PhaG-mediated synthesis of Poly(3-hydroxyalkanoates) consisting of medium-chain-length constituents from nonrelated carbon sources in recombinant Pseudomonas fragi

Recently, a new metabolic link between fatty acid de novo biosynthesis and biosynthesis of poly(3-hydroxy-alkanoate) consisting of medium-chain-length constituents (C(6) to C(14)) (PHA(MCL)), catalyzed by the 3-hydroxydecanoyl-[acyl-carrier-protein]:CoA transacylase (PhaG), has been identified in Pseudomonas putida (B. H. A. Rehm, N. Krüger, and A. Steinbüchel, J. Biol. Chem. 273:24044-24051, 1998). To establish this PHA-biosynthetic pathway in a non-PHA-accumulating bacterium, we functionally coexpressed phaC1 (encoding PHA synthase 1) from Pseudomonas aeruginosa and phaG (encoding the transacylase) from P. putida in Pseudomonas fragi. The recombinant strains of P. fragi were cultivated on gluconate as the sole carbon source, and PHA accumulation to about 14% of the total cellular dry weight was achieved. The respective polyester was isolated, and GPC analysis revealed a weight average molar mass of about 130,000 g mol(-1) and a polydispersity of 2.2. The PHA was composed mainly (60 mol%) of 3-hydroxydecanoate. These data strongly suggested that functional expression of phaC1 and phaG established a new pathway for PHA(MCL) biosynthesis from nonrelated carbon sources in P. fragi. When fatty acids were used as the carbon source, no PHA accumulation was observed in PHA synthase-expressing P. fragi, whereas application of the beta-oxidation inhibitor acrylic acid mediated PHA(MCL) accumulation. The substrate for the PHA synthase PhaC1 is therefore presumably directly provided through the enzymatic activity of the transacylase PhaG by the conversion of (R)-3-hydroxydecanoyl-ACP to (R)-3-hydroxydecanoyl-CoA when the organism is cultivated on gluconate. Here we demonstrate for the first time the establishment of PHA(MCL) synthesis from nonrelated carbon sources in a non-PHA-accumulating bacterium, employing fatty acid de novo biosynthesis and the enzymes PhaG (a transacylase) and PhaC1 (a PHA synthase).     

The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources

We recently identified the phaG(Pp) gene encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase in Pseudomonas putida, which directly links the fatty acid de novo biosynthesis and polyhydroxyalkanoate (PHA) biosynthesis. An open reading frame (ORF) of which the deduced amino acid sequence shared about 57% identity with PhaG from P. putida was identified in the P. aeruginosa genome sequence. Its coding region (herein called phaG(Pa)) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The resulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phaG(Pp)-negative mutant P. putida PhaG(N)-21. The PHA was composed of 5 mol% 3-hydroxydodecanoate, 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate and 5 mol% 3-hydroxyhexanoate. Furthermore, an isogenic phaG(Pa) knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaG(Pa) mutant did not show any difference in growth rate, but PHA accumulation from gluconate was decreased to about 40% of wild-type level, whereas from fatty acids wild-type level PHA accumulation was obtained. These data suggested that PhaG from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity and strongly enhances the metabolic flux from fatty acid de novo synthesis towards PHA(MCL) synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known.     

Accumulation of polyhydroxyalkanoate from styrene and phenylacetic acid by Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of converting the aromatic hydrocarbon styrene, its metabolite phenylacetic acid, and glucose into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen (as sodium ammonium phosphate) is supplied to the growth medium. PHA accumulation occurs to a low level when the nitrogen concentration drops below 26.8 mg/liter and increases rapidly once the nitrogen is no longer detectable in the growth medium. The depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the rate of substrate utilization and biochemical activity of whole cells grown on styrene, phenylacetic acid, and glucose. However, the efficiency of carbon conversion to PHA dramatically increased once the nitrogen concentration dropped below 26.8 mg/liter in the growth medium. When supplied with 67 mg of nitrogen/liter, the carbon-to-nitrogen (C:N) ratios that result in a maximum yield of PHA (grams of PHA per gram of carbon) for styrene, phenylacetic acid, and glucose are 28:1, 21:1, and 18:1, respectively. In cells grown on styrene and phenylacetic acid, decreasing the carbon-to-nitrogen ratio below 28:1 and 21:1, respectively, by increasing the nitrogen concentration and using a fixed carbon concentration leads to lower levels of PHA per cell and lower levels of PHA per batch of cells. Increasing the carbon-to-nitrogen ratio above 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, by decreasing the nitrogen concentration and using a fixed carbon concentration increases the level of PHA per cell but results in a lower level of PHA per batch of cells. Increasing the carbon and nitrogen concentrations but maintaining the carbon-to-nitrogen ratio of 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, results in an increase in the total PHA per batch of cells. The maximum yields for PHA from styrene, phenylacetic acid, and glucose are 0.11, 0.17, and 0.22 g of PHA per g of carbon, respectively.     

Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of converting the aromatic hydrocarbon styrene, its metabolite phenylacetic acid, and glucose into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen (as sodium ammonium phosphate) is supplied to the growth medium. PHA accumulation occurs to a low level when the nitrogen concentration drops below 26.8 mg/liter and increases rapidly once the nitrogen is no longer detectable in the growth medium. The depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the rate of substrate utilization and biochemical activity of whole cells grown on styrene, phenylacetic acid, and glucose. However, the efficiency of carbon conversion to PHA dramatically increased once the nitrogen concentration dropped below 26.8 mg/liter in the growth medium. When supplied with 67 mg of nitrogen/liter, the carbon-to-nitrogen (C:N) ratios that result in a maximum yield of PHA (grams of PHA per gram of carbon) for styrene, phenylacetic acid, and glucose are 28:1, 21:1, and 18:1, respectively. In cells grown on styrene and phenylacetic acid, decreasing the carbon-to-nitrogen ratio below 28:1 and 21:1, respectively, by increasing the nitrogen concentration and using a fixed carbon concentration leads to lower levels of PHA per cell and lower levels of PHA per batch of cells. Increasing the carbon-to-nitrogen ratio above 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, by decreasing the nitrogen concentration and using a fixed carbon concentration increases the level of PHA per cell but results in a lower level of PHA per batch of cells. Increasing the carbon and nitrogen concentrations but maintaining the carbon-to-nitrogen ratio of 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, results in an increase in the total PHA per batch of cells. The maximum yields for PHA from styrene, phenylacetic acid, and glucose are 0.11, 0.17, and 0.22 g of PHA per g of carbon, respectively.     

Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli.

Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the beta-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.     

Bacterial synthesis of polyhydroxyalkanoates containing aromatic and aliphatic monomers by Pseudomonas putida CA-3

Pseudomonas putida CA-3 has the ability to accumulate to high levels unique polyhydroxyalkanoate (PHA) heteropolymers composed of aromatic and aliphatic monomers. The majority of monomers are aromatic making up 98% of the polymer. (R)-3-hydroxyphenylvalerate and (R)-3-hydroxyphenylhexanoate are the most abundant monomers found in polymers accumulated from phenylalkanoic acids with an uneven and even number of carbons on the acyl side chain respectively. PHAs accumulated from phenylvaleric and phenylhexanoic acid were partially crystalline while all other PHAs were amorphous. Significant differences in the yield and PHA content of the cells occurred when different phenylalkanoic acids were supplied as growth substrates. Increasing the initial concentration of the growth substrate increased both the PHA content of the cells and the overall yield (g PHA/g carbon supplied) of PHA accumulated by P. putida CA-3 cells. The highest PHA content (% cell dry wt.) from an aromatic carbon source was 59% when 15mM phenylvaleric acid was supplied as the sole source of carbon and energy. This corresponded to a maximum PHA yield of 0.42 g PHA/g carbon supplied. In and attempt to increase the level of PHA accumulated from related growth substrates acrylic acid was added to the growth medium. However, the addition of various concentrations of acrylic acid to the growth medium had either no effect or decreased the PHA content of the cell accumulated from phenylalkanoic acids by P. putida CA-3.     

Taxonomic study of bacteria isolated from natural mineral waters: proposal of Pseudomonas jessenii sp. nov. and Pseudomonas mandelii sp. nov

The taxonomic position of 23 strains isolated from mineral waters and previously grouped in the authentic pseudomonads on the basis of a phenotypic analysis (cluster IX, subclusters XIIIa and XIIIc of VERHILLE, S., ELOMARI, M., COROLER, L., IZARD, D., LECLERC, H. (Syst. Appl. Microbiol, 20, 137-149, 1997) has been genotypically further studied in the present work. On the basis of hybridization results, these strains were gathered into two new genomic groups for which we propose the names of Pseudomonas jessenii sp. nov. (Type strain CIP 105274) and Pseudomonas mandelii sp. nov. (Type strain CIP 105273). Deoxyribonucleic acid relatedness levels showed homologies ranging from 78 to 100% for Pseudomonas jessenii and from 77 to 100% for Pseudomonas mandelii. Furthermore, hybrization rates with 66 representative well characterized species or only partially characterized species of the genus Pseudomonas were below 53%, with delta Tm values of 7 degrees C and more. The mol% G + C content ranged from 57 to 58. The two new species presented basic morphological characteristics common to all pseudomonads. Various phenotypic features, such as denitrification, growth at 4 degrees C or 41 degrees C, trigonelline assimilation, alpha-L-glutamyl-L-histidine arylarmidase activity, growth on benzoate and meso-tartrate were found to differentiate Pseudomonas jessenii from Pseudomonas mandelii and from other Pseudomonas species. Pseudomonas jessenii encompassed a total of 9 strains from both phenotypic groups IX and XIIIa. Pseudomonas mandelii clustered a total of 13 strains from both phenotypic groups IX and XIIIc. Their clinical significance is unknown. The 16S rDNA of each type strain was sequenced and compared with the known sequences of the representative strains of the genus Pseudomonas. A phylogenetic tree was constructed to determine the intrageneric relationships within the genus Pseudomonas.     

Identification of 5-hydroxyhexanoic acid, 4-hydroxyheptanoic acid and 4-hydroxyoctanoic acid as new constituents of bacterial polyhydroxyalkanoic acids

 A recombinant strain of Pseudomonas putida GPp104 (pHP1014::E146), which expressed the polyhydroxyalkanoic acid (PHA) synthase of Thiocapsa pfennigii exhibiting an unusual substrate specificity at a high level was incubated in two-stage batch or fed-batch accumulation experiments with 5-hydroxyhexanoic acid (5HHx) as carbon source in the second cultivation phase, copolyesters of 3-hydroxybutyric acid (3HB) plus 5HHx, or of 3HB, 3-hydroxyhexanoic acid (3HHx) plus 5HHx were accumulated as revealed by gas-chromatographic and ¹³C-NMR spectroscopic analysis. When the recombinant P. putida GPp104 was incubated with 4-hydroxyheptanoic acid (4HHp) as carbon source in the second cultivation phase, a copolyester consisting of 3HB, 3-hydroxyvaleric acid and 3- and 4-hydroxyheptanoic acid accumulated. Providing 4-hydroxyoctanoic acid as carbon source in the second cultivation phase led to the accumulation of a polyester that contained 1–2 mol% 4-hydroxyoctanoic acid besides 3-hydroxyoctanoic acid, 3HHx, 3-hydroxyvaleric acid and 3HB. In addition to PHA containing these new constituents, PHA with 4-hydroxyvaleric acid was accumulated from laevulinic acid. Eleven strains from five genera have been also analysed for their ability to utilize different carbon sources for colony growth, which might serve as potential precursors for the biosynthesis of PHA with unusual constituents. Although most of the carbon sources were utilized by some strains for colony growth, accumulation experiments gave no evidence for the accumulation of new PHA by these wild-type strains. Received: 22 April/Received revision: 23 May 1996/Accepted: 2 June 1996     

Increasing the yield of MCL-PHA from nonanoic acid by co-feeding glucose during the PHA accumulation stage in two-stage fed-batch fermentations of Pseudomonas putida KT2440

A method was developed to increase the yield of MCL-PHA from nonanoic acid in the PHA accumulation phase. Pseudomonas putida KT2440 was grown on glucose until ammonium-limitation was imposed. In the second (accumulation) stage, either glucose, nonanoic acid, or a mixture of these carbon and energy sources was supplied. Since the medium-chain-length poly-3-hydroxyalkanoate (MCL-PHA) subunits produced are unique for each carbon source, their relative contribution to PHA yield could be calculated. Y(C7+C9)/NA was 0.254 mol mol(-1) during PHA synthesis from nonanoic acid. Y(C8+C10)/G was only 0.057 mol mol(-1) during PHA synthesis from glucose. When nonanoic acid and glucose were fed together, Y(C7+C8)/NA almost doubled to 0.450 mol mol(-1) while Y(C8+C10)/G decreased to 0.011 mol mol(-1). These results demonstrate that substantial savings can be obtained by feeding glucose with substrates that are good for PHA production but much more expensive than glucose.     

Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.     

Photosynthetic and quasi-photosynthetic bacteria

Abstract Nine different bacterial strains that utilise phenylacetic acid as the only carbon and energy source were isolated from samples of different geographical origin. The isolates were characterised taxonomically and physiologically. Evidence is presented that in all the isolates as well as in four previously isolated control strains with the ability to utilize phenylacetic acid, the enzyme phenylacetate-CoA ligase is specifically induced during growth on phenylacetic acid. The Michaelis constant ( K _m) in one Pseudomonas strain was sufficiently low (-1 mM) to suggest that the enzyme may have a role in phenylacetic acid metabolism.     

Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, beta-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.     

应用聚合酶链式反应快速特异鉴定假单胞菌和伯克霍尔德氏菌(R)-3-羟基酯酰载酯蛋白-辅酶A转酰基酶基因

聚羟基脂肪酸酯(PHA)是一类具有广泛应用前景的可降解生物塑料。因其可以以葡萄糖等廉价底物直接发酵生产PHA而日益受到重视。目前的研究表明在积累中长链PHA的假单胞菌中,由phaG基因编码的(R)-3-羟基酯酰载酯蛋白-辅酶A转酰基酶(PhaG)起关键作用,但目前为止对该蛋白还知之甚少。通过聚合酶链式反应(PCR)建立了一种快速、特异鉴定phaG基因的方法,应用该方法成功地从两株积累不同PHA的假单胞菌Pseudomonas stutzeri 1317和Pseudamanas nitroreducens 0802中分别克隆得到phaG基因,并在phaG基因突变株Pseudomonas putida PHAGx-21中表达成功。同时,还首次报道了从非假单胞菌菌株Burkholderia caryophylli AS 1．2741中鉴定得到phaG基因,提示PhaG介导的中长链PHA合成途径作为一种通用的代谢模式在细菌中广泛存在,为进一步实现从廉价的非相关底物合成中长链PHA提供了必要的分子生物学基础。     

Sequence and transcriptional studies of five clustered flagellin genes from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The Escherichia coli 3-ketoacyl-ACP reductase gene (fabGEc) was cloned using a PCR technique to investigate the metabolic link between fatty acid metabolism and polyhydroxyalkanoate (PHA) production. Three plasmids respectively harboring fabGEc and the poly-3-hydroxyalkanoate synthesis genes phaCAc and phaC1Ps from Aeromonas caviae and Pseudomonas sp. 61-3 respectively were constructed and introduced into E. coli HB101 strain. On a two-stage cultivation using dodecanoate as the sole carbon source, recombinant E. coli HB101 strains harboring fabGEc and phaC genes accumulated PHA copolymers (about 8 wt% of dry cell weight) consisting of several (R)-3-hydroxyalkanoate units of C4, C6, C8, and C10. It has been suggested that overexpression of the fabGEc gene leads to the supply of (R)-3-hydroxyacyl-CoA for PHA synthesis via fatty acid degradation.     

Inactivation of isocitrate lyase leads to increased production of medium-chain-length poly(3-hydroxyalkanoates) in Pseudomonas putida

Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two- to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant.     

Polyhydroxyalkanoate (PHA) production using waste vegetable oil by Pseudomonas sp. strain DR2

To produce polyhydroxyalkanoate (PHA) from inexpensive substrates by bacteria, vegetable-oil-degrading bacteria were isolated from a rice field using enrichment cultivation. The isolated Pseudomonas sp. strain DR2 showed clear orange or red spots of accumulated PHA granules when grown on phosphate and nitrogen limited medium containing vegetable oil as the sole carbon source and stained with Nile blue A. Up to 37.34% (w/w) of intracellular PHA was produced from corn oil, which consisted of three major 3-hydroxyalkanoates; octanoic (C8:0, 37.75% of the total 3-hydroxyalkanoate content of PHA), decanoic (C10:0, 36.74%), and dodecanoic (C12:0, 11.36%). Pseudomonas sp. strain DR2 accumulated up to 23.52% (w/w) of PHAMCL from waste vegetable oil. The proportion of 3- hydroxyalkanoate of the waste vegetable-oil-derived PHA [hexanoic (5.86%), octanoic (45.67%), decanoic (34.88%), tetradecanoic (8.35%), and hexadecanoic (5.24%)] showed a composition ratio different from that of the corn-oil-derived PHA. Strain DR2 used three major fatty acids in the same ratio, and linoleic acid was the major source of PHA production. Interestingly, the production of PHA in Pseudomonas sp. strain DR2 could not occur in either acetate- or butyrate-amended media. Pseudomonas sp. strain DR2 accumulated a greater amount of PHA than other well-studied strains (Chromobacterium violaceum and Ralstonia eutropha H16) when grown on vegetable oil. The data showed that Pseudomonas sp. strain DR2 was capable of producing PHA from waste vegetable oil.     

Production of polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from an oil remaining from biotechnological rhamnose production

Screening experiments identified several bacteria which were able to use residual oil from biotechnological rhamnose production as a carbon source for growth. Ralstonia eutropha H16 and Pseudomonas oleovorans were able to use this waste material as the sole carbon source for growth and for the accumulation of polyhydroxyalkanoic acids (PHA). R. eutropha and P. oleovorans accumulated PHA amounting to 41.3% and 38.9%, respectively, of the cell dry mass, when these strains were cultivated in mineral salt medium with the oil from the rhamnose production as the sole carbon source. The accumulated PHA isolated from R. eutropha consisted of only 3-hydroxybutyric acid, whereas the PHA isolated from P. oleovorans consisted of 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxy decanoic acid, and 3-hydroxydodecanoic acid. The composition was confirmed by gas chromatography of the isolated polyesters. Batch and fed-batch cultivations in stirred-tank reactors were done. Received: 15 June 1999 / Received revision: 10 August 1999 / Accepted: 13 August 1999     

Polyhydroxyalkanoate production by antarctic soil bacteria isolated from Casey Station and Signy Island

Polyhydroxyalkanoate (PHA) is a family of biopolymers produced by some bacteria and is accumulated intracellularly as carbon and energy storage material. Fifteen PHA-producing bacterial strains were identified from bacteria isolated from Antarctic soils collected around Casey Station (66°17'S, 110°32'E) and Signy Island (60°45'S, 45°36'W). Screening for PHA production was carried out by incubating the isolates in PHA production medium supplemented with 0.5% (w/v) sodium octanoate or glucose. 16S rRNA gene sequence analysis revealed that the isolated PHA-producing strains were mainly Pseudomonas spp. and a few were Janthinobacterium spp. All the isolated Pseudomonas strains were able to produce medium-chain-length (mcl) PHA using fatty acids as carbon source, while some could also produce mcl-PHA by using glucose. The Janthinobacterium strains could only utilize glucose to produce polyhydroxybutyrate (PHB). A Pseudomonas isolate, UMAB-40, accumulated PHA up to 48% cell dry mass when utilizing fatty acids as carbon source. This high accumulation occurred at between 5°C and 20°C, then decreased with increasing temperatures. Highly unsaturated mcl-PHA was produced by UMAB-40 from glucose. Such characteristics may be associated with the ability of UMAB-40 to survive in the cold.