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.     

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.     

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.     

High NH3N tolerance of a cheR2-deletion Rhodobacter capsulatus mutant for photo-fermentative hydrogen production using cornstalk

A mutant, JL1601, with a cheR2 gene deletion was constructed with Rhodobacter capsulatus JL1 as the wild type strain. The effects of pH, ammonia nitrogen concentration and light intensity on the photo-fermentative hydrogen production performance of JL1601 were studied. Suitable pH range for hydrogen production with the cheR2^- mutant migrated from faintly acid to weak alkaline condition, and R.capsulatus JL1601 (cheR2^-) showed higher NH₃N tolerance. The mutant performed better H₂ production performance than the wildtype under the same illumination condition. The maximum H₂ yield and rate of R.capsulatus JL1601 (cheR2^-) obtained to be 4406.7 ± 45.9 mL/L and 54.1 ± 1.6 mL/(L·h) with the mixture of acetate and butyrate as the carbon sources, which were increased by 36.3% and 12.3% in comparison to those of wildtype, respectively. Practical experiments using acid-enzymatic pretreated cornstalk as the carbon source indicated that, the maximum hydrogen yields by the mutant JL1601 (cheR2^-) was up to 224.85 ± 5.18 mL-H₂/g-cornstalk, which increased by 171.43% compared with that of wild type strain.     

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.     

Effect of changing temperature on anaerobic hydrogen production and microbial community composition in an open-mixed culture bioreactor

The temperature effect (37–65 °C) on H₂ production from glucose in an open-mixed culture bioreactor using an enrichment culture from a hot spring was studied. The dynamics of microbial communities was investigated by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). At 45 and 60 °C the H₂ production was the highest i.e. 1.71 and 0.85 mol H₂/mol glucose, respectively. No H₂ was produced at temperatures 50 and 55 °C. At 37–45 °C, H₂ production was produced by butyrate type fermentation while fermentation mechanism changed to ethanol type at 60 °C. Clostridium species were dominant at 37–45 °C while at 50–55 °C and 60 °C the culture was dominated by Bacillus coagulans and Thermoanaerobacterium, respectively. In the presence of B. Coagulans the metabolism was directed to lactate production. The results show that the mixed culture had two optima for H₂ production and that the microbial communities and metabolic patterns promptly changed according to changing temperatures.     

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.     

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.     

Increased hydrogen photoproduction by means of a sulfur-deprived Chlamydomonas reinhardtii D1 protein mutant

The photoproduction of H₂ was studied in a sulfur-deprived Chlamydomonas reinhardtii D1 mutant that carried a double amino acid substitution. The leucine residue L159 was replaced by isoleucine, and the asparagine N230 was replaced by tyrosine (L159I-N230Y). Phenotypic characterization of the mutant showed some interesting features compared to its wild type, namely: (1) a lower chlorophyll content; (2) a higher photosynthetic capacity and higher relative quantum yield of photosynthesis; (3) a higher respiration rate; (4) a very high conversion of violaxanthin to zeaxanthin during H₂ production; (5) a prolonged period of H₂ production. In standard conditions, the mutant produced more than 500 ml of H₂, that is, more than one order of magnitude greater than its wild type, and about 5-times greater than the CC124 strain that was used for comparison. The better performance of the mutant was mainly the result of a longer production period. Biogas produced contained up to 99.5% H₂.     

Photoheterotrophic growth of purple non-sulfur bacteria on Tris Acetate Phosphate Yeast extract (TAPY) medium and its hydrogen productivity in light under nitrogen deprivation

In this study, an active photoheterotrophic growth of purple non-sulfur bacteria (PNSB) on Tris Acetate Phosphate Yeast extract (TAPY) medium is reported. TAPY medium is a modified TAP medium supplemented with filter sterilized yeast extract (0.3 g L⁻¹) and vitamin B12. Heterotrophic growth of PNSB on nitrogen replete TAPY medium in dark could be obtained but much slower than that in light where cells could grow but without formation of pigments. The medium showed high potency for hydrogen production under nitrogen deprivation in light by Rhodobacter sphaeroides DSM 5864. Through using TAPY medium, 1 mole of acetate provided as glacial acetic acid produced 0.819 mole of hydrogen gas by R. sphaeroides on nitrogen deprived TAPY medium (initial pH 7 adjusted by HCl) with a maximum H₂ production rate of (0.669 mmol H₂ h⁻¹ L⁻¹) obtained at 42 h after start of fermentation. Repeated-batch hydrogen production could be achieved with high efficiency for three cycles by supplementing the nitrogen deprived culture with filter sterilized sodium acetate at the end of the log hydrogen production phase of each cycle. The medium was also applicable for photoheterotrophic growth in light and heterotrophic growth in dark of other PNSB namely Rhodobacter capsulatus JCM-21090 and Rhodospirillum rubrum DSM 467. Although R. rubrum could actively grow on TAPY medium under nitrogen deprivation in light, it was the lowest in hydrogen production compared to R. sphaeroides and R. capsulatus. The active growth of R. rubrum on nitrogen deprived TAPY medium suggests its possible use for producing biodegradable plastic polymers better than hydrogen. The results suggest that it's possible to use TAPY medium for growth and producing variable bioproducts by PNSB in future studies and applications. The repeated-batch hydrogen production by R. sphaeroides on nitrogen deprived TAPY medium is promising for large-scale applications of hydrogen production industry by only sequential supply with sodium acetate to the culture for three rounds of batch fermentation. This is the first report in using TAPY medium for growth and hydrogen production by PNSB.     

Impairment of NADH dehydrogenase for increased hydrogen production and its effect on metabolic flux redistribution in wild strain and mutants of Enterobacter aerogenes

An NADH dehydrogenase encoded by the nuo cluster was isolated and impaired by knocking out the nuoB gene in Enterobacter aerogenes to examine its effect on hydrogen production. Three nuoB-deleted mutant strains were constructed from the wild-type strain E. aerogenes IAM1183 and two recombinant strains, IAM1183-A (ΔhycA) and IAM1183-O (ΔhybO), from which the hycA and hybO genes had already been deleted previously, respectively. Compared with the performance of the wild-type strain, the overall hydrogen production of the mutants IAM1183-B (ΔnuoB), IAM1183-AB (ΔhycA/ΔnuoB) and IAM1183-BO (ΔhybO/ΔnuoB) was increased by 49.2%, 54.0%, and 52.4% in batch culture, respectively. The hydrogen yields from glucose by the three mutants IAM1183-B, IAM1183-AB, IAM1183-BO were 1.38, 1.49, and 1.39 mol H₂/mol glucose, respectively, while it was 1.16 mol H₂/mol glucose in the wild-type strain. Metabolic flux analysis indicated that all three mutants exhibited reduced fluxes to lactate production, and enhanced fluxes toward the generation of hydrogen, acetate, ethanol, succinate and 2,3-butanediol. Both the formate pathway and the NADH pathway contributed to increased hydrogen production in the mutant strains. The assay of 4 NADH-mediated enzyme activities (H₂ase, LDH, ADH and BDDH) was in accordance with the redistributions of the metabolic fluxes in the mutant strains.     

Escherichia coli hydrogenase 4 (hyf) and hydrogenase 2 (hyb) contribution in H2 production during mixed carbon (glucose and glycerol) fermentation at pH 7.5 and pH 5.5

Molecular hydrogen (H₂) production by Escherichia coli was studied during mixed carbon sources (glucose and glycerol) fermentation at pH 7.5 and pH 5.5. H₂ production rate (V_H2) by bacterial cells grown on mixed carbon was assayed with either adding glucose (glucose assay) or glycerol (glycerol assay) and compared with the cells grown on sole carbon (glucose or glycerol only) and appropriately assayed. Wild type cells grown on mixed carbon, in the assays with adding glucose, produced H₂ at pH 7.5 with the same level as in the cells grown on glucose only. At pH 7.5 V_H2 in fhlA single and fhlA hyfG double mutants decreased ∼6.5 and ∼7.9 fold, respectively. In wild type cells grown on mixed carbon V_H2 at pH 5.5 was lowered ∼2 fold, compared to the cells grown on glucose only. But in hyfG and hybC single mutants V_H2 was decreased ∼2 and ∼1.6 fold, respectively. However, at pH 7.5, in the assays with glycerol, V_H2 was low, when compared to the cells grown on glycerol only. At pH 5.5 in the assays with glycerol V_H2 was absent. Moreover, V_H2 in wild type cells was inhibited by 0.3 mM N,N^′-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F₀F₁-ATPase, in a pH dependent manner. At pH 7.5 in wild type cells V_H2 was decreased ∼3 fold but at pH 5.5 the inhibition was ∼1.7 fold. At both pHs in fhlA mutant V_H2 was totally inhibited by DCCD. Taken together, the results obtained indicate that at pH 7.5, in the presence of glucose, glycerol can also be fermented. They point out that Hyd-4 mainly and Hyd-2 to some extent contribute in H₂ production by E. coli during mixed carbon fermentation at pH 5.5 whereas Hyd-1 is only responsible for H₂ oxidation.     

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.     

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.     

Evidence for hydrogenase-4 catalyzed biohydrogen production in Escherichia coli

Biohydrogen production by Escherichia coli during fermentation of the mixture of glycerol, glucose and formate at different pH values was studied. Employing mutants lacking large subunits of different hydrogenases (Hyd), it was reported that, at pH 7.5, H₂ production was produced except in a hyaB hybC hycE triple mutant, thus suggesting compensatory H₂-producing functions of the Hyd enzymes. Activity of Hyd-4 was revealed in glucose assays at pH 7.5 in the triple mutant whereby 62% of the wild type level of H₂ production was derived from Hyd-4. In formate assays, it was shown, that, first, the hyaB hybC double mutant had a H₂ production ～3 fold higher than wild type, indicating that Hyd-1 and Hyd-2 oxidize H₂, and second, that at pH 5.5, Hyd-4 and Hyd-3 were responsible for H₂ production. These findings are significant when applying various carbon sources such as sugars, alcohol and organic acids for biohydrogen production.     

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.