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.     

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.     

Biohydrogen production by Rhodobacter capsulatus Hup mutant in pilot solar tubular photobioreactor

In this study, a pilot solar tubular photobioreactor was successfully implemented for fed batch operation in outdoor conditions for photofermentative hydrogen production with Rhodobacter capsulatus (Hup⁻) mutant. The bacteria had a rapid growth with a specific growth rate of 0.052 h⁻¹ in the batch exponential phase and cell dry weight remained in the range of 1–1.5 g/L throughout the fed batch operation. The feeding strategy was to keep acetic acid concentration in the photobioreactor at the range of 20 mM by adjusting feed acetate concentration. The maximum molar productivity obtained was 0.40 mol H₂/(m³ h) and the yield obtained was 0.35 mol H₂ per mole of acetic acid fed. Evolved gas contained 95–99% hydrogen and the rest was carbon dioxide by volume.     

Clostridium strain co-cultures for biohydrogen production enhancement from condensed molasses fermentation solubles

An anaerobic continuous-flow hydrogen fermentor was operated at a hydraulic retention time of 8 h using condensed molasses fermentation solubles (CMS) substrate of 40 g-COD/L. Serum bottles were used for seed micro-flora cultivation and batch hydrogen fermentation tests (CMS substrate concentrations of 10–160 g-COD/L). Three hydrogen-producing bacterial strains Clostridium sporosphaeroides F52, Clostridium tyrobutyricum F4 and Clostridium pasteurianum F40 were isolated from the seed fermentor and used as the seeding microbes in single and mixed-culture cultivations for determining their hydrogen productivity. These strains possessed specific hydrogenase genes that could be detected from CMS-fed hydrogen fermentors and were major hydrogen producers. C. pasteurianum F40 was the dominant strain with a high hydrogen production rate while C. sporosphaeroides F52 may play a main role in degrading carbohydrate and glutamate. These strains could be co-cultivated as a symbiotic mixed-culture process to enhance hydrogen productivity. C. pasteurianum F40 or C. tyrobutyricum F4 co-culture with the glutamate-utilizing strain C. sporosphaeroides F52 efficiently enhanced hydrogen production by 12–220% depending on the substrate CMS concentrations.     

Aspects of the metabolism of hydrogen production by Rhodobacter sphaeroides

Photosynthetic bacteria are favorable candidates for biological hydrogen production due to their high conversion efficiency and versatility in the substrates they can utilize. For large-scale hydrogen production, an integrated view of the overall metabolism is necessary in order to interpret results properly and facilitate experimental design. In this study, a summary of the hydrogen production metabolism of the photosynthetic purple non-sulfur (PNS) bacteria will be presented.Practically all hydrogen production by PNS bacteria occurs under a photoheterotrophic mode of metabolism. Yet results show that under certain conditions, alternative modes of metabolism—e.g. fermentation under light deficiency—are also possible and should be considered in experimental design.Two enzymes are especially critical for hydrogen production. Nitrogenase promotes hydrogen production and uptake hydrogenase consumes hydrogen.Though a wide variety of substrates can be used for growth, only a portion of these is suitable for hydrogen production. The efficiency of a certain substrate depends on factors such as the activity of the TCA cycle, the carbon-to-nitrogen ratio, the reduction-state of that material and the conversion potential of the substrate into alternative metabolites such as PHB.All these individual components of the hydrogen production interact and are subject to strict regulatory controls. An overall scheme for the hydrogen production metabolism is presented.     

Disruption of multidrug resistance protein gene of Rhodobacter capsulatus results in improved photoheterotrophic hydrogen production

Rhodobacter capsulatus (R. capsulatus), which is a typical purple nonsulfur photosynthetic bacterium, is able to produce hydrogen under photosynthetic condition. A mutant of R. capsulatus named MC122 was obtained by Tn5 transposon mutagenesis. The transposon mutant had improved photoheterotrophic hydrogen production performance using acetic acid as substrate and its mutation site was located by sequencing the rescued plasmid containing the transposon insertion from the genome of the mutant. It was found for the first time that disruption of the multidrug resistance protein (mdtB) gene resulted in improved hydrogen production.     

State estimation of a batch hydrogen production process using the photosynthetic bacteria Rhodobacter capsulatus

This paper addresses the problem of estimating the states of an anaerobic photosynthetic process used for biohydrogen production by the photosynthetic bacterium Rhodobacter capsulatus. The process is described by a non-linear, time-discrete model and the state estimation is solved using an observer based on the Moving-Horizon State Estimation Method (MHSE). This approach is based on the minimization of a criterion (a non-linear function), in this case, the difference between the estimated output and the measured output of the system over a considered time horizon, where the solution is computed by using a numerical interval method. The observer was successfully applied to hydrogen production by R. capsulatus strain B10 in a batch process.     

Significance of carbon to nitrogen ratio on the long-term stability of continuous photofermentative hydrogen production

The stable and optimized operation of photobioreactors (PBRs) is the most challenging task in photofermentative biological hydrogen production. The carbon to nitrogen ratio (C/N) in the feed is a critical parameter that significantly influences microbial growth and hydrogen production. In this study, the effects of changing the C/N ratio to achieve stable biomass and continuous hydrogen production using fed-batch cultures of Rhodobacter capsulatus YO3 (uptake hydrogenase deleted, hup-) were investigated. The experiments were carried out in 8 L panel PBRs operated in indoor conditions under continuous illumination and controlled temperature. Culture media containing different acetate (40-80 mM) and glutamate (2-4 mM) concentrations were used to study the effects of changing the C/N ratio on biomass growth and hydrogen production. Stable biomass concentration of 0.40 g dry cell weight per liter culture (gDCW/L_c) and maximum hydrogen productivity of 0.66 mmol hydrogen per liter culture per hour (mmol/L_c/h) were achieved during fed-batch operation with media containing 40 mM acetate and 4 mM glutamate, C/N = 25, for a period of over 20 days. A study on the effect of biomass recycling on biomass growth and hydrogen production showed that the feedback of cells into the photobioreactor improved biomass stability during the fed-batch operation but decreased hydrogen productivity.     

Enhancing photo-fermentative hydrogen production by Rhodobacter sphaeroides KD131 and its PHB synthase deleted-mutant from acetate and butyrate

Purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides KD131 wild type (wt) and its PHB synthase deleted-mutant P1 were evaluated for hydrogen (H₂) production from acetate and butyrate, the most abundant liquid end products of dark fermentation. In the presence of glutamate (8 mM), 60 mM of acetate and 30 mM of butyrate were degraded down to 41.5% and 24.0%, respectively, and achieved a H₂ yield (HY) of 0.65 mol H₂/mol acetate- and 2.50 mol H₂/mol butyrate-consumed, while 30 mM succinate exhibited an HY of 3.29 mol H₂/mol substrate-consumed. The order of HY observed was inversely related to poly-(3-hydroxybutyrate) (PHB) content and pH increase in the broth. When mutant P1 was used, in spite of depressed cell growth and lower substrate degradation compared to those observed in strain KD131 wt, higher H₂ production was observed, achieving around two-fold increase of HY in both acetate and butyrate. A pH control to 7.0 during fermentation was effective in increasing substrate degradation and decreasing PHB content, thereby significantly increasing H₂ production. When pH was controlled to 7.0, strain KD131 wt evolved more H₂ by 2.36 and 1.70 folds in the acetate- and succinate-medium, respectively, compared to those observed in without pH control. The highest H₂ production was observed when the mutant P1 was photo-fermented with a pH control to 7.0 in the medium containing acetate-(NH₄)₂SO₄. It seemed that pH control had an effect not only on the depressed production of PHB but also on soluble microbial products and secondary metabolites, which would compete with H₂ production in expending reducing power.     

Design of a new biosensor for algal H2 production based on the H2-sensing system of Rhodobacter capsulatus

The H₂-sensing system of Rhodobacter capsulatus was engineered to elicit a fluorescent response upon cell exposure to H₂. The system is surprisingly sensitive to H₂ and is capable of detecting levels of H₂ down to 200 pM in solution, which approximates the background concentration of H₂ in water exposed to the earth’s atmosphere. The response was roughly linear between 0.3 and 300 ppm V of added headspace H₂ and gave a K_app of 142 nM H_2, when cells were grown anaerobically for 12 h in the presence of H₂. Hydrogen-sensing R. capsulatus cells were grown fermentatively in the dark in co-culture with Chlamydomonas reinhardtii on microtiter plates and the bacteria fluoresced in proportion to H₂ production by the algae. This represents a promising, high-throughput assay for H₂ production in algal libraries, and an enhanced capability for developing H₂ as a clean and renewable fuel.     

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.     

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.     

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.     

Development of a PCR assay for the detection of nifH and nifD genes in indigenous photosynthetic bacteria

Molybdenum (Mo) nitrogenases consist of two components: dinitrogenase reductase (encoded by nifH) and the dinitrogenase or MoFe protein (encoded by nifDK). Nitrogenase enzyme of photosynthetic bacteria is responsible for hydrogen production. Therefore, primers were designed for the nitrogenase gene only. In this study, two primers (ND and NH) were designed after comparative genomic analysis of nifH and nifD gene sequences from public databases. The designed primers were used for the amplification of nifH and nifD genes to detect nitrogenase genes in photosynthetic bacteria. Initial detection was done using a monoplex Polymerase Chain Reactions (PCRs) followed by optimization of the PCR protocols. Subsequently, a duplex PCR was designed for amplification and detection of nifH and nifD genes in indigenous photosynthetic bacteria. Evaluation of the duplex PCR on six samples isolated from Palm Oil Mill Effluent (POME) showed that only four isolates contained both the nifH and nifD genes, indicating that these isolates were potential hydrogen-producing bacteria. PCR detection provides a rapid and efficient pre-identification of potential photosynthetic bacterial hydrogen producers.     

Light shade bands for the improvement of solar hydrogen production efficiency by Rhodobacter sphaeroides RV

At mid-day, the sunlight provides excessive energy for photo-hydrogen production by photosynthetic bacteria. Conversion efficiency from light energy to hydrogen decreased under high illumination. To overcome this problem, we examined a method to spatial dispersion of the high illumination. The new photobioreactor using the light shade bands set on the surface of the reactor was developed for efficient hydrogen production. Spatial dispersion gave remarkable effect on conversion efficiency under the excessive light condition. Indoors, the 1.0 cm width of light shade bands gave the best conversion efficiency (2.1%). Actual use of the sunlight, the 1.5 cm width of light shade bands provided the best conversion efficiency (1.4%). Light inhibition was successfully suppressed by the light shade bands in both experiments. The dispersed light energy could be used for other energy conversion device as solar cells.     

Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16

Lignocellulosic biomass, if properly hydrolyzed, can be an ideal feedstock for fermentative hydrogen production. This work considered the pretreatment of corn stover (CS) using a dilute acid hydrolysis process and studied its fermentability for hydrogen production by the strain Thermoanaerobacterium thermosaccharolyticum W16. The effects of sulfuric acid concentration and reaction time in the hydrolysis stage of the process were determined based on a 2² central composite experimental design with respect to maximum hydrogen productivity. The optimal hydrolysis conditions to yield the maximum quantity of hydrogen by W16 were 1.69% sulfuric acid and 117 min reaction time. At these conditions, the hydrogen yield was shown to be 3305 ml H₂ L⁻¹ medium, which corresponds to 2.24 mol H₂ mol⁻¹ sugar. The present results indicate the potential of using T. thermosaccharolyticum W16 for high-yield conversion of CS hemicellulose into bio-H₂ integrated with acid hydrolysis.     

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.