Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides

Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides was studied in batch experiments. Results showed that in all batches hydrogen was produced after a lag phase of about 10 h; pure culture of R. sphaeroides produced hydrogen at rates substantially lower than C. butyricum. In co-culture systems, R. sphaeroides even with cell populations 5.9 times higher still could not compete with C. butyricum for glucose. In co-culture systems, R. sphaeroides syntrophically interacted with C. butyricum, using the acetate and butyrate produced by the latter as substrate for hydrogen production. Hydrogen production was ceased in all batches when the pH was lowered to the level of pH 6.5, resulting from the accumulation of fatty acids. It was also demonstrated in this study that fluorescence in situ hybridization (FISH) was an effective means for the quantification of the relative abundance of individual bacteria in a co-culture system.     

Photo-fermentative hydrogen gas production from dark fermentation effluent of acid hydrolyzed wheat starch with periodic feeding

Hydrogen gas production by photo-fermentation of dark fermentation effluent of acid hydrolyzed wheat starch was investigated at different hydraulic residence times (HRT = 1-10 days). Pure Rhodobacter sphaeroides (NRRL B-1727) culture was used in continuous photo-fermentation by periodic feeding and effluent removal. The highest daily hydrogen gas production (85 ml d⁻¹) was obtained at HRT = 4 days (96 h) while the highest hydrogen yield (1200 ml H₂ g⁻¹ TVFA) was realized at HRT = 196 h. Specific and volumetric hydrogen formation rates were also the highest at HRT = 96 h. Steady-state biomass concentrations and biomass yields increased with increasing HRT. TVFA loading rates of 0.32 g L⁻¹ d⁻¹ and 0.51 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate, respectively. Hydrogen gas yield obtained in this study compares favorably with the relevant literature reports probably due to operation by periodic feeding and effluent removal.     

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.     

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.     

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.     

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.     

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.     

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.     

Efficient hydrogen gas production from cassava and food waste by a two-step process of dark fermentation and photo-fermentation

A two-step process of sequential anaerobic (dark) and photo-heterotrophic fermentation was employed to produce hydrogen from cassava and food waste. In dark fermentation, the average yield of hydrogen was approximately 199 ml H₂ g⁻¹ cassava and 220 ml H₂ g⁻¹ food waste. In subsequent photo-fermentation, the average yield of hydrogen from the effluent of dark fermentation was approximately 611 ml H₂ g⁻¹ cassava and 451 ml H₂ g⁻¹ food waste. The total hydrogen yield in the two-step process was estimated as 810 ml H₂ g⁻¹ cassava and 671 ml H₂ g⁻¹ food waste. Meanwhile, the COD decreased greatly with a removal efficiency of 84.3% in cassava batch and 80.2% in food waste batch. These results demonstrate that cassava and food waste could be ideal substrates for bio-hydrogen production. And a two-step process combining dark fermentation and photo-fermentation was highly improving both bio-hydrogen production and removal of substrates and fatty acids.