The influence of process parameters on the characteristics of polyhydroxyalkanoates produced by mixed cultures

The characterization of polyhydroxyalkanoates (PHA) produced by mixed cultures is fundamental for foreseeing the possible final applications of the polymer. In this study PHA produced under aerobic dynamic feeding (ADF) conditions are characterized. The PHA produced shows a stable average molecular weight ([symbol: see text]) in the range (1.0-3.0) x 10(6), along three years of reactor operation. Attempts to improve the amount of PHA produced did not introduce significant variations on the values [symbol: see text]. Along this period, the polydispersity indices (PDI) were between 1.3 and 2.2. The use of different carbon sources allowed the tailoring of polymer composition: homopolymers of poly(3-hydroxybutyrate), P(3HB), were obtained with acetate and butyrate, whereas a mixture of acetate and propionate, and propionate and valerate, gave terpolymers of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 2-methyl-3-hydroxyvalerate (2M3HV). All of these PHA had [symbol: see text] between 2.0 x 10(6) and 3.0 x 10(6). Thermal characterization of the produced polymers showed values of glass transition temperature, melting temperature, melting enthalpy, and crystallinity slightly lower than those obtained for PHA from pure cultures. The introduction of a purification step during the polymer extraction process allowed the elimination of possible contaminants but did not significantly improve the polymer quality.     

Metabolic engineering for microbial production and applications of copolyesters consisting of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates

Poly(hydroxyalkanoate)s (PHAs) are a class of microbially synthesized polyesters that combine biological properties, such as biocompatibility and biodegradability, and non-bioproperties such as thermoprocessability, piezoelectricity, and nonlinear optical activity. PHA monomer structures and their contents strongly affect the PHA properties. Using metabolic engineering approaches, PHA structures and contents can be manipulated to achieve controllable monomer and PHA cellular contents. This paper focuses on metabolic engineering methods to produce PHA consisting of 3-hydroxybutyrate (3HB) and medium-chain-length 3-hydroxyalkanoates (3HA) in recombinant microbial systems. This type of copolyester has mechanical and thermal properties similar to conventional plastics such as poly(propylene) and poly(ethylene terephthalate) (PET). In addition, pathways containing engineered PHA synthases have proven to be useful for enhanced PHA production with adjustable PHA monomers and contents. The applications of PHA as implant biomaterials are briefly discussed here. In the very near term, metabolic engineering will help solve many problems in promoting PHA as a new type of plastic material for many applications.     

Bioconversion of Glycerine Pitch into a Novel Yellow-Pigmented P(3HB-co-4HB) Copolymer: Synergistic Effect of Ammonium Acetate and Polymer Characteristics

Glycerine pitch waste generated from oleochemical industry was exploited as a carbon source for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) copolymer production by a novel, yellow-pigmented bacterium Cupriavidus sp. USMAHM13 to improve the economics of microbial polyhydroxyalkanoate production and to establish a feasible waste management approach. Medium optimization using response surface methodology through shake-flask fermentation had led to the accumulation of P(3HB-co-51%4HB) copolymer using a combination of glycerine pitch (10 g/l), 1,4-butanediol (8.14 g/l), and ammonium acetate (2.39 g/l). P(3HB-co-4HB) copolymers with 4HB monomer compositions ranged from 3 to 40 mol% were obtained through batch fermentation in a bioreactor using different concentrations of ammonium acetate. The copolymers exhibited a wide range of material properties depending on the monomer composition and type of carbon sources. P(3HB-co-40%4HB) was a typical random copolymer, whereas other P(3HB-co-4HB) produced were blend copolymers. Carotenoid pigment which was produced simultaneously with the polymer production was found to have negligible effect on the mechanical and thermal properties of the P(3HB-co-4HB) copolymer films.     

Preparation and Characterization of Polyhydroxyalkanoates Macroporous Scaffold Through Enzyme-Mediated Modifications

Polyhydroxyalkanoates (PHAs) are hydrophobic biodegradable thermoplastics that have received considerable attention in biomedical applications due to their biocompatibility, mechanical properties, and biodegradability. In this study, the degradation rate was regulated by optimizing the interaction of parameters that influence the enzymatic degradation of P(3HB) film using response surface methodology (RSM). The RSM model was experimentally validated yielding a maximum 21 % weight loss, which represents onefold increment in percentage weight loss in comparison with the conventional method. By using the optimized condition, the enzymatic degradation by an extracellular PHA depolymerase from Acidovorax sp. DP5 was studied at 37 °C and pH 9.0 on different types of PHA films with various monomer compositions. Surface modification of scaffold was employed using enzymatic technique to create highly porous scaffold with a large surface to volume ratio, which makes them attractive as potential tissue scaffold in biomedical field. Scanning electron microscopy revealed that the surface of salt-leached films was more porous compared with the solvent-cast films, and hence, increased the degradation rate of salt-leached films. Apparently, enzymatic degradation behaviors of PHA films were determined by several factors such as monomer composition, crystallinity, molecular weight, porosity, and roughness of the surface. The hydrophilicity and water uptake of degraded salt-leached film of P(3HB-co-70%4HB) were enhanced by incorporating chitosan or alginate. Salt-leached technique followed by partial enzymatic degradation would enhance the cell attachment and suitable for biomedical as a scaffold.     

Biosynthesis and biocompatibility of biodegradable poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Poly(3-hydroxybutyrate), PHB, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB), consisting of 0-94% mole fraction of 4HB content, were produced in high content by Cupriavidus necator strain A-04. The carbon sources used for PHB production included sugars made locally in Thailand: refined sugarcane, brown sugarcane, rock sugar, toddy palm sugar and coconut palm sugar. The switching of the ratios of carbon to nitrogen, together with the ratios of fructose to 1,4-butanediol, were applied to P(3HB-co-4HB) production in fed-batch cultures. Optimal P(3HB-co-4HB) production was achieved with 112 g biomass and 73 g P(3HB-co-4HB) with 38% mole fraction of 4HB content. Next, P(3HB-co-4HB) with a 0, 5, 24, 38 and 64% mole fraction of 4HB content were purified and prepared as plastic films. The mechanical properties and biocompatibility of these films were tested and compared with commercial PHB, polystyrene (PS) and polyvinylchloride (PVC) prepared without additives. The results demonstrated that PHB had thermal and mechanical properties similar to those of commercial PHB. The P(3HB-co-4HB) polymers possessed melting temperature and glass transition temperature values higher than those reported previously. The mechanical properties were compared with those of PS and PVC. The in vitro biocompatibility was assessed using L929, human dermal fibroblast and Saos-2 human osteosarcoma cells. The cytotoxicity results and scanning electron micrographs showed that P(3HB-co-4HB) films have good surface characteristics and can promote cell attachment, proliferation and differentiation. Combined with their good mechanical properties, P(3HB-co-4HB) polymers possess potential usefulness for biomaterial applications in artificial skin tissue support and orthopedic support.     

FUNCTIONAL POLYHYDROXYALKANOATES SYNTHESIZED BY MICROORGANISMS*

Many bacteria have been found to synthesize a family of polyesters termed polyhydroxyalkanoate, abbreviated asPHA. Some interesting physical properties of PHAs such as piezoelectricity, non-linear optical activity, biocompatibility andbiodegradability offer promising applications in areas such as degradable packaging, tissue engineering and drag delivery.Over 90 PHAs with various structure variations have been reported and the number is still increasing. The mechanicalproperty of PHAs changes from brittle to flexible to elastic, depending on the side-chainlength of PHA. Many attempts havebeen made to produce PHAs as biodegradable plastics using various microorganisms obtained from screening naturalenvironments, genetic engineering and mutation. Due to the high production cost, PHAs still can not compete with the non-degradable plastics, such as polyethylene and polypropylene. Various processes have been developed using low cost rawmaterials for fermentation and an inorganic extraction process tbr PHA purification. However, a super PHA production strainmay play the most critical role for any large-scale PHA production. Our recent study showed that PHA synthesis is acommon phenomenon among bacteria inhabiting various locations, especially oil-contaminated soils. This is very importantfor finding a suitable bacterial strain for PHA production. In fact, PHA production strains capable of rapid growth and rapidPHA synthesis on cheap molasses substrate have been found on molasses contaminated soils. A combination of novelproperties and lower cost will allow easier commercialization of PHA for many applications.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

Molecular weight characterization of poly[(R)-3-hydroxybutyrate] synthesized by genetically engineered strains of Escherichia coli

This study investigated the relationship of growth conditions, host strains and molecular weights of poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesized by genetically engineered Escherichia coli. Various PHA synthases belonging to types I-IV enzymes were expressed in E. coli JM109 under the same experimental conditions, and the molecular weights of the polymers were characterized by gel permeation chromatography. The results demonstrate that P(3HB) polymers have varied molecular weights and polydispersities dependent on the characteristics of the individual PHA synthase employed. P(3HB) with high number-average molecular weights (M_n) [(1.5-4.0) × 10⁶] and narrow polydispersities (1.6-1.8) were synthesized by PHA synthases from Ralstonia eutropha (type I), Delftia acidovorans (type I) and Allochromatium vinosum (type III). Contrary to these, P(3HB) with relatively low M_n [(0.17-0.79) × 10⁶] and broad polydispersities (2.2-9.0) were synthesized by PHA synthases from Aeromonas caviae (type I), Pseudomonas sp. 61-3 (type II) and Bacillus sp. INT005 (type IV). Furthermore, the molecular weights of P(3HB) synthesized under various culture conditions, in various hosts of E. coli and by mutants of PHA synthase were characterized. It was found that, in addition to culture pH [Kusaka et al. Appl Microbiol Biotechnol 1997;47:140], other variances such as culture temperature, host strain and use of mutants are effective in changing polymer molecular weight.     

Facile Synthesis of Poly(hydridocarbyne): A Precursor to Diamond and Diamond‐like Ceramics

Polycarbynes have previously been shown to be polymeric precursors to diamond and diamond‐like carbon. Here, we report an incredibly simple method for producing one of these polymers, poly(hydridocarbyne). The method simply requires chloroform, electricity, a solvent and an electrolyte. Since the polymer is soluble, the production of diamond objects of any shape is feasible. It is hoped that the ease of the synthesis will make these types of polymers accessible to scientists from all disciplines and that the potential applications for this material, which range from electrical to biomedical, are finally realized.     

Physicochemical properties of multicomponent polyhydroxyalkanoates: Novel aspects

The physicochemical properties such as the degree of crystallinity and temperature and molecularmass characteristics of a number of polyhydroxyalkanoates of various chemical composition synthesized on a complex carbon substrate by bacteria Cupriavidus eutrophus В10646 have been investigated. Two-, three-, and four-component copolymer samples have different sets and ratios of monomers with various lengths of carbon chains: 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HH), 3-hydroxy-4-methyl valerate (3H4MV), and diethylene glycol (DEG). It has been shown that weight-average molar mass М _w and polydispersity vary in a wide range with no correlation existing with the composition of copolymer polyhydroxyalkanoates and that thermal stability is preserved in the temperature interval between the melting temperature and the thermal degradation temperature from 100 to 120–140°С. The composition and ratio of monomers most notably affect the degree of crystallinity of polyhydroxyalkanoates. Significant differences between the degrees of crystallinity of three- and four-component polyhydroxyalkanoates have been found for the first time. The degree of crystallinity for copolymers P(3HB/3HV/4HB) is 9–22%, and the degree of crystallinity for copolymers P(3HB/3HV/3HH) and P(3HB/3GV/3H4MV) is 41–63%; this value is close to the degree of crystallinity for diblock copolymers P(3HB)/DEG, which is 56–69%. For the four-component copolymers P(3HB/3GV/4HB/3HH), the degree of crystallinity is 30–41%. The values of М _w for the copolymers P(3HB/DEG) are inhomogeneous and the polymers contain fractions uneven with respect to molecular mass: a high-molecular-mass polymer (М _w from 2700 to 4900 kDa) and a low-molecular-mass polymer (М _w = 46–167 kDa). For the copolymers P(3HB)/DEG and P(3HB/3HV/3H4MV), two peaks are observed in the region of melting with the gap between these peaks being 4–20°С. All of the types of copolymer samples, regardless of the monomer ratio, show an increase in elongation at break against the background of a decrease in tensile stress and Young’s modulus, with these effects being pronounced to different extents. On the whole, the properties of multicomponent polyhydroxyalkanoates differ appreciably.     

Unusual change in molecular weight of polyhydroxyalkanoate (PHA) during cultivation of PHA-accumulating Escherichia coli

This study aimed to investigate the factors affecting molecular weight of poly[(R)-3-hydroxybutyrate] [P(3HB)] when polyhydroxyalkanoate (PHA) synthase (PhaRC_Bsp) from Bacillus sp. INT005 was used for P(3HB) synthesis in Escherichia coli JM109. It was found that the molecular weight of P(3HB) decreased with time in mid- and late-phase of culture and was strongly affected by culture temperature. At 37 °C culture temperature, the molecular weight of P(3HB) rapidly decreased from 4.4 × 10⁵ to 4.8 × 10⁴ with culture time, whereas it was almost unchanged at 25 °C. Kinetic analysis suggested that the decrease in molecular weight of P(3HB) was due to random scission of the polymer chain. The decrease in molecular weight of P(3HB) was not observed when PHA synthases other than PhaRC_Bsp were expressed. This study sheds light on the unique behaviour in molecular weight change of P(3HB) that is synthesized by E. coli expressing PhaRC_Bsp.     

Characterization of biosynthesized P(3HB-co-3HA)s swellable in organic solvents

Polyhydroxyalkanoate (PHA) copolymers consisting of (R)-3-hydroxybutyrate (3HB) and medium-chain-length (R)-3-hydroxyalkanoate (3HA), P(3HB-co-3HA), are usually solved in chloroform. However, we found that some of the P(3HB-co-3HA) aged for more than 1 month under ambient conditions were not solved in chloroform, but instead swelled when the 3HA fraction was over 14 mol%. On the basis of differential scanning calorimetry and wide-angle x-ray diffraction analyses, we predicted that swellable P(3HB-co-3HA) contained numerous P(3HB) microcrystals, which may form physical crosslinks between adjacent PHA polymer chains.     

Characterization of partially transesterified poly(beta-hydroxyalkanoate)s by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used for the characterization of a partially transesterified poly(beta-hydroxyalkanoate) (PHA), a polymer produced by the bacterial strain Alcaligenes eutrophus with saponified vegetable oils as the sole carbon sources. The transesterification was carried out separately under acidic and basic conditions to obtain PHA oligomers weighing <10 kDa. The intact oligomers were detected in their cationized forms, [M + Na]+ and [M + K]+, by MALDI-TOFMS. A composition analysis, using the MALDI-TOF spectra, indicated that the oligomers obtained via acid catalysis contained a methyl 3-hydroxybutyrate end group, and those obtained by base catalysis had a methyl crotonate (olefinic) end group. In addition to hydroxybutyrate (HB), the oligomers were found to contain a small percentage of hydroxyvalerate, which was independently confirmed by gas chromatography/mass spectrometry. In comparison, analysis of a commercial PHA polymer, transesterified under identical conditions, showed only the presence of HB, i.e., a pure poly(HB) homopolymer.     

Biosynthesis and Characterization of Poly (3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) Terpolymer with Various Monomer Compositions by <Emphasis Type="Italic">Cupriavidus</Emphasis> sp. USMAA2-4

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] terpolymer was produced using Cupriavidus sp. USMAA2-4 via one-step cultivation process through combination of various carbon sources such as 1,4-butanediol or γ-butyrolactone with either 1-pentanol, valeric acid, or 1-propanol. Oleic acid was added to increase the biomass production. The composition of 3HV and 4HB monomers were greatly affected by the concentration of 1,4-butanediol and 1-pentanol. Terpolymers with 3HV and 4HB molar fractions ranging from 2 to 41 mol.% and 5 to 31 mol.%, respectively, were produced by varying the concentration of carbon precursors. The thermal and mechanical properties of the terpolymers containing different proportions of the constituent monomers were characterized using gel permeation chromatography (GPC), DSC, and tensile machine. GPC analysis showed that the molecular weights (M _w) of the terpolymer produced were within the range of 346 to 1,710 kDa. The monomer compositions of 3HV and 4HB were also found to have great influences on the thermal and mechanical properties of the terpolymer P(3HB-co-3HV-co-4HB) produced.     

Design of novel plasticizers for nylon: from molecular modeling to experimental verification

Polyamides are semi‐crystalline polymers useful in a wide range of applications in the plastics industry. Some applications require higher flexibility and workability of the polyamides. Therefore, plasticizers are added to ease compounding and processing procedures and produce the desired product properties. The goal of the present work was to study experimentally a series of four esters of 4‐hydroxybenzoate with linear side chain as plasticizers for the random copolymer nylon 66/6 and to compare the experimental results to computational results that were obtained in a previous work. The plasticizers used were the methyl, ethyl, propyl, and butyl esters of 4‐hydroxybenzoate (M4HB, E4HB, P4HB, and B4HB, respectively). Four properties of the plasticizer‐polymer blends were examined: area under the loss modulus curve; impact energy; the decrease in Tg, which is related to the plasticization efficiency, and static modulus that serves as an indication to the mechanical properties or the polymer. It was found that P4HB and B4HB offer the optimal combination of plasticization efficiency and mechanical properties and that M4HB is inferior to the other three plasticizers, as was predicted computationally. This study verifies that computational design of plasticizers for nylon is valid and can serve as an important tool to develop new plasticizers specifically and new additives to polymers generally. Copyright © 2016 John Wiley & Sons, Ltd.     

Mutation effects of a conserved alanine (Ala510) in type I polyhydroxyalkanoate synthase from Ralstonia eutropha on polyester biosynthesis

Type I polyhydroxyalkanoate (PHA) synthases, as represented by Ralstonia eutropha enzyme (PhaC(Re)), have narrow substrate specificity toward (R)-3-hydroxyacyl-coenzyme A with acyl chain length of C3-C5 to yield PHA polyesters. In this study, saturation point mutagenesis of a highly conserved alanine at position 510 (A510) in PhaC(Re) was carried out to investigate the effects on the polymerization activity and the substrate specificity for in vivo PHA biosynthesis in bacterial cells. A series of saturation mutants were first applied for poly[(R)-3-hydroxybutyrate] homopolymer synthesis in Escherichia coli and R. eutropha PHB(-)4 (PHA negative mutant) cells to assess the polymerization activity. All mutants showed quantitatively similar polymerization activities when R. eutropha PHB(-)4 was used for assay, whereas several mutants such as A510P showed low activities in E. coli. Further analysis has revealed that majority of mutants synthesize polyesters with higher molecular weights than the wild-type. In particular, substitution by acidic amino acids, A510D(E), led to remarkable increases in molecular weights. Subsequently, PHA copolymer synthesis from dodecanoate (C12 fatty acid) was examined. The copolymer compositions were varied depending on the mutants used. Significant increased fractions of long monomer units (C6 and C8) in PHA copolymers were observed for three mutants [A510M(Q,C)]. From these results, the mutations at this potion are beneficial to change the molecular weight of polyesters and the substrate specificity of PhaC(Re). Molecular weight distributions of PHA polymers synthesized by the wild-type enzyme (PhaC(Re)) and its mutants.     

Enhancement of Stress Tolerance in the Polyhydroxyalkanoate Producers without Mobilization of the Accumulated Granules

Poly(3-hydroxybutyrate) [P(3HB)], a polymer belonging to the polyhydroxyalkanoate (PHA) family, is accumulated by numerous bacteria as carbon and energy storage material. The mobilization of accumulated P(3HB) is associated with increased stress and starvation tolerance. However, the potential function of accumulated copolymer such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] remained unknown. In this study, Delftia acidovorans DS 17 was used to evaluate the contributions of P(3HB) and P(3HB-co-3HV) granules during simulated exogenous carbon deprivation on cell survival by transferring cells with PHAs to carbon-free mineral salt medium supplemented with 1 % (w/v) nitrogen source. By mobilizing the intracellular P(3HB) and P(3HB-co-3HV) at 11 and 40 mol% 3HV compositions, the cells survived starvation. Surprisingly, D. acidovorans containing P(3HB-co-94 mol% 3HV) also survived although the mobilization was not as effective. Similarly, recombinant Escherichia coli pGEM-T::phbCAB _Cn (harboring the PHA biosynthesis genes of Cupriavidus necator) containing P(3HB) granules had a higher viable cell counts compared to those without P(3HB) granules but without any P(3HB) mobilization when exposed to oxidative stress by photoactivated titanium dioxide. This study provided strong evidence that enhancement of stress tolerance in PHA producers can be achieved without mobilization of the previously accumulated granules. Instead, PHA biosynthesis may improve bacterial survival via multiple mechanisms.     

Enzymatic transformation of bacterial polyhydroxyalkanoates into repolymerizable oligomers directed towards chemical recycling

The enzymatic transformation into an oligomer was carried out with the objective of developing the chemical recycling of bacterial polyesters. Poly(R-3-hydroxyalkanoate)s (PHAs), such as poly[(R-3-hydroxybutyrate)-co-12%(R-3-hydroxyhexanoate)] and poly[(R-3-hydroxybutyrate)-co-12%(R-3-hydroxyvalerate)], were degraded by granulated Candida antarctica lipase B immobilized on hydrophilic silica (lipase GCA) in a diluted organic solvent at 70 degrees C. The degradation products were cyclic oligomers having a molecular weight of a few hundreds. The obtained cyclic oligomer was readily repolymerized by the same lipase (lipase GCA) to produce the corresponding polyester in a concentrated solution. The cyclic oligomer was copolymerized with epsilon-caprolactone using lipase to produce the corresponding terpolymers having an Mw of 21,000. This is the first example of the enzymatic chemical recycling of bacterial PHAs using lipase. Poly(R-3-hydroxybutyrate) [P(3HB)] was also degraded into the linear-type R-3HB monomer to trimer by P(3HB)-depolymerase (PHBDP) in phosphate buffer at 37 degrees C. The degradation using PHBDP required a longer reaction time compared with the lipase-catalyzed degradation in organic solvent. The monomer composition of the oligomer depended on the origin of the PHBDP. The R-3HB monomer was predominately produced by PHBDP from Pseudomonas stutzeri, while the R-3HB dimer was produced by PHBDP from Alcaligenes faecalis T1. Repolymerization of these oligomers by lipase in concentrated organic solvent produced a relatively low-molecular-weight P(3HB) (e.g., Mw=2,000). Degradation of P(3HB) by lipase in organic solvent into repolymerizable cyclic oligomer and degradation of P(3HB) by PHBDP in buffer into hydroxy acid type R-3HB dimer.     

Modeling mechanical properties of polyhydroxyalkanoate during degradation in animal tissue

Polyhydroxyalkanoates (PHAs) are a family of biodegradable and biocompatible polymers produced by several species microorganisms that possess favorable mechanical properties (e.g. strength and elongation properties). Different types of PHA polymers have been used in medical applications. However, in order to better understand the use of this polymer in the different applications, a thorough understanding of the kinetics of in vivo degradation is one of the major requirements. In this study, poly(3‐hydroxybutyrate) (PHB) was subcutaneously implanted in mice and incubated for 2, 4, 8, or 16 weeks. After removal from the animal, the strength, elongation, mass loss, and enthalpy of the PHB were tested for each time point. From these data, a mathematical model was generated by Rayleigh's method of dimensional analysis, where polymer strength over tissue contact time could be predicted. To prove the model, previous data obtained by our group were used: poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) [P(HB‐co‐HHx)] incubation in the presence of human embryonic kidney cells (HEK). It was found that the developed model was aligned with experimental results, could predict the strength of the polymer when in contact with cells, and the predicted strength follows the trend of the experimental data. Also, the dimensionless constant (K) value associated with the model is different for both experiments, where this constant, produced via experimental data, is used for construction of a homogeneous equation. Copyright © 2017 John Wiley & Sons, Ltd.     

13C and 1H amorphous linewidths in semicrystalline poly(4-hydroxybutyrate) and poly(3-co-4-hydroxybutyrate) above Tg by high resolution and solid-state NMR experiments

The high-resolution ¹³C and ¹H nuclear magnetic resonance (NMR) linewidths of semi-crystalline poly(4-hydroxybutyrate), P4HB, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate), (P3/4HB-18, 18% 4HB units) in the amorphous phase and in the melt are studied as a function of temperature and magnetic field strength. Measurements of the ¹³C spin-spin relaxation times under the same experimental conditions show that the natural line-width is a minor contributor to the line-broadening observed in the ¹³C spectra of the solid polymers. A variety of coherent averaging solid-state NMR methods are used to examine possible contributions from various line-broadening mechanisms. It is shown that magnetic susceptibility and chemical shift dispersion are the major factors for the broadening of the proton and carbon resonances of P4HB in the amorphous phase and the melt, respectively. Incomplete motional narrowing due to a slow motional mode restricted in amplitude by the presence of crystallites and/or chain constraints was found to be the major line-broadening factor for P3/4HB-18 in the amorphous phase. Correlations between crystalline morphology, physical and mechanical properties, and polymer chain dynamics are discussed, along with the way these factors affect the NMR linewidth data presented. © 1996 John Wiley & Sons, Inc.