Bioreduction and biocrystallization of palladium by Desulfovibrio desulfuricans NCIMB 8307

The reduction of Pd(II) to Pd(0) was accelerated by using the sulfate-reducing bacterium Desulfovibrio desulfuricans NCIMB 8307 at the expense of formate or H(2) as electron donors at pH 2-7. With formate no reduction occurred at pH 2, but with H(2) 50% of the activity was retained at pH 2, with the maximum rate (1.3-1.4 micromol min(-1) mg dry cells(-1)) seen at pH 3-7, which was similar to the rate with formate at neutral pH. Excess nitrate was inhibitory to Pd(II) reduction using formate, but not H(2). Chloride ion was inhibitory as low as 100 mM using formate but with H(2) only ca. 25% inhibition was observed at 500 mM Cl(-) and H(2) was concluded to be the electron donor of choice for the potential remediation of industrial wastes. Deposited Pd was visible on the cells using transmission and scanning electron microscopy and analysis by energy dispersive X-ray microanalysis (EDAX) identified the deposit as Pd, confirmed as Pd(0) by X-ray powder diffraction analysis (XRD). The crystal size of the biodeposited Pd(0) was determined to be only 50% of the size of Pd(0) crystals manufactured chemically from Pd(II) at the expense of H(2) and, unlike the chemically manufactured material, the biocrystal size was independent of the pH. The "biological" Pd(0) functioned as a superior chemical catalyst in a test reaction which liberated hydrogen from hypophosphite. Pd, and also Pt and Rh, could be recovered by resting cell suspensions under H(2) from an industrial processing wastewater, suggesting a possible future application of bioprocessing technology for precious metals.     

Tetrachloroethene dehalorespiration and growth of Desulfitobacterium frappieri TCE1 in strict dependence on the activity of Desulfovibrio fructosivorans

Tetrachloroethene (PCE) dehalorespiration was investigated in a continuous coculture of the sulfate-reducing bacterium Desulfovibrio fructosivorans and the dehalorespiring Desulfitobacterium frappieri TCE1 at different sulfate concentrations and in the absence of sulfate. Fructose (2.5 mM) was the single electron donor, which could be used only by the sulfate reducer. With 2.5 mM sulfate, the dehalogenating strain was outnumbered by the sulfate-reducing bacterium, sulfate reduction was the dominating process, and only trace amounts of PCE were dehalogenated by strain TCE1. With 1 mM sulfate in the medium, complete sulfate reduction and complete PCE dehalogenation to cis-dichloroethene (cis-DCE) occurred. In the absence of sulfate, PCE was also completely dehalogenated to cis-DCE, and the population size of strain TCE1 increased significantly. The results presented here describe for the first time dehalogenation of PCE by a dehalorespiring anaerobe in strict dependence on the activity of a sulfate-reducing bacterium with a substrate that is exclusively used by the sulfate reducer. This interaction was studied under strictly controlled and quantifiable conditions in continuous culture and shown to depend on interspecies hydrogen transfer under sulfate-depleted conditions. Interspecies hydrogen transfer was demonstrated by direct H(2) measurements of the gas phase and by the production of methane after the addition of a third organism, Methanobacterium formicicum.     

Tetrachloroethene Dehalorespiration and Growth of Desulfitobacterium frappieri TCE1 in Strict Dependence on the Activity of Desulfovibrio fructosivorans

Tetrachloroethene (PCE) dehalorespiration was investigated in a continuous coculture of the sulfate-reducing bacterium Desulfovibrio fructosivorans and the dehalorespiring Desulfitobacterium frappieri TCE1 at different sulfate concentrations and in the absence of sulfate. Fructose (2.5 mM) was the single electron donor, which could be used only by the sulfate reducer. With 2.5 mM sulfate, the dehalogenating strain was outnumbered by the sulfate-reducing bacterium, sulfate reduction was the dominating process, and only trace amounts of PCE were dehalogenated by strain TCE1. With 1 mM sulfate in the medium, complete sulfate reduction and complete PCE dehalogenation to cis-dichloroethene (cis-DCE) occurred. In the absence of sulfate, PCE was also completely dehalogenated to cis-DCE, and the population size of strain TCE1 increased significantly. The results presented here describe for the first time dehalogenation of PCE by a dehalorespiring anaerobe in strict dependence on the activity of a sulfate-reducing bacterium with a substrate that is exclusively used by the sulfate reducer. This interaction was studied under strictly controlled and quantifiable conditions in continuous culture and shown to depend on interspecies hydrogen transfer under sulfate-depleted conditions. Interspecies hydrogen transfer was demonstrated by direct H₂ measurements of the gas phase and by the production of methane after the addition of a third organism, Methanobacterium formicicum.     

Biomass-supported palladium catalysts on Desulfovibrio desulfuricans and Rhodobacter sphaeroides

A Rhodobacter sphaeroides-supported dried, ground palladium catalyst ("Rs-Pd(0)") was compared with a Desulfovibrio desulfuricans-supported catalyst ("Dd-Pd(0)") and with unsupported palladium metal particles made by reduction under H2 ("Chem-Pd(0)"). Cell surface-located clusters of Pd(0) nanoparticles were detected on both D. desulfuricans and R. sphaeroides but the size and location of deposits differed among comparably loaded preparations. These differences may underlie the observation of different activities of Dd-Pd(0) and Rs-Pd(0) when compared with respect to their ability to promote hydrogen release from hypophosphite and to catalyze chloride release from chlorinated aromatic compounds. Dd-Pd(0) was more effective in the reductive dehalogenation of polychlorinated biphenyls (PCBs), whereas Rs-Pd(0) was more effective in the initial dehalogenation of pentachlorophenol (PCP) although the rate of chloride release from PCP was comparable with both preparations after 2 h.     

Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains

During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed.Glycerol also supported growth of three out of four classical Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.Abbreviations DHA dihydroxyacetone - DHAP dihydroxyacetone phosphate - G3P glycerol 3-phosphate - GAP glyceraldehyde 3-phosphate - 3-PGA 3-phosphoglycerate - 2-PGA 2-phosphoglycerate - 2,3-DPGA 2,3-diphosphoglycerate - PEP phosphoenolpyruvate - DH dehydrogenase - GK glycerol kinase - DHAK dihydroxyacetone kinase - TIM triosephosphate isomerase - PGK 3-phosphoglycerate kinase - PK pyruvate kinase - LDH lactate dehydrogenase - DTT dithiotreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - BV²⁺/BV⁺ oxidized/reduced benzylviologen - PMS phenazine methosulfate - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide     

Oxidation of benzaldehydes to benzoic acid derivatives by three Desulfovibrio strains

Desulfovibrio vulgaris Marburg, "Desulfovibrio simplex" XVI, and Desulfovibrio sp. strain MP47 used benzaldehydes such as vanillin, 3,4,5-trimethoxybenzaldehyde, protocatechualdehyde, syringaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, and 2-methoxybenzaldehyde as electron donors for sulfate reduction and carbon dioxide and/or components of yeast extract as carbon sources for cell synthesis. The aldehydes were oxidized to their corresponding benzoic acids. The three sulfate reducers oxidized up to 7 mM vanillin and up to 4 mM p-anisaldehyde. Higher concentrations of vanillin or p-anisaldehyde were toxic. In addition, pyridoxal hydrochloride and o-vanillin served as electron donors for sulfate reduction. Salicylaldehyde, pyridine-2-aldehyde, pyridine-4-aldehyde, and 4-hydroxy-3-methoxybenzylalcohol were not oxidized. No molecular hydrogen was detected in the gas phase. The oxidized aldehydes were not further degraded.     

Fermentative production of chiral acetoin by wild-type Bacillus strains

Abstract

In recent years, there have been many studies on producing acetoin by microbial fermentation, while only a few studies have focused on chiral acetoin biosynthesis. The weight assignment method was first applied to balance the chiral purity (expressed as the enantiomeric excess value) and the titer of acetoin. Bacillus sp. H-18W, a thermophile, was selected from seven Bacillus strains for chiral acetoin production. To lower the cost of the fermentation medium, soybean meal was used as a feedstock. Four kinds of frequently used commercial proteinases with different active sites were tested for the hydrolyzation of the soybean meal, and the combination of the acidic proteinase and the neutral proteinase showed the best results. In a fermentation medium containing 100?g L^?1 glucose and 200?g L^?1 hydrolysate, Bacillus sp. H-18W produced 21.84?g L^?1 acetoin with an ee value of 96.25% at 60?h. This is the first report of using a thermophilic strain to produce chiral acetoin by microbial fermentation. Thermophilic fermentation can reduce the risk of bacterial contamination and can save cooling water. Using soybean meal hydrolysate and glucose as feedstocks, this work provides an economical and alternative method for the production of chiral pure acetoin.     

Intraspecies variability of Desulfovibrio desulfuricans strains determined by the genetic profiles

Fifteen (soil and intestinal) strains of Desulfovibrio desulfuricans species were typed by PCR method with the use of primers specific for repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) sequences. As a result, characteristic DNA fingerprints for the strains were obtained. Moreover, the genetic profiles were found to be useful for typing and distinguishing the strains of D. desulfuricans. According to cluster analysis, PCR with primers complementary to the sequences REP appeared to be slightly more discriminatory than PCR with ERIC primers for the investigated strains. Distinct fingerprint patterns of two isolates derived from the same patient pointed to the different origin of both strains.     

Utilization of D-xylose by wild-type strains of Salmonella typhimurium.

Enzyme studies of strains of Salmonella typhimurium representing biotypes that utilized D-xylose rapidly (xylose strong) or slowly (xylose weak) showed that they were different in the utilization of D-xylose because the xylose-weak strains were deficient in the transport of D-xylose. This observation is consistent with the idea that strains of the different xylose-weak biotypes, e.g. biotypes 17 to 32, were descended from strains of xylose-strong types, particularly from biotype 1.     

Oxidation of benzaldehydes to benzoic acid derivatives by three Desulfovibrio strains.

Desulfovibrio vulgaris Marburg, "Desulfovibrio simplex" XVI, and Desulfovibrio sp. strain MP47 used benzaldehydes such as vanillin, 3,4,5-trimethoxybenzaldehyde, protocatechualdehyde, syringaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, and 2-methoxybenzaldehyde as electron donors for sulfate reduction and carbon dioxide and/or components of yeast extract as carbon sources for cell synthesis. The aldehydes were oxidized to their corresponding benzoic acids. The three sulfate reducers oxidized up to 7 mM vanillin and up to 4 mM p-anisaldehyde. Higher concentrations of vanillin or p-anisaldehyde were toxic. In addition, pyridoxal hydrochloride and o-vanillin served as electron donors for sulfate reduction. Salicylaldehyde, pyridine-2-aldehyde, pyridine-4-aldehyde, and 4-hydroxy-3-methoxybenzylalcohol were not oxidized. No molecular hydrogen was detected in the gas phase. The oxidized aldehydes were not further degraded.     

Xylulose fermentation by mutant and wild-type strains of Zygosaccharomyces and Saccharomyces cerevisiae

Anaerobic xylulose fermentation was compared in strains of Zygosaccharomyces and Saccharomyces cerevisiae, mutants and wild-type strains to identify host-strain background and genetic modifications beneficial to xylose fermentation. Overexpression of the gene (XKS1) for the pentose phosphate pathway (PPP) enzyme xylulokinase (XK) increased the ethanol yield by almost 85% and resulted in ethanol yields [0.61 C-mmol (C-mmol consumed xylulose)⁻¹] that were close to the theoretical yield [0.67 C-mmol (C-mmol consumed xylulose)⁻¹]. Likewise, deletion of gluconate 6-phosphate dehydrogenase (gnd1Δ) in the PPP and deletion of trehalose 6-phosphate synthase (tps1Δ) together with trehalose 6-phosphate phosphatase (tps2Δ) increased the ethanol yield by 30% and 20%, respectively. Strains deleted in the promoter of the phosphoglucose isomerase gene (PGI1) – resulting in reduced enzyme activities – increased the ethanol yield by 15%. Deletion of ribulose 5-phosphate (rpe1Δ) in the PPP abolished ethanol formation completely. Among non-transformed and parental strains S. cerevisiae ENY. WA-1A exhibited the highest ethanol yield, 0.47 C-mmol (C-mmol consumed xylulose)⁻¹. Other non-transformed strains produced mainly arabinitol or xylitol from xylulose under anaerobic conditions. Contrary to previous reports S. cerevisiae T23D and CBS 8066 were not isogenic with respect to pentose metabolism. Whereas, CBS 8066 has been reported to have a high ethanol yield on xylulose, 0.46 C-mmol (C-mmol consumed xylulose)⁻¹ (Yu et al. 1995), T23D only formed ethanol with a yield of 0.24 C-mmol (C-mmol consumed xylulose)⁻¹. Strains producing arabinitol did not produce xylitol and vice versa. However, overexpression of XKS1 shifted polyol formation from xylitol to arabinitol. Received: 2 July 1999 / Accepted in revised form: 12 October 1999     

The accumulation of bacteriochlorophyll precursors by mutant and wild-type strains of Rhodopseudomonas spheroides

1. Two mutant strains of Rhodopseudomonas spheroides, which are blocked in the synthesis of bacteriochlorophyll, accumulate pigments. These have been tentatively identified as magnesium 2,4-divinylphaeoporphyrin a₅ monomethyl ester and the magnesium derivative of 2-devinyl-2-hydroxyethyl-phaeophorbid a, formed by mutant 2/73 and 2/21 respectively. 2. Maximum extracellular production of these pigments occurs when suspensions of the organisms are incubated with low aeration in a growth medium containing iron and supplemented with glycine, succinate, methionine and Tween 80. 3. Concomitant protein synthesis is required for pigment production by the mutants from glycine and succinate but this requirement is less marked when δ-aminolaevulic acid is the substrate. 4. In the absence of Tween 80, a considerable proportion of the total pigment is retained within the cells and appears in the particulate fraction of cell-free extracts. 5. Suspensions of the parent strain containing δ-aminolaevulic acid can be made to accumulate extracellular pigments which are tentatively identified as magnesium protoporphyrin monomethyl ester and the magnesium derivative of 2-devinyl-2-hydroxyethyl-phaeophorbid a. 6. Maximum production occurs with cells incubated photosynthetically after a period of oxygen repression of bacteriochlorophyll synthesis. Formation of the phaeophorbid derivative is enhanced by 8-azaguanine or 5-fluorouracil, or by adenine deficiency in a nutritional mutant; Tween 80 is also needed and iron is essential. 7. Synthesis of bacteriochlorophyll might possibly involve the participation of lipoprotein-bound intermediates, which may be formed at the initial stage of condensation between glycine and succinyl-CoA to give δ-aminolaevulic acid.     

Metabolism of l-alanine in Desulfotomaculum ruminis and two marine Desulfovibrio strains

The degradation of l-alanine by three strains of sulfate-reducing bacteria that can grow with l-alanine as an energy source was investigated. In Desulfotomaculum ruminis and most likely also in two marine Desulfovibrio strains alanine is converted to pyruvate via an NAD-dependent alanine dehydrogenase. D. ruminis contained high activities of soluble NADH and NADPH dehydrogenases. In the marine strains the activities were much lower and the NADH dehydrogenase was partly associated with the membrane fraction.     

Differences between pectic enzymes produced by laboratory and wild-type strains of Saccharomyces cerevisiae

The laboratory strain of S. cerevisiae, IM1-8b, showed pectolytic activity in the presence of either glucose, fructose, or sucrose as the carbon source, but not with galactose. The enzyme activity was rapidly lost with shaking. The optimum pH and temperature for activity were 4.5 and 45°C, respectively. The enzyme was an endopolygalacturonase, since it preferentially hydrolysed pectate over pectin and decreased the viscosity of a 5% polygalacturonic solution by about 30% in 30min producing oligogalacturonic acid and digalacturonic acid as end-products.     

Uptake and phosphorylation of 2-deoxy-D-glucose by wild-type and single-kinase strains of Saccharomyces cerevisiae

The role of phosphorylation in sugar transport in baker's yeast was studied using 2-deoxy-D-glucose. In wild-type baker's yeast, 2-deoxy-D-glucose is accumulated as a mixture of the free sugar and several derivatives. Pool labeling experiments, designed to determine the temporal order of appearance of labeled 2-deoxy-D-glucose in the intracellular pools, have confirmed previous reports that 2-deoxy-D-glucose first appears in the sugar phosphate pool. Such results are consistent with a transport associated phosphorylation mechanism. Since wild-type yeasts contain three enzymes which could participate in such a process, hexokinase isozymes PI and PII and glucokinase, pool labeling experiments were carried out with single-kinase mutant strains containing only one of these enzymes. Results similar to those for wild-type strains were obtained for all three single-kinase strains, suggesting that if transport associated phosphorylation does occur in baker's yeast, it is not a function of the specific kinase present in the cell. While the results of the pool labeling experiments are consistent with a transport associated phosphorylation mechanism for 2-deoxy-D-glucose, caution is urged in interpreting the results of experiments with whole cells where problems of compartmentation and multiple pools are difficult to assess.     

Glycerol catabolism in wild-type and mutant strains ofPseudomonas aeruginosa

Glycerol uptake, glycerol kinase (EC 2.7.1.30) and glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) activities are specifically induced during growth ofPseudomonas aeruginosa PAO on either glycerol or glycerol-3-phosphate. Mutants of strain PAO unable to grow on both glycerol and glycerol-3-phosphate were isolated. Mutant PFB 121 was deficient in an inducible, membrane-bound, pyridine nucleotide-independent, glycerol-3-phosphate dehydrogenase activity and PFB 82 was deficient in glycerol uptake and glycerol kinase and glycerol-3-phosphate dehydrogenase activities. Each mutant spontaneously reverted to wild phenotype, which indicates that each contained a single genetic lesion. These results demonstrate that membrane-bound, inducible glycerol-3-phosphate dehydrogenase is required for catabolism of both glycerol and glycerol-3-phosphate and provide suggestive evidence for a single regulatory locus that controls the synthesis of glycerol uptake, glycerol kinase, and glycerol-3-phosphate dehydrogenase inP. aeruginosa.     

Production of wild-type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

This study focused on the growth of Saccha-romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP)(2) and (WP)(4), respectively. Different cultivation and strategies were tested in a 2-L shake flask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg(-1) with activity per protein broth (specific activity) of 266 Umg(-1), whereas cutinase-(WP)(2) was secreted with a specific cell activity of 2.1 Umg(-1) with a specific activity of 200 Umg(-1), and cutinase-(WP)(4) with a specific cell activity of 0.7 Umg(-1) with a specific activity of 15 Umg(-1). The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture).     

Energy-linked potassium uptake by mitochondria from wild-type and poky strains of Neurospora crassa.

Mitochondria from Neurospora crassa, like mammalian mitochondria, carry out rapid, energy-linked K+ uptake and H+ release in the presence of valinomycin. The maximal rate of K+ uptake was about 1.0 mumol/mg of mitochondrial protein per min and was seen at valinomycin concentrations in the range of 100 to 200 mug per mg of mitochondrial protein and at K+ concentrations of 4 mM or above. Uptake could be supported either by substrate oxidation or by adenosine 5'-triphosphate (ATP), and was inhibited in the former case by antimycin or cyanide, in the latter case by oligomycin, and in both cases by 2,4-dinitrophenol. Mitochondria from the cytochrome-deficient mutant poky carried out substrate-driven K+ uptake at reduced rates, but oligomycin-sensitive, ATP-driven K+ uptake at rates about 60% greater than those shown by wild-type mitochondria. This result is consistent with the recent finding (Mainzer and Slayman 1976) that poky contains elevated amounts of oligomycin-sensitive mitochondrial adenosine 5'-triphosphatase activity.     

Phosphomannomutase activity in wild-type and alginate-producing strains ofPseudomonas aeruginosa

Phosphomannomutase (PMM) activity was detected in the soluble cytoplasmic fraction of crude extracts of both mucoid (alginate-producing) and nonmucoid strains ofPseudomonas aeruginosa. The enzyme activity was concentrated and partially purified from cell extracts of mucoid strain V388 by precipitation with ammonium sulfate and by molecular exclusion chromatography. These preparations catalyzed the conversion of mannose 1-phosphate to mannose 6-phosphate in a coupled assay system that contained commercial phosphomannoisomerase, phosphoglucoisomerase, and glucose 6-phosphate dehydrogenase. Catalytic activity in this system was strictly dependent on the presence of glucose 1,6-diphosphate (apparent Km, 150 M) and exhibited a pH optimum of around 9 in Bicine-NaOH buffer. PMM exhibited an apparent Km of 60 M for mannose 1-phosphate, but concentrations greater than 150 M caused significant inhibition. Specific activities of PMM were consistently higher in the soluble fractions of mucoid strains (1.2–3.6 nmol/min/mg protein) than of nonmucoid strains (0.2–0.6 nmol/min/mg protein).