Pseudomonas Pseudoflava Research Articles

Production of polyhydroxyalkanoates (PHA) by pseudomonas from waste products

Polyhydroxyalkanoate commercial manufacture is hampered by high production costs (PHAs). Up to 50% of the total process expenses might be attributed to the medium used for cultivation. With this research, the researchers hoped to determine if acidogenic fermentation of food waste may yield volatile fatty acids as low-cost carbon sources for bacterial growth essential to make polyhydroxyalkanoates (PHAs). The bacterium Pseudomonas pseudoflava was used to examine the generation of polyhydroxyalkanoates (PHA) from waste food at varied carbon concentrations (4-55g/l). Bacteria produced the most PHA at a concentration of 19 g/l (52.5 percent) and removed the most carbon at a concentration of 7 g/l (67.6 percent). Various analytical methods were used to examine the synthesized PHA's structure, molecular weight, and thermal characteristics, among other things. According to the results of this study, organic carbon persisting in wastewaters may be degraded and converted into PHA by using P. pseudoflava.

Polyhydroxyalkanoates (PHA) production from synthetic waste using Pseudomonas pseudoflava: PHA synthase enzyme activity analysis from P. pseudoflava and P. palleronii

Synthetic wastewater (SW) at various carbon concentrations (5–60g/l) were evaluated for polyhydroxyalkanoates (PHA) production using the bacteria Pseudomonas pseudoflava. Bacteria showed highest PHA production with 20g/l (57±5%), and highest carbon removal at 5g/l (74±6%) concentrations respectively. Structure, molecular weight, and thermal properties of the produced PHA were evaluated using various analytical techniques. Bacteria produced homo-polymer [poly-3-hydroxybutyrate (P3HB)] when only acetate was used as carbon source; and it produced co-polymer [poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV)] by addition of co-substrate propionate. PHA synthase, the enzyme which produce PHA was extracted from two bacterial strains i.e., P. pseudoflava and P. palleronii and its molecular weight was analysed using SDS-PAGE. Protein concentration, and PHA synthase enzyme activity of P. pseudoflava and P. palleronii was carried out using spectrophotometer. Results denoted that P. pseudoflava can be used for degradation of organic carbon persistent in wastewaters and their subsequent conversion into PHA.

Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava

BackgroundThe aim of this study is determine the relative sensitivity of a panel of seven polyhydroxyalkanoate producing bacteria to a panel of seven lignocellulosic-derived fermentation inhibitors representing aliphatic acids, furans and phenolics. A further aim was to measure the polyhydroxybutyrate production of select organisms on lignocellulosic-derived monosaccharides arabinose, xylose, glucose and mannose.FindingsWe examined the sensitivity of seven polyhydroxyalkanoate producing bacteria: Azohydromonas lata, Bacillus megaterium, Bacillus cereus, Burkholderia cepacia, Pseudomonas olevorans, Pseudomonas pseudoflava and Ralstonia eutropha, against seven fermentation inhibitors produced by the saccharification of lignocellulose: acetic acid, levulinic acid, coumaric acid, ferulic acid, syringaldehyde, furfural, and hyroxymethyfurfural. There was significant variation in the sensitivity of these microbes to representative phenolics ranging from 0.25-1.5 g/L coumaric and ferulic acid and between 0.5-6.0 g/L syringaldehyde. Inhibition ranged from 0.37-4 g/L and 0.75-6 g/L with acetic acid and levulinic acid, respectively. B. cepacia and P. pseudoflava were selected for further analysis of polyhydroxyalkanoate production.ConclusionsWe find significant differences in sensitivity to the fermentation inhibitors tested and find these variations to be over a relevant concentration range given the concentrations of inhibitors typically found in lignocellulosic hydrolysates. Of the seven bacteria tested, B. cepacia demonstrated the greatest inhibitor tolerance. Similarly, of two organisms examined for polyhydroxybutyrate production, B. cepacia was notably more efficient when fermenting pentose substrates.

Biosynthesis of copolyesters by Hydrogenophaga pseudoflava from various lactones

Hydrogenophaga pseudoflava (formerly Pseudomonas pseudoflava) was able to accumulate a large amount of copolyesters when grown on mixed substrates of glucose and lactones in a batch fermentation. Lactones such as γ-butyrolactone, γ-valerolactone, and higher analogues generally did not support cell growth when used as the sole carbon source. Co-feeding of lactones with glucose enhanced the utilization of lactones for both copolyester accumulation and cell growth. The copolyester from the cells grown on the mixed substrates of glucose (10 g/L) and γ-valerolactone (1–3 mL/L) was poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), while cells grown with γ-butyrolactone (1–3 mL/L) as a cosubstrate produced poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)). The values of parameter D, calculated from the NMR dyad-sequence data for polymer samples obtained after 72 h of cultivation, showed lactone concentration dependences differing among lactones. The time-course data obtained from growth on the cosubstrates of γ-valerolactone (2 mL/L) and glucose (10 g/L) revealed that 3HV-rich copolymers were synthesized in the early growth phase, and the 3HB-rich fraction steadily increased in the later accumulation phase and then peaked at 80 h when γ-valerolactone was depleted. These polyhydroxyalkanoate accumulation profiles suggested a high D value of the final product, whose value was determined to be 3.25. γ-Valerolactone was consumed faster than γ-butyrolactone. The difference between the assimilation behavior of the two lactones was discussed in relation to the heterogeneity of the final copolyester products. A correlation between NMR microstructure and the physiology of polyhydroxyalkanoate accumulation was observed.Key words: microstructural heterogeneity, bacterial copolyesters, lactones, Hydrogenophaga pseudoflava.

Secretion of human epidermal growth factor (EGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium, Pseudomonas pseudoflava, carrying broad-host-range EGF secretion vector pKSEGF2

We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted EGF into the culture medium constitutively. Four hydrogen-utilizing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcaligenes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested. In these transconjugated hydrogen-utilizing bacteria, P. pseudoflava carrying pKSEGF2 grew autotrophically and secreted EGF, confirmed by Western blot (immunoblot) analysis, into the culture medium constitutively.

水素酸化脱窒細菌の比脱窒速度に及ぼす諸因子の検討

Autotrophic denitrification was studied using hydrogen gas as the hydrogen donor. It was confirmed that microorganisms which have the ability of autotrophic denitrification using hydrogen gas are distributed widely. It was concluded that Alcaligenes eutrophus (ATCC 17697), Paracoccus denitrificans (IFO 13301) and Pseudomonas pseudoflava (ATCC 33668) can reduce nitrate to nitrogen gas in the presence of hydrogen gas. The effect of pH value for autotrophic denitrification of these bacteria was not significant within the range of 7 to 8. The effect of temperature on the specific denitrification rate was also examined and the calculated Arrhenius constant was within the range of 44.1 to 63.2 kJ·mole-1. The specific denitrification rate of A. eutrophus and P. pseudoflava was above 1 mgN·mgSS-1·d-1 at 15 degree centigrade. The specific denitrification activity was constant above 10 kPa of partial pressure of hydrogen gas and the Michaelis-Menten half-velocity constant for hydrogen gas was found to be less than 3% of saturation.

A model system for a fluorometric biosensor using permeabilized Zymomonas mobilis or enzymes with protein confined dinucleotides

Using permeabilized Zymomonas mobilis or glucose-fructose oxidoreductase isolated from this microorganism a model system for biosensors with a protein confined NADP(H) cofactor for the determination of glucose, fructose, gluconolactone, and sorbitol was developed. Either permeabilized microorganisms containing the oxidoreductase or the pure enzyme were confined via membrane separation in a small measuring chamber, that was integrated into a flow injection analysis system (FIA). The measuring principle was the monitoring of the NAD(P)H fluorescence, excited at 360 nm and measured at 450 nm. NADP(H), which is confined in the protein complex, was oxidized or reduced during the enzymatic reactions and the changes in the fluorescence intensity were related to the substrate concentration. The sensitivity of the system covered a range from 0.001 to 100 g/L of the analyte depending on substrate and operating conditions. The applicability of this model system for bioprocess monitoring was proved using samples from a Pseudomonas pseudoflava cultivation.

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.

Hydrogenophaga, a New Genus of Hydrogen-Oxidizing Bacteria That Includes Hydrogenophaga flava comb. nov. (Formerly Pseudomonas flava), Hydrogenophaga palleronii (Formerly Pseudomonas palleronii), Hydrogenophaga pseudoflava (Formerly Pseudomonas pseudoflava and "Pseudomonas carboxydoflava"), and Hydrogenophaga taeniospiralis (Formerly Pseudomonas taeniospiralis)

The relationships of the yellow-pigmented hydrogen-oxidizing species Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas taeniospiralis, and “Pseudomonas carboxydoflava,” which are all members of the acidovorans ribosomal ribonucleic acid (rRNA) complex in rRNA superfamily III, were studied by using deoxyribonucleic acid (DNA):rRNA hybridization, immunotyping, numerical analysis of biochemical and auxanographic features, polyacrylamide gel electrophoresis of cellular proteins, numerical analysis of fatty acid patterns, and DNArDNA hybridization. Our results show that these five yellow-pigmented hydrogen-oxidizing Pseudomonas species are more closely related to each other than to other taxa belonging to the acidovorans rRNA complex. We propose the transfer of these species to a new genus, Hydrogenophaga, with the following four species: Hydrogenophaga flava (formerly Pseudomonas flava), Hydrogenophaga pseudoflava (to accommodate both Pseudomonas pseudoflava and “Pseudomonas carboxydoflava”), Hydrogenophaga taeniospiralis (formerly Pseudomonas taeniospiralis), and Hydrogenophaga palleronii (formerly Pseudomonas palleronii). The type species is H. flava, with monotype strain DSM 619 (= LMG 2185 = CCUG 1658). Because H. flava grows slowly and unreliably, but is genotypically and protein electrophoretically very similar to H. pseudoflava, the latter species can be used as an alternative reference taxon for the new genus. The type strains of H. pseudoflava, H. taeniospiralis, and H. palleronii are strains GA3 (= LMG 5945 = CCUG 13799), DSM 2082 (= LMG 7170 = CCUG 15921), and Stanier 362tl (= LMG 2366tl = CCUG 20334), respectively.

Nitrogen Fixation by New Strains of Pseudomonas pseudoflava and Related Bacteria

Five representative bacterial strains resembling Pseudomonas pseudoflava, chosen from among forty strains isolated by three different procedures, were characterized by phenotypic and genotypic analyses. They were shown to belong to the species P. pseudoflava, or to a distinct, but closely related, cluster. In contrast to the initial isolates of this species, most of the new strains were able to fix N2, as shown by their growth in nitrogen-free medium and by the acetylene reduction test. Moreover, DNA regions of these new strains were homologous to Klebsiella pneumomae nifHDK genes, confirming the presence of nitrogenase genes. The nif genes are probably chromosomally located, since no plasmid was detected in these strains. The presence of complete but defective nif genes in P. pseudoflava GA3 (the type strain, which does not fix N2) was excluded, as no homology was found between K. pneumoniae nif genes and P. pseudoflava GA3 total DNA. The isolation conditions seem to determine the selection of P. pseudoflava N2-fixing strains, since selection for H2-chemolithoautotrophs gave no N2-fixing isolates, whereas H2-chemolithoheterotrophic selection conditions (with combined nitrogen) yielded a majority of N2 fixers.

Colonization and mineralization of palmitic acid byPseudomonas pseudoflava

Pseudomonas pseudoflava and palmitic acid were used to investigate the role of bacterial colonization in the degradation of waterinsoluble organic compounds. Mineralization was measured by trapping the(14)CO2 produced from the labeled substrate, and colonization of the surface of the solid organic chemical was determined by epifluorescence microscopy. In a medium containing solid palmitic acid,P. pseudoflava mineralized the organic substrate at a logarithmic rate. Mineralization was evident before colonization of the surface of the chemical was detected. The rate of appearance of single cells and/or aggregations of cells on the surface of the palmitic acid was essentially the same as the doubling time of free cells in solution. At about 50 hours, mineralization and colonization of the surface stopped. In a salts solution containing solid palmitic acid,P. pseudoflava grew logarithmically in the solution and biphasically on the surface of the palmitic acid. We suggest that the bacterium first metabolizes soluble palmitic acid and later colonizes the solid when the substrate in solution has been depleted.

ESR properties of membrane-bound hydrogenases from aerobic hydrogen bacteria

Analysis of the membrane-bound hydrogenase of Alcaligenes eutrophus, strain H16, indicated a content of 7–9 Fe, 7–9 labile sulphide and 0.6–0.7 nickel atoms per molecule of 98 kDa. The protein, as prepared, gave a complex electron-spin resonance (ESR) spectrum in the oxidized state at low temperatures (below 30 K), with average g value greater than 2.0. This was interpreted as being due to the interaction of [3Fe-χS] or [4Fe-4S] 3+ cluster and another paramagnetic centre. At higher temperatures a rhombic spectrum, attributred to Ni(III), was observed. On treatment with mercaptoethanol, or reduction to a redox potential below +160 mV, the low-temperature spectrum changed into a narrow signal at g = 2.02, with increased amplitude. The g = 2.02 signal was reduced reversibly with a midpoint potential of 40 ± 30 mV. The complex spectrum could be restored if the enzyme was rapidly reduced and reoxidized, but not under the conditions of the redox titration, when a broader signal appeared instead. The reductive titration was accompanied by a 3–7-fold increase in enzymic activity. On further reduction of A. eutrophus H16 hydrogenase a complex spectrum with average g value less than 2.0 was observed. This indicate the presence of a [4Fe-45] 1+ cluster, interacting with either another such cluster or another paramagnetic centre. This cluster or clusters were estimated to have a midpoint potential of −90 ± 30 mV. The ESR spectra of the hydrogenases of A. eutrophus, type strain, and Pseudomonas pseudoflava, in the oxidized and reduced states, were similar to those of the A. eutrophus H16 enzyme, indicating that they have a similar arrangement of redox centres. The characteristic spectra of the reduced e indicate that they represent a new type of membrane-bound hydrogenase.

Ubiquinones (Coenzyme Q) in hydrogen oxidizing bacteria

The isoprenoid quinone content and composition of several strains of the following hydrogen oxidizing bacteria were studied: Xanthobacter autotrophics, Xanthobacter flavus, Paracoccus denitrificans, Pseudomonas pseudoflava, P. carboxydovorans, P. carboxydo-hydrogena. All strains studied contained ubiquinone (UQ) in concentrations between 0.7 and 2.8 μmol/g bacterial dry weight. No menaquinones were found. UQ of all strains (except one) contained side chain lengths of 10isoprenoid units (UQ-10), two strains contained in addition UQ-8. UQ-9 was found in Pseudomonas carboxydohydrogena only.

Occurrence of Phosphatidylcholine in Hydrogen‐Oxidizing Bacteria

15 strains of hydrogen-oxidizing bacteria were grown heterotrophically, harvested during the stationary phase of growth, and analyzed for their principal phospholipids. All strains - with the exception of Corynebacterium autotrophicum strain SA 32 and Pseudomonas pseudoflava - contained phosphatidylcholine as a major constituent. It is concluded that the presence of phosphatidylcholine is neither characteristic of a peculiar bacterial genus or family, nor is it absolutely correlated to the ability to oxidize hydrogen. The phosphatidylcholines of all strains contain C19 cyclopropane acid which is, in some strains, predominantly located at C-2 position of the glycerol moiety.

Characterization and Comparison of the DNAs of the Three Closely Related Bacteriophages gd, ge and gf with the Genome DNA of the Hydrogen-oxidizing Host Strain Pseudomonas pseudoflava GA3

The double-stranded (ds)DNAs of the three closely related temperate Pseudomonas pseudoflava bacteriophages gd, ge and gf were studied biochemically and biophysically. The GC content of the DNA was 67.4 +/- 0.5% and differed only slightly from that of the host P. pseudoflava. By electron microscopic length measurements a mol. wt. of 26.1 X 10(6) to 26.7 X 10(6) was calculated for the three bacteriophage DNAs. Homogeneity of the bacteriophage DNAs was further demonstrated by specific cleavage with restriction endonucleases WcoRI and HindIII. It was concluded that the three homo-immune bacteriophages are identical. The genome size of the host P. pseudoflava GA3 was 3.7 X 10(9) as calculated from optical renaturation rate measurement with Xanthomonas pelargonii reference DNA. The bacteriophage gd genome thus amounts to 0.7% of the chromosome of this bacterial host.

Deoxyribonucleic Acid Relationships Among Hydrogen-Oxidizing Strains of the Genera Pseudomonas, Alcaligenes, and Paracoccus

Optical determination of deoxyribonucleic acid (DNA)-DNA reassociation kinetics was applied to the classification of 32 selected strains of hydrogen-oxidizing bacteria belonging to the genera Pseudomonas, Alcaligenes, and Paracoccus. The renaturation studies revealed a high intraspecies DNA homology for some strains of the species Pseudomonas palleronii, Pseudomonas pseudoflava, and Alcaligenes paradoxus, supporting former taxonomic concepts of different authors. The 12 denitrifying strains belonging to the genus Paracoccus were shown to be interrelated at various levels of percent degree of binding, and at least two clusters of very high genome-DNA relatedness have been found. DNA-DNA reassociation kinetics were also used for the calculation of the average molecular weight of genome DNA. The genome molecular weight of the hydrogen-oxidizing bacteria investigated in this study ranged from 3 X 109 to 5 X 109.

Biological, Serological and Biochemical Investigations of Three Closely Related Temperate Bacteriophages Specific for Pseudomonas pseudoflava

Summary A close relationship between three temperate phages (gd, ge and gf), specific for three strains of the newly described species Pseudomonas pseudoflava was revealed by an identical pattern of structural proteins, by serological cross-reactions and by the finding of cross-immunity and cross-resistance. In addition attachment of the phages to the pili of their hosts and the presence of a restriction and modification mechanism was indicated.

Pseudomonas pseudoflava, a New Species of Hydrogen-Oxidizing Bacteria: Its Differentiation from Pseudomonas flava and Other Yellow-Pigmented, Gram-Negative, Hydrogen-Oxidizing Species

Five new strains of yellow-pigmented, gram-negative, motile, hydrogen-oxidizing bacteria were isolated; each served as a host for simultaneously isolated bacteriophages. These isolates and two additional strains were compared with other gram-negative, hydrogen-oxidizing bacteria with respect to morphology; nutritional and biochemical properties; growth parameters; cytochrome content; pigment production; susceptibility to bacteriophages, bacteriostatic agents, and antibiotics; deoxyribonucleic acid base composition; and deoxyribonucleic acid-deoxyribonucleic acid homology. Six of the strains were characterized by a high degree of interstrain similarity and were found to be related to Pseudomonas flava. However, due to basic differences between these strains and P. flava, the former are regarded as comprising a new species for which, because of its moderate relationship to P. flava, the name Pseudomonas pseudoflava is proposed. The type strain of P. pseudoflava, GA3, has been deposited with the Deutsche Sammlung von Mikroorganismen in Göttingen under the number DSM 1034.

Micromorphology of Gram-negative hydrogen bacteria

The cell morphology, the arrangement and fine structure of flagella and the piliation of the following Gram-negative aerobic hydrogen bacteria have been studied: Alcaligenes eutrophus, Alcaligenes paradoxus, Alcaligenes ruhlandii, Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas facilis, Aquaspirillum autotrophicum, Paracoccus denitrificans, Corynebacterium autotrophicum, and strains MA 2 and SA 35. The identity of the bacteria was examined by their substrate spectra and type of flagellation. Three types of flagellar fine structure were differentiated. The presence of pili was noted in strains of Alcaligenes paradoxus, Pseudomonas flava, P.pseudoflava, P.palleronii, and P.facilis.