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.     

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.     

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.     

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.     

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 AI crude extract on PHB accumulation and hydrogen photoproduction in Rhodobacter sphaeroides

Poly-β-hydroxy butyric acid (PHB) accumulation and gaseous H₂ release are regarded as alternatives for expending reducing power. Some researchers suggested that quorum-sensing system affects PHB accumulation in Rhodobacter sphaeroides, but whether the system plays regulation role between hydrogen producing and PHB synthesis is still unknown. By adding autoinducer of R. sphaeroides into its culture solutions, measuring its total hydrogen production, PHB content and PHB synthase activity, the function of quorum-sensing on PHB accumulation and hydrogen production was preliminarily investigated. Compared with the control, the total gas productions in experimental groups increased accompanying slight decrease of PHB contents, which was partially caused by the reduction of PHB synthase activities. Biolog tests indicated the carbon source utilization profiles, especially those involving fatty acids and butanoate metabolism, had partly changed after exogenous signal molecules added. These results suggest that quorum-sensing is involved in signal regulation between PHB accumulation and hydrogen production in R. sphaeroides.     

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.     

Relationship between molecular hydrogen production,proton transport and the F0F1-ATPase activity in Rhodobacter sphaeroides strains from mineral springs

This article aims to study hydrogen production and proton transport in two strains of purple non-sulfur bacterium Rhodobacter sphaeroides isolated from mineral springs of Armenia. This bacterium is able to grow and produce molecular hydrogen (H₂) in anaerobic conditions upon illumination. Along with H₂ production, a marked decrease in redox potential and the alkalization of the medium have been observed; the latter might be the evidence of proton influx. H₂ production and alkalization of the medium by whole cells both are suppressed by the F₀F₁-ATPase inhibitors – N,N′-dicyclohexylcarbodiimide (DCCD), sodium azide (NaN₃) and protonophore – carbonyl cyanide m-chlorophenylhydrazone (CCCP). Membrane vesicles of two strains of R. sphaeroides demonstrate ATPase activity, inhibited by DCCD and NaN₃, but not by CCCP. These results indicate a relationship between H₂ production, proton transport and the F₀F₁-ATPase activity that might be a pathway to regulate bacterial activity under anaerobic conditions.     

Growth characteristics and hydrogen production by Rhodobacter sphaeroides using various amino acids as nitrogen sources and their combinations with carbon sources

Some amino acids (alanine, asparagine, glutamate, glycine, proline, and tyrosine) were used as nitrogen sources in combination with carbon sources (succinate and malate) to study growth properties and H₂ production by purple non-sulfur bacterium Rhodobacter sphaeroides strains A-10 and D-3. Both strains produced H₂ in succinate–glutamate and malate–glutamate media. Succinate was a better carbon source than malate. In comparison with strain D-3, strain A-10 was able to utilize proline, alanine or tyrosine as nitrogen sources in succinate medium and to produce H₂. Both strains were unable to produce H₂ in the presence of asparagine or glycine as nitrogen sources. N,N′-dicyclohexylcarbodiimide, the F₀F₁-ATPase inhibitor, led to marked inhibition of H₂ production activity of R. sphaeroides. The results suggest that the R. sphaeroides cells growth can be achieved by the use of a large diversity of substrates but only some of them can increase the H₂ production rate.     

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.     

Application of immobilized Rhodobacter sphaeroides bacteria in hydrogen generation process under semi-continuous conditions

The non-modified and modified (AEAPTMS) VitraPOR^®Filter porous glasses with thickness of 6.8 mm and diameter of 100 mm were applied as carriers for immobilization of Rhodobacter sphaeroides O.U. 001 bacteria. Two different glasses with pores of 40–100 and 100–160 μm were applied. The Van Niel's medium was applied for bacteria growth, whereas hydrogen generation reaction was performed with modified Biebl and Pfennig medium. Our own construction of Flat Plate Photobioreactor (FPP) with capacity of 200 cm³ operating under semi-continuous conditions was applied in this study. The maximum hydrogen production rate obtained was 0.059 dm³ H₂/dm³/h, while the maximum hydrogen yield was 4.2 mol H₂/mol malic acid. System was relatively stable because each series lasted about 3 months. Then the hydrogen production decreased. In order to introduce amine groups on the surface of porous glass, modification with AEAPTMS (3-(2-aminoethyl)aminopropyl)trimetoxysilane was performed. Obtained results proved that both modified and non-modified porous glasses are appropriate materials for stable immobilization of microbiological culture. The best activity and stability was achieved with porous glass matrix representing larger pores, whereas modification of the surface of these matrices with amine improved the amount of immobilized cells but did not improve the hydrogen yield.     

Brewery wastewaters in photobiological hydrogen generation in presence of Rhodobacter sphaeroides O.U. 001

Rhodobacter sphaeroides O.U. 001 (concentration of inoculum-0.36 g dry wt/l) and brewery wastewaters were applied in photobiogeneration of hydrogen under illumination of 116 W/m². The best results were obtained with filtered wastewaters sterilized at 120 °C for 20 min and maximal concentration of waste in medium equal 10% v/v. The main product in generated biogas was hydrogen (90%). After sterilization the amount of generated hydrogen was tripled (from 0.76 to 2.2 l H₂/l medium), whereas waste concentration of 10% v/v resulted in the best substrate yield (0.22 l H₂/l of waste). Under these conditions the amount of generated hydrogen was 2.24 l H₂/l medium and light conversion efficiency reached value of 1.7%. The modified Gompertz equations served in modeling of the kinetics of the studied process.     

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.     

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.     

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).     

Sustained hydrogen production from formate using immobilized recombinant Escherichia coli SH5

Biohydrogen is an ideal energy carrier for mobile chemical fuel cells, but its use is often limited by unavailability of sustained H₂ production system(s). Here, we developed a compact system for H₂ production from formate based on immobilized cells of recombinant Escherichia coli SH5. Three different matrices were tested as immobilization medium, among which agar showed the best performance in mechanical stability and permeability of substrate(s) and/or gaseous products (H₂ and CO₂). To explore and optimize the H₂ production capability of the immobilized cells, the conditions for cell immobilization including cell loading and agar concentration as well as the factors affecting H₂ production rate such as temperature, pH, and substrate concentration were studied in detail. A maximum volumetric production rate of 2.4 L H₂ L⁻¹ h⁻¹ was obtained when the immobilized cells were incubated with 350 mM sodium formate at pH 6.5 and 37 °C. Periodic supplementation of 200 mM formate with 20 mM glucose at pH 6.5 maintained the high H₂ production rate for a prolonged period of 10 h. We believe that our process can be developed for sustained H₂ production and is applicable to the operation of fuel cells in small-scale.     

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using fed-batch operation based on ORP level

In this study, a new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process by Rhodobacter sphaeroides ZX-5. In order to obtain the high hydrogen yield, photo-hydrogen production by fed-batch culture with on-line oxidation-reduction potential (ORP) feedback control was investigated. Meanwhile, the effects of feeding malic acid concentration and pH adjustment on the growth and hydrogen production of R. sphaeroides ZX-5 were studied. In the entire fed-batch culture, biomass (i.e., OD₆₆₀) rapidly increased up to 1.79 within 18 h, and then OD₆₆₀ value stayed constant within a range of 1.85-2.18 until the end of the photo-fermentation. The cumulative hydrogen volumes in each phase of fed-batch process were 2339, 1439, 1328, and 510 ml H₂/l-culture, respectively. Throughout the entire repeated fed-batch photo-fermentation, the maximum substrate conversion efficiency of 73.03% was observed in the first fed-batch process, obviously higher than that obtained from batch culture process (59.81%). In addition, compared to the batch culture, a much higher maximum hydrogen production rate (102.33 ml H₂/l h) was achieved during fed-batch culture. The results demonstrated that photo-hydrogen production using fed-batch operation based on ORP feedback control is a favorable choice of sustainable and feasible strategy to improve phototrophic hydrogen production efficiency.     

Evaluation of hydrogen production by Rhodobacter sphaeroides O.U.001 and its hupSL deficient mutant using acetate and malate as carbon sources

Rhodobacter sphaeroides O.U.001 is one of the candidates for photobiological hydrogen production among purple non-sulfur bacteria. Hydrogen is produced by Mo-nitrogenase from organic acids such as malate or lactate. A hupSL in frame deletion mutant strain was constructed without using any antibiotic resistance gene. The hydrogen production potential of the R. sphaeroides O.U.001 and its newly constructed hupSL deleted mutant strain in acetate media was evaluated and compared with malate containing media. The hupSL⁻R. sphaeroides produced 2.42 l H₂/l culture and 0.25 l H₂/l culture in 15 mM malate and 30 mM acetate containing media, respectively, as compared to the wild type cells which evolved 1.97 l H₂/l culture and 0.21 l H₂/l culture in malate and acetate containing media, correspondingly. According to the results, hupSL⁻R. sphaeroides is a better hydrogen producer but acetate alone does not seem to be an efficient carbon source for photoheterotrophic H₂ production by R. sphaeroides.