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.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from various carbon sources by batch-fed cultures ofAlcaligenes eutrophus

Copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced at 30°C from various carbon sources byAlcaligenes eutrophus under batch-fed growth conditions. The production of P(3HB-co-3HV) from butyric and pentanoic acids was effective under nitrogenlimited conditions, and the conversion of carbon sources into copolyester was as high as 56 wt% at a C/N molar ratio of 40. In contrast, under excess-nitrogen conditions (C/N<10), cell growth was good, while P(3HB-co-3HV) production was partially inhibited. The production of P(3HB-co-3HV) from fructose and propionic acid was almost completely inhibited under excess-nitrogen conditions.     

Accumulation of Polyhydroxyalkanoates by Rhizobacteria Underneath Nickel-Hyperaccumulators from Serpentine Ecosystem

Nickel-resistant bacteria isolated from underneath Ni-hyperaccumulators growing on serpentine soils were screened for production of polyhydroxyalkanoates. These rhizobacteria accumulated poly-3-hydroxybutyric acid [P(3HB)] accounting 3.9–67.7% of cell dry weight during growth in gluconate and/or glucose. Cupriavidus pauculus KPS 201 utilized only gluconate and accumulated about 67.7% P(3HB) while, Bacillus firmus AND 408 utilized both carbon sources for polymer synthesis. The isolates being resistant to Ni also accumulated substantial amount of P(3HB) when grown in presence of the heavy metal and this was revealed by transmission electron microscopic studies. Although B. firmus AND 408 produced only P(3HB) at higher concentrations of gluconate, C. pauculus KPS 201 synthesized copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) [P(3HB-co-3HV)]. In presence of 0.8% gluconate and 4 mM Ni, KPS 201 cells produced PHA amounting 81% CDW, which contained 76 and 24 mol% 3HB and 3HV monomers, respectively.     

Degradation of Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Some Soil Aspergillus spp.

Systematic screening of 45 soil fungi for degradation polyhydroxyalkanoic acids (PHAs) has led to the selection of 6 potent Aspergillus isolates belonging to A. flavus, A. oryzae, A. parasiticus, and A. racemosus. Degradation of PHAs as determined by tube assay method revealed that these Aspergillus spp. were more efficient in degrading poly(3-hydroxybutyrate) [P(3HB)] compared to copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (P3HB-co-16% 3HV). Moreover, the extent of degradation in mineral base medium was much better than those in complex organic medium. For all the Aspergillus spp. tested, maximum degradation was recorded at a temperature of 37°C with significant inhibition of growth. The optimum pH range for degradation was 6.5–7.0 with degradation being maximum at pH 6.8. The extent of polymer degradation increased with increase in substrate concentration, the optimum concentration for most of the cultures being 0.4% and 0.2% (w/v) for P(3HB) and P(3HB-co-16%3HV) respectively. Supplementation of the degradation medium with additional carbon sources exerted significant inhibitory effect on both P(3HB) and P(3HB-co-16%3HV) degradation.     

Large-scale production of poly(3-hydroxyvaleric acid) by fermentation ofChromobacterium violaceum,processing, and characterization of the homopolyester

A fed-batch process was developed, which allowed biotechnological production of the homopolyester poly(3-hydroxyvaleric acid) [poly(3HV)], in a mineral salts medium containing valeric acid as carbon source and complex nutrients as supplements byChromobacterium violaceum at a 10- and 300-L fermentation scale. This process yielded up to 40 g dry cell matter per L fermentation broth, and the cells contained up to 70% (w/w) poly(3HV). Poly(3HV), which was extracted from the cells with chloroform and was precipitated from this solvent with ethanol, was processed to test bars by injection molding or by press processing and to fibers by melt spinning. The unprocessed and processed poly(3HV) material was characterized with respect to the molecular weight and with respect to thermal, rheological, and mechanical properties. It was shown that it is possible to process biodegradable poly(3HV) thermoplastically and to obtain a polymer suitable for applications with low strength requirements.     

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.     

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.     

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.     

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.     

Increased 3HV Concentration in the Bacterial Production of 3-Hydroxybutyrate (3HB) and 3-Hydroxyvalerate (3HV) Copolymer with Acid-Digested Rice Straw Waste

Bacterial synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymer [P(3HB-co-3HV)] using the hydrolysate of rice straw waste as a carbon source was affected by the composition of the hydrolysate, which depends highly on the rice straw pretreatment condition. Acid digestion with 2 % sulfuric acid generated larger production of P(3HB-co-3HV) than 6 % sulfuric acid, but 3HV concentration in the copolymer produced with 2 % acid hydrolysate was only 8.8 % compared to 18.1 % with 6 % acid hydrolysate. To obtain a higher 3HV mole fraction for enhanced flexibility of the copolymer, an additional heating was conducted with the 2 % acid hydrolysate after removal of residual rice straw. As the additional heating time increased a higher concentration of levulinic acid was generated, and consequently, the mole fraction of 3HV in P(3HB-co-3HV) increased. Among the conditions tested (i.e., 20-, 40-, 60-min), 60-min additional heating following 2 % sulfuric acid digestion achieved the highest 3HV mole fraction of 22.9 %. However, a longer heating time decreased the P(3HB-co-3HV) productivity, probably due to the increased intermediates concentrations acting as inhibitors in the hydrolysates. Therefore, the use of additional heating needs to consider both the increase in the 3HV mole fraction and the decrease in the P(3HB-co-3HV) productivity.     

Pseudomonas lemoignei has five poly(hydroxyalkanoic acid) (PHA) depolymerase genes: A comparative study of bacterial and eukaryotic PHA depolymerases

Four polyhydroxyalkanoate (PHA) depolymerases were purified from the culture fluid ofPseudomonas lemoignei: poly(3-hydroxybutyrate) (PHB), depolymerase A (M _r , 55,000), and PHB depolymerase B (M _r , 67,000) were specific for PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) as substrates. The third depolymerase additionally hydrolyzed poly(3-hydroxyvalerate) (PHV) at high rates (PHV depolymerase;M _r , 54,000). The N-terminal amino acid sequences of the three purified proteins, of a fourth partially purified depolymerase (PHB depolymerase C), and of the PHB depolymerases ofComamonas sp. were determined. Four PHA depolymerase genes ofP. lemoignei (phaZ1,phaZ2,phaZ3, andphaZ4) have been cloned inEscherichia coli, and the nucleotide sequence ofphaZ1 has been determined recently (D. Jendrossek, B. Müller, and H. G. Schlegel,Eur. J. Biochem. 218, 701–710, 1993). In this study the nucleotide sequences ofphaZ2 andphaZ3 were determined.PhaZ1,phaZ2, andphaZ4 were identified to encode PHB depolymerase C, PHB depolymerase B, and PHV depolymerase, respectively.PhaZ3 coded for a novel PHB depolymerase ofP. lemoignei, named PHB depolymerase D. None of the four genes harbored the PHB depolymerase A gene, which is predicted to be encoded by a fifth depolymerase gene ofP. lemoignei (phaZ5) and which has not been cloned yet. The deduced amino acid sequences ofphaZ1–phaZ3 revealed high homologies to each other (68–72%) and medium homologies to the PHB depolymerase gene ofAlcaligenes faecalis T₁ (25–34%). Typical leader peptide amino acid sequences, lipase consensus sequences (Gly-Xaa-Ser-Xaa-Gly), and unusually high proportions of threonine near the C terminus were found in PhaZ1, PhaZ2, and PhaZ3. Considering the biochemical data of the purified proteins and the amino acid sequences, PHA depolymerases ofP. lemoignei are most probably serine hydrolases containing a catalytical triad of Asp, His, and Ser similar to that of lipases. A comparison of biochemical and genetic data of various eubacterial and one eukaryotic PHA depolymerases is provided also.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

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.     

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.     

Proposed mechanism for bacterial metabolism of polyacrylate

A possible aerobic degradative pathway for polyacrylate was examined with trimer (1,3,5-pentane tricarboxylic acid; PTCA)-utilizing bacteria. A few metabolic products from PTCA accumulated in culture filtrates and reaction mixtures of washed cells. Fraction A was detected as a main metabolite by high-performance liquid chromatography. A small amount of fraction B was concomitant with fraction A. Another fraction, C, was also detected. These compounds were suggested by liquid chromatography-mass spectrometry analyses to be 1,3,5-(1- or 2-pentene)tricarboxylic acid (fraction A or B) and 1,3,5-(2-oxopentane)tricarboxylic acid (fraction C). Fraction A was quickly further metabolized by washed cells, but fraction B was only gradually degraded. From these results, the metabolic pathway for polyacrylate is suggested to be quite similar to-oxidation for saturated fatty acids. The degradation of PTCA by washed cells was slower than that by growing cells and was inhibited by 5 mM NaN₃. This suggests that the metabolism is linked to a respiratory chain or energy-producing system of bacteria which can aerobically assimilate PTCA.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

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.     

Municipal Wastes Treatment and Production of Polyhydroxyalkanoate by Modified Two-Stage Batch Reactor

The municipal wastes were utilized as substrate for polyhydroxyalkanoate (PHA) using two strains of Bacillus licheniformis (PHAs-007, wild type and M2-12, mutant). Municipal wastes were subjected to separate wastewater and biosolid. Municipal biosolid was digested by anaerobic bacteria thereafter only the supernatant with soluble organic compounds was subjected into the PHA-producing reactor containing municipal wastewater. The mutant strain M2-12 gave the highest value of biomass (42.0 ± 2.0 g/L) and PHA concentration (37.4 ± 1.0 g/L with 88.9 % of dry cell weight, DCW) and reduced 76.5 % of soluble chemical oxygen demand after 60 h of cultivation. The value of pH, biochemical oxygen demand and total solid of the reclaimed wastewater after PHA recovery was 7.1, 20 and 97 mg/L, respectively. Moreover, the polymers produced by both strains of B. licheniformis were characterized. The resultant polymer from B. licheniformis PHAs-007 and M2-12 cultivated in the PHA-producing reactor was identified as poly-3-hydroxybutyrate-co-3-hydroxyvalerate [P(3HB-co-3HV)] and poly-3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB)], respectively. The results suggesting that the production of PHA by municipal wastes is feasible thus the PHA production stage can be integrated in waste treatment to produce PHA and treated municipal wastes at the same time.     

Synthesis and biodegradation of poly(γ-butyrolactone-co-l-lactide)

Biodegradable polyesters were synthesized by ring-opening copolymerization of -butyrolactone (BL) and its derivatives withl-lactide (LLA). Although tetraphenyl tin was the main catalyst used, other organometallic catalysts were used as well.¹H and¹³C NMR spectra showed that poly(BL-co-LLA)s were statistical and that their number-average molecular weights were as high as 7×10⁴. The maximum BL content obtained from copolymerization BL/LLA was around 17%. TheT _m andT _g values of the copolymers showed a gradual depression with an increase in BL content. NoT _m was obtained for the copolymers containing more than 13 mol% BL. The biodegradability of the copolyesters was evaluated by enzymatic hydrolysis and nonenzymatic hydrolysis tests. The enzymatic hydrolysis was carried out at 37°C for 24 h using lipases fromRhizopus arrhizus andR. delemar. Hydrolyses by both lipases showed that an increase in BL content of the copolymer resulted in enhanced biodegradability. Nonenzymatic accelerated hydrolysis of copolymers at 70°C was found to increase proportionally to their exposure time. The hydrolysis rate of these copolymers was considerably faster than that of PLLA. The higher hydrolyzability was recorded for the BL-rich copolymers. The copolymerization of -methyl--butyrolactone (MBL) or -ethyl--butyrolactone (EBL) with LLA resulted in relatively LA-rich copolymers.     

Enzymatic degradation of poly([R,S] β-hydroxybutyrate)

The synthetic analogue of a bacterially produced polyester, poly(-hydroxybutyrate) (PHB) was synthesized from racemic -butyrolactone using anin situ trimethyl aluminum-water catalyst. The polymer was fractionated into samples differing in molecular weight and isotactic diad content. The latter was closely related to degree of crystallinity. The biodegradation of these fractions were examined by monitoring mass loss over time in the presence of anAlcaligenes faecalis T1 extracellular bacterial poly(-hydroxybutyrate) depolymerase. The fraction with high isotactic diad tacticity content showed little or no degradation over a 50 hour incubation period, whereas the fraction of intermediate isotactic diad content degraded in a continuous steady fashion at a rate that was less than that for bacterial PHB. The low isotactic diad fraction underwent a rapid initial degradation, followed by no further mass loss. The presence of stereoblocks in the polymer structure of the various fractions was an influence on the degree of susceptibility towards degradation and is related to sample crystallinity.     

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.