Degradation of poly(3-hydroxybutyrate), PHB,by bacteria and purification of a novel PHB depolymerase fromComamonas sp.

Bacteria capable of growing on poly(3-hydroxybutyrate), PHB, as the sole source of carbon and energy were isolated from various soils, lake water, activated sludge, and air. Although all bacteria utilized a wide variety of monomeric substrates for growth, most of the strains were restricted to degrade PHB and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, P(3HB-co-3HV). Five strains were also able to decompose a homopolymer of 3-hydroxyvalerate, PHV. Poly(3-hydroxyoctanoate), PHO, was not degraded by any of the isolates. One strain, which was identified asComamonas sp., was selected, and the extracellular depolymerase of this strain was purified from the medium by ammonium sulfate precipitation and by chromatography on DEAE-Sephacel and Butyl-Sepharose 4B. The purified PHB depolymerase was not a glycoprotein. The relative molecular masses of the native enzyme and of the subunits were 45,000 or 44,000, respectively. The purified enzyme hydrolyzed PHB, P(3HB-co-3HV), and—at a very low rate—also PHV. Polyhydroxyalkanoates, PHA, with six or more carbon atoms per monomer or characteristic substrates for lipases were not hydrolyzed. In contrast to the PHB depolymerases ofPseudomonas lemoignei andAlcaligenes faecalis T₁, which are sensitive toward phenylmethylsulfonyl fluoride (PMSF) and which hydrolyze PHB mainly to the dimeric and trimeric esters of 3-hydroxybutyrate, the depolymerase ofComamonas sp. was insensitive toward PMSF and hydrolyzed PHB to monomeric 3-hydroxybutyrate indicating a different mechanism of PHB hydrolysis. Furthermore, the pH optimum of the reaction catalyzed by the depolymerase ofComamonas sp. was in the alkaline range at 9.4.     

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.     

Purification of Aspergillus fumigatus (Pdf1) Poly(β-hydroxybutyrate) (PHB) Depolymerase Using a New, Single-Step Substrate Affinity Chromatography Method: Characterization of the PHB Depolymerase Exhibiting Novel Self-Aggregation Behavior

The extracellular poly(-hydroxybutyrate) (PHB) depolymerase of Aspergillus fumigatus Pdf1 was purified by a new, simple, one-step affinity chromatography method using the substrate PHB. The purified enzyme was glycosylated, with the molecular mass of 40 KD, and exhibited a novel self-aggregation behavior by means of hydrophobic interaction that was resolved by Triton X-100 (TX-100) pretreatment of enzyme and also TX-100 incorporation in the native gel. The apparent K _m value of purified enzyme for PHB was 119 g/mL and 3-hydroxybutyrate was detected as the main endproduct of PHB hydrolysis. The depolymerase was insensitive to phenylmethyl sulfonyl fluoride (PMSF), sodium azide, ethylenediaminetetraacetic acid (EDTA), and para-chloromercuric benzoic acid (PCMB), but was inactivated by dithioerythritol (DTT) and showed specificity for short chain-length poly(-hydroxyalkanoates) (PHAs) such as PHB, poly(hydroxyvalerate) (PHV), and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Medium-chain-length PHA failed to get hydrolyzed. The enzyme, however, exhibited strong cross reactivity with the Comamonas sp. PHB depolymerase antibodies, but not with PHV depolymerase antibodies of Pseudomonas lemoignei. Southern hybridization and dot blot analysis of A. fumigatus Pdf1 genomic DNA with alkaline phosphatase labeled probes of P. lemoignei PHB and PHV depolymerase genes revealed no homology, although the enzyme hydrolyzed both PHB and PHV.     

Microscopic Visualization of the Enzymatic Degradation of Poly(3HB-co-3HV) and Poly(3HV) Single Crystals by PHA Depolymerases From Pseudomonas lemoignei

As a complement to previous studies of the enzymatic degradation of folded chain lamellar single crystals of polyhydroxyalkanoates, single crystals of a number of polyhydroxyalkanoates were partially degraded with depolymerases from Pseudomonas lemoignei and examined by transmission electron microscopy. Single crystals of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate), bacterial poly(3-hydroxyvalerate), and synthetic poly(3-hydroxybutyrate) with 88% isotactic diads were degraded using purified extracellular PHA-depolymerases from P. lemoignei: PHB-depolymerase A, PHB-depolymerase B, and depolymerases from recombinant E. coli: PHB-depolymerase PhaZ4 (PHB-depolymerase E), PHB-depolymerase PhaZl (PHB-depolymerase C), and PHB-depolymerase PhaZ5 (PHB-depolymerase A). In contrast to previous results with single crystals of bacterial PHB, the predominant effect observed with all crystals was a significant narrowing of the lamellae. This suggests an edge attack mechanism which because of lateral disorder of the crystals leads to a narrowing of the crystalline lamellae as opposed to the splintering effect previously observed. The model suggested for the degradation of single crystals of bacterial PHB by PHB-depolymerases is refined to include the effects of lateral disorder caused by the introduction of valerate or repeat units of opposite stereochemistry into the single crystal.     

Properties of a Poly(3-hydroxybutyrate) Depolymerase from Penicillium funiculosum

A poly(3-hydroxybutyrate) (PHB) depolymerase was purified from a fungus, Penicillium funiculosum (IFO6345), with phenyl-Toyopearl and its properties were compared with those of other PHB depolymerases. The molecular mass of the purified enzyme was estimated at about 33 kDa by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The pH optimum and pI were 6.5 and 6.5, respectively. The purified protein showed affinity to Con A-Sepharose, indicating that it is a glycoprotein. Diisopropylfluorophosphate and dithiothreitol inhibited the depolymerase activity completely. The N-terminal amino acid sequence of the purified enzyme was TALPAFNVNPNSVSVSGLSSGGYMAAQL, which contained a lipase box sequence. This purified enzyme is one of the extracellular PHB depolymerase which belong to serine esterase. The purified enzyme showed relatively strong hydrolytic activity against 3-hydroxybutyrate oligomers compared with its PHB-degrading activity. PHB-binding experiments showed that P. funiculosum depolymerase has the weakest affinity for PHB of all the depolymerases examined.     

Biochemical and Genetic Characterization of an Extracellular Poly(3-Hydroxybutyrate) Depolymerase from Acidovorax Sp. Strain TP4

To determine the properties of enzymes from bacteria that degrade polypropiolactone (PPL), we isolated 13 PPL-degrading bacteria from pond water, river water, and soil. Nine of these strains were identified as Acidovorax sp., three as Variovorax paradoxus, and one as Sphingomonas paucimobilis. All the isolates also degraded poly(3-hydroxybutyrate) (PHB). A PPL-degrading enzyme was purified to electrophoretical homogeneity from one of these bacteria, designated Acidovorax sp. TP4. The purified enzyme also degraded PHB. The molecular weight of the enzyme was estimated as about 50,000. The enzyme activity was inhibited by diisopropylfluorophosphate, dithiothreitol, and Triton X-100. The structural gene of the depolymerase was cloned in Escherichia coli. The nucleotide sequence of the cloned DNA fragment contained an open reading frame (1476 bp) specifying a protein with a deduced molecular weight of 50,961 (491 amino acids). The deduced overall sequence was very similar to that of a PHB depolymerase of Comamonas acidovorans YM1609. From these results it was concluded that the isolated PPL-degrading enzyme belongs to the class of PHB depolymerases. A conserved amino acid sequence, Gly-X₁-Ser-X₂-Gly (lipase box), was found at the N-terminal side of the amino acid sequence. Site-directed mutagenesis of the TP4 enzyme confirmed that ²⁰Ser in the lipase box was essential for the enzyme activity. This is the first report of the isolation a PHB depolymerase from Acidovorax.     

Characterization of a poly(3-hydroxybutyrate) depolymerase fromaureobacterium saperdae: Active site and kinetics of hydrolysis studies

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase was purified fromAureobacterium saperdae cultural medium by using hydrophobic interaction chromatography. The isolated enzyme was composed of a single polypeptide chain with a molecular mass of 42.7 kDa as determined by SDS-PAGE and by native gel filtration on TSK-HW-55S. The enzyme was not a glycoprotein. Its optimum activity occurred at pH 8.0 and it showed a broad pH stability, ranging from pH 3 to pH 11.N-Bromosuccinamide and 2-hydroxy-5-nitrobenzyl bromide completely inactivated the enzyme, suggesting the involvement of tryptophan residues at the active site of the protein. The enzyme was very sensitive to diisopropyl fluorophosphate and diazo-dl-norleucine methyl ester, showing the importance of serine and carboxyl groups. The modification of cysteine residues byp-hydroxy mercuricbenzoate did not cause a loss of activity, whereas dithiothreitol rapidly inactivated the enzyme, revealing the presence of disulfide bonds.A saperdae depolymerase acted on the surface layer of PHB films and the degradation proceeded by surface erosion releasing monomers and dimers of 3-hydroxybutric acid. The degradation of PHB films byA. saperdae depolymerase was partially inhibited in the presence of excess amounts of enzyme. This phenomenon, already observed by Mukaiet al. with poly(hydroxyalkanoates) depolymerases fromAlcaligenes faecalis, Pseudomonas pickettii, andComamonas testosteroni, was analyzed according to the kinetic model proposed by these authors. The experimental data evidenced a general agreement with the kinetic model, although higher initial degradation rates were found withA. saperdae depolymerase.     

Molecular structure of extracellular poly(3-hydroxybutyrate) depolymerase fromAlcaligenes faecalis T1

The amino acid sequence of a peptide containing an active serine was examined with poly(3-hydroxybutyrate) (PHB) depolymerase ofAlcaligenes faecalis T1. The sequence Cys-Asn-Ala-Trp-Ala-Gly-Ser-Asn-Ala-Gly-Lys was obtained. This amino acid sequence around the active serine does not fit any reported sequence of other esterases and proteases. On the other hand, a segment of the amino acid sequence of PHB depolymerase ofA. faecalis was homologous to the type III sequence of fibronectin. Similar sequences have been reported in some type of bacterial chitinase and cellulases, and PHB depolymerase seems to have an overall similarity to these bacterial extracellular hydrolases.     

Characterization and enzymatic degradation of a cross-linked bacterial polyester

The bacterial polyester, poly(-hydroxybutyrate-co--hydroxyvalerate) (PHB/V), was cross-linked with 1, 5, 7, 10, 20, and 30 wt% benzoyl peroxide by thermal decomposition reactions. Solvent extractions were carried out to determine the cross-linked fractions of the films. The sol/gel data were used to estimate cross-link densities. Films of PHB/V cross-linked with 10% benzoyl peroxide were placed in contact with purified depolymerase A secreted byP. lemoignei. These samples exhibited weight loss rates which were half that of un-cross-linked PHB/V, but the network was degraded completely by the enzyme. The results of this study suggest that anendo-type enzymatic degradation may occur, in addition to theexo-type activity, which is normally presumed to occur with theP. lemoignei depolymerase system.     

Topographical imaging as a means of monitoring biodegradation of poly(hydroxyalkanoate) films

Poly(hydroxyalkanoates) (PHAs) are a class of bacterially-derived polymers that are naturally biodegradable through the action of extracellular depolymerase enzymes secreted by a number of different bacteria and fungi. In this paper we describe the development of topographical imaging protocols (by both scanning electron microscopy; SEM, and confocal microscopy; CM) as a means of monitoring the biodegradation of solution cast films of poly(3-hydroxybutanoate-co-3-hydroxyhexanoate) (P3HB/3HHx) and medium-chain-length (mcl-) PHA. Pseudomonas lemoignei and Comamonas P37C were used as sources for PHA depolymerase enzymes as these bacteria are known to degrade at least one of the polymers in question. SEM revealed the bacterial colonization of the film surfaces while CM permitted the comparative assessment of the roughness of the film surfaces upon exposure to the two bacterial strains. By dividing the total surface area of the film (A′) by the total area of the scan (A) it was possible to monitor biodegradation by observing differences in the topography of the film surface. Prior to inoculation, P3HB/3HHx films had an A′/A ratio of 1.06. A 24-h incubation with P. lemoignei increased the A′/A ratio to 1.47 while a 48- and 120-h incubation with Comamonas resulted in A′/A ratios of 1.16 and 1.33, respectively. These increases in the A′/A ratios over time demonstrated an increase in the irregularity of the film surface, indicative of PHA polymer breakdown. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Bacterial Poly(Hydroxyalkanoate) Polymer Production from the Biodiesel Co-product Stream

A co-product stream from soy-based biodiesel production (CSBP) containing glycerol, fatty acid soaps, and residual fatty acid methyl esters (FAME) was utilized as a fermentation feedstock for the bacterial synthesis of poly(3-hydroxybutyrate) (PHB) and medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers. Pseudomonas oleovorans NRRL B-14682 and P. corrugata 388 grew and synthesized PHB and mcl-PHA, respectively, when cultivated in up to 5% (w/v) CSBP. In shake flask culture, P. oleovorans grew to 1.3 ± 0.1 g/L (PHA cellular productivity = 13–27% of the bacterial cell dry weight; CDW) regardless of the initial CSBP concentration, whereas P. corrugata reached maximum cell yields of 2.1 g/L at 1% CSBP, which tapered off to 1.7 g/L as the CSBP media concentration was increased to 5% (maximum PHA cellular productivity = 42% of the CDW at 3% CSBP). While P. oleovorans synthesized PHB from CSBP, P. corrugata produced mcl-PHA consisting primarily of 3-hydroxyoctanoic acid (C_8:0; 39 ± 2 mol%), 3-hydroxydecanoic acid (C_10:0; 26 ± 2 mol%) and 3-hydroxytetradecadienoic acid (C_14:2; 15 ± 1 mol%). The molar mass (M_n) of the PHB polymer decreased by 53% as the initial CSBP culture concentration was increased from 1% to 5% (w/v). In contrast, the M_n of the mcl-PHA polymer produced by P. corrugata remained constant over the range of CSBP concentrations used.     

Purification and Properties of a Poly (β-hydroxybutyrate) Depolymerase From Penicillium sp.

An extracellular poly (β-hydroxybutyrate) (PHB) depolymerase was purified from a Penicillium sp. DS9701-09a by centrifugation, ultrafiltration, precipitation and gel filtration chromatography. The specific activity of the purified enzyme was 37.9-folds higher than that of the culture supernatant and the recovery yield was 11.8%. The PHB deploymerase molecular mass was 44.8 kDa from analysis of both Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometer. The isoelectric point of 6.7 for the enzyme was determined by a two-dimensional electrophoresis. The optimum enzyme activity was observed at a temperature of 50 °C and pH 5.0. The apparent K _m of the enzyme was found to be 1.35 mg/mL. The PHB depolymerase consisted of 16 kinds of normal amino acids. The secondary structure of the enzyme was determined by CD spectrum. α-helix and β-turn were found to be 66% and 34% for the enzyme without ammonium sulphite. Chemical inhibition on the PHB depolymerase activity was examined and EDTA was found to have a significantly inhibitory effect.     

Application of recombinant gene technology for production of polyhydroxyalkanoic acids: Biosynthesis of poly(4-hydroxybutyric acid) homopolyester

Screening of a large number of bacteria revealed several strains, which utilize 1,4-butanediol and/or 4-hydroxybutyric acid (4HB) as a carbon source for growth and for synthesis of polyhydroxyalkanoic acids (PHA) containing 4HB as one constituent among others (mostly 3-hydroxybutyric acid). However, none of the wild-type strains investigated in this study was able to produce a homopolyester consisting solely of 4HB. Only several poly(3-hydroxybutyric acid)-leaky mutants ofAlcaligenes eutrophus strain JMP222 synthesized poly(4HB) homopolyester, which amounted to approximately 10% (w/w) of the cellular dry matter. If the PHA synthase structural gene ofA. eutrophus strain H16 was expressed in these mutants, the amount of poly(4HB) was increased to approximately 30% (w/w). The occurrence of poly(4HB) was demonstrated by gas chromatographic as well as¹H and¹³C nuclear magnetic resonance spectroscopic analysis.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

Enhanced production of poly(3-hydroxybutyrate) by filamentation-suppressed recombinantEscherichia coli in a defined medium

Fed-batch cultures of recombinantEscherichia coli strains were carried out for the production of poly(3-hydroxybutyric acid) (PHB) in a chemically defined medium. TheE. coli strains used were XL1-Blue, harboring pSYL105, a stable high-copy number plasmid containing theAlcaligenes eutrophus polyhydroxyalkanoate (PHA) genes, and XL1-Blue, harboring pSYL107, which is pSYL105 containing theE. coli ftsZ gene to suppress filamentation. With XL1-Blue(pSYL105) the final cell mass and PHB concentration obtained in 62 h were 102 and 22.5 g/L, respectively. Fed-batch culture of XL1-Blue(pSYL107) under identical conditions resulted in a final cell mass and PHB concentration of 127.5 and 48.2 g/L, respectively. The PHB contents obtained with XL1-Blue(pSYL105) and XL1-Blue(pSYL107) were 22.1 and 37.8%, respectively. Therefore, PHB was more efficiently produced in a defined medium by employing filamentation-suppressed recombinantE. coli.     

Enzymatic Degradation and Aminolysis of Microbial Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Single Crystals

Solution-grown single crystals of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] were hydrolyzed by polyhydroxybutyrate (PHB) depolymerase from Ralstonia pickettii T1. Enzymatic degradation proceeded from the edges of lamellar crystals, yielding serrated contour and small crystal fragments. Gel permeation chromatography analysis revealed that the molecular weights of the crystals decreased during enzymatic degradation, suggesting that the enzymatic hydrolysis of chain-folding regions at the crystal surfaces occurred in addition to the enzymatic degradation at crystal laterals or edges. After P(3HB-co-4HB) single crystals were aminolysed in 20% aqueous methylamine solution to remove the folded-chain regions and enzymatic degradation by lipase from Rhizopus oryzae to remove 4HB components at crystal surfaces of single crystal aminolyzed, it was found that a small amount (up to ca. 2 mol%) of 4HB component can be incorporated into the P(3HB) mother crystal lattice irrespective of the 4HB content.     

Poly(hydroxyalkanoate) Biosynthesis from Crude Alaskan Pollock (Theragra chalcogramma) Oil

Six strains of Pseudomonas were tested for their abilities to synthesize poly(hydroxyalkanoate) (PHA) polymers from crude Pollock oil, a large volume byproduct of the Alaskan fishing industry. All six strains were found to produce PHA polymers from hydrolyzed Pollock oil with productivities (P; the percent of the cell mass that is polymer) ranging from 6 to 53% of the cell dry weight (CDW). Two strains, P. oleovorans NRRL B-778 (P = 27%) and P. oleovorans NRRL B-14682 (P = 6%), synthesized poly(3-hydroxybutyrate) (PHB) with number average molecular weights (M_n) of 206,000 g/mol and 195,000 g/mol, respectively. Four strains, P. oleovorans NRRL B-14683 (P = 52%), P. resinovorans NRRL B-2649 (P = 53%), P. corrugata 388 (P = 43%), and P. putida KT2442 (P = 39%), synthesized medium-chain-length PHA (mcl-PHA) polymers with M_n values ranging from 84,000 g/mol to 153,000 g/mol. All mcl-PHA polymers were primarily composed of 3-hydroxyoctanoic acid (C_8:0) and 3-hydroxydecanoic acid (C_10:0) amounting to at least 75% of the total monomers present. Unsaturated monomers were also present in the mcl-PHA polymers at concentrations between 13% and 16%, providing loci for polymer derivatization and/or crosslinking. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Purification and Properties of Extracellular Poly(3-hydroxybutyrate) Depolymerase Produced by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> 202

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase produced by a thermotolerant fungal soil isolate, Aspergillus fumigatus 202, was purified and characterized. Maximum PHB depolymerase production was obtained at the end of 48 h with initial medium pH 7.0 and 45 °C in Bushnell Haas Minerals medium containing PHB as sole source of carbon. The PHB depolymerase was purified using size exclusion chromatography to a fold purification of 20.62 and 61.62% yield. SDS-PAGE and isoelectric focusing revealed the molecular weight and pI of the purified enzyme as 63,744 Da and 4.2, respectively. N-terminal amino acid sequence of purified enzyme was HAXDAYLVK. This non-glycosylated enzyme was most active at pH 9.0 and 45 °C. Purified enzyme was inactivated by N-bromosuccinimide and dithiothreitol suggesting the involvement of tryptophan residues and disulfide bonds at its active site. Nonionic detergents like Tween 20, Tween 80 and Triton X-100 inhibited the enzyme activity. Ions like Ca⁺² and Mg⁺² (5 mM) increased the enzyme activity 1.5 times. Fe⁺² effectively inhibited the enzyme activity to 88% whereas Hg⁺² completely inhibited the enzyme.     

Synthesis of Short-chain-length/Medium-chain-length Polyhydroxyalkanoate (PHA) Copolymers in Peroxisome of the Transgenic Arabidopsis Thaliana Harboring the PHA Synthase Gene from Pseudomonas sp. 61-3

In this paper, the photosynthetic production of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymers is reported. The wild-type and highly active doubly mutated PHA synthase 1 (S325T/Q481K, abbreviated ST/QK) genes from Pseudomonas sp. 61-3 were introduced into Arabidopsis thaliana. Peroxisome targeting signal 1 (PTS1) was used to target PHA synthases into the peroxisome to synthesize PHA from the intermediates of the β-oxidation pathway. The transgenic Arabidopsis produced PHA copolymers consisting of 40–57 mol% 3-hydroxybutyrate, 21–49 mol% 3-hydroxyvalerate, 8–18 mol% 3-hydroxyhexanoate, and 2–8 mol% 3-hydroxyoctanoate. The maximum PHA contents were 220μ g/g cell dry weight (cdw) in leaves, and 36μ g/g cdw in stems, respectively. The expression of the ST/QK mutated PHA synthase in leaves gene did not lead to significant difference in PHA content and monomer composition of PHAs, compared to the wild-type PHA synthase gene, suggesting that the supply of monomers may be a rate-determining step of PHA biosynthesis in the peroxisome. However, in stems, there were significant differences dependent on whether the wild-type or ST/QK mutated PHA synthase was expressed. These results suggest that tissue-specific monomer availability is important in determining the final mol% composition of PHA copolymers produced by the peroxisome in plants.     

A method for the isolation of microorganisms producing extracellular long-side-chain poly (β-hydroxyalkanoate) depolymerase

A simple method was developed for the preparation of an autoclavable, long-side-chain poly (-hydroxyalkanoate) (LSC-PHA) colloidal suspension, which was used as a substrate for enzymatic degradation and to prepare agar overlay plates for the isolation of microorganisms producing extracellular LSC-PHA depolymerase. Six cultures producing extracellular LSC-PHA depolymerase were isolated from a composted hydrocarbon-contaminated soil. All were pseudomonads or related bacteria. All (with the possible exception ofXanthomonas maltophilia) could produce LSC PHA. Except forX. maltophilia none could hydrolyze poly (-hydroxybutyrate). Screening of sevenPseudomonas strains known to accumulate LSC PHA showed that all were negative for extracellular LSC-PHA depolymerase production. It was concluded that extracellular LSC-PHA depolymerase producers are found mostly in the genusPseudomonas but that they are relatively uncommon.     

Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in compost

The microbial degradation of tensile test pieces made of poly(3-hydroxybutyrate) [P(3HB)] or copolymers with 10% [P(3HB-co-10%3HV)] and 20% [P(3HB-co-20%3HV)] 3-hydroxyvaleric acid was studied in small household compost heaps. Degradation was measured through loss of weight (surface erosion) and changes in molecular weight and mechanical strength. It was concluded, on the basis of weight loss and loss of mechanical properties, that P(3HB) and P(3HB-co-3HV) plastics were degraded in compost by the action of microorganisms. No decrease inM _w could be detected during the degradation process. The P(3HB-co-20%3HV) copolymer was degraded much faster than the homopolymer and P(3HB-co-10%3HV). One hundred nine microbial strains capable of degrading the polymersin vitro were isolated from the samples used in the biodegradation studies, as well as from two other composts, and identified. They consisted of 61 Gram-negative bacteria (e.g.,Acidovorax facilis), 10 Gram-positive bacteria (mainlyBacillus megaterium), 35Streptomyces strains, and 3 molds.