Hydrogen production by co-cultures of Lactobacillus and a photosynthetic bacterium, Rhodobacter sphaeroides RV

Hydrogen production with glucose by using co-immobilized cultures of a lactic acid bacterium, Lactobacillus delbrueckii NBRC13953, and a photosynthetic bacterium, Rhodobacter sphaeroides RV, in agar gels was studied. Glucose was converted to hydrogen gas in a yield of 7.1 mol of hydrogen per mole of glucose at a maximum under illuminated conditions.     

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.     

A newly isolated Rhodobacter sphaeroides HY01 with high hydrogen production performance

A newly isolated strain, HY01, was identified to be Rhodobacter sphaeroides by phylogenetic analysis. Some DNA sequence data indicate that it is highly similar to R. sphaeroides ATCC 17029. The effects of initial pH values, nitrogen sources and carbon sources on hydrogen production were studied. The results showed that pH 7.25 is optimum for its hydrogen production. Among the nitrogen sources of glutamic acid, yeast extract, glycine, ethanolamine and l-aspartic acid, the maximum hydrogen production rate of 98.0 ± 6.2 mL/(Lh) using 10 mM glutamic acid and maximum hydrogen yield of 7012.5 ± 150 mL/L using 5 mM glutamic acid was obtained. Maximum hydrogen production rates of 148.7 ± 4.6 mL/(Lh) and 94.8 mL/(Lh) were obtained under 10 klux and 5 klux light intensity using 7 mM glutamic acid as nitrogen source, respectively. Compared with the reported data, it shows high hydrogen production performance and is a good candidate for further study.     

Culture conditions affecting H2 production by phototrophic bacterium Rhodobacter sphaeroides KD131

The effect of culture conditions on photo-H₂ production was investigated using the photosynthetic bacterium Rhodobacter sphaeroides KD131. When the initial cell concentrations were either below or above a threshold of 0.56 g-dcw/L, the H₂ production decreased due to an imbalance between the biomass and the substrate. Malate- and succinate-fed cultures exhibited the highest substrate conversion to H₂ production, whereas more than 85% of the substrate was utilized for cell growth in acetate- and butyrate-fed cultures. Compared with (NH₄)₂SO₄, glutamate as a nitrogen source was more appropriate for the initial H₂ production, but inhibited H₂ evolution during extended cultivation due to released NH₄⁺ ion. Even though the KD131 strain grew well under slightly acidic conditions, the pH value should be maintained in a neutral range in order to enhance H₂ production. The highest H₂ yield of 3.65 mol-H₂/mol-succinate was achieved when the KD131 strain grew in the succinate–glutamate medium with an initial cell concentration of 0.56 g-dcw/L and the pH level controlled to 7.5.     

Enhancement on hydrogen production performance of Rhodobacter sphaeroides HY01 by overexpressing fdxN

Ferredoxin I (FdI), encoded by fdxN gene, is proved to be the main electron donor of nitrogenase for hydrogen production. In this work, fdxN gene overexpression was implemented in a mutant MHY01, which was constructed by inserting fdxN gene into the hupSL region in Rhodobacter sphaeroides HY01 genome. Its photo-fermentative H₂ production performance was studied. The results showed that the expression level of fdxN and nitrogenase activity in MHY01 (hupSL::fdxN) were enhanced by 177% and 61.7% respectively compared with that of wild type HY01. Using 25 mM acetate and 34 mM butyrate as carbon source and 6 mM l-glutamate as nitrogen source, the maximum H₂ production rate was 156.1 mL/(L·h), which was increased by 50.7% compared with that of HY01. The maximum H₂ production rates of MHY01 were enhanced by 30.0%, 52.5% and 50.7% compared with those obtained from HY01 at the inoculation size of 5%, 10% and 15% respectively. The results suggested that overexpressing fdxN could enhance the nitrogenase activity and H₂ production performance of purple non-sulfur bacteria. The abundancy of ferredoxin I might limit the efficiency of electron transfer flux associated with the biohydrogen production process.     

An electricity production study by Rhodobacter sphaeroides

Microbial fuel cells are a type of bio-electrochemical system which can capture electrons produced by microorganisms. In this study, it is aimed to increase the electrogenic capacity of photosynthetic microbial fuel cells in a newly designed fuel cell. Rhodobacter sphaeroides, non-sulfur purple bacterium, was grown in anodic part of the fuel cells against permanganate as a cathodic electrolyte. Platine and graphite were used as the anodic and cathodic electrode, consequently. The distance between two electrodes was 1.5 cm. The concentrations of cathodic electrolyte were optimized. By the end, it is made to reach the highest anode potential (1.006 V) and electrogenic capacity (851.82 μA) in 5 mM permanganate concentration. The internal resistance was calculated as 1170 Ω. In these conditions, the current density with 2.1 cm² cathodic surface is 405.63 mA/m². These are the highest value of electricity generation potential of R. sphaeroides within the known PMFCs.     

Repeated-batch production of hydrogen using Rhodobacter sphaeroides S10

Photofermentation of acid hydrolyzed oil palm empty fruit bunch is reported for hydrogen production in repeated-batch fermentations using the bacterium Rhodobacter sphaeroides S10. Photofermentations were carried out at 35 °C at an incident light level of 10 klux. At specified times, different specified volumes of the culture broth were removed and replaced with an equal volume of the fresh medium. The initial mixed carbon (glucose, xylose, acetic acid) content in the medium of the repeated-batch reactors was adjusted to 20 mM. The kinetics of hydrogen production were evaluated in repeated-batch fermentations carried out in various ways: different volume exchange levels, different switch times from batch to repeated-batch operation, and different cycle times.     

The effect of cycA overexpression on hydrogen production performance of Rhodobacter sphaeroides HY01

The gene cycA encodes a periplasmic protein, cytochrome c₂ (cyt c₂), which dominates electron transfer from the membrane-bound ubiquinol: cyt c₂ oxidoreductase (cyt bc₁) to the photosynthetic reaction center, contributing to the production of transmembrane proton potential and then the synthesis of ATP. For photosynthetic bacteria, the total energy supply for light anaerobic growth and hydrogen production comes from photophosphorylation. As a result, the key protein encoding gene plays an important role in hydrogen production. To figure out the specific effect of cycA expression level on H₂ production ability of Rhodobacter sphaeroides HY01, cycA-expression plasmids derived from pRK415 and pBBR1MCS-2 were constructed and then crossed into the parent strain R. sphaeroides HY01 for H₂ production test. And further verification by RT-PCR suggested that there was about 20% enhancement of cycA expression level by pBBR1MCS-2 where the H₂ production performance of corresponding strain was improved by 6–8% compared with blank control. In contrast, cycA expression level was about 3.4 folds by vector pRK415 compared with control strain, but corresponding strains showed slightly depressed H₂ production performance. Besides, the mutant XJ01 with cycA gene overexpressing by 70% in the genome of HY01(hupSL:cycA) also showed positive effect on hydrogen production performance. The results demonstrated that slightly overexpression of cycA could enhance the hydrogen production rate, but too much higher level of cycA-expression could show negative effect on H₂ production performance of R. sphaeroides HY01.     

Statistical optimization of biohydrogen production from sucrose by a co-culture of Clostridium acidisoli and Rhodobacter sphaeroides

Statistically based experimental designs were applied to optimize the fermentation process parameters for hydrogen (H₂) production by co-culture of Clostridium acidisoli and Rhodobacter sphaeroides with sucrose as substrate. An initial screening using the Plackett–Burman design identified three factors that significantly influenced H₂ yield: sucrose concentration, initial pH, and inoculum ratio. These factors were considered to have simultaneous and interdependent effects. A central composite design and response surface analysis were adopted to further investigate the mutual interactions among the factors and to identify the values that maximized H₂ production. The optimal substrate concentration, initial pH, and inoculum ratio of C. acidisoli to R. sphaeroides were 11.43 g/L sucrose, 7.13, and 0.83, respectively. Using these optimal culture conditions, substrate conversion efficiency was determined as 10.16 mol H₂/mol sucrose (5.08 mol H₂/mol hexose), which was near the expected value of 10.70 mol H₂/mol sucrose (5.35 mol H₂/mol hexose).     

Remarkable enhancement on hydrogen production performance of Rhodobacter sphaeroides by disrupting spbA and hupSL genes

The redox balance and bacteriochlorophyll (Bchl) synthesis are both significant to hydrogen generation in photosynthetic bacteria. In this study, spbA and hupSL genes were knocked out from the genome of Rhodobacter sphaeroides HY01. The UV–vis spectra showed that the Bchl contents of spbA mutants were enhanced under photosynthetic conditions. The hydrogen yields of WH04 (hupSL⁻) and WSH10 (spbA⁻, hupSL⁻) mutants increased by 19.4%, 21.8%, and the maximum hydrogen evolution rates increased by 29.9% and 55.0% respectively using glutamate as sole nitrogen source. The maximum hydrogen production rate of WSH10 was up to 141.9 mL/(L·h). The nifH expression levels of the mutants and the wild type supported the correlation between hydrogen production and nitrogenase activity. The results demonstrate that disruption of spbA in R. sphaeroides can partially derepress the ammonium inhibition in nitrogenase activity, and indicate that spbA is a negative regulator in nitrogenase synthesis in the presence of ammonium.     

Synergistic effects and optimization of photo-fermentative hydrogen production of Rhodobacter sphaeroides DSM 158

The synergistic effects and optimization of pH, carbon-to-nitrogen ratio (C/N), and light intensity (I) on the photo-fermentative hydrogen production of Rhodobacter sphaeroides 158 DSM and light conversion efficiency have been investigated under different conditions of pH (6.5–8); C/N (15–35); and light intensity (35–185 W m^?2). Response surface methodology (RSM) and Box-Behnken experimental design (BBD) were used to identify the optimum values of the three key parameters of pH, C/N, and I, based on the impact on hydrogen production potential (HPP), hydrogen production rate (HPR), and light conversion efficiency $\eta$. With desirability value of 0.91, the optimum values of 7.4, 27.5, and 126 W m^?2 were identified for pH, C/N, and I respectively, with HPP, HPR and $\eta$ reaching 960 mL L^?1, 41.74 mL L^?1 h^?1, and 0.31 respectively. Regression analysis indicated a good fit between experimental and model data. The study showed that both C/N ratio and I have crucial and significant effect on the HPP, HPR and $\eta$, followed by pH, the synergistic effect of pH–I and C/NI on the light conversion efficiency ($\eta$) was significant while pH C/N was insignificant. The results and analysis obtained could be very useful for better optimizing the photo-fermentative hydrogen production.     

Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture

In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as inclusion bodies the solubility of the protein increased significantly when the expression temperature shifted from 37 °C to 30 °C. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and 218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were suggested and were used to increase bacterial hydrogen production.     

Optimization of photosynthetic hydrogen production from acetate by Rhodobacter sphaeroides RV

In this study, hydrogen production by Rhodobacter sphaeroides RV from acetate was investigated. Ammonium sulphate and sodium glutamate were used to study the effects of nitrogen sources on photosynthetic hydrogen production. The results showed the optimal concentrations for ammonium sulphate and sodium glutamate were in the range of 0.4–0.8 g/L. Orthogonal array design was applied to optimize the hydrogen-producing conditions of the concentrations of yeast, FeSO₄ and NiCl₂. The theoretical optimal condition for hydrogen production was as follow: yeast 0.1 g/L, FeSO₄ 100 mg/L and NiCl₂ 20 mg/L.     

Effect of carbon sources on the photobiological production of hydrogen using Rhodobacter sphaeroides RV

Rhodobacter sphaeroides RV was employed to produce hydrogen for the photo-fermentation of sole (acetate, propionate, butyrate, lactate, malate, succinate, ethanol, glucose, citrate and sodium carbonate) and compound carbon sources (malate and succinate, lactate and succinate). The concentrations of sole carbon sources on hydrogen production were investigated in batch assays at 0.8 g/L sodium glutamate and the maximum hydrogen yield was 424 mmol H₂/mol-substrate obtained at 0.8 g/L sodium propionate. The maximum hydrogen yield reached 794 mmol H₂/mol-substrate for 2.02 g lactate and 2.0 g succinate as the compound carbon source. The results showed hydrogen production for the compound carbon source was better than the sole carbon source.     

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.     

Function of glucose catabolic pathways in hydrogen production from glucose in Rhodobacter sphaeroides 6016

Purple non-sulfur (PNS) bacteria can convert volatile fatty acids into hydrogen with a high substrate conversion efficiency. However, when PNS bacteria utilize sugars as a carbon source, such as glucose and sucrose, the substrate conversion efficiency is relatively low. In order to investigate the contributions of the glucose catabolic pathways in Rhodobacter sphaeroides 6016 to its hydrogen production, the cfxA gene from the Embden–Meyerhof–Parnas (EMP) pathway, edd from the Entner–Doudoroff (ED) pathway, and kdg from the semi-phosphorylative ED bypass were knocked out to construct the mutant strains edd⁻, cfxA⁻, and kdg⁻, respectively. Additionally, two of these three genes were knocked out to construct the mutant strains kdg⁻edd⁻, kdg⁻cfxA⁻, and cfxA⁻edd⁻. Hydrogen productions by these mutant strains were compared to that of the wild type strain 6016 using 25 mM glucose as a carbon source. Compared to 6016, variations in hydrogen production and growth were detected in the edd mutant strains (kdg⁻edd⁻, cfxA⁻edd⁻, and edd⁻), while no obvious changes were detected in the others. Notably, the kdg⁻edd⁻ mutant did not produce hydrogen, and its maximum growth was 70% less than that of R. sphaeroides 6016. These results indicate that the ED pathway and semi-phosphorylative ED bypass have a governing impact on cell growth and hydrogen production from glucose in R. sphaeroides 6016. The potential synergistic function of the ED pathway and semi-phosphorylative ED bypass and the reasons for the low hydrogen yield from sugar carbon sources in R. sphaeroides 6016 are discussed.     

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using a novel strategy — shaking and extra-light supplementation approach

Biohydrogen has gained attention due to its potential as a sustainable alternative to conventional methods for hydrogen production. In this study, the effect of light intensity as well as cultivation method (standing- and shaking-culture) on the cell growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated in 38-ml anaerobic photobioreactor with RCVBN medium. Thus, a novel shaking and extra-light supplementation (SELS) approach was developed to enhance the phototrophic H₂ production by R. sphaeroides ZX-5 using malate as the sole carbon source. The optimum illumination condition for shaking-culture by strain ZX-5 increased to 7000–8000 lux, markedly higher than that for standing-culture (4000–5000 lux). Under shaking and elevated illumination (7000–8000 lux), the culture was effective in promoting photo-H₂ production, resulting in a 59% and 56% increase of the maximum and average hydrogen production rate, respectively, in comparison with the culture under standing and 4000–5000 lux conditions. The highest hydrogen-producing rate of 165.9 ml H₂/l h was observed under the application of SELS approach. To our knowledge, this record is currently the highest hydrogen production rate of non-immobilized purple non-sulphur (PNS) bacteria. This optimal performance of photo-H₂ production using SELS approach is a favorable choice of sustainable and economically feasible strategy to improve phototrophic H₂ production efficiency.     

Molecular hydrogen production by nitrogenase of Rhodobacter sphaeroides and by Fe-only hydrogenase of Rhodospirillum rubrum

The genes coding for two PII-like proteins, GlnB and GlnK, which play key roles in repressing the nitrogenase expression in the presence of ammonium ion, were interrupted from the chromosome of Rhodobacter sphaeroides. The glnB–glnK mutant exhibits the less ammonium ion-mediated repression for nitrogenase compared with its parental strain, which results in more H₂ accumulation by the mutant under the conditions. Rhodospirillum rubrum produces H₂ by both nitrogenase and hydrogenase. R. rubrum containing the recombinant pRK415 with an insert of hydC coding for its own Fe-only hydrogenase showed twofold higher accumulation of H₂ in the presence of pyruvate under photoheterotrophic conditions, which was not observed in the absence of pyruvate. The same was true with R. rubrum containing the recombinant pRK415 cloned with hydA coding for Fe-only hydrogenase of Clostridium acetobutylicum. Thus, Fe-only hydrogenase requires pyruvate as an electron donor for the production of H₂.