Selective inhibitors for continuous non-axenic hydrogen production by Rhodobacter capsulatus

To produce H₂ continuously by photosynthetically grown Rhodobacter capsulatus in non-axenic anaerobic reactors, the interaction between the phototroph and possible contaminants was studied and the ecological competitiveness of the Rhodobacter spp. in nitrogen-limited conditions was determined. Experimental test runs showed that blue-green and green algae, sulphate-reducing, acetogenic and methanogenic bacteria significantly interfere with the net amounts of H₂ produced by photobacteria. Therefore, inhibitors to control the growth of those contaminants selectively were screened. By applying a combination of chloroxuron (10mg/l) and cycloheximide (10mg/l) against algae, isohumulones (30 bitterunits/l) and molyb-date (0.5g/l) against sulphate-reducing bacteria and isohumulones and chloroform (10 mg/l) against acetogens and methanogens, photoreactors could be operated in a non-axenic way and continued to produce hydrogen gas at rates depending on the feed quality varying from 333 to 676 ml H₂l reactor/d, for a period of 116d without apparent interference from other microbial contaminants. These findings have a considerable potential for facilitating the isolation of organo-phototrophs and the production of H₂ by these bacteria.     

Long-term biological hydrogen production by agar immobilized Rhodobacter capsulatus in a sequential batch photobioreactor

In this study, agar immobilization technique was employed for biological hydrogen production using Rhodobacter capsulatus DSM 1710 (wild type) and YO3 (hup-mutant) strains in sequential batch process. Different agar and glutamate concentrations were tested with defined nutrient medium. Agar concentration 4% (w/v) and 4 mM glutamate were selected for bacterial immobilization in terms of rate and longevity of hydrogen production. Acetate concentration was increased from 40 to 60—100 and 60 mM gave best results with both bacterial strains immobilized in 4% (w/v) agar. Cell concentration was increased from 2.5 to 5 mg dcw mL⁻¹ agar and it was found that increasing cell concentration of wild-type strain caused decrease in yield and productivity while these parameters improved by increasing cell concentration of mutant strain. Also, the hydrogen production time has extended from 17 days up to 60 days according to the process conditions and parameters. Hydrogen production by immobilized photosynthetic bacteria is a convenient technology for hydrogen production as it enables to produce hydrogen with high organic acid concentrations comparing to suspended cultures. Besides, immobilization increases the stability of the system and allowed sequential batch operation for long-term application.

     

Inhibition of aconitase and fumarase by nitrogen compounds in Rhodobacter capsulatus

High levels of aconitase and fumarase activities were found in Rhodobacter capsulatus E1F1 cells cultured with nitrate as the sole nitrogen source either under light-anaerobic or dark-aerobic conditions. Both activities were strongly and reversibly inhibited in vitro by nitrite or nitric oxide, whereas nitrate or hydroxylamine showed a lower effect. Other enzymes of the tricarboxylic acids cycle such as malate dehydrogenase or isocitrate dehydrogenase were not affected by these nitrogen compounds. When growing on nitrate in the dark R. capsulatus E1F1 cells accumulated nitrite intracellularly, so that an in vivo inhibition of aconitase and fumarase could account for the strong inhibition of growth observed in the presence of nitrite under dark-aerobic conditions.Abbreviations ACO aconitase - FUM fumarase - MDH malate dehydrogenase - ICDH isocitrate dehydrogenase - TCA tricarboxylic acid     

Methylammonium uptake by Rhodobacter capsulatus

The ammonium uptake system of Rhodobacter capsulatus B100 was examined using the ammonium analog methylammonium. This analog was not transported when cells were grown aerobically on ammonium. When cultured on glutamate as a nitrogen source, or when nitrogen-starved, cells would take up methylammonium. Therefore, in cells grown under nitrogen-limiting conditions, a second system of ammonium uptake (or a modified form of the first) is present which is distinguished by its capacity for transporting the analog in addition to ammonium. The methylammonium uptake system exhibited saturation kinetics with a K _m of 22 M and a V _max of about 3 nmol per min · mg protein. Ammonium completely inhibited analog transport with a K _i in the range of 1 M. Once inside the cell methylammonium was rapidly converted to -N-methylglutamine; however, a small concentration gradient of methylammonium could still be observed. Kinetic parameters reflect the effects of assimilation.The methylammonium uptake system was temperature and pH dependent, and inhibition studies indicated that energy was required for the system to be operative. A glutamine auxotroph (G29) lacking the structural gene for glutanime synthetase did not accumulate the analog, even when nitrogen starved. The Nif^- mutant J61, which is unable to express nitrogenase structural genes, also did not transport methylammonium, regardless of the nitrogen source for growth. However, the mutant exhibited wild-type ammonium uptake and glutamine synthetase activity. These data suggest that transport of ammonium is required for growth on limited nitrogen and is under the control of the Ntr system in R. capsulatus.Abbreviations CCCP carbonyl cyanide-m-chlorophenyl hydrazone - CHES cyclohexylaminoethanesulfonic acid - DMSO dimethyl sulfoxide - GMAD -N-methylglutamine - GS glutamine synthetase - MES 2-(N-morpholino) ethanesulfonic acid - MSX methionine-Dl-sulfoximine - pCMB p-chloromercuribenzoate - Tricine N-tris(hydroxymethyl)methylglycine     

Light intensity distribution in the externally illuminated cylindrical photo-bioreactor and its application to hydrogen production by Rhodobacter capsulatus

The light distribution in the externally illuminated cylindrical photo-bioreactor for production of hydrogen by a photosynthetic bacterium Rhodobacter capsulatus ST-410 was estimated. The estimation was performed on the basis of the Matsuura and Smith's diffuse model [1]. In the diffuse model, the incident light rays are assumed to proceed in every direction and the local intensity is calculated as the sum of the intensities of light. Since Lambert-Beer's law, extensively used in photometry, was not useful for explaining the decrease in the intensity of light by the biomass, an empirical expression was used. The measurement of the intensities from every direction was conducted in an externally illuminated cylindrical photo-bioreactor having an inner diameter of 60mm and a working volume of 550ml. The obtained results confirmed our estimation. The light distribution was applied to estimate the hydrogen production by R. capsulatus ST-410 using the same photo-bioreactor. The overall hydrogen-production rate was successfully estimated.     

Effects of light intensity distribution on growth of Rhodobacter capsulatus

For cultivation of photosynthetic cells under defined light intensity distributions, the repeated batch culture, in which a part of culture broth containing grown cells was repeatedly replaced at predetermined time intervals with a fresh medium to keep the cell concentration constant at an initial value, was employed. By use of this method the effects of the light intensity distribution on the growth characteristics of Rhodobacter capsulatus were studied. Unexpected decreases in the specific growth rate were observed in culture of R. capsulatus at high cell concentrations and a long light path length. Big differences in the light intensities of lightly and darkly illuminated portions in photobioreactors, which reflects the light intensity distribution, seemed to cause this phenomenon, which must be taken into consideration for stable growth of photosynthetic cells.     

Acetate as a carbon source for hydrogen production by photosynthetic bacteria

Hydrogen is a clean energy alternative to fossil fuels. Photosynthetic bacteria produce hydrogen from organic compounds by an anaerobic light-dependent electron transfer process. In the present study hydrogen production by three photosynthetic bacterial strains (Rhodopseudomonas sp., Rhodopseudomonas palustris and a non-identified strain), from four different short-chain organic acids (lactate, malate, acetate and butyrate) was investigated. The effect of light intensity on hydrogen production was also studied by supplying two different light intensities, using acetate as the electron donor. Hydrogen production rates and light efficiencies were compared. Rhodopseudomonas sp. produced the highest volume of H2. This strain reached a maximum H2 production rate of 25 ml H2 l(-1) h(-1), under a light intensity of 680 micromol photons m(-2) s(-1), and a maximum light efficiency of 6.2% under a light intensity of 43 micromol photons m(-2) s(-1). Furthermore, a decrease in acetate concentration from 22 to 11 mM resulted in a decrease in the hydrogen evolved from 214 to 27 ml H2 per vessel.     

Clustering of genes necessary for hydrogen oxidation in Rhodobacter capsulatus.

Three cosmids previously shown to contain information necessary for the expression of uptake of hydrogenase in Rhodobacter capsulatus were found to be present in a cluster on the chromosome. Earlier genetic experiments suggested the presence of at least six genes essential for hydrogenase activity that are now shown to be in a region of approximately 18 kb that includes the structural genes for the enzyme. A potential response regulator gene was sequenced as a part of the hup gene region.     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine     

Influence of nitrogen source on hydrogen generation by a photosynthetic bacterium

Axenic culture of photosynthetic purple sulphur bacterium, isolated from a paper mill effluent, grew best with ammonium chloride as nitrogen source. Cells grown with N₂ or ammonium chloride produced hydrogen with initial lag periods of 24 and 48 h, respectively. The maximum amount of hydrogen was evolved by cells grown with N₂ and with malate as electron donor.R. Lakshmi is with the Microbial Physiology Laboratory, Department of Botany, and H. Polasa is with the Department of Microbiology, Osmania University, Hyderabad 500 007, India.     

豆粕水解液为氮源发酵产脂肪酶的研究

研究了以豆粕水解液作为氮源,假丝酵母Candida sp．99—125发酵生产脂肪酶的过程。分析水解时间对于产酶的影响,对比豆粕水解前后作为氮源发酵时的产酶规律。在30L发酵罐中批次发酵酶活最高可达6000IU／mL,采用豆油反馈流加之后,发酵脂肪酶活力可达8500IU／mL。     

Synthesis of bacteriochlorophyll a by the purple nonsulfur bacterium Rhodobacter capsulatus

The ability of purple nonsulfur bacteria Rhodobacter capsulatus B10 to synthesize bacteriochlorophyll under phototrophic and dark conditions was studied. The modes for cultivation in the dark with oxygen limitation in a continuous culture at D = 0.1 h(-1) were selected. The yield of biomass reached 20 g/l; the bacteriochlorophyll a output of the process amounted to 16.6 mg/l h(-1).