Biodegradability studies of polyhydroxyalkanoate (PHA) film produced by a marine bacteria using Jatropha biodiesel byproduct as a substrate

Polyhydroxyalkanoates are water-insoluble, hydrophobic polymers and can be degraded by microorganisms that produce extracellular PHA depolymerase. The present work was aimed to evaluate the degradability of Polyhydroxyalkanoate film produced by Halomonas hydrothermalis using Jatropha biodiesel byproduct as a substrate. PHB films were subjected to degradation in soil and compared with the synthetic polymer (acrylate) and blend prepared using the synthetic polymer (acrylate) and PHB. After 50 days, 60% of weight loss in PHB film and after 180 days 10% of blended film was degraded while no degradation was found in the synthetic film. Scanning electron microscopy and confocal microscopy revealed that after 50 days the PHB film and the blended film became more porous after degradation while synthetic film was not porous. The degradative process was biologically mediated which was evident by the control in which the PHB films were kept in sterile soil and the films showed inherent integrity over time. The TGA and DSC analysis shows that the melting temperatures were changed after degradation indicating physical changes in the polymer during degradation.     

Microbial degradation of poly-β-hydroxybutyrate using landfill soils

The population of poly-β-hydroxybutyrate-degrading microorganisms and the biodegradation of PHB in local landfill soils were examined in vitro and in vivo. Forty-two PHB-degraders consisting of 12 bacteria, 25 actinomycetes and 5 moulds were isolated. The total PHB-degraders averaged 4.7 × 10⁷ and 20 × 10⁴ colony forming units (cfu)/g for San Mateo wet and dry soils, respectively, and 2.3 × 10⁷ and 8.5 × 10⁴ cfu/g for Carmona wet and dry samples, respectively. The PHB-degraders formed 0–59% of the total microbial population in San Mateo and 8–42% in Carmona. Complete (100%) degradation of PHB powder was observed for Chryseomonas-27 and Aspergillus-39 on day 5 in shake flask culture and for Streptomyces-4 on day 7. Burial test in landfill soils showed a 90–91% weight loss of PHB film strips within four weeks; the weight loss of polypropylene film strips was up to 0.12% only. Scanning electron micrographs of degraded films revealed the attachment of microbial cells and fungal mycelium and spores on the surfaces. Holes and cavities were also noted due to the microbial degradation processes.     

Anaerobic Biodegradation of Poly-3-Hydroxybutyrate (PHB) by Sulfate Reducing Bacterium Desulfotomaculum sp.

Poly-3-hydroxybutyrate (PHB) film pieces were degraded by sulfate reducing Desulfotomaculum sp. incubated under anaerobic laboratory conditions. Degradation started with adherence of the microbial cells and followed by formation of black colonies on the film surface. Scanning electron microscopic (SEM) observations revealed the presence of bacteria and formation of small holes on the film. After 60 days of incubation at 30°C, 10 % weight loss in polymer and 13 % sulfate reduction in the medium was observed. According to gel permeation chromatography (GPC) analysis, the molecular weight of the PHB decreased after 30 days and did not decrease further at a more extended incubation period. Loss of weight of PHB does not seem to be correlated with molecular weight decrease.     

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids.

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Effects of culture conditions on the production of polyhydroxyalkanoates by Azotobacter chroococcum H23 in media containing a high concentration of alpechín (wastewater from olive oil mills) as primary carbon source

Large amounts of homopolymers containing beta-hydroxybutyrate (PHB) and copolymers containing beta-hydroxyvalerate (P[HB-co-HV]) are produced by Azotobacter chroococcum strain H23 when growing in culture media amended with alpechín (wastewater from olive oil mills) as the sole carbon source. Copolymer was formed when valerate (pentanoate) was added as a precursor to the alpechín medium, but it was not formed with the addition of propionate as a precursor. A. chroococcum formed homo- and copolymers of polyhydroxyalkanoates (PHAs) up to 80% of the cell dry weight, when grown on NH(4)(+)-medium supplemented with 60% (v/v) alpechín, after 48 h of incubation at 100 rev min(-1) and 30 degrees C. Production of PHAs by strain H23 using alpechín looks promising, as the use of a cheap substrate for the production of these materials is essential if bioplastics are to become competitive products.     

Effect of temperature and cycle length on microbial competition in PHB-producing sequencing batch reactor

The impact of temperature and cycle length on microbial competition between polyhydroxybutyrate (PHB)-producing populations enriched in feast-famine sequencing batch reactors (SBRs) was investigated at temperatures of 20 °C and 30 °C, and in a cycle length range of 1–18 h. In this study, the microbial community structure of the PHB-producing enrichments was found to be strongly dependent on temperature, but not on cycle length. Zoogloea and Plasticicumulans acidivorans dominated the SBRs operated at 20 °C and 30 °C, respectively. Both enrichments accumulated PHB more than 75% of cell dry weight. Short-term temperature change experiments revealed that P. acidivorans was more temperature sensitive as compared with Zoogloea. This is particularly true for the PHB degradation, resulting in incomplete PHB degradation in P. acidivorans at 20 °C. Incomplete PHB degradation limited biomass growth and allowed Zoogloea to outcompete P. acidivorans. The PHB content at the end of the feast phase correlated well with the cycle length at a constant solid retention time (SRT). These results suggest that to establish enrichment with the capacity to store a high fraction of PHB, the number of cycles per SRT should be minimized independent of the temperature.     

Biodegradability and mechanical properties of poly-(beta-hydroxybutyrate-co-beta-hydroxyvalerate)-starch blends.

Wheat starch granules and poly-(beta-hydroxybutyrate-co-beta-hydroxyvalerate) [P(HB-co-HV), (19.1 mol% HV)] were blended at 160 degrees C. Increasing the starch content from 0 to 50% (wt/wt) decreased the tensile strength of P(HB-co-HV) from 18 MPa to 8 MPa and diminished flexibility as Young's modulus increased from 1,525 MPa to 2,498 MPa, but overall mechanical properties of the polymer remained in a useful range. A mixed microbial culture required more than 20 days to degrade 150-microns-thick samples of 100% P(HB-co-HV), whereas samples containing 50% (wt/wt) starch disappeared in fewer than 8 days. Starch granules degraded before P(HB-co-HV) did. Aerobic degradation proceeded more rapidly than anaerobic degradation.     

The fate of ‘biodegradable’ plastics in municipal leaf compost

Summary Blends of starch with polypropylene, starch with polyethylene, polycaprolactone with polyethylene, and a copolymer of -hydroxybutyrate and -hydroxyvalerate (PHB/V) were exposed to degrading leaves in a municipal leaf composting operation. Every month for 6 months, duplicate samples were analyzed for changes in weight and tensile properties, and many of these samples were further analyzed for changes in molecular weight and surface morphology. All results were compared to controls which were incubated for 6 months in moist, sterile leaves at a leaf compost temperature. Very little change was noted for any of the polyolefin blends over the 6-month period. In contrast, PHB/V samples showed massive deterioration with substantial weight loss. Although there was a decrease in molecular weight and a loss of tensile properties in leaf-exposed PHB/V films, the sterile control films also showed similar changes, but without weight loss. Of the microbial isolates from film surfaces, only fungi possessed PHB/V depolymerase activity.     

Degradation of natural and synthetic polyesters under anaerobic conditions

Often, degradability under anaerobic conditions is desirable for plastics claimed to be biodegradable, e.g. in anaerobic biowaste treatment plants, landfills and in natural anaerobic sediments. The biodegradation of the natural polyesters poly(beta-hydroxybutyrate) (PHB), poly(beta-hydroxybutyrate-co-11.6%-beta-hydroxyvalerate) (PHBV) and the synthetic polyester poly(epsilon-caprolactone) (PCL) was studied in two anaerobic sludges and individual polyester degrading anaerobic strains were isolated, characterized and used for degradation experiments under controlled laboratory conditions. Incubation of PHB and PHBV films in two anaerobic sludges exhibited significant degradation in a time scale of 6-10 weeks monitored by weight loss and biogas formation. In contrast to aerobic conditions, PHB was degraded anaerobically more rapidly than the copolyester PHBV, when tested with either mixed cultures or a single strained isolate. PCL tends to degrade slower than the natural polyesters PHB and PHBV. Four PHB and PCL degrading isolates were taxonomically identified and are obviously new species belonging to the genus Clostridium group I. The depolymerizing enzyme systems of PHB and PCL degrading isolates are supposed to be different. Using one isolated strain in an optimized laboratory degradation test with PHB powder, the degradation time was drastically reduced compared to the degradation in sludges (2 days vs. 6-10 weeks).     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Production of poly-β-hydroxybutyrate from methanol: characterization of a new isolate of Methylobacterium extorquens

Summary Poly--hydroxybutyric acid (PHB) and similar bacterial polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of methanol, one of the cheapest noble substrates available, may help to reduce the cost of producing such bioplastics. As a first step, a culture collection of 118 putative methylotrophic microorganisms was obtained from various soil samples without any laboratory enrichment step to favour culture diversity. The most promising culture was selected based on rapidity of growth and PHB accumulation and later identified as Methylobacterium extorquens. This isolate was obtained from soml contaminated regularly with used oil products for some 40 years. Concentrations of methanol greater than 8 g/l affected growth significantly and the methanol concentration was optimal at 1.7 g/l. PHB concentrations averaged 25–30% (w/v) of dry weight under non-optimized conditions. Controlling methanol concentration, using an open-loop configuration, led to biomass levels of 9–10 g/l containing 30–33% PHB while preventing methanol accumulation. The new isolate was also able to produce the co-polymer PHB/poly--hydroxyvalerate (PHV) using the mixture methanol + valerate. The PHV-to-PHB ratio was about 0.2 at the end of the fermentation. An average molecular mass varying between 2 and 3 × 10⁵ Da was obtained for three PHB samples using two different measurement methods.Publication number NRCC No. 33672 Offprint requests to: D. Groleau     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9.

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Regulating the molar fraction of 4-hydroxybutyrate in Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by biological fermentation and enzymatic degradation

The regulation of 4-hydroxybutyrate (4HB) molar fraction in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] of a local isolate Cupriavidus sp. USMAA1020 was attempted by employing a feeding strategy through fed-batch fermentation in 100-L fermenter. The growth of Cupriavidus sp. USMAA1020 was enhanced by frequently feeding carbon and nitrogen at a ratio of 5 (C/N 5) using a DO-stat with cascade mode at 20% (v/v) dissolved oxygen (DO). The feeding of C/N 5 and the use of the DO-stat mode were able to regulate the 4HB composition from 0–67 mol% by sequential feeding of γ-butyrolactone and supplementing oleic acid. A high 4HB molar fraction of 67 mol% with a PHA concentration of 5.2 g/L was successfully obtained by employing this feeding strategy. Notably, enzymatic degradation carried out enhanced the 4HB composition of the copolymer synthesized. PHB depolymerase enzyme from Acidovorax sp. was used to degrade this P(3HB-co-70-mol%4HB) copolymer and the 4HB composition could be increased up to 83 mol%. The degradation process was observed by monitoring the time-dependent change in the weight loss of copolymer films. The percentage of weight loss of solvent-cast film increased proportionally up to 19% within 3 h, whereas salt-leached films showed 90% of weight loss within 3 h of incubation and were completely degraded by 4 h. The molecular weight (M _n ) of the films treated with enzyme demonstrated a slight decrease. SEM observation exhibited a rough surface morphology of the copolymer degraded with depolymerase enzyme.     

The recovery of poly(3-hydroxybutyrate) by using dispersions of sodium hypochlorite solution and chloroform

Summary To take advantage of both differential digestion by hypochlorite and solvent extraction, we used dispersions of sodium hypochlorite solution and chloroform in the recovery of microbial PHB. The treatment with hypochlorite alone caused such severe degradation and the molecular weight decreased drastically with increasing hypochlorite concentration. However, using the dispersion, the degradation of PHB was markedly diminished owing to theshielding effect of chloroform. In this case, we could obtain PHB of above 97% purity with a number average molecular weight of 1,000,000 comparable to the original molecular weight of 1,200,000.     

Growth of Azotobacter vinelandii UWD in Fish Peptone Medium and Simplified Extraction of Poly-β-Hydroxybutyrate

Azotobacter vinelandii UWD was grown in a fermentor with glucose medium with and without 0.1% fish peptone (FP) in batch and fed-batch cultures for the production of the natural bioplastic poly-β-hydroxybutyrate (PHB). Strain UWD formed PHB five times faster than cell protein during growth in glucose and NH₄⁺, but PHB synthesis stopped when NH₄⁺ was depleted and nitrogen fixation started. When FP was added to the same medium, PHB accumulated 16 times faster than cell protein, which in turn was inhibited by 40%, and PHB synthesis was unaffected by NH₄⁺ depletion. Thus, FP appeared to be used as a nitrogen source by these nitrogen-fixing cells, which permitted enhanced PHB synthesis, but it was not a general growth stimulator. The addition of FP to the medium led to the production of large, pleomorphic, osmotically sensitive cells that demonstrated impaired growth and partial lysis, with the leakage of DNA into the culture fluid, but these cells were still able to synthesize PHB at elevated rates and efficiency. When FP was continuously present in fed-batch culture, the yield in grams of polymer per gram of glucose consumed was calculated to range from 0.43 g/g, characteristic of nongrowing cells, to an unprecedented 0.65 g/g. Separation of an FP-free growth phase from an FP-containing growth phase in fed-batch culture resulted in better growth of these pleomorphic cells and good production of PHB (yield, 0.32 g/g). The fragility of these cells was exploited in a simple procedure for the extraction of high-molecular-weight PHB. The cells were treated with 1 N aqueous NH₃ (pH 11.4) at 45°C for 10 min. This treatment removed about 10% of the non-PHB mass from the pellet, of which 60 to 77% was protein. The final product consisted of 94% PHB, 2% protein, and 4% nonprotein residual mass. The polymer molecular weight (1.7 × 10⁶ to 2.0 × 10⁶) and dispersity (1.0 to 1.9) were not significantly affected (P = 0.05) by this treatment. In addition, the NH₃ extraction waste could be recycled in the fermentation as a nitrogen source, but it did not promote PHB production like FP. A scheme for improved downstream extraction of PHB as well as the merits of using pleomorphic cells in the production of bioplastics is discussed.     

The use of an acetoacetyl‐CoA synthase in place of a β‐ketothiolase enhances poly‐3‐hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C₄ plants for the production of poly[(R)‐3‐hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield‐potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing β‐ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl‐CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB‐producing sugarcane containing the β‐ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10⁶ Da. These results are a major step forward in engineering a high biomass C₄ grass for the commercial production of PHB.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Characterization of a thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2

Poly-3-hydroxybutyrate (P3HB) degradation capabilities of a novel bacterium, Anoxybacillus gonensis G2, were investigated. Both changes on film surfaces of the solution-cast films monitored by scanning electron microscopy (SEM) and weight loss up to 24% after 72 h exposure to A. gonensis G2 cultures indicated secretion of an active esterase responsible for the degradation of P3HB films. Kinetic parameters, Vmax and Km for the esterase activity of crude enzyme from A. gonensis G2 in the presence of p-nitrophenylbutyrate as substrate were observed as 50 U/L and 0.125 mM, respectively, in 50 mM phosphate buffer, pH 7.5 at 60 degrees C. The stimulation of the activity by Ca2+ is an evidence for the requirement of Ca2+ as a cofactor for the enzyme activity which is a characteristic for lipases/esterases. Inhibition of the esterase activity by metal chelating agents such as ethylenediamine tetraacetate, azide and cyanide has also supported the requirement of a metal ion for the activity. The thermal and pH stability profiles for the enzyme showed that the thermophilic bacterium A. gonensis G2 secretes an extracellular thermoalkalophilic PHB depolymerase active at 60 degrees C, and stable at this temperature for 120 min at pH 7.5 and for 24 h at pH 7.5-9.5 range at 4 degrees C by retaining over 75% of its initial activities.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.