Hydrogen production properties of Rhodobacter capsulatus with genetically modified redox balancing pathways

Rhodobacter capsulatus produces molecular hydrogen under the photoheterotrophic growth condition with reduced carbon sources (organic acids). Under this condition, ubiquinol pool is over reduced and excess reducing equivalents are primarily consumed via the reduction of CO₂ through the Calvin–Benson–Bassham (CBB) pathway, the dimethylsulfoxide reductase (DMSOR) system or by the reduction of protons into hydrogen gas with the use of nitrogenase to maintain a balanced intracellular oxidation-reduction potential (redox balance). In order to investigate the effect of redox balancing pathways on nitrogenase-dependent hydrogen production, CO₂ fixation was blocked by inactivating the phosphoribulokinase (PRK) of CBB pathway in wild type (MT1131), uptake-hydrogenase deficient strain (YO3), and cyt cbb₃ oxidase and uptake-hydrogenase deficient double mutant (YO4) strains. The hydrogen production properties of newly generated strains deficient in the CBB pathway were analyzed and compared with wild type strains. The obtained data indicated that, the total hydrogen production was increased slightly in CBB deficient mutant of YO3 and YO4 (4.7% and 12.5% respectively). Moreover, the maximum hydrogen production rate was increased by 13.3% and 12.7% for CBB deficient mutant of MT1131 and YO3 respectively. It was also observed that under the photoheterotrophic growth condition with ammonium as a nitrogen source, PRK deficient strains gave photoheterotrophically competent ammonium insensitive revertants.     

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.     

Effect of inactivation of genes involved in ammonium regulation on the biohydrogen production of Rhodobacter capsulatus

Hydrogen production by nitrogenase is an energetically expensive process for the cell, hence strictly controlled at different levels. Ammonium is one of the substances regulating nitrogenase activity. The key proteins in the regulation of nitrogenase by ammonium are two regulatory proteins; GlnB and GlnK. In order to increase hydrogen production of Rhodobacter capsulatus DSM1710 (wild type strain) grown on agricultural materials/wastes, ammonium inhibition of nitrogenase enzyme has to be eliminated. In this study, GlnB and GlnK were targeted to be inactivated by in frame site-directed mutagenesis. The glnB mutant R. capsulatus (GP1 strain) was obtained at the end of mutagenesis studies. In the case of glnK, the suicide vector was constructed and delivered into the cells. However, glnK mutant could not be obtained.The effect of ammonium on the growth and hydrogen production of R. capsulatus GP1 was investigated and compared with DSM1710. Both DSM1710 and GP1 strains were effectively utilized acetate. The mutation did not affect cell growth significantly at different ammonium levels. Ammonium negatively affected hydrogen production of GP1 strain as well as the DMS1710. However, hydrogen production was significantly low in GP1 strain. The ammonium inhibition of hydrogen production could not be removed in glnB mutant probably due to the presence of an active GlnK protein in the cell. Therefore, GlnK has much more important role in the ammonium dependent control of nitrogenase than GlnB does. The growth and hydrogen production kinetics of R. capsulatus DSM1710 and GP1 were modelled. They were shown to fit to Logistic Model and Modified Gompertz Model, respectively.     

Genetically engineered deletion in the N-terminal region of nifA1 in R. capsulatus to enhance hydrogen production

NifA is the primary activator of nitrogenase, and the N-terminal domain of nifA is sensitive to ammonium concentration. In this work, a mutant Rhodobacter capsulatus ZX01 with a genetically engineered deletion in the N-terminal region of nifA1 was constructed by employing overlap extension PCR to mitigate the inhibition of ammonium on nitrogenase expression in photosynthetic bacteria. The effects of different ammonium ion concentrations on the growth and photo-fermentative hydrogen production performance of wild-type strain R. capsulatus SB1003 and mutant ZX01 with glucose and volatile fatty acids as the carbon sources were studied, respectively. When the ratio of NH₄⁺-N was 20% and 30%, the hydrogen yield of the mutant ZX01 was enhanced by 14.8% and 20.9% compared with that of R. capsulatus SB1003 using 25 mM acetic acid and 34 mM butyric acid as the carbon source, respectively. In comparison, using 30 mM glucose as the carbon source, the hydrogen yield of ZX01 was increased by 17.7% and 22.2% compared with that of R. capsulatus SB1003 when the ratio of NH₄⁺-N was 20% and 30%, and the nitrogenase activity of ZX01 was also enhanced by 38.0% and 47.6%, respectively. When using 10 mM NH₄⁺ as a single nitrogen source, ZX01 showed a 2.6-fold increase in H₂ production. These results indicated that ZX01 demonstrated higher ammonium tolerance and better hydrogen production performance than the wild-type. The deletion in the N-terminal region of nifA1 could partially de-repress the nitrogenase activity inhibited by ammonium.     

Photofermentative hydrogen production using dark fermentation effluent of sugar beet thick juice in outdoor conditions

In the present study, photofermentative hydrogen production on thermophilic dark fermentation effluent (DFE) of sugar beet thick juice was investigated in a solar fed-batch panel photobioreactor (PBR) using Rhodobacter capsulatus YO3 (hup⁻) during summer 2009 in Ankara, Turkey. The DFE was obtained by continuous dark fermentation of sugar beet thick juice by extreme thermophile Caldicellulosiruptor saccharolyticus and it contains acetate (125 mM) and NH₄⁺ (7.7 mM) as the main carbon and nitrogen sources, respectively. The photofermentation process was done in a 4 L plexiglas panel PBR which was daily fed at a rate of 10% of the PBR volume. The DFE was diluted 3 times to adjust the acetate concentration to approximately 40 mM and supplemented with potassium phosphate buffer, Fe and Mo. In order to control the temperature, cooling was provided by recirculating chilled water through a tubing inside the reactor. Hydrogen productivity of 1.12 mmol/L_c/h and molar yield of 77% of theoretical maximum over consumed substrate were attained over 15 days of operation. The results indicated that Rb. capsulatus YO3 could effectively utilize the DFE of sugar beet thick juice for growth and hydrogen production, therefore facilitating the integration of the dark and photo-fermentation processes for sustainable biohydrogen production.     

Bioproduction of hydrogen by Rhodobacter capsulatus KU002 isolated from leather industry effluents

A preliminary study on photoproduction of hydrogen by Rhodobacter capsulatus KU002 isolated from leather industry effluents under different cultural conditions with various carbon and nitrogen sources was investigated. Hydrogen production was measured using a Gas chromatograph. Lactate promoted more amounts of hydrogen production under anaerobic light conditions and aerobic light conditions. Cumulative hydrogen production by the organism was recorded at various time intervals. Incubation period of 120 h was optimum for production of hydrogen. pH 7.0 ± 0.2 was optimum for production of hydrogen by growing cells, while pH 7.5 ± 0.26 for resting cells. l-cystine was a good nitrogen source for production of hydrogen. Growing cells produced more amount of hydrogen than resting cells. Glutamine was a poor nitrogen source for hydrogen production by Rb. capsulatus. Significance of the above results in the presence of existing literature is discussed.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum TERI S7 from oil reservoir flow pipeline

Thermophilic dark fermentative hydrogen producing bacterial strain, TERI S7, isolated from an oil reservoir flow pipeline located in Mumbai, India, showed 98% identity with Thermoanaerobacterium thermosaccharolyticum by 16S rRNA gene analysis. It produced 1450–1900 ml/L hydrogen under both acidic and alkaline conditions; at a temperature range of 45–60 °C. The maximum hydrogen yield was 2.5 ± 0.2 mol H₂/mol glucose, 2.2 ± 0.2 mol H₂/mol xylose and 5.2 ± 0.2 mol H₂/mol sucrose, when the respective sugars were used as carbon source. The cumulative hydrogen production, hydrogen production rate and specific hydrogen production rate by the strain TERI S7 with sucrose as carbon source was found to be 1704 ± 105 ml/L, 71 ± 6 ml/L/h and 142 ± 13 ml/g/h respectively. Major soluble metabolites produced during fermentation were acetic acid and butyric acid. The strain TERI S7 was also observed to produce hydrogen continuously up to 48 h at pH 3.9.     

Amelioration of photofermentative hydrogen production from molasses dark fermenter effluent by zeolite-based removal of ammonium ion

One of the challenges in the development of integrated dark and photofermentative biological hydrogen production systems is the presence of ammonium ions in dark fermentation effluent (DFE). Ammonium strongly inhibits the sequential photofermentation process, and so its removal is required for successful process integration. In this study, the removal of ammonium ions from molasses DFE using a natural zeolite (clinoptilolite) was investigated. The samples were treated with batch suspensions of Na-form clinoptilolite. The ammonium ion concentration could be reduced from 7.60 mM to 1.60 mM and from 12.30 mM to 2.40 mM for two different samples. Photofermentative hydrogen production on treated and untreated molasses DFE samples were investigated in batch photobioreactors by an uptake hydrogenase deleted (hup⁻) mutant strain of Rhodobacter capsulatus. Maximum hydrogen productivities of 1.11 mmol H₂/L_c·h and 1.16 mmol H₂/L_c·h and molar yields of 79% and 90% were attained in the treated DFE samples, while the untreated samples resulted in no hydrogen production. The results showed that ammonium ions in molasses DFE could be effectively removed using clinoptilolite by applying a cost-effective, simple batch process.     

Modelling of hydrogen production in batch cultures of the photosynthetic bacterium Rhodobacter capsulatus

The photosynthetic bacterium, Rhodobacter capsulatus, produces hydrogen under nitrogen-limited, anaerobic, photosynthetic culture conditions, using various carbon substrates. In the present study, the relationship between light intensity and hydrogen production has been modelled in order to predict both the rate of hydrogen production and the amount of hydrogen produced at a given time during batch cultures of R. capsulatus. The experimental data were obtained by investigating the effect of different light intensities (6000–50,000 lux) on hydrogen-producing cultures of R. capsulatus grown in a batch photobioreactor, using lactate as carbon and hydrogen source. The rate of hydrogen production increased with increasing light intensity in a manner that was described by a static Baly model, modified to include the square of the light intensity. In agreement with previous studies, the kinetics of substrate utilization and growth of R. capsulatus was represented by the classical Monod or Michaelis–Menten model. When combined with a dynamic Leudekong–Piret model, the amount of hydrogen produced as a function of time was effectively predicted. These results will be useful for the automatization and control of bioprocesses for the photoproduction of hydrogen.     

Long-term stable hydrogen production from acetate using immobilized Rhodobacter capsulatus in a panel photobioreactor

Biological hydrogen production is attractive since renewable resources are utilized for hydrogen production. In this study, a novel panel photobioreactor (1.4 L) was constructed from Plexiglas with a network of nylon fabric support for agar immobilized bacteria complex. Two strains of Rhodobacter capsulatus DSM 1710 wild-type strain and Rhodobacter capsulatus YO3 (hup⁻, uptake hydrogenase deleted mutant) with cell concentrations of 2.5 and 5.0 mg dcw/mL agar, respectively were entrapped by 4% (w/v) of agar. The system was operated for 72–82 days in a sequential batch mode utilizing acetate as substrate at 30 °C under continuous illumination. Immobilization increased the stability of the photobioreactors by reducing the fluctuations in pH. The pH remained between 6.7 and 8.0 during the process. Both hydrogen yield and productivity were higher in immobilized photobioreactors compared to suspended culture. The highest hydrogen productivities of 0.75 mmol H₂/L/h and 1.3 mmol H₂/L/h were obtained by R. capsulatus DSM1710 and R. capsulatus YO3 respectively.     

Improved photo – Hydrogen production by transposon mutant of Rhodobacter capsulatus with reduced pigment

The light shielding effect of photosynthetic bacteria in photo fermentation process, which is caused by high content of pigment, hinders their hydrogen production rates under intense light irradiation. In order to mitigate this effect and improve hydrogen production efficiency, it is necessary to screen mutants that hold less pigment content. In this study, a mini-Tn5 transposon encoded plasmid pRL27 was employed for transposon mutagenesis of Rhodobacter capsulatus SB1003. A mutant named MC1417 showed significant lower light absorbance from 330 nm to 900 nm compared to its parental strain by UV–visible spectra, and its bacteriochlorophyll a content was reduced by 38%. The results showed that its photo fermentative hydrogen production was improved by 50.5% on the basis of BChl a content using acetate and butyrate as carbon source under intense light irradiation, indicating that it is effective on improving hydrogen production by repressing the pigment biosynthesis. DNA sequencing and BLAST in NCBI Genebank showed that the mutation occurred within its pucDE gene.     

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).     

Concentration-dependent effects of metronidazole,inhibiting nitrogenase,on hydrogen photoproduction and proton-translocating ATPase activity of Rhodobacter sphaeroides

Rhodobacter sphaeroides MDC6522 is able to produce hydrogen (H₂) during photofermentation. Metronidazole (2-methyl-5-nitroimidazole-1-ethanol) has been shown to affect bacterial growth under anaerobic nitrogen-limited conditions in a concentration-dependent manner (within the range of 0.1–2 mM) by increasing lag phase duration and decreasing growth rate. The addition of metronidazole into the growth medium resulted in a delayed decrease of redox potential (E_h): by the addition of 0.1 mM metronidazole E_h decreased to −590 ± 25 mV, whereas in the presence of 2 mM E_h drop down was to −175 ± 15 mV. H₂ production during R. sphaeroides growth disappeared in the presence of metronidazole. By addition of 0.5 mM metronidazole H₂ yield was ∼8 fold lower in comparison with control; whereas the bacterium was unable to produce H₂ in the presence of 1–2 mM. The effects of metronidazole on nitrogenase-dependent H₂ production by R. sphaeroides might be explained by change of general photosynthetic electron transport with metronidazole as an alternative electron acceptor instead of nitrogenase. ATPase activity of membrane vesicles was determined with and without N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F₀F₁-ATPase. It was revealed that DCCD-inhibited ATPase activity increased in the presence of metronidazole. It is possible that this effect may be resulted in either by direct affect of metronidazole on the F₀F₁-ATPase, or by its effect on E_h regulating ATPase activity.     

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.     

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.     

Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal biomass as feedstock

In this work, a carbohydrate-rich microalga, Chlorella vulgaris ESP6, was grown photoautotrophically to fix the CO₂. The resulting microalgal biomass was hydrolyzed by acid or alkaline/enzymatic treatment and was then used for biohydrogen production with Clostridium butyricum CGS5. The C. vulgaris biomass could be effectively hydrolyzed by acid pretreatment while similar hydrolysis efficiency was achieved by combination of alkaline pretreatment and enzymatic hydrolysis. The biomass of C. vulgaris ESP6 containing a carbohydrate content of 57% (dry weight basis) was efficiently hydrolyzed by acid treatment with 1.5% HCl, giving a reducing sugars (RS) yield of nearly 100%. C. butyricum CGS5 could utilize RS from C. vulgaris ESP6 biomass to produce hydrogen without any additional organic carbon sources. The optimal conditions for hydrogen production were 37 °C and a microalgal hydrolysate loading of 9 g RS/L with pH-controlled at 5.5. Under the optimal conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1476 ml/L, 246 ml/L/h, and 1.15 mol/mol RS, respectively. The results demonstrate that the C. vulgaris biomass has the potential to serve as effective feedstock for dark fermentative H₂ production.     

Hydrogen production performance by Enterobacter cloacae KBH3 isolated from termite guts

Two out of six bacterial isolates obtained from the guts of Globitermes sp. termites were identified as hydrogen-producing bacteria. One isolate, Enterobacter cloacae KBH3, was characterised using the BIOLOG identification system and 16S rRNA gene analysis. In a batch fermentation study to evaluate its growth in defined medium, E. cloacae KBH3 produced 154 ml H₂ per litre medium with approximately 50% hydrogen content. The carbon utilisation results suggest that E. cloacae KBH3 have the potential to be a good hydrogen producer. This strain is also able to produce hydrogen within a wide range of temperatures (28–40 °C) and pH (4.5–8). In several fermentation runs, the pH of the culture dropped from 6.5 to 5.36 within the first 3 h, which was mostly due to the biosynthesis of formate. An increase of cumulative hydrogen production was recorded as well as a decrease in the concentration of formate, indicating the importance of the formate pathway for hydrogen production. The highest rate of hydrogen production of 180.74 ml H₂/l/h was achieved when lactate and acetate were at their highest concentrations. Most of the hydrogen gas was produced during the exponential growth phase, and the biogas continued to be produced during the stationary phase. The specific growth rate was calculated to be 0.224 per hour while the hydrogen yield was 1.8 mol of hydrogen per mol of glucose. At the end of the batch study, the highest cumulative hydrogen production was 2404 ml H₂ per litre of fermentation medium.     

Optimization of photo-hydrogen production based on cheese whey spent medium

Cheese whey wastewater diluted to 10 g lactose/L was initially subjected to dark-fermentation by Enterobacter aerogenes MTCC 2822, and the VFAs-rich spent medium (acetic acid 1900 mg/L, butyric acid 537 mg/L, and traces of propionic acid) was subjected to photo-fermentation through enrichment by Ni²⁺ (0–8 μmol/L), Fe²⁺ (0–100 μmol/L) or Mg²⁺ (0–15 mmol/L) in batch mode by Rhodopseudomonas BHU 01 strain. The maximum cumulative H₂ production (144 ml) and yield (58 mmol) was obtained at 4 μmol Ni²⁺/L. Likewise, Fe²⁺ (60 μmol/L) resulted in maximum cumulative H₂ production (139 ml) and yield (56 mmol). Nevertheless, 6 mmol of Mg²⁺ did not significantly affect H₂ production (110 ml) or yield (44 mmol); the latter value in close proximity with the control (37 mmol). The concomitant reduction in COD was maximum (15.61%) for 4 μmol Ni²⁺/L, followed by 15.33% for 60 μmol Fe²⁺/L, and the least for 6 mmol Mg²⁺/L (14.5%). The observations suggest the role of Fe²⁺ and Ni²⁺ in regulation of nitrogenase and hydrogenase, while that of Mg²⁺ mainly in the biosynthesis of photopigment bacteriochlorophyll (Bchl).