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.     

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.     

Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710

Photofermentative hydrogen production is influenced by several parameters, including feed composition, pH levels, temperature and light intensity. In this study, experimental results obtained from batch cultures of Rhodobacter capsulatus DSM 1710 were analyzed to locate the maximum levels for the rate and yield of hydrogen production with respect to temperature and light intensity. For this purpose, a 3^k general full factorial design was employed, using temperatures of 20, 30 and 38 °C and light intensities of 100, 200 and 340 W/m². ANOVA results confirmed that these two parameters significantly affect hydrogen production. Surface and contour plots of the regression models revealed a maximum hydrogen production rate of 0.566 mmol H₂/L/h at 27.5 °C and 287 W/m² and a maximum hydrogen yield of 0.326 mol H₂/mol substrate at 26.8 °C and 285 W/m². Validation experiments at the calculated optima supported these findings.     

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.     

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.     

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.     

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.     

Effect of changes in the level of light harvesting complexes of Rhodobacter sphaeroides on the photoheterotrophic production of hydrogen

Increase in levels of photosynthetic spectral complexes by maintaining the plasmids harboring DNA encoding puhA, pufBA, or pucBAC in trans in Rhodobacter sphaeroides resulted in decrease of the photoheterotrophic production of H₂. However, removal of B875 or B800–850 light-harvesting (LH) complexes affected H₂ production differently. Lack of B875 complex following in-frame deletion of pufBA (mutant PUF1) not only slowed photoheterotrophic growth but also decreased H₂ production, indicative of the essential requirement of the complex for LH process. However, the pucBA-deleted mutant, PUC1 lacking of B800–850 complex, increased H₂ production in comparison with its parental cell by approximately twofold, given irradiated with light (10 W/m²) saturating the growth of wild type. The H₂ production of PUC1 did not increase in proportion to the light intensity, which is also observed with wild type. Thus, we suggest that light is not limited for the H₂ production of the cells under the experimental conditions employed in this work, but the cellular energy to be used for the formation of B800–850 complex may flow into the metabolism leading to the H₂ production in PUC1.     

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.     

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.     

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.     

Evaluation of hydrogen production by Rhodobacter sphaeroides O.U.001 and its hupSL deficient mutant using acetate and malate as carbon sources

Rhodobacter sphaeroides O.U.001 is one of the candidates for photobiological hydrogen production among purple non-sulfur bacteria. Hydrogen is produced by Mo-nitrogenase from organic acids such as malate or lactate. A hupSL in frame deletion mutant strain was constructed without using any antibiotic resistance gene. The hydrogen production potential of the R. sphaeroides O.U.001 and its newly constructed hupSL deleted mutant strain in acetate media was evaluated and compared with malate containing media. The hupSL⁻R. sphaeroides produced 2.42 l H₂/l culture and 0.25 l H₂/l culture in 15 mM malate and 30 mM acetate containing media, respectively, as compared to the wild type cells which evolved 1.97 l H₂/l culture and 0.21 l H₂/l culture in malate and acetate containing media, correspondingly. According to the results, hupSL⁻R. sphaeroides is a better hydrogen producer but acetate alone does not seem to be an efficient carbon source for photoheterotrophic H₂ production by R. sphaeroides.     

Expression of aldehyde dehydrogenase gene increases hydrogen production from low concentration of acetate by Rhodobacter sphaeroides

Rhodobacter sphaeroides RV (RV) is a hydrogen-producing bacterium exhibiting the highest yield of hydrogen production from organic acids such as lactate and acetate, which are the byproducts of hydrogen fermentation by hydrogen-producing anaerobic bacteria. Co-fermentation of the RV strain with anaerobic bacteria is an efficient method of hydrogen production. However, less than 21 mM acetate is produced by the anaerobic bacteria, which is too low for efficient hydrogen production by the RV strain; it requires approximately 75 mM acetate. In this study, 2 distinct isozymes of aldehyde dehydrogenase from Rhodospirillum rubrum were separately overexpressed in the RV strain. The recombinant RV strains that were designated as RVAD1 and RVAD2, exhibited 13-fold higher ALDH activities than the wild-type RV strain. Hydrogen yields of both of the recombinant strains were 1.4-fold higher than that of the RV strain in 21 mM acetate. In 43 mM acetate, the RVAD1 strain showed higher yield, though the RVAD2 strain showed lower yield as compared to that of the RV strain. In 64 mM acetate and all concentrations of lactate tested (21, 43 and 64 mM), the yields of the recombinant strains were lower than those of the RV strain. The intact (empty) expression plasmid increased the ALDH activity and had little effect on the hydrogen production in acetate, however, it decreased the production in lactate. At the beginning of the fermentation process, when very little hydrogen had been produced, the recombinant strains expressing the ALDH gene consumed smaller amounts of acetate compared to the wild-type strain. We have discussed the effects of ALDH on hydrogen production in this report.     

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.