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.     

Physical and Thermal Analysis of the Degradation of Poly(3-Hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-Hydroxybutyrate) Coated Paper in a Constructed Soil Medium

The degradation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) coated brown Kraft paper and its components in a constructed soil environment was investigated. Soil burial tests were carried out over 8 weeks. Weight loss measurements, photographic analysis, environmental scanning electron microscopy (ESEM), dynamic mechanical analysis (DMA) and differential scanning calorimetry (DSC) were conducted to assess the physical, structural, mechanical and thermal behavior before and after the soil burial test. Paper showed the highest physical degradation and weight loss. With respect to the control samples, the stiffness of the partially degraded samples decreased. The overall crystallinity of the biopolymer and the coated paper was affected significantly by burial. The pure biopolymer’s weight loss was substantially enhanced when coated on paper. This result reveals a possible increased microbial population in the coated paper relative to the pure biopolymer.     

Accumulation of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) by mutants and recombinant strains ofAlcaligenes eutrophus

Alcaligenes eutrophus accumulated a terpolyester of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), and 4-hydroxyvaleric acid (4HV) during cultivation with 4HV as carbon and energy source under nitrogen starvation. The polyester accumulated by wild-type strains under these conditions contained 4HV at a molar fraction of approximately 5 mol% only. A catabolic pathway of 4HV was postulated, which included the activation of 4HV to 4HV-CoA and a conversion of 4HV-CoA to 3HV-CoA. Tn5::mob-induced mutants were isolated fromA. eutrophus HF39, which were affected in 4HV and/or valeric acid catabolism. Among 83 mutants were 27 4HV-negative or 4HV-leaky mutants; two mutants were identified which accumulated a terpolyester with a molar fraction of 10.1 to 22.7 mol% 4HV. In addition, a further increase in the molar fraction of 4HV in poly(3HB-co-3HV-co-4HV) and a two- to fourfold increase in the PHA synthase activity were monitored in these mutants or others and also in HF39, if the cells were complemented with the hybrid plasmid pHP1014::PP1, which contained the PHA biosynthesis genes ofA. eutrophus H16. Application of mutagenesis plus recombinant DNA techniques resulted in the accumulation of a terpolyester with up to 30 mol% 4HV and with approximately equimolar fractions of 3HB, 3HV, and 4HV.     

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.     

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.     

In Vivo and In Vitro Degradation Studies for Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyhexanoate) Biopolymer

Studies have shown that the copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] possesses favorable mechanical properties for use in medical supplies and products (e.g., sutures, scaffolds, bone plates). One of the major under-addressed issues associated with the use of biodegradable, bio-based PHA polymers in resorbable medical products is the correlation between the mechanical properties and the in vivo material degradation over time. In this study, P(HB-co-17 mol% HHx) matrices were mechanically tested after either incubation in cultures of human embryonic kidney cells (HEK) for in vitro degradation studies for up to 4 weeks, or inserted into Danio rerio (zebrafish) tissues for in vivo degradation studies for up to 7 weeks. The mechanical properties and scanning electron microscopy (SEM) images of the degraded materials were examined and later correlated to understand the degradation phenomenon. Our results show that Young’s modulus of P(HB-co-17 mol%HHx) during in vitro studies decreased from 3.26 to 2.42 GPa within 4 weeks, and in vivo breakdown resulted in a significant decrease in Young’s modulus with a decrease from 3.26 to 0.51 GPa and a mass loss of 59 % within 7 weeks. SEM images showed the development of pores and cracks on the surface of the material over time. Plasticization and recrystallization were observed and likely play a role in the alteration of mechanical properties.     

Anti-biofilm Activity of Solvent-Cast and Electrospun Polyhydroxyalkanoate Membranes Treated with Lysozyme

Biofilms consist of groups of microorganisms that adhere to surfaces, such as wound and implant surfaces, making it difficult to prevent or remove their formation by antibiotic treatment, due to the innate resistance of the biofilm. Effective treatments of medical biofilms are limited. Polyhydroxyalkanoate (PHA) is a biodegradable and biocompatible polymer that is a suitable alternative to petroleum based polymers for use as a raw material for medical applications. In this study, membranes of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] containing different HHx monomer contents were used due to its porosity and flexibility, and different sheets were prepared by solvent-casting and electrospinning methods. The sheets were loaded with lysozyme in order to measure the maximum amount of protein adsorption and to examine the ability of immobilized enzyme to inhibit biofilm formation and detach previously established biofilms. Our results have shown maximum loading of 16.1 µg enzyme per 9.5 mm³ discs, and these sheets are effective for inhibiting biofilm formation. Also, lysozyme loaded, eletrospun sheets were observed to more effectively inhibit biofilm formation, as compared to solvent-cast sheets. Based on this study, P(HB-co-HHx) sheets are a suitable material for being used as a potential raw material for fabrication of wound dressings to be used in anti-biofilm treatments.     

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.     

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.     

Microbial Degradation of Poly(3-Hydroxybutyrate-co-4-Hydroxybutyrate) in Soil

The microbial degradation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) copolymers with different 4HB molar fraction were investigated in soil for 60?days. In the degradation process, changes in weight loss and molecular weight were periodically measured to determine the biodegradability; the surface morphology also was observed using scanning electron microscopy and polarizing optical microscopy. The results showed that the rate of degradation of P3/4HB depends strongly on its crystallinity and surface morphology. Enzymatic degradation, which proceeded via surface erosion mechanisms, was observed mainly during the degradation period in soil. The amorphous interspherulitic regions and crystal center were prone to degrade.     

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.     

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.     

Thermal Properties and Biodegradability Studies of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

For investigating the relationship between thermal properties and biodegradability of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), several films of PHBV containing different polyhydroxyvalerate (HV) fractions were subjected to degradation in different conditions for up to 49 days. Differential scanning calorimetry (DSC), thermogravimetry (TG), specimen weight loss and scanning electron microscopy (SEM) were performed to characterize the thermal properties and enzymatic biodegradability of PHBV. The experimental results suggest that the degradation rates of PHBV films increase with decreasing crystallinity; the degradability of PHBV occurring from the surface is very significant under enzymatic hydrolysis; the crystallinity of PHBV decreased with the increase of HV fraction in PHBV; and no decrease in molecular weight was observed in the partially-degraded polymer.     

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.     

Environmental Degradation of Blends of Atactic Poly[(R,S)-3-hydroxybutyrate] with Natural PHBV in Baltic Sea Water and Compost with Activated Sludge

Degradation of atactic poly[(R,S)-3-hydroxybutyrate] (a-PHB) binary blends with natural poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 12 mol% of 3HV units), has been investigated and compared with plain PHBV in the compost containing activated sludge and under marine exposure conditions in the dynamic water of the Baltic Sea. Characteristic parameters of compost and the Baltic Sea water were monitored during the incubation period (6 weeks) and their influence on the degree of biodegradation is discussed. After specified degradation times of the experiments the weight loss of the samples, surface changes, changes in molecular weight and polydispersity as well as changes of the composition and thermo-mechanical properties of the blends have been evaluated. Macroscopic observations of the samples were accompanied by investigations using optical microscopy, size-exclusion chromatography (SEC), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) and tensile testing. The degree of degradation of blends of a-PHB with PHBV depends on the blend composition and environmental conditions. In both environments studied the weight loss of plain PHBV was more significant than changes the molecular weight. In both environments only enzymatic degradation of the blends, which proceeds via surface erosion mechanisms, was observed during the incubation period.     

Soil Biodegradation of PHBV/Peach Palm Particles Biocomposites

There is great interest in developing eco-friendly green biocomposites from plant-derived natural fibers and crop-derived bioplastics attributable to their renewable resource-based origin and biodegradable nature. Fully biodegradable composites, made from both biodegradable polymeric matrices and natural fibers, should be advantageous in some applications, such as one way packaging. Polyhydroxyalkanoates (PHAs) are naturally occurring biodegradable polymers produced from a wide range of microorganisms, with poly(3-hydroxybutyrate) P(3HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) being important examples of PHAs. In this work, biocomposites of PHBV consisting of a PHBV matrix incorporating peach palm particles (PPp), [i.e., 100/0, 90/10, 80/20 and 75/25 (%w/w) PHBV/PPp] were processed by injection molding at 160 °C. The effect of PPp loading on the thermal and the mechanical properties, as well as on the morphological behavior of the PHBV/PPp biocomposites was investigated. Soil biodegradation tests were carried out by burying specimen beakers containing aged soil and kept under controlled temperature and humidity in accordance with ASTM G160-98. Degradation of the biocomposites was evaluated by visual analysis, scanning electron microscopy (SEM) and thermogravimetric analysis (TGA) following test exposures of up to 5 months. The addition of PPp reduced the maximum strength and the elongation at break of the biocomposites. On the other hand, the Young’s modulus improved with the PPp content. Micrographs of the fracture surfaces following tensile strength testing revealed a large distance between the PHBV matrix and PPp particles although a low interaction is expected. Where measured, these distances tended increase as the PPp content of the biocomposites increased. Soil biodegradation tests indicated that the biocomposites degraded faster than the neat polymer due to the presence of cavities that resulted from introduction of the PPp and that degradation increased with increasing PPp content. These voids allowed for enhanced water adsorption and greater internal access to the soil-borne degrader microorganisms.     

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.     

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.     

Effects of higher-order structure of poly(3-hydroxybutyrate) on its biodegradation. II. Effects of crystal structure on microbial degradation

As one of a series of studies concerning the relationship between the higher-order structure and the biodegradability of a biodegradable plastic, the effects of the crystal structure of the plastic on microbial degradation were investigated. Bacterial poly(d-(–)-3-hydroxybutyrate) (PHB) films which had a wide range of crystallinity were prepared by the melt-quenching method. Results of the microbial degradation indicated that the development of crystallinity evidently depressed the microbial degradability. From scanning electron microscopy (SEM) observations, it is suggested that the microbial degradation proceeded in at least two manners. One was preferential degradation of the amorphous region leaving the crystalline lamellae intact, which was considered to be a homogeneous enzymatic degradation over the surface. The other was nonpreferential spherical degradation on the surface. The SEMs indicate that the spherical holes were the result of colonization by degrading bacteria. The holes varied in size and number with the change of crystal structure. Therefore, it is considered that the crystal structure of PHB also influenced the physiological behavior of the degrading bacteria on the PHB surface.     

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.