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PubMed2001

Properties, modifications and applications of biopolyesters.

Poly(hydroxyalkanoates) (PHAs), of which poly(hydroxybutyrate) (PHB) is the most common, can be accumulated by a large number of bacteria as energy and carbon reserve. Due to their biodegradability and biocompatibility these optically active biopolyesters may find industrial applications. A general overview of the physical and material properties of PHAs, alongside with accomplished applications and new developments in this field is presented in this chapter. The properties of PHAs are dependent on their monomer composition and therefore it is of great interest that recent research has revealed that, in addition to PHB, a large variety of PHAs can be synthesized microbially. The monomer composition of PHAs depends on the nature of the carbon source and microorganism used. PHB is a typical highly crystalline thermoplastic whereas medium chain length PHAs are elastomers with low melting points and a relatively lower degree of crystallinity. By (chemical) modification of the PHAs, the ultimate properties of the materials can be adjusted even further, when necessary. Applications that have been developed from PHB and related materials (e.g. Biopol) can be found in very different application areas and cover packaging, hygienic, agricultural and biomedical products. Recent application developments based on medium chain length PHAs range from high solid alkyd-like paints to pressure sensitive adhesives, biodegradable cheese coatings and biodegradable rubbers. Technically, the prospects for PHAs are very promising. When the price of these materials can be further reduced, application of biopolyesters will also become economically very attractive.

Bacteria↗
PubMed2004

Clarification of interactions among microorganisms and development of co-culture system for production of useful substances.

Co-culture systems containing two microorganisms for the production of useful substances are described. We developed a novel co-culture system composed of two fermentors and two microfiltration modules. The proposed co-culture system allowed regulation of the dissolved oxygen concentration at a level suitable for an individual microorganism in each fermentor, as well as the successful exchange of culture medium between two fermentors. By co-culture, using a combination of Pichia stipitis and Saccharomyces cerevisiae, ethanol was produced efficiently from a mixture of glucose and xylose. Moreover, the useful probiotic cells were simultaneously produced with a high productivity by our co-culture using a combination of Bifidobacterium and Propionibacterium. Kefiran production by Lactobacillus kefiranofaciens alone under the culture conditions, established by mimicking the presence and activities of yeast cells in kefir grains, was also investigated. The results obtained showed that under the culture conditions established by mimicking the actions of yeast cells on L. kefiranofaciens in kefir grains, the amount of kefiran produced was enhanced, even when the lactic acid bacterium alone was used.

Bacteria↗
PubMed1993

Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea.

From a genomic library of Thiocystis violaceae strain 2311 in lambda L47, two adjacent EcoRI restriction fragments of 5361 base pairs (bp) and of 1978 bp were cloned. The 5361-bp EcoRI restriction fragment hybridized with a DNA fragment harbouring the Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthase operon (phbCAB) and restored the ability to synthesize and accumulate PHA in PHA-negative mutants derived from A. eutrophus. The nucleotide sequence analysis of both fragments revealed five open-reading frames (ORFs); at least three of them are probably relevant for PHA biosynthesis. The amino acid sequences of the putative proteins deduced from these genes indicate that they encode a beta-ketothiolase [phbATv, relative molecular mass (M(r)) 40850], which exhibited 87.3% amino acid identity with the beta-ketothiolase from Chromatium vinosum. The amino acid sequences of the putative proteins deduced from ORF2Tv (M(r) 41450) and phbCTv (M(r) 39550), which were located upstream of and antilinear to phbATv, exhibited 74.7% and 87.6% amino acid identity, respectively, with the corresponding gene products of C. vinosum. Downstream of and antilinear to phbCTv was located ORF5, which encodes for a protein of high relative molecular mass (M(r) 76428) of unknown function. With respect to the divergent organisation of ORF2Tv and phbCTv on one side and of phbATv on the other side and from the homologies of the putative gene products, this region of the T. violaceae genome resembled very much the corresponding region of C. vinosum, which was identified recently.

Amino Acid Sequence↗
PubMed1986

The effect of the dissolved oxygen concentration and anabolic limitations on the behaviour of Rhizobium ORS571 in chemostat cultures.

Chemostat cultures of Rhizobium ORS571 limited by the supply of oxygen or an anabolic substrate contained poly-beta-hydroxybutyrate (PHB). Low amounts of PHB (about 10%) were present in ammonia- or nitrate-limited cultures; higher amounts were found in Mg++-limited cultures (about 20%) and in oxygen-limited nitrogen-fixing cultures (37%). A method is described to calculate YATP values (g PHB-free biomass . mol-1 ATP) from the Ysucc values (g dry wt . mol-1 succinate) measured. Ysucc and YATP values in cultures limited by the supply of an anabolic substrate and in the oxygen-limited ammonia-assimilating culture were much lower than the values found in the PHB-free succinate-limited cultures. This shows that uncoupling of growth and energy production occurred. Therefore, H2/N2 ratio (mol hydrogen formed per mol nitrogen fixed) in nitrogen-fixing cultures could not be calculated from the comparison of the YATP value found in the nitrogen-fixing culture and the value found in the corresponding ammonia-assimilating culture. Although the optimal dissolved oxygen concentration (d.o.c.) for nitrogen-fixing cultures of Rhizobium ORS571 is 5 or 10 microM, nitrogen-fixing cultures could be obtained up to a d.o.c. of 40 microM. Not only nitrogenase but also hydrogenase was active at this d.o.c. However, accumulation of PHB (10%) may indicate that cultures grown at unfavourable oxygen concentrations (15-40 microM O2) were N-limited rather than energy-limited, which may be the result of partial inactivation or repression of nitrogenase at a higher d.o.c.

Adenosine Triphosphate↗
PubMed1984

Microbial aspects of the cockroach hindgut.

The cockroach hindgut has a complex, active microbiota, a large portion of which is associated with the chitinous gut wall. It provides a different environment from that of termites and other insects which are dependent on their hindgut microbiota for the digestion of cellulose. The pH of the midgut of Eublaberus posticus was not as high as it is in insects with a primarily cellulolytic nutrition. The hindgut of E. posticus was highly methanogenic, normal adults producing typically 10-25 mumol of methane per hour. The hindgut contained large amounts of the storage products polyphosphate and poly-beta-hydroxybutyrate. Dilution series on nonselective medium yielded 100 times more obligately anaerobic colonies than facultatively anaerobic colonies. The most common facultative isolates were Klebsiella oxytoca, Citrobacter freundii and Enterobacter agglomerans. Treatment of E. posticus with metronidazole caused a dedifferentiation of the different regions of the hindgut. One region of the hindgut is characterized by its visibly black color, a unique microbiota, and electron dense deposits in electron micrographs. Chemical determinations showed high concentrations of ferrous and sulfide ions in the region. X-ray microprobe analysis showed that some of the electron dense deposits consisted of iron, sulfur and lower amounts of copper, aluminium, and chromium associated with ruthenium red staining material. Spectra of other deposits revealed only silicon, which was not associated with ruthenium red.

Anaerobiosis↗
PubMed1981

Cellular glycogen, beta-1,2,-glucan, poly beta-hydroxybutyric acid and extracellular polysaccharides in fast-growing species of Rhizobium.

Synthesis of acidic exopolysaccharides, neutral cellular polysaccharides and poly-beta-hydroxybutyric acid (PHB) by Rhizobium is strongly dependent on cultural conditions and the strains used. Exopolysaccharide production by R. leguminosarum, R. Phaseoli and R. trifolii closely parallels growth, whereas R. meliloti mainly excretes (low mol wt) polysaccharides when cell propagation is limited by lack of a necessary growth element (nitrogen) and an excess of carbon source is still present in the medium. In all strains, accumulation of cellular glycogen, beta-1,2-glucan and PHB is initiated only under growth-limiting conditions. When the external carbon source is exhausted, glycogen and PHB are metabolized by the cells, sustaining their longevity and thus act as true reserve materials; on the other hand, beta-1,2-glucan and excreted polysaccharides are not utilized on further incubation of the culture. Differences exist in the nature and relative amounts of the products synthesized by strains of different species of Rhizobium. R. leguminosarum, R. phaseoli and R. trifolii synthesize a uronic containing exopolysaccharide, PHB and/or glycogen, non-metabolizable capsular polysaccharide and low amounts of beta-1,2-glucan. R. meliloti synthesizes a uronic acid-free exopolysaccharide, PHB and /or glycogen and high concentrations of beta-1,2-glucan. Exopolysaccharides, beta-1,2-glucan and glycogen preparations were obtained by isolation and purification from cells of fast-growing species of Rhizobium and chemically characterized.

Glucans↗
PubMed1991

Production of poly-D(-)-3-hydroxybutyrate and poly-D(-)-3-hydroxyvalerate by strains of Alcaligenes latus.

Alcaligenes latus strains can accumulate poly-D(-)-3-hydroxybutyrate (PHB) up to about 85% of cell dry weight. The abilities to store poly-D(-)-3-hydroxyvalerate (PHV) of three strains of A. latus were investigated. With Na-propionate as PHV precursor, strain A. latusDSM 1122 had better PHV accumulation ability than strains A. latus DSM 1123 and 1124. Strain A. latus DSM 1123 could store PHV when Na-valerate but not Na-propionate served as the PHV precursor. PHB and PHV accumulation by A. latus DSM 1124 rapidly increased when propionic acid and acetic acid were together added to the fermentor. This increase was not obtained in the culture shaker flask and fermentor growing the same strain when Na-propionate alone served as a PHV precursor.

Acetates↗
PubMed2001

Poly-3-hydroxybutyrate production by Azotobacter chroococcum.

Thirty-seven soil isolates and mutants of Azotobacter chroococcum tested for poly-3-hydroxybutyrate (PHB) production using Sudan black B staining method were found to be positive. One mutant showed a higher number of PHB-producing cells and maximum number of granules per cell. Using 2% glucose and 15 mmol/L ammonium acetate, PHB production was found to be maximum at 36 and 48 h of growth under submerged cultivation and under stationary cultivation, respectively. PHB production was found to be higher on sucrose and commercial sugar (as carbon sources) as compared to glucose and mannitol. As commercial sugar is cheaper than sucrose it was selected as carbon source for PHB production, that being found to be maximum at 1% concentration. Inorganic nitrogen sources seemed to have no stimulatory effect on the production of PHB. However, ammonium acetate (15 mmol/L) was found to be best for PHB production. Peptone (0.2%) gave a better yield of PHB under both growth conditions. Using all optimized conditions, PHB production was studied in ten selected strains. Two of them were found to be best PHB producers under both growth conditions, one producing 621 and 740 micrograms/g dry mass under submerged cultivation and under stationary cultivation, respectively, while the second one produced 589 and 733 micrograms/g.

Azo Compounds↗
PubMed2001

Growth-associated production of poly-3-hydroxybutyrate by Bacillus mycoides.

Bacillus mycoides strain RIJ B-017, a growth-associated poly-3-hydroxybutyrate (PHB) producer was grown on sucrose-containing media. PHB accumulated in cells up to 72% of dry cell mass. The overall maximum value of PHB yield (Yp/s) and productivities (Qp and qp) 250 mgp/gs, 120 mgp L-1 h-1 and 30 mgp gx-1 h-1, respectively, were obtained at 15 g/L sucrose. Differential scanning calorimeter heating curve showed two peaks, one at 95.9 degrees C and another at 165.4 degrees C with a shoulder around 154.6 degrees C. The viscosity-average molar mass in chloroform at 27 degrees C was 505 kDa. The carbon content of PHB was 55.4% of the mass.

Bacillus↗
PubMed1998

Cloning and sequence analysis of the poly (3-hydroxyalkanoic acid)-synthesis genes of Pseudomonas acidophila.

Pseudomonas acidophila can grow with CO2 as a sole carbon source by the possession of a recombinant plasmid that clones genes that confer chemolithoautotrophic growth ability derived from the H2-oxidizing bacterium Alcaligenes hydrogenophilus. H2-oxidizing bacteria produce poly(3-hydroxybutyric acid) (PHB) from CO2, but recombinant P. acidophila can produce the more useful biopolymer poly(3-hydroxyalkanoic acid) (PHA). In this study, the pha genes of P. acidophila were cloned and a sequence analysis was carried out. A gene library was constructed using the cosmid vector pVK102. A recombinant cosmid carrying the pha genes was selected by the complementation of a PHB-negative mutant of Alcaligenes eutrophus H16. The resulting recombinant cosmid pIK7 contained a 14.8-kb DNA insert. Subcloning was done. and the recombinant plasmid pEH74 was selected by hybridization with the A. eutrophus H16 pha genes. Escherichia coli possessing pEH74 produced PHB, indicating that pEH74 contained the pha genes of P. acidophila. The nucleotide sequences of the PHA-synthesis genes phaA (beta-ketothiolase), phaB (acetoacetyl-CoA reductase), and phaC (PHA synthase) in pEH74 were determined. The homologies of phaA, phaB, and phaC between P. acidophila and A. eutrophus H16 were 64.7, 76.1 and 56.6%, respectively.

Acetyl-CoA C-Acyltransferase↗
PubMed1995

Production of poly(hydroxyalkanoic acid).

Poly(hydroxyalkanoic acid) [PHA] is accumulated by numerous microorganisms as an energy reserve material under unbalanced growth conditions in the presence of excess carbon source. In spite of being a good candidate for biodegradable thermoplastics, their high price compared with conventional plastics currently in use has limited their availability in a wide range of applications. With the aim of reducing the high production cost of PHA, much effort is currently being devoted to improve productivity by employing various microorganisms and by developing efficient culture techniques. Several processes recently developed and employed for the production of PHA by various bacteria are described.

Acyltransferases↗
PubMed2001

Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus ruber and identification of granule-associated proteins.

Triacylglycerol granules synthesized and accumulated by Rhodococcus opacus and Rhodococcus ruber were isolated by glycerol density gradient centrifugation. Whereas only one type of granule could be isolated from R. opacus, two types of granules with different specific densities were isolated from R. ruber. Both types of R. ruber granules showed a similar content of triacylglycerols and poly(3-hydroxybutyrate- co-3-hydroxyvalerate), but the protein profiles of both types were significantly different. The granules with the lower specific density were colorless; the granules with the higher specific density had a deep orange pigmentation. Solubilization studies revealed three different groups of granule-associated proteins: (1) unspecifically bound proteins, (2) relatively weakly associated proteins, and (3) proteins that resisted solubilization by treatment with 2 M NaCl, 2% (w/v) Triton X-114, 6 M guanidinium hydrochloride, up to 8% (w/v) SDS, and proteolytic digestion. The strong association of proteins of the last group suggested that these may play a specific role in the synthesis or mobilization of storage lipids or in the structure of the granules. The N-terminal amino acid sequences of the most tightly bound proteins were obtained. Proteins of low molecular weight with striking sequence similarity to the ribosomal protein L7 from various actinomycetes were always copurified with the granules.

Amino Acid Sequence↗
PubMed2002

A possible role of poly-3-hydroxybutyric acid in antibiotic production in Streptomyces.

The occurrence of poly-3-hydroxybutyric acid (PHB) in 12 different strains of the genus Streptomyces was investigated. Gas chromatographic estimation indicated that all the strains produced PHB and the range of maximum PHB accumulation was between 1.5 and 11.8% dry cell weight. PHB was isolated from Streptomyces coelicolor A3(2) M145 and characterized using Fourier transform-infrared (FT-IR) spectroscopy. The correlation between PHB utilization and antibiotic production in S. coelicolor A3(2) M145, was studied; results indicated a possible role of PHB as a carbon reserve material used for antibiotic production.

Anthraquinones↗
PubMed2003

Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-beta-hydroxybutyrate synthesis.

The lipids poly-beta-hydroxybutyrate (PHB) and alkylresorcinols are the major metabolic products of Azotobacter vinelandii cysts. Cysts are formed in less than 0.01% of late stationary phase cells grown on sucrose. Culturing vegetative cells in n-butanol or beta-hydroxybutyrate induces encystment. After induction of encystment, PHB rapidly accumulates in large granules. Then, the cells begin the synthesis of alkylresorcinols that replace the phospholipids in the membranes and are components of the exine, the outer layer of the cyst envelope. Vegetative cells do not synthesize alkylresorcinols. We report here the effect of mutations in the phbBAC operon, coding for the enzymes of the PHB biosynthetic pathway, on the synthesis of alkylresorcinols and cyst formation. The phb mutations did not impair the capacity to form mature cysts. However, the cysts formed by these strains posses a thicker exine layer and a higher content of alkylresorcinols than the cysts formed by the wild-type strain. A blockage of PHB synthesis caused by phb mutations resulted in the synthesis of alkylresorcinols and encystment even under non-inducing conditions. We propose that, as a consequence of the blockage in the PHB biosynthetic pathway, the acetyl-CoA and reducing power pools are increased causing the shift to lipid metabolism required for the synthesis of alkylresorcinols and cyst formation.

Azotobacter vinelandii↗
PubMed2003

Poly beta-hydroxybutyrate depolymerase (PhaZ) in Azospirillum brasilense and characterization of a phaZ mutant.

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-hydroxybutyrate (PHB) under sub-optimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments. PHB depolymerase (PhaZ) is an essential enzyme in PHB degradation. The phaZ gene was identified in Azospirillum brasilense, cloned, sequenced, and shown to be located on the chromosome. Insertion of a kanamycin-resistant cassette within phaZ of A. brasilense resulted in a phaZ mutant that was unable to degrade PHB; however, carbon source utilization was similar in both the wild-type and the mutant strain. The ability of the wild-type to endure starvation conditions, ultraviolet irradiation, heat, and osmotic shock, and to grow in the presence of hydrogen peroxide was higher than that of the mutant strain. By contrast, the ability of the phaZ mutant strain to endure desiccation was higher than that of the wild-type strain. No differences between the strains were seen in their ability to endure sonication, or to survive in carrier materials used for soil inoculants. In addition, motility was the same between the two strains, whereas cell aggregation and exopolysaccharide production were higher in the wild-type than in the phaZ mutant strain.

Amino Acid Sequence↗
PubMed2004

Thermotolerant poly(3-hydroxybutyrate)-degrading bacteria from hot compost and characterization of the PHB depolymerase of Schlegelella sp. KB1a.

Eighteen gram-negative thermotolerant poly(3-hydroxybutyrate) (PHB)-degrading bacterial isolates ( T(max) approximately 60 degrees C) were obtained from compost. Isolates produced clearing zones on opaque PHB agar, indicating the presence of extracellular PHB depolymerases. Comparison of physiological characteristics and determination of 16S rRNA gene sequences of four selected isolates revealed a close relatedness of three isolates (SA8, SA1, and KA1) to each other and to Schlegelella thermodepolymerans and Caenibacterium thermophilum. The fourth strain, isolate KB1a, showed reduced similarities to the above-mentioned isolates and species and might represent a new species of Schlegelella. Evidence is provided that S. thermodepolymerans and C. thermophilum are only one species. The PHB depolymerase gene, phaZ, of isolate KB1a was cloned and functionally expressed in Escherichia coli. Purified PHB depolymerase was most active around pH 10 and 76 degrees C. The DNA-deduced amino acid sequence of the mature protein (49.4 kDa) shared significant homologies to other extracellular PHB depolymerases with a domain substructure: catalytic domain type 2-linker domain fibronectin type 3-substrate-binding domain type 1. A catalytic triad consisting of S(20), D(104), and H(138) and a pentapeptide sequence (GLS(20)AG) characteristic for PHB depolymerases (PHB depolymerase box, GLSXG) and for other serine hydrolases (lipase box, GXSXG) were identified.

Amino Acid Motifs↗
PubMed2004

Studies on the biodegradability of polythioester copolymers and homopolymers by polyhydroxyalkanoate (PHA)-degrading bacteria and PHA depolymerases.

The biodegradability of microbial polythioesters (PTEs), a novel class of biopolymers which were discovered recently and can be produced by polyhydroxyalkanoate (PHA)-accumulating bacteria, was studied. Using poly(3-hydroxybutyrate- co-3-mercaptopropionate) [poly(3HB- co-3MP)] as sole carbon source for screening, 22 new bacterial strains were isolated and characterized. Interestingly, none of the PHA-degrading bacteria was able to utilize the homopolymer poly(3MP) as a carbon source for growth or to form clear zones on poly(3MP)-containing agar plates. The extracellular PHA depolymerases from two strains ( Schlegelella thermodepolymerans, Pseudomonas indica K2) were purified to electrophoretic homogeneity and biochemically characterized. The PHA depolymerase of S. thermodepolymerans exhibited a temperate optimum of about 75 degrees C to 80 degrees C and was stable at 70 degrees C for more than 24 h. Regarding the substrate specificities of the PHA depolymerase of S. thermodepolymerans, enzyme activities decreased significantly with increasing 3MP content of the copolymer substrates. Interestingly, no activity could be detected with homoPTEs consisting only of 3MP or of 3-mercaptobutyrate. Similar results were obtained with the PHA depolymerases PhaZ2, PhaZ5 and PhaZ7 of Paucimonas lemoignei which were also investigated. The PHA depolymerase of Ps. indica K2 did not cleave any of the investigated polymers containing 3MP. Gas chromatography, infrared and (1)H-NMR spectrometry and matrix-assisted laser desorption/ionization time-of-flight analysis revealed that 3MPs containing oligomers were enriched in the water-insoluble fraction remaining after partial digestion of poly(3HB- co-3MP) by purified poly(3HB) depolymerase of S. thermodepolymerans. In contrast, 3HB was enriched in the water-soluble fraction, which also contained 3HB- co-3MP dimer obtained by partial digestion of this copolymer by the enzyme. This study clearly indicates that PHA depolymerases are specific for oxoester linkages of PHAs and that the thioester bonds of PTEs cannot be cleaved by this type of enzyme.

Bacteria↗