The photosynthetic hydrogen production performance of a newly isolated Rhodobacter capsulatus JL1 with various carbon sources

Rhodobacter capsulatus is a photosynthetic bacterium with the ability to produce H₂ under photosynthetic condition. In this study, a new strain JL1 isolated from lake water was identified as Rhodobacter capsulatus by phylogenetic analysis of 16S ribosomal DNA (rDNA) sequence. Initial medium pH and l-glutamate (nitrogen source) concentration were optimized. At optimum pH 7.0 and 7 mmol/L l-glutamate, R. capsulatus JL1 could grow and produce hydrogen on the carbon sources of acetate, butyrate, glucose, xylose and fructose with the maximum substrate to H₂ conversion efficiencies of 67.5%, 26.6%, 46.1%, 46.2% and 46.6%, respectively. The maximum H₂ production rate, 124 ± 0.6 mL/(L·h), was obtained using 20 mmol-glucose/L as the carbon source. The addition of appropriate acetic acid to the tests with low concentration of glucose was able to improve the H₂ yield. Under the optimum operation parameters, the maximum H₂ yield and H₂ production rate of R. capsulatus JL1 from 16.4 g-corn straw/L-culture were 2966.5 ± 43.2 mL/L and 71.1 ± 4.5 mL/(L·h), while the chemical oxygen demand (COD) removal rate was up to 49.6%. This study indicates that R. capsulatus JL1 can serve as good candidate strain for H₂ production with organic waste water as well as effluent of dark-fermentation.     

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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

Single-stage photo-fermentative hydrogen production from hydrolyzed straw biomass using Rhodobacter sphaeroides

The hydrogen production utilizing photosynthetic and anaerobic bacteria in two-stage approach has many drawbacks, such as shortage of raw materials and complexity of operations. Accordingly, we aimed to develop a simple one-stage H₂ production protocol using the depolymerization of maize straw cellulose as a cheap carbon source. R. sphaeroides HY01 and its mutant (Hup^?) were studied regarding their H₂ production under different culture conditions. Further study using two model sugars, their combination, and straw hydrolysate as carbon sources was conducted to determine the effects of substrate on H₂ production. When using the straw hydrolysate as carbon source, the pH remained in a range of 7.1–7.6, whereas it dropped to 5.4–7.4 when using the model sugars, and the former biomass value was greater. The H₂ production performance of the mutant was significantly better than that of HY01. One-step photo-fermentative H₂ production was superior when using straw hydrolysate as opposed to the simple model sugars, and its yield was up to 4.62 mol H₂·mol^?1 reducing sugar.     

Photo-fermentative hydrogen production performance of a newly isolated Rubrivivax gelatinosus YP03 strain with acid tolerance

Screening and excavating new photosynthetic bacteria with excellent hydrogen production performance is extremely important for improving the photo-fermentative hydrogen production. A new photosynthetic bacterium YP03 was isolated and identified to be Rubrivivax gelatinosus by morphological characterization and phylogenetic analysis. The effects of several key factors on hydrogen production performance were carried out. The results indicated that YP03 strain showed a preference for the carbon sources, and 5375 ± 398 mL/L of maximum hydrogen yield was obtained using butyrate medium. Meanwhile, YP03 strain could use several nitrogen sources to produce hydrogen, and glutamic acid was the optimum nitrogen source for hydrogen produced. Furthermore, YP03 exhibited better hydrogen production performance at initial pH 7.0, reaction temperature 33 °C and light intensity 5000 lux, and the maximum hydrogen production rate was 108.3 ± 12.4 mL/(Lh), which was relatively high compared with the previous reports by R. gelatinosus. Especially, the proper pH for hydrogen production by YP03 ranged from weak acid to neutral (6.5–7.0) and it still could produce hydrogen at pH 5.5 showing the characteristic of acid tolerance. It suggested that YP03 is a potential candidate for the integration of dark- and photo-fermentative hydrogen production. These findings contribute to our understanding of YP03 strain and provide a prospective photosynthetic bacterium for efficient hydrogen production in future research.     

Isolation,characterization and optimization of photo-hydrogen production conditions by newly isolated Rhodobacter sphaeroides KKU-PS5

A purple non-sulfur (PNS) photosynthetic bacterium was isolated from an upflow anaerobic sludge blanket (UASB) bioreactor for methane production and was identified as Rhodobacter sphaeroides KKU-PS5 (GenBank Accession no. KC481702) by 16s rRNA gene sequence analysis. Strain KKU-PS5 could utilize glucose, xylose, fructose, arabinose, malate, succinate, acetate, butyrate, lactate and D-mannitol for growth and hydrogen production. Malate was a preferred carbon source while glutamate and Aji-L (waste from the process of crystallizing monosodium glutamate) were the preferred nitrogen sources. The ability to utilize Aji-L as a low-cost nitrogen supplement for photo-biohydrogen production by the strain KKU-PS5 is considered as its desirable characteristic. The threshold substrate concentration of malate was 30 mmol/L. The optimum conditions for hydrogen production from malate were an initial pH of 7.0, FeSO₄ concentration of 4 mg/L, temperature of 30 °C and light intensity of 6 klux. Under the optimum conditions, the maximum hydrogen production, the hydrogen yield (HY) and the hydrogen production rate (HPR) of 1330 mL-H₂/L, 3.80 mol-H₂/mol-malate, and 11.08 mL-H₂/L h, respectively, were achieved. Hydrogen production under a dark/light cycle led to a decreased HY and HPR in comparison to continuous illumination.     

Impact of cultural conditions on photoproduction of hydrogen by Allochromatium sp. GSKRLMBKU-01 isolated from marine water of Visakhapatnam

In the present study, photoproduction of hydrogen by Allochromatium sp. strain GSKRLMBKU-01 isolated from marine water was measured under different cultural conditions. Hydrogen production was measured by using a Gas chromatography using argon gas as a carrier. Among different carbon and nitrogen sources used, succinate induced maximum hydrogen (5.68 ± 0.27 mL) production by immobilized cells, while free cells recorded 4.24 ± 0.30 mL of hydrogen under anaerobic light conditions. Immobilization of cells resulted in increased production of hydrogen. Ammonium chloride promoted more amounts of hydrogen production (2.68 ± 0.29 mL) by free cells, whereas glycine enhanced the hydrogen production upto 4.82 ± 0.36 mL in immobilized cells. In formic acid and urea, less hydrogen production was observed in both free and immobilized cells compared to other sources. Cumulative hydrogen production by the bacterium was recorded with the progress in incubation period. Incubation period of 192 h, pH of 7.0 and temperature at 30 °C were found to be optimum for the maximum hydrogen production. Significance of the above results was discussed in light of existing literature.     

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.     

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.     

Effect of substrate concentration on continuous Photo-fermentative hydrogen production from lactate using Rhodobacter sphaeroides

The information on continuous operation and the use of actual waste as a feedstock are essential for the practical application of photo-fermentative H₂ production. For the first 200 days, continuous H₂ production from lactate was attempted using purple non-sulfur (PNS) bacteria, Rhodobacter sphaeroides KD131, under an illumination of 110 W/m². During the continuous operation, 30% of the fermenter volume was replaced by fresh feedstock once a day, and substrate concentration was gradually increased from 5 mM to 30 mM. H₂ production was negligible at 5 mM, which was ascribed to the fact that the electrons contained in lactate were mostly consumed for cell growth and soluble microbial products (SMPs) production. As lactate concentration increased, H₂ production gradually increased and reached a maximum at 20 mM, showing a substrate conversion efficiency (SCE) of 38%, a H₂ yield of 2.3 mol H₂/mol lactate_added, and a H₂ production rate of 309 mL H₂/L-fermenter/d. Further increases of lactate concentration resulted in a drop of H₂ production (<1.0 mol H₂/mol lactate_added). When the feedstock was changed to actual waste obtained from a 1-day lactate fermentation of food waste, stable H₂ production was maintained, but showed a decreased SCE of 24%. It was speculated that the low performance was due to the fact that actual waste contained not only pure lactate but also other organic compounds that could not be utilized by PNS bacteria. In addition, compared to feeding with pure lactate, the electron consumption to the cell growth was higher in feeding with actual waste, which led to the lower performance.     

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.