.4%. Our data were compared with hydrogen production by Chlamydomonas reinhardtii. In this communication, the relationship between physiological behaviour, biochemical characteristic (starch and protein) and rates gas production (O₂ and H₂) was also specified.     

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.