Erythromycin production by Streptomyces erythreus entrapped in calcium alginate beads

Summary Mycelia of Streptomyces erythreus were immobilized in calcium alginate beads and employed for production of erythromycin. Compared to conventional and washed mycelial fermentation, the average specific productivity of immobilised mycelia was superior.     

Ammonium uptake in Rhodopseudomonas capsulata

Investigations of the uptake of ammonium (NH ₄ ⁺ ) by Rhodopseudomonas capsulata B100 supported the presence of an NH ₄ ⁺ transport system. Experimentally NH ₄ ⁺ was determined by electrode or indophenol assay and saturation kinetics were observed with two apparent K _m's of 1.7 M and 11.1 M (pH 6.8, 30°) and a V _max at saturation of 50–60 nmol/min·mg protein. The optimum pH and temperature were 7.0 and 33° C, respectively. The Q₁₀ quotient was calculated to be 1.9 at 100 M NH ₄ ⁺ , indicating enzymatic involvement. In contrast to the wild type, B100, excretion of NH ₄ ⁺ , not uptake, was observed in a glutamine auxotroph, R. capsulata G29, which is derepressed for nitrogenase and lacks glutamine synthetase activity. G29R1, a revertant of G29, also took up NH ₄ ⁺ at the same rate as wild type and had fully restored glutamine synthetase activity. Partially restored derivatives, G29R5 and G29R6, grew more slowly than wild type on NH ₄ ⁺ as the nitrogen source, remained derepressed for nitrogenase in the presence of NH ₄ ⁺ , and displayed rates of NH ₄ ⁺ uptake in proportion to their glutamine synthetase activity. Ammonium uptake and glutamine synthetase activity were also restored in R. capsulata G29 exconjugants which had received the plasmid pPS25, containing the R. capsulata glutamine synthetase structural gene. These data suggest that NH ₄ ⁺ transport is tightly coupled to assimilation.Abbreviations used CHES cyclohexylaminoethanesulfonic acid - GS glutamine synthetase - SDS sodium dodecylsulfate     

Nickel uptake in Rhodopseudomonas capsulata

Nickel uptake system was investigated with a wild-type cell of Rhodopseudomonas capsulata and two mutants lacking uptake hydrogenase (Hup^-). Wild type cells grown photoheterotrophically incorporated ⁶³Ni²⁺ by a high affinity system. The uptake system had a pH of 7.0 and a temperature optimum of 28°C. Both Mg²⁺ and Co²⁺ ions severely repressed the uptake of Ni²⁺. Nickel transport was also inhibited by metabolic inhibitors including cyanide, azide, 2,4-dinitrophenol and m-chlorophenyl carbonylcyanidehydrazone. These data imply that Ni²⁺ uptake system occurs by the energy-linked system for Mg²⁺ transport. The intracellular distribution of ⁶³Ni²⁺ in Hup^- cells showed the same pattern as that of wild-type cells, indicate that the Hup^- strains have no deficiency in Ni²⁺ transport.Abbreviations CCCP m-chlorophenyl carbonylcyanidehydrazone - HEPES N-2-hydroxylethylpiperazine-N-2-ethane-sulfuric acid - HOQNO 2-n-nonly-4-hydroxyquinoline-N-oxide - TMA tetramethylammonium hydroxide     

Photopigments in Rhodopseudomonas capsulata cells grown anaerobically in darkness.

The phototrophic bacterium Rhodopseudomonas capsulata can obtain energy for dark anaerobic growth from sugar fermentations dependent on accessory oxidants such as trimethylamine-N-oxide or dimethyl sulfoxide. Cells grown for one to two subcultures in this fashion, with fructose as the energy source, showed approximately a twofold increase in bacteriochlorophyll content (per milligram of cell protein) and developed extensive intracytoplasmic membranes in comparison with cells grown photosynthetically at saturating light intensity. Cells harvested from successive anaerobic dark subcultures, however, showed progressively lower pigment contents. After ca. 20 transfers, bacteriochlorophyll and carotenoids were barely detectable, and the amount of intracytoplasmic membrane diminished considerably. Spontaneous mutants incapable of producing normal levels of photosynthetic pigments arose during prolonged anaerobic dark growth. Certain mutants of this kind appear to have a selective advantage over wild-type cells under fermentative growth conditions. Of four pigment mutants characterized (two being completely unable to produce bacteriochlorophyll), only one retained the capacity to grow photosynthetically.     

Glycerol assimilation by a mutant of Rhodopseudomonas capsulata

A spontaneous mutant of Rhodopseudomonas capsulata, capable of growth on glycerol, has been isolated. The mutant requires CO(2) or malate to assimilate glycerol photosynthetically. This requirement is not manifested aerobically. Glycerokinase (EC 2.7.1.30) and pyridine nucleotide-independent l-alpha-glycerophosphate dehydrogenase (EC 1.1.2.1) activities appear coincidently with the metabolism of glycerol, suggesting that this organism employs these enzymes for glycerol dissimilation.     

Growth Inhibition of Rhodopseudomonas capsulata by Methylmercury Acetate

Growth of Rhodopseudomonas capsulata was inhibited in a bacteriostatic manner by as little as 10-minus 8 M methylmercury acetate (MeHgAc) in unsupplemented synthetic liquid medium or when cells were exposed to 8.0 nm of MeHgAc per mg of cell protein in a single exposure.     

H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: production and utilization of H2 by resting cells.

Photoproduction of H2 and activation of H2 for CO2 reduction (photoreduction) by Rhodopseudomonas capsulata are catalyzed by different enzyme systems. Formation of H2 from organic compounds is mediated by nitrogenase and is nto inhibited by an atmosphere of 99% H2. Cells grown photoheterotrophically on C4 dicarboxylic acids (with glutamate as N source) evolve H2 from the C4 acids and also from lactate and pyruvate; cells grown on C3 carbon sources, however, are inactive with the C4 acids, presumably because they lack inducible transport systems. Ammonia is known to inhibit N2 fixation by photosynthetic bacteria, and it also effectively prevents photoproduction of H2; these effects are due to inhibition and, in part, inactivation of nitrogenase. Biosynthesis of the latter, as measured by both H2 production and acetylene reduction assays, is markedly increased when cells are grown at high light intensity; synthesis of the photoreduction system, on the other hand, is not appreciably influenced by light intensity during photoheterotrophic growth. The photoreduction activity of cells grown on lactate + glutamate (which contain active nitrogenase) is greatly activated by NH4+, but this effect is not observed in cells grown with NH4+ as N source (nitrogenase repressed) or in a Nif- mutant that is unable to produce H2. Lactate, malate, and succinate, which are readily used as growth substrates by R. capsulata and are excellent H donors for photoproduction of H2, abolish photoreduction activity. The physiological significances of this phenomenon and of the reciprocal regulatory effects of NH4+ on H2 production and photoreduction are discussed.     

Enzymes of serine biosynthesis in Rhodopseudomonas capsulata

Rhodopseudomonas capsulata has been shown to possess all the enzymatic activities of both the phosphorylated and nonphosphorylated pathways of serine biosynthesis. In addition there was an active serine hydroxymethyltransferase which catalyzed the reversible interconversion of serine and glycine. In cells grown photosynthetically with malate as the carbon source, the activities of the phosphorylated pathway enzymes were substantially higher than the analogous reactions of the nonphosphorylated sequence. l-Serine (1 mm) caused approximately 60%, inhibition of the first enzyme of the phosphorylated route, 3-phosphoglyceric acid dehydrogenase, but was less effective in inhibiting the last enzyme, phosphoserine phosphatase. Glycine also exerted a regulatory effect on this pathway but it was not as potent an inhibitor as serine. The inhibitions caused by serine and glycine were simply additive; there was no evidence of concerted feedback inhibition of the phosphorylated pathway by these amino acids.     

Glycerol utilization by a mutant of Rhodopseudomonas capsulata.

The glycerol-catabolizing enzymes of a mutant of Rhodopseudomonas capsulata were found to be constitutive and modulated coordinately, although apparently not functional in the presence of malate. No difference in glycerol permeation was found between the mutant and wild type.     

Kluyveromyces lactis cells entrapped in Ca-alginate beads for the continuous production of a heterologous glucoamylase

Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source. Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium. Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree. The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product. In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed. The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus. The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.     

Stimulation by light of nitrogenase synthesis in cells of Rhodopseudomonas capsulata growing in N-limited continuous cultures

In cells of Rhodopseudomonas capsulata growing in nitrogen-limited continuous culture the nitrogenase-specific activity was found to be closely dependent on the light intensity. As light intensity, measured with a photodiode immersed in the culture, was varied stepwise from 1000 to 7000 lux, the nitrogenase activity, measured at steady state, increased gradually up to 5-fold. Shifting light intensity from 1200 to 7000 lux resulted in a sharp rise in nitrogenase activity which doubled within the first two hours. The determination by immunoassays of the intracellular levels of each nitrogenase component revealed that the light-dependent stimulation of nitrogenase activity was correlated with the accumulation of the nitrogenase enzyme inside the cells. Under high illumination, nitrogenase represented up to 40% of the cytoplasmic proteins. The specific activities of each component in intact cells, calculated on the basis of their relative concentration in the cells and on in vivo nitrogenase assays, appeared roughly constant and hardly affected by changes of light intensity. The specific activity of the Fe protein was about 7-fold higher in intact cells than in the purified state. The ratio of the two nitrogenase components remained fairly constant and close to one, irrespective of the light intensity to which cells were exposed. These results demonstrate that in nitrogen-limited grown cells of Rps. capsulata light brings about an induction or a derepression of nitrogenase synthesis the extent of which is dependent on light intensity.     

Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata.

Hydrogen evolution and consumption by cell and chromatophore suspensions of the photosynthetic bacterium Rhodopseudomonas capsulata was measured with a sensitive and specific mass spectrometric technique which directly monitors dissolved gases. H2 production by nitrogenase was inhibited by acetylene and restored by carbon monoxide. An H2 evolution activity coupled with HD formation and D2 uptake (H-D exchange) was unaffected by C2H2 and CO. Cultures lacking nitrogenase activity also exhibited H-D exchange activity, which was catalyzed by a membrane-bound hydrogenase present in the chromatophores of R. capsulata. A net hydrogen uptake, mediated by hydrogenase, was observed when electron acceptors such as CO2, O2, or ferricyanide were present in the medium.     

Dissimilatory nitrate and dimethylsulphoxide reduction in Rhodopseudomonas capsulata

Abstract The electron flow to the dissimilatory nitrate reductase (NRII), and dimethylsulphoxide (DMSO) oxidoreductase in Rhodopseudomonas capsulata strains was studied. Our results support the view that DMSO reduction, like dissimilatory nitrate reduction was linked to the electron transfer chain and probably coupled to energy conservation.     

Optimization of glutamate concentration and pH for H production from volatile fatty acids by Rhodopseudomonas capsulata

AIMS: This study attempted to employ response surface methodology (RSM) to evaluate the effects of glutamate concentration and pH on H(2) production from volatile fatty acids by Rhodopseudomonas capsulata. METHODS AND RESULTS: A mixture of acetate, propionate and butyrate was used as a carbon source for the H(2) production by R. capsulata. The H(2) yield and H(2) production rate were strongly affected by the glutamate concentration, pH and their interaction. The predicted maximum H(2) yield of 0.534 was obtained when glutamate concentration and pH were 6.56 mmol l(-1) and 7.29 respectively. On the contrary, the maximum H(2) production rate of 18.72 ml l(-1) h(-1) was achieved at a glutamate concentration of 7.01 mmol l(-1) and pH 7.31. CONCLUSIONS: Taking H(2) yield and H(2) production rate together into account, a glutamate concentration of 6.56-7.01 mmol l(-1) and pH of 7.29-7.31 should be selected for H(2) production from a mixture of acetate, propionate and butyrate by R. capsulata. SIGNIFICANCE AND IMPACT OF THE STUDY: The RSM was a useful tool for maximizing H(2) production by photosynthetic bacteria (PSB).     

H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures.

Purple photosynthetic bacteria produce H2 from organic compounds by an anaerobic light-dependent electron transfer process in which nitrogenase functions as the terminal catalyst. It has been established that the H2-evolving function of nitrogenase is inhibited by N2 and ammonium salts, and is maximally expressed in cells growing photoheterotrophically with certain amino acids as sources of nitrogen. In the present studies with Rhodopseudomonas capsulata, nutritional factors affecting the rate and magnitude of H2 photoproduction in cultures growing with amino acid nitrogen sources were examined. The highest H2 yields and rates of formation were observed with the organic acids: lactate, pyruvate, malate, and succinate in media containing glutamate as the N source; under optimal conditions with excess lactate, H2 was produced at rates of ca. 130 ml/h per g(dry weight) of cells. Hydrogen production is significantly influenced by the N/C ratio in the growth substrates; when this ratio exceeds a critical value, free ammonia appears in the medium and H2 is not evolved. In the "standard" lactate + glutamate system, both H2 production and growth are "saturated" at a light intesity of ca. 600 ft-c (6,500 lux). Evolution of H2, however, occurs during growth at lithe intensities as low as 50 to 100 ft-c (540 to 1,080 lux), i.e., under conditions of energy limitation. In circumstances in which energy conversion rate and supplies of reducing power exceed the capacity of the biosynthetic machinery, energy-dependent H2 production presumably represents a regulatory device that facilitates "energy-idling." It appears that even when light intensity (energy) is limiting, a significant fraction of the available reducing power and adenosine 5'-triphosphate is diverted to nitrogenase, resulting in H2 formation and a bioenergetic burden to the cell.     

The effect of temperature and light intensity on hydrogen gas production by different Rhodopseudomonas capsulata strains

Summary Six strains of Rhodopseudomonas capsulata were tested for their ability for anaerobic light-dependent hydrogen gas production from acetate in different incubation temperatures and light intensities.Certain strains show a higher efficiency of acetate conversion to H₂ at higher temperatures and higher light intensities, others on the other hand are insensitive or even show the opposite effect.