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PubMed1964

ATMOSPHERIC NITROGEN FIXATION BY METHANE-OXIDIZING BACTERIA.

Davis, J. B. (Socony Mobil Oil Co., Inc., Dallas, Tex.), V. F. Coty, and J. P. Stanley. Atmospheric nitrogen fixation by methane-oxidizing bacteria. J. Bacteriol. 88:468-472. 1964.-Methane-oxidizing bacteria capable of fixing atmospheric nitrogen were isolated from garden soil, pond mud, oil field soil, and soil exposed to natural gas, indicating a rather wide prevalence in nature. This may explain the high concentration of organic nitrogen commonly found in soils exposed to gas leakage from pipelines or natural-gas seeps. Added molybdenum was a requirement for growth in a nitrogen-free mineral salts medium. All nitrogen-fixing, methane-oxidizing bacteria isolated were gram-negative, nonsporeforming, usually motile rods. Colonies were light yellow, yellow, or white. The most common isolate, which formed light-yellow colonies, is referred to as Pseudomonas methanitrificans sp. n., and is distinguished from Pseudomonas (Methanomonas) methanica by nitrogen-fixing ability and a preponderance of poly-beta-hydroxybutyrate in the cellular lipid fraction.

Bacteria↗
PubMed1965

MORPHOLOGICAL CHANGES IN POLY-BETA-HYDROXYBUTYRATE GRANULES ASSOCIATED WITH DECREASED SUSCEPTIBILITY TO ENZYMATIC HYDROLYSIS.

Merrick, J. M. (State University of New York, Buffalo), D. G. Lundgren, and R. M. Pfister. Morphological changes in poly-beta-hydroxybutyrate granules associated with decreased susceptibility to enzymatic hydrolysis. J. Bacteriol. 89:234-239. 1965.-A complex enzyme system obtained from extracts of Rhodospirillum rubrum cells hydrolyzes poly-beta-hydroxybutyric acid (PHB) contained in native PHB granules isolated from Bacillus megaterium. A labile factor associated with the granules and necessary for depolymerization is easily destroyed by various chemical and physical treatments. Granules inactivated by these treatments were examined in an electron microscope. In all cases, the distinct morphological appearance of native granules was altered. Morphological changes were mainly characterized by membrane fragmentation, loss of coalescence, and surface alterations. These observations suggest that native PHB granules possess definite structural features, disruption of which results in decreased susceptibility of the polymer to enzymatic hydrolysis.

Bacillus megaterium↗
PubMed1965

CHARACTERIZATION OF POLY-BETA-HYDROXYBUTYRATE EXTRACTED FROM DIFFERENT BACTERIA.

Lundgren, D. G. (Syracuse University, Syracuse, N.Y.), R. Alper, C. Schnaitman and R. H. Marchessault. Characterization of poly-beta-hydroxybutyrate extracted from different bacteria. J. Bacteriol. 89:245-251. 1965.-Poly-beta-hydroxybutyrate (PHB) from different bacterial genera was studied with regard to its crystal structure, infrared absorption, intrinsic viscosity, and electron microscopy. All PHB samples precipitated from dilute chloroform solution gave identical X-ray diffractograms confirming uniformity of crystal structure, and uniformity of molecular structure, based on the similarity of the recorded infrared spectra, was also established. Crystal morphology was also similar, showing the reported "lath" shape structure for purified polymer from Bacillus cereus. Intrinsic viscosity ranged from 0.04 to 11.5 depending upon the polymer treatment; polymer molecular weights, based upon viscometry, could be estimated to range from 1,000 to 250,000. It is concluded that the same basic molecule is involved in all PHB present in the bacterial kindgom.

Azotobacter↗
PubMed1965

CHARACTERISTICS AND INTERMEDIATES OF SHORT-TERM C-14-O-2 INCORPORATION DURING RIBOSE OXIDATION BY HYDROGENOMONAS FACILIS.

McFadden, B. A. (Washington State University, Pullman), and H. R. Homann. Characteristics and intermediates of short-term C(14)O(2) incorporation during ribose oxidation by Hydrogenomonas facilis. J. Bacteriol. 89:839-847. 1965.-Ribose-grown cells of Hydrogenomonas facilis, which had been suspended in growth medium and were oxidizing ribose, were exposed to HC(14)O(3) (-) of high specific activity. The uptake was proportional to cell mass. Short-term uptake (less than 2 min) was completely inhibited by 10(-3)m 2,4-dinitrophenol (DNP) or by <4 x 10(-6)mm-chlorocarbonyl cyanide phenylhydrazone, and to the extent of 42% by 5 x 10(-5)m DNP. The following observations were made in kinetic studies (8, 16, 35, 67, 96, and 181 sec) of fixation in the presence of ribose. Glutamate was extensively labeled in periods up to 3 min. It was one of the major early products, containing 30% of the label at 8 sec. The sugar phosphate fraction was not detectably labeled at 8 or 16 sec, but its C(14)-content increased rapidly to 27% at 35 sec and then slowly decreased. Label in phosphoglycerate, phosphoenolpyruvate, and alanine did not appear until 35 sec, and did not exceed about 7, 2, and 3%, respectively, of the total extracted radioactivity. Adenosine triphosphate and adenosine diphosphate were heavily labeled after fixation in a pilot study for 125 sec. Although considerable radioactivity incorporated during the pilot study was intractable by the extraction procedure employed, virtually no C(14) was found in the residue in poly-beta-hydroxybutyric acid. A large number of amino acids and organic acids and some organic phosphates were not detectably labeled in any of the experiments. Omission of ribose greatly diminished incorporation, particularly into glutamate.

2,4-Dinitrophenol↗
PubMed1965

NICKEL-DEPENDENT CHEMOLITHOTROPHIC GROWTH OF TWO HYDROGENOMONAS STRAINS.

Bartha, R. (University of Washington, Seattle), and E. J. Ordal. Nickel-dependent chemolithotrophic growth of two Hydrogenomonas strains. J. Bacteriol. 89:1015-1019. 1965.-The trace element requirements for growth of facultative chemolithotrophic Hydrogenomonas strains H1 and H16 were investigated under both autotrophic and heterotrophic conditions. The organisms were grown in a mineral medium, rendered deficient in trace elements by extraction with 8-hydroxyquinoline and chloroform, and, in some cases, by coprecipitation with copper. The organic substrates, succinate and fumarate, used for heterotrophic growth were treated in a similar fashion. Acetate and butyrate were purified by redistillation. It was found that iron alone was required for heterotrophic growth (optimal concentration, 1.5 x 10(-6)m Fe(+++)), but cells grown chemolithotrophically on molecular hydrogen required the addition of nickel. The yield of protein was proportional to the nickel added, reaching a maximum at 3 x 10(-7)m Ni(++). Manganese, cobalt, copper, and zinc, alone or in combination, failed to substitute for nickel in the experiments with Hydrogenomonas. Although nickel is required specifically for the chemolithotrophic growth of Hydrogenomonas, nickel deficiency did not affect: (i) the synthesis or activation of hydrogenase, (ii) the Knallgas reaction, (iii) the assimilation of CO(2) by resting cells, or the synthesis of the storage material poly-beta-hydroxybutyric acid. It is suggested that nickel participates in some reaction involved in CO(2) fixation by growing cells.

Acetates↗
PubMed1965

INFLUENCE OF AERATION AND OF PANTOTHENATE ON GROWTH YIELDS OF ZYMOMONAS MOBILIS.

Belaïch, Jean-Pierre (Centre National de la Recherche Scientifique, Marseille, France), and Jacques C. Senez. Influence of aeration and of pantothenate on growth yields of Zymomonas mobilis. J. Bacteriol. 89:1195-1200. 1965.-The growth yields and rates of Zymomonas mobilis were measured in aerobic and anaerobic cultures on glucose medium containing yeast extract, amino acids, or ammonium chloride as the nitrogen source. In the absence of yeast extract, pantothenate was required. The growth yield and rate of the cultures in synthetic (amino acids) or minimal (NH(4)Cl) medium supplemented with pantothenate corresponded only to about one-half the "normal" values obtained in the presence of yeast extract, suggesting a situation of energetically uncoupled growth. Attempts to restore normal growth by the addition of various compounds were unsuccessful. Aeration of the cultures resulted in a partial oxidation of ethyl alcohol to acetate, but did not modify the growth yield nor the division time. Both aerobic and anaerobic cells, however, contained cytochrome c and a cytochrome oxidase of the a(2) type, which was completely inhibited by 10(-4)m cyanide. In anaerobically grown cells, an additional cytochrome of the b type was present. The absence of a Pasteur effect suggests that the transfer of electrons by the respiratory chain of Z. mobilis may not be coupled with oxidative phosphorylation. Aeration had no effect on the catalase content of the cells. As shown by C(14)-glucose incorporation, 2 to 3% of the glucose metabolized was assimilated by the cells in both synthetic and rich complex medium. No intracellular glycogen nor poly-beta-hydroxybutyrate was accumulated when growth was limited by nitrogen or by phosphate in the presence of excess glucose.

Amino Acids↗
PubMed1985

Quantitative determination of microbial activity and community nutritional status in estuarine sediments: evidence for a disturbance artifact.

In estuarine sediments with a high degree of vertical heterogeneity in reduced substrate and terminal electron acceptor concentrations, the method of exposure of the microbiota to labeled substrates can introduce a "disturbance artifact" into measures of metabolic activity. The detection of this artifact is based on quantitative measurement of the relative rates of incorporation of [14C]acetate into phospholipid fatty acids (PLFA) and endogenous storage lipid, poly-beta-hydroxyalkanoate (PHA). Previous studies have shown that PLFA synthesis measures cellular growth and that PHA synthesis measures carbon accumulation (unbalanced growth). The "disturbance artifact" of exposure to [14C]acetate was demonstrated by comparing injection of a core with the usual or pore-water replacement or slurry techniques. Only injection of labeled substrate allowed detection of preassay disturbance of the sediment with a garden rake. The raking increased PLFA synthesis with little effect to differences in concentration or distribution of [14C]acetate in the 10-min incubation. Bioturbation induced by sand dollar feeding in estuarine sediment could be detected in an increased PLFA/PHA ratio which was due to decreased PHA synthesis if the addition of labeled substrate was by the injection technique. Addition of labeled precursors to sediment by slurry or pore-water replacement induces greater disturbance artifacts than injection techniques.

Acetates↗
PubMed1987

Cellular incorporation of poly-beta-hydroxybutyrate into plasma membranes of Escherichia coli and Azotobacter vinelandii alters native membrane structure.

Under growth-limiting conditions or conditions which mediate genetic transformation, Escherichia coli and Azotobacter vinelandii incorporate poly-beta-hydroxybutyrate into their plasma membranes. Genetic transformation competence of both bacteria increased in proportion to the concentration of membrane poly-beta-hydroxybutyrate. The effects of this lipid polymer on membrane structure were investigated by freeze-fracture electron microscopy. Before poly-beta-hydroxybutyrate incorporation, freeze-fracture revealed a typical mosaic of particles and pits on both concave and convex surfaces of the plasma membrane. As the cells incorporated the lipid polymer into the membrane, transformability developed and small semiregular plaques which possessed shallow particles were seen. These plaques grew in size and frequency as the membrane poly-beta-hydroxybutyrate concentrations and transformability increased.

Azotobacter↗
PubMed1995

Low molecular weight complexed poly(3-hydroxybutyrate): a dynamic and versatile molecule in vivo.

It is increasingly clear that poly(3-hydroxybutyrate) (PHB) is not just an inert storage polymer, confined to certain bacteria, but a ubiquitous, interactive, solvating biopolymer involved in important physiological functions. Low molecular weight PHB, complexed to other macromolecules (c-PHB), is widely distributed in biological cells, being found in representative organisms of nearly all phyla. Complexation modifies the physical and chemical properties of c-PHB, allowing it to pervade aqueous as well as hydrophobic regions of the cell, and as a result c-PHB can be found in cytoplasm and intracellular fluids as well as in membranes and lipoproteins. The lipidic homopolymer associates with other macromolecules primarily via its ester carbonyl oxygens, which can act as hydrogen-bond acceptors or as ligands for coordinate bonds to cations. The spacing of the electron-donating groups along the flexible backbone allows for multiple bonding interactions, and forms the basis for the ability of c-PHB to bind to proteins, or to form ion-conducting complexes with salts. The singular ability of c-PHB to dissolve salts and facilitate their transfer across hydrophobic barriers defines a potential physiological niche for c-PHB in cell metabolism.

Hydroxybutyrates↗
PubMed1995

In situ biodegradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in natural waters.

The biodegradation of samples of poly(3-hydroxybutyrate)(P(3HB)), poly(3-hydroxybutyrate-co-10%-3-hydroxyvalerate)(P(3HB-co-10 %-3HV)), and poly 3-hydroxybutyrate-co-20%-3-hydroxyvalerate)(P(3HB-co-20%- 3HV)) was investigated in situ in natural waters. The degradation was studied by decrease in mass, molecular weight, and tensile strength. In two freshwater ponds the polymers were slowly degraded. After half a year of submersion the mass loss was less than 7%. After 358 days in a freshwater canal, 34% mass loss was recorded for the homopolymer, and 77% for the P(3HB-co-10%-3-HV) samples, while the P(3HB-co-20%-3HV) samples had completely disappeared. In seawater in the harbour of Zeebrugge, P(3HB) samples lost 31% of their initial mass, and the copolymers 49-52%, within 270 days. In all of these environments, the degradation rate was faster during the summer, when the temperature of the water was higher. No relevant changes in molecular weight could be detected, indicating that the degradation took place only at the surface of the samples. The degradation resulted in considerable loss of tensile strength of the copolymer samples. Ninety-two microorganisms, mainly bacteria, able to degrade P(3HB) in polymer overlayer plates, were isolated and identified by fatty acid analysis. The isolates from one freshwater pond belonged mainly to the bacterial genus Acidovorax, while the microorganisms from the other freshwater pond belonged to various bacterial genera, to Streptomyces, and to the mould genus Penicillium. Most of the 31 bacterial isolates from seawater were identified as Alteromonas haloplanktis. The results demonstrate that P(3HB) and P(3HB-co-3HV) samples are effectively biodegradable in natural waters under real-life conditions and reveal the biodiversity of the microflora responsible for this biodegradation.

Bacteria↗
PubMed1995

Biodegradation of cyclic and substituted linear oligomers of poly(3-hydroxybutyrate).

Cyclic oligo(3-hydroxybutyrate), oligo(3-HB), was synthesized and purified, resulting in oligolides that contained three to seven (R)-3-hydroxybutyrate units (triolides up to heptolides). In addition, linear 3-HB octamers obtained as either tert-butyl or methyl esters were substituted with different end groups at the hydroxy end. The hydroxy terminus was replaced by either a benzyloxy, trifluoroacetoxy, crotonyloxy (S)-3-hydroxybutyryloxy, or fluorenylmethylcarbonyloxy (FMOC) group. P(3-HB) hairpin loops occurred on the surface of certain regions of the polymer, especially of lamellar crystallites. Cyclic 3-HB oligomers provide a model system for these loops. It is assumed that they provide attachment points for the depolymerizing enzymes. All of the (R)-oligolides tested were degraded except the (R)-triolide. Triolides were not degraded, suggesting that enzymatic attack was prevented presumably by steric hindrance on the rigid ring system. Unsubstituted linear octamers were degraded. Biodegradation was prevented when the hydroxy terminus was protected by the FMOC group, but was not dependent on a free hydroxy terminal group; all other protecting groups did not prevent degradation. Substitution of the carboxy end of a methyl or tert-butyl ester group did not influence biodegradation.

Biodegradation, Environmental↗
PubMed1995

The function of ackA and pta genes is necessary for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli.

In Escherichia coli carrying the poly(3-hydroxyalkanoate) (PHA) biosynthesis pathway on a plasmid (pha+), the function of the ackA (acetate kinase) and pta (phosphotransacetylase) genes is necessary for efficient incorporation of 3-hydroxyvalerate (3-HV) into the copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)). Recombinant pha+ E. coli fadR atoC(Con) strains possessing mutations in ackA, pta, or both ackA and pta exhibited substantially reduced levels of 3-HV formation. Conversely, the same strains carrying the ackA gene on a multicopy plasmid exhibited an increase in 3-HV formation concomitant with a large increase in acetate kinase activity. However, if the strain possessing the multicopy ackA+ plasmid was mutant at the pta locus, it lost the ability to incorporate significant amounts of 3-HV into P(3HB-co-3HV). In addition to the ackA pta pathway, there is an inducible activity that can also mediate the incorporation of 3-HV into P(3HB-co-3HV). This pathway is repressed by glucose and is not normally operative in P(3HB-co-3HV) production in recombinant pha+ E. coli strains that are grown using glucose as the major carbon source. It appears likely that this activity is due to an inducible acetyl-CoA synthetase that converts propionate to propionyl-CoA.

Acetate Kinase↗
PubMed1995

Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studies.

A number of Escherichia coli strains including K12, B, W, XL1-Blue, DH5 alpha, HB101, JM109, and C600 were transformed with the stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid biosynthesis genes, and were subsequently compared for their ability to synthesize and accumulate poly(3-hydroxybutyric acid) (PHB). The rate of PHB synthesis, the extent of PHB accumulation, and PHB yield from glucose varied considerably from one strain to another. Strains XL1-Blue and B harboring pSYL105 synthesized PHB at the highest rate to a final concentration of ca. 7 g/L in complex medium containing 20 g glucose/L. With an aim to reduce the cost of the medium, the effect on PHB accumulation of supplementing a defined medium with complex nitrogen sources was examined. A PHB concentration of 81 g/L could be obtained in 41 h from a pH-stat fed-batch culture of XL1-Blue(pSYL105) in a semidefined medium. When the availability of acetyl-CoA was increased by supplementing the medium with complex nitrogen sources, amino acids, or oleic acid, PHB synthesis by recombinant E. coli was enhanced.

Alcaligenes↗
PubMed1995

High-level poly(beta-hydroxybutyrate) production in recombinant Escherichia coli in sugar-free, complex medium.

The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50-80% PHB/cell dry mass when the cells are grown in Luria-Bertani medium alone or supplemented with 1% glucose (w/v), respectively. To further stimulate PHB formation independent of additional carbon source in Luria-Bertani medium, molecular methods have been applied to provide efficient E. coli transcription and translation signals for the PHB synthase gene (phbC). The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain. The T7 gene 10 ribosome binding site is utilized for translational initiation. PHB production in E. coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the beta-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter-operator region and also carrying separately the phbC gene with its natural promoter sequence. In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E. coli when grown in Luria-Bertani medium without glucose supplement.

Acetyl-CoA C-Acyltransferase↗
PubMed1995

Morphology and biodegradability of a binary blend of poly((R)-3-hydroxybutyric acid) and poly((R,S)-lactic acid).

The miscibility, morphology, and biodegradability of a binary blend of bacterial poly((R)-3-hydroxybutyric acid)(P((R)-3HB);Mn = 300,000) with atactic poly((R,S)-lactic acid)(P((R,S)-LA);Mn = 9,000) were studied by means of differential scanning calorimetry, optical microscopy, scanning electron microscopy, and hydrolysis with an without enzyme. Differential scanning calorimetry revealed that a P((R)-3HB)-P((R,S)-LA) blend had a single glass-transition temperature for all proportions of the components. The spherulites of P((R)-3HB) were volume filled in the blend films, indicating the inclusion of amorphous P((R,S)-LA) within the spherulites. The spherulitic growth rate decreased with an increase in the content of P((R,S)-LA). These results indicate that the P((R)-3HB)-P((R,S)-LA) blend is miscible in the melt and in the amorphous state. The enzymatic hydrolysis of P((R)-3HB)-P((R,S)-LA) blend films was carried out at 37 degrees C for 19 h in 0.1 M potassium phosphate buffer (pH 7.4) with an extracellular poly(hydroxybutyrate) depolymerase from Alcaligenes faecalis T1. The rate of enzymatic surface erosion decreased with increasing P((R,S)-LA) content in the blend films. The simple hydrolysis of P((R)-3HB)-P((R,S)-LA) blend films without enzyme was also conducted at 37 degrees C in a 0.01 M potassium phosphate buffer (pH 7.4) for 150 days. The hydrolytic scission of P((R)-3HB) polymer chains was accelerated by blending with P((R,S)-LA). However, the rate of enzymatic hydrolysis was much faster than the rate of nonenzymatic hydrolysis.

Alcaligenes↗
PubMed1997

Interaction between poly-3-hydroxybutyrate-co-3-hydroxyvalerate and a denitrifying Pseudomonas strain.

In a laboratory-scale system, dentrification activity of a heterotrophic microbial starter culture changed when different lots of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) were used as the solid carbon source in the heterotrophic denitrification reactor. In this study, possible influences of physical and chemical properties of commercially produced P(HB-co-HV) (Biopol) on biofilm formation and metabolic activity of a denitrifying starter culture were investigated. These parameters indicate the polymers' suitability for the application as the matrix substance in the bioreactor. No differences in microstructure were detected between the different lots of polymers. Growth inhibitory effects by chemical additives were found in the case of triacetine, which was included as a plasticizer in seven of eight tested lots. The amount of hydroxyvaleric acid in the polymer was not assumed to affect denitrification activity. Relevant differences could be detected regarding primary adhesion of the starter culture Pseudomonas sp. strain 2nIII. It showed good adsorption properties to hydrophobic substances with a dependence on precultivation conditions. Pseudomonas sp. strain 2nIII degraded poly-3-hydroxybutyrate acid homopolymer and P(HB-co-HV) copolymers but was unable to break up poly-3-hydroxyvaleric acid. A possible reason for these findings is the substrate specifity of the polyhydroxyalkanoate depolymerase.

Adsorption↗