Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal

Pure culture of Rhodobacter sphaeroides (NRRL- B1727) was used for continuous photo-fermentation of volatile fatty acids (VFA) present in the dark fermentation effluent of ground wheat starch. The feed contained 1950 ± 50 mg L⁻¹ total VFA with some nutrient supplementation. Hydraulic residence time (HRT) was varied between 24 and 120 hours. The highest steady-state daily hydrogen production (55 ml d⁻¹) and hydrogen yield (185 ml H₂ g⁻¹ VFA) were obtained at HRT = 72 hours (3 days). Biomass concentration increased with increasing HRT. Volumetric and specific hydrogen formation rates were also maximum at HRT = 72 h. High extent of TVFA fermentation at HRT = 72 h resulted in high hydrogen gas production.     

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.     

Bio-hydrogen production and the F0F1-ATPase activity of Rhodobacter sphaeroides: Effects of various heavy metal ions

Rhodobacter sphaeroides MDC 6521 isolated from Arzni mineral springs in Armenia is able to produce bio-hydrogen (H₂) in anaerobic conditions upon illumination in the presence of various metal ions. The significant aspect in regulation of H₂ production by these bacteria and its energetics is the requirement for F₀F₁-ATPase, the main membrane enzyme responsible for generation of proton motive force under anaerobic conditions. In order to determine the mediatory role of F₀F₁ in H₂ production, the effects of various metal ions (Mn²⁺, Mg²⁺, Fe²⁺, Ni²⁺, and Mo⁶⁺) on N,N′-dicyclohexylcarbodiimide inhibited ATPase activity of R. sphaeroides membrane vesicles were investigated. These ions in appropriate concentrations considerably enhanced H₂ production, which was not observed in the absence of Fe²⁺, indicate the requirement for Fe²⁺. The R. sphaeroides membrane vesicles demonstrated significant ATPase activity. In the absence of Fe²⁺ inhibition (∼80%) of ATPase activity was observed, which was increased by addition of metal ions. A higher ATPase activity was detected in the presence of Fe²⁺ (80 μM) and Mo⁶⁺ (16 μM). These results indicate a relationship between the F₀F₁-ATPase activity and H₂ production that might be a significant pathway to provide novel evidence of a requirement for F₀F₁-ATPase in H₂ production by R. sphaeroides.     

Ni (II) and Mg (II) ions as factors enhancing biohydrogen production by Rhodobacter sphaeroides from mineral springs

Various metal ions play a key role in biohydrogen (H₂) production by phototrophic bacteria through incorporation into or stimulating the responsible enzymes and/or related pathways. The Ni (II) and Mg (II) ions effects on growth and H₂ production by Rhodobacter sphaeroides strain MDC6521 isolated from mineral springs in Armenia were established. The highest growth specific rate was obtained with 4–6 μM Ni²⁺ and 5 mM Mg²⁺. pH of the growth medium changed from 7.0 to 9.2–9.4 during the bacterial growth up to 72 h in spite of Ni²⁺ added but pH increased in different manner with Mg²⁺. In the presence of 2–4 μM Ni²⁺ external oxidation-reduction potential (ORP) decreased to more negative values (−800 ± 15 mV). This decrease of ORP indicated ∼2.7-fold enhanced H₂ yield (9.80 mmol L⁻¹) with Ni²⁺ compared with the control (without Ni²⁺). The H₂ yield determined in the medium with Mg²⁺ was ∼2.2 fold higher than that with 1 mM Mg²⁺. These results reveal new regulatory ways to improve H₂ production by R. sphaeroides those were depending on Ni²⁺ and Mg²⁺ of different concentrations.     

Yeast extract as an effective nitrogen source stimulating cell growth and enhancing hydrogen photoproduction by Rhodobacter sphaeroides strains from mineral springs

The suitability and limitation of yeast extract as nitrogen source to support cell growth and to enhance hydrogen photoproduction by Rhodobacter sphaeroides strains MDC6521 and MDC6522 isolated from mineral springs in Armenia was investigated during the anaerobic growth. Yeast extract (2 g L⁻¹) was indicated to be an effective nitrogen source for bacterial cell growth stimulation and enhanced H₂ production (compared to glutamate). Both strains followed similar growth patterns in medium with yeast extract as nitrogen source and succinate or malate as carbon source. The highest growth rate was obtained for bacterial cells with yeast extract: the latter added gave a stimulated (2–3.5 fold) growth rate than using glutamate. R. sphaeroides suspension oxidation–reduction potential (ORP), which was measured with a platinum electrode, decreased down to low negative values with nitrogen source for both strains. ORP decreased down to more negative values (−610 ± 25 mV) in the presence of yeast extract than when adding glutamate (−405 ± 15 mV) compared to the control (without nitrogen source addition): the significant decrease of ORP indicated enhanced (∼6 fold) H₂ yield. The noticeable ORP decrease measured with the titanium-silicate electrode and simultaneously the increase of extracellular pH ([pH]_out) were observed; ORP was more negative at alkaline [pH]_out. Thus, the optimal culture conditions with nitrogen and carbon sources for bacterial growth stimulation and enhanced H₂ production were established. The ORP decrease together with the increase of [pH]_out point out a significant role of reduction processes in cell growth and ability of bacteria to live.     

Kinetic analysis of photosynthetic growth,hydrogen production and dual substrate utilization by Rhodobacter capsulatus

Rhodobacter capsulatus is purple non-sulfur (PNS) bacterium which can produce hydrogen and CO₂ by utilizing volatile organic acids in presence of light under anaerobic conditions. Photofermentation by PNS bacteria is strongly affected by temperature and light intensity. In the present study we present the kinetic analysis of growth, hydrogen production, and dual consumption of acetic acid and lactic acid at different temperatures (20, 30 and 38 °C) and light intensities (1500, 2000, 3000, 4000 and 5000 lux). The cell growth data fitted well to the logistic model and the cumulative hydrogen production data fitted well to the Modified Gompertz Model. The model parameters were affected by temperature and light intensity. Lactic acid was found to be consumed by first order kinetics. Rate of consumption of acetic acid was zero order until most of the lactic acid was consumed, and then it shifted to first order. The results revealed that the optimum light intensities for maximum hydrogen production were 5000 lux for 20 °C and 3000 lux for 30 °C and 38 °C.     

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.     

Effect of physico-chemical parameters on biohydrogen production and growth characteristics by batch culture of Rhodobacter sphaeroides CIP 60.6

In this paper, Rhodobacter sphaeroides CIP 60.6 strain was newly used for the biohydrogen production in a perfectly shaken column photobioreactor, grown in batch culture under anaerobic and illumination conditions, to investigate the effects of some physico-chemical parameters in microbial hydrogen photofermentation. Luedeking–Piret model was considered for the data fitting to find out the mode of hydrogen generation and the relationship between the cell growth and hydrogen production. The results show that, both growth cells and resting cells can produce hydrogen at light intensities greater or equal to 2500 lux, however, at the weak intensities hydrogen is a metabolite associated to growth. Growth rate and hydrogen production rate increase with the increasing of light intensity. Moreover, hydrogen production rate become higher in stationary phase than that in logarithmic phase, with the enhancement of light intensity. Maximum hydrogen production rate obtained was 39.88 ± 0.14 ml/l/h, at the optimal conditions (4500–8500 lux). Modified Gompertz equation was applied for the data fitting to verify the accuracy and the agreement of the model with experimental results. It is revealed that, in the modified Gompertz equation, the lag time represents time for which hydrogen production becomes maximal, not the beginning time of hydrogen production. The stop of stirring reduced hydrogen production rate and created unstable hydrogen production in reactor. The pH ranges of 7.5 ± 0.1 were the favorable pH for hydrogen production.