Biosynthesis of poly(3-hydroxyalkanoates) from threonine.

A series of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) was biosynthesized by Alcaligenes eutrophus from an amino acid, threonine. The 3HV content of these polyesters ranged from less than 0.1% to 30%.     

Gas-chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria

Summary The accuracy and reproducibility of the gas-chromatographic method for the analysis of PHB and PHA in whole cells of Alcaligenes eutrophus H16 and Pseudomonas putida KT2442 were determined. It was found that for analysis of PHA the methanolysis time in the assay had to be increased to 4 h. Accuracy of the PHB and PHA assay were 0.018 mg and 0.304 mg respectively, based on estimation of the measurement error.     

Direct biosynthesis of poly(3-hydroxyalkanoates) bearing epoxide groups

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas cichorii YN2 cultured with C6–C12 1-alkenes was studied. PHAs containing repeating units with terminal epoxide groups were obtained when C7–C12 1-alkenes were fed separately as the only carbon source, but no epoxidized unit was detected when 1-hexene was fed. The content of epoxidized units in the polymers was in the range of 4–20 mol%, which was not dependent on the C atom length of the 1-alkene used as a substrate. The polymers produced undergo a glass transition at around −40 °C, and number average molecular weights were in the range of 1 50 000–2 00 000 as determined by GPC relative to polystyrene, with M_w/M_n ratios of 1.9–2.5. As an intermediate, the corresponding 1,2-epoxyalkane was found in the culture medium. According to this result, the epoxidation of the 1-alkene is the initial step in the synthetic pathway of the epoxy unit in the polymer.     

The accumulation of poly(3-hydroxyalkanoates) in Rhodobacter sphaeroides

In recent years industrial interest has been focussed on the evaluation of poly(3-hydroxyalkanoates) (PHA) as potentially biodegradable plastics for a wide range of technical applications. Studies have been carried out in order to optimize growth and culture conditions for the intracellular formation of PHA in the phototrophic, purple, non-sulfur bacterium Rhodobacter sphaeroides. Its potential to produce polyesters other than poly(3-hydroxybutyrate) (PHB) was investigated. On an industrial scale, the use of photosynthetic bacteria could harness sunlight as an energy source for the production of these materials. R. sphaeroides was grown anaerobically in the light on different carbon sources. Under nitrogenlimiting conditions a PHA content of up to 60 to 70% of the cellular dry weight was detected. In all of the cases studied, the storage polymer contained approximately 98 mol% of 3-hydroxybutyrate (HB) and 2 mol% 3-hydroxyvalerate (HV) monomer units. Decreasing light intensities did not stimulate PHA formation. Compared to Rhodospirillum rubrum (another member of the family of Rhodospirillaceae), R. sphaeroides showed a limited flexibility in its ability to form PHA with varying monomer unit compositions.     

Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic.

Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.     

Formation of poly(3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria

The formation of poly(3-hydroxyalkanoic acid), PHA, by various strains of chemolithotrophic and phototrophic bacteria has been examined. Chemolithotrophic bacteria were grown aerobically under nitrogen-limiting conditions on various aliphatic organic acids. Phototrophic bacteria were grown anaerobically in the light on a nitrogen-rich medium and were subsequently transferred to a nitrogen-free medium containing acetate, propionate, valerate, heptanoate or octanoate as carbon source. All 41 strains investigated in this study were able to synthesize and accumulate PHA. All 11 strains of chemolithotrophic bacteria and all 15 strains belonging to the non-sulfur purple bacteria synthesized a polymer, which contained 3-hydroxy-valerate (3HV) beside 3-hydroxybutyrate (3HB), if the cells were cultivated in the presence of propionate, valerate or heptanoate. Many non-sulfur purple bacteria synthesized copolyesters of 3HB and 3HV even with acetate as carbon source. In contrast, most sulfur purple bacteria did not incorporate 3HV at all. Among 15 strains tested, only Chromatium vinosum strain 1611, C. purpuratum strain BN5500 and Lamprocystis roseopersicina strain 3112 were able to synthesize polyesters containing 3HV with propionate, valerate or heptanoate as carbon source.     

Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

Determination of solubility parameters for poly(3-hydroxyalkanoates).

The three dimensional solubility parameters defined by Hansen are based on dispersion forces between structural units, interaction between polar groups and hydrogen bonding. For polar polymers such as poly(3-hydroxyalkanoates), P(3HA), this approach was used to obtain the three coordinates of a solubility parameter in terms of: a dispersion part, a polar part and a hydrogen bonding part. Thirty-eight different solvents for poly(3-hydroxybutyrate), PHB, which are mentioned in the literature are examined by this method and the theoretical predictions are compared with the experimental reports. Another overall comparison between PHA polymers provides their Hansen and Hildebrand parameters for side chain lengths up to C13. In this series a linear progression in calculated solubility parameters with side chain length was found. An Appendix provides information and data on calculation of the solubility parameters. While the solubility information is limited and only covers homopolymers, it should help to highlight some of the contradictions regarding PHB solubility. This semi-empirical approach is only valid for amorphous polymers hence crystallinity effects, which are important with PHB, as well as molecular weight effects still require analysis.     

Effect of poly(3-hydroxyalkanoates) as natural polymers on mesenchymal stem cells

Mesenchymal stem cells (MSCs) are stromal multipotent stem cells that can differentiate into multiple cell types, including fibroblasts, osteoblasts, chondrocytes, adipocytes, and myoblasts, thus allowing them to contribute to the regeneration of various tissues, especially bone tissue. MSCs are now considered one of the most promising cell types in the field of tissue engineering. Traditional petri dish-based culture of MSCs generate heterogeneity, which leads to inconsistent efficacy of MSC applications. Biodegradable and biocompatible polymers, poly(3-hydroxyalkanoates) (PHAs), are actively used for the manufacture of scaffolds that serve as carriers for MSC growth. The growth and differentiation of MSCs grown on PHA scaffolds depend on the physicochemical properties of the polymers, the 3D and surface microstructure of the scaffolds, and the biological activity of PHAs, which was discovered in a series of investigations. The mechanisms of the biological activity of PHAs in relation to MSCs remain insufficiently studied. We suggest that this effect on MSCs could be associated with the natural properties of bacteria-derived PHAs, especially the most widespread representative poly(3-hydroxybutyrate) (PHB). This biopolymer is present in the bacteria of mammalian microbiota, whereas endogenous poly(3-hydroxybutyrate) is found in mammalian tissues. The possible association of PHA effects on MSCs with various biological functions of poly(3-hydroxybutyrate) in bacteria and eukaryotes, including in humans, is discussed in this paper.     

Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates)

Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D(1)) and 0.16 h(-1) in the second fermentor (D(2)) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D(1) and D(2) over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans were found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L(-1) h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermentor. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes.     

Efficient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans: economic considerations

Poly(3-hydroxyalkanoates) (PHA) have the potential to become a biodegradable alternative for conventional plastics. In order to produce PHA at competitive costs in comparison with commonly used plastics, efficient PHA production systems will have to be developed. Poly(3-hydroxybutyrate) fermentations are well developed and in actual use on an industrial scale; medium-chain-length PHA (mcl-PHA) production is less well described, although the vast majority of all PHA known today are mcl-PHA. This paper compares and describes mcl-PHA production systems with respect to the volumetric productivity, the cellular PHA content and the polymer yield on carbon substrates. Nitrogen was shown to be the most effective limitation to trigger PHA formation in P. oleovorans after different nutrient limitations had been compared. By using an economic model for the calculation of PHA production costs, we show that it should be possible to produce octane-based mcl-PHA on a large scale (more than 1000 tonnes/year) at costs below U.S. $ 10 kg⁻¹. Received: 4 April 1997 / Accepted: 20 May 1997     

Production of poly(3-hydroxyalkanoates) by Pseudomonas putida KT2442 in continuous cultures

 The synthesis of poly(3-hydroxyalkanoates) (PHA) by Pseudomonas putida KT2442 growing on long-chain fatty acids was studied in continuous cultures. The effects of the growth rate on the biomass and polymer concentration were determined and it was found that the PHA concentrations decreased with increasing growth rates. The highest volumetric productivity was 0.13 g PHA l^-1 h^-1 at a specific growth rate (μ) of 0.1 h^-1. The molecular mass of the polymer remained constant at all growth rates but changes in the monomeric composition of the PHA synthesized were observed. Variation of the carbon to nitrogen (C/N) ratio of the substrate feed at μ=0.1 h^-1 revealed optimal PHA formation at C/N=20 mol/mol. In order to optimize PHA production P. putida KT2442 was cultivated to high cell densities in oxygen-limited continuous cultures. In this way a maximum biomass concentration of 30 g/l containing approximately 23% PHA was achieved. This corresponds to a volumetric productivity of 0.69 g  l^-1 h^-1. Received: 14 December 1995 / Received revision: 18 April 1996 / Accepted: 22 April 1996     

High cell density cultivation ofPseudomonas oleovorans for the production of poly(3-hydroxyalkanoates)

Fed-batch culture ofPseudomonas oleovorans was carried out for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) using octanoate as a carbon source. Octanoate and the salt solution containing ammonium sulfate and magnesium sulfate were intermittently fed in the course of fermentation. Cell mass and PHA concentrations of 42.8 and 16.8 g/L, respectively, could be obtained in 40 h. The PHA content and the PHA productivity were 39.2% and 0.42 g PHA/L-h, respectively. The yields of cell mass and PHA were 0.71 g dry cell mass/goctanoate and 0.28 g PHA/g octanoate, respectively. Therefore, octanoate can be used for the production of MCL-PHAs to a high, concentration with high productvity.     

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

[This corrects the article on p. 3236 in vol. 59.].     

Carboxylate-induced degradation of poly(3-hydroxybutyrate)s

This communication shows that thermal degradation of poly(3-hydroxybutyrate)s (PHBs) is induced by carboxylate groups via a newly proposed E1cB mechanism. In PHBs with end groups in the form of carboxylic acid salts with Na+, K+, and Bu4N+ counterions, the proposed mechanism explains the dependence of thermal stability on the size of the counterion. The degradation via intermolecular alpha-deprotonation by carboxylate is suggested to be the main PHB decomposition pathway at moderate temperatures. The results of the present study show the ability to control the degradation and stability of poly(3-hydroxybutyrate)s as well as of their blends via chemical structure and concentration of the carboxylate polymer end groups.     

Characterization by mass spectrometry of poly(3-hydroxyalkanoates) produced by Rhodospirillum rubrum from 3-hydroxyacids.

The sequence distributions of two microbial copolyesters obtained by fermentation of Rhodospirillum rubrum, grown with 3-hydroxyhexanoic or 3-hydroxyheptanoic acids, were determined by analyzing the oligomers prepared by partial pyrolysis or partial methanolysis of these copolyesters using fast atom bombardment mass spectrometry (FAB-MS). Oligomers up to pentamers were identified in the case of partial pyrolysis and up to tetradecamers in the case of partial methanolysis. The comparison between the experimental and calculated peak intensities of FAB mass spectra allows the calculation of compositions and sequence distributions, which in these copolyesters follow Bernoullian statistics, indicating that they are random terpolyesters.     

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

The microbial degradation of tensile test pieces made of poly(3-hydroxybutyrate) [P(3HB)] or a copolymer of 90% 3-hydroxybutyric acid and 10% 3-hydroxyvaleric acid was studied in soils incubated at a constant temperature of 15, 28, or 40 degrees C for up to 200 days. In addition, hydrolytic degradation in sterile buffer at temperatures ranging from 4 to 55 degrees C was monitored for 98 days. Degradation was measured through loss of weight (surface erosion), molecular weight, and mechanical strength. While no weight loss was recorded in sterile buffer, samples incubated in soils were degraded at an erosion rate of 0.03 to 0.64% weight loss per day, depending on the polymer, the soil, and the incubation temperature. The erosion rate was enhanced by incubation at higher temperatures, and in most cases the copolymer lost weight at a higher rate than the homopolymer. The molecular weights of samples incubated at 40 degrees C in soils and those incubated at 40 degrees C in sterile buffer decreased at similar rates, while the molecular weights of samples incubated at lower temperatures remained almost unaffected, indicating that molecular weight decrease is due to simple hydrolysis and not to the action of biodegrading microorganisms. The degradation resulted in loss of mechanical properties. From the samples used in the biodegradation studies, 295 dominant microbial strains capable of degrading P (3HB) and the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer in vitro were isolated and identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Starch graft poly(methyl acrylate) loose-fill foam: preparation, properties and degradation

Starch graft poly(methyl acrylate) (S-g-PMA) was prepared by ceric ion initiation of methyl acrylate in an aqueous corn starch slurry (prime starch) which maximized the accessibility of the starch for graft polymerization. A new ceric ion reaction sequence was established as starch-initiator-methyl acrylate followed by addition of a small amount of ceric ion solution when the graft polymerization was almost complete to quench the reaction. As a result of this improved procedure, no unreacted methyl acrylate monomer remained, and thus, essentially no ungrafted poly(methyl acrylate) homopolymer was formed in the final grafted product. Quantities of the high purity S-g-PMA so prepared in pilot scale were converted to resin pellets and loose-fill foam by single screw and twin screw extrusion. The use of prime starch significantly improved the physical properties of the final loose-fill foam, in comparison to foam produced from regular dry corn starch. The S-g-PMA loose-fill foam had compressive strength and resiliency comparable to expanded polystyrene but higher bulk density. The S-g-PMA loose-fill foam also had better moisture and water resistance than other competitive starch-based materials. Studies indicated that the starch portion in S-g-PMA loose-fill foam biodegraded rapidly, whereas poly(methyl acrylate) remained relatively stable under natural environmental conditions.     

Crystalline-structure-dependent enzymatic degradation of polymorphic poly(3-hydroxypropionate)

The crystalline structure dependence of enzymatic degradation behavior was investigated for the polymorphic poly(3-hydroxypropionate) (P3HP), which has a basic backbone chemical structure of bacterial poly(3-hydroxyalkanoate)s (P3HAs). The P3HP films consisting of the beta-, gamma-, and/or delta-form crystal were cast or melt-crystallized as reported previously (Macromolecules 2005, 38, 6455; Macromolecules 2006, 39, 194-203) by controlling the molecular weight, crystallization temperature, and/or temperature of the melt. Their thermal properties, crystalline structures, morphologies, and (13)C solid spin-lattice relaxation dynamics were characterized by the differential scanning calorimetry, the wide-angle X-ray diffraction, the small-angle X-ray scattering (SAXS), and the (13)C solid-state NMR spectra (SNMR), respectively. Both the crystallinities and the lamellar thicknesses of P3HP films were found to decrease roughly in the order of beta-form > (or approximately) gamma-form > delta-form. From previous work, which indicates that the P3HA enzymatic degradation depends only on the degree of crystallinity and the lamellar thickness, their enzymatic degradation rates are then expected to increase in the order of beta-form < (or approximately) gamma-form < delta-form. Unexpectedly, their experimental P3HP enzymatic degradation rates in the presence of P3HA depolymerase isolated from Ralstonia pickettii T1 increase in the reverse order, i.e., delta-form < gamma-form < beta-form. The weight loss rate of the delta-form film is almost 1 order of magnitude smaller than that of the fastest degraded beta-form film. It is then strongly indicated that the crystalline structure plays a strikingly decisive role in the enzymatic degradation of P3HP. In particular, only when the conformation of crystalline chain accords with that of the bacterial poly(3-hydroxybutyrate) (P3HB) sample, i.e., the 2 1 helix conformation, is the P3HP sample degraded as slow as the P3HB sample. The inherent reason responsible for the unique P3HP enzymatic degradation behavior has been further clarified by comparing the molecular interaction and dynamics of polymorphic P3HP crystals.     

Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) by Comamonas testosteroni during cultivation on vegetable oils

Comamonas testosteroni has been studied for its ability to synthesize and accumulate medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) during cultivation on vegetable oils available in the local market. Castor seed oil, coconut oil, mustard oil, cotton seed oil, groundnut oil, olive oil and sesame oil were supplemented in the mineral medium as a sole source of carbon for growth and PHAs accumulation. The composition of PHAs was analysed by a coupled gas chromatography/mass spectroscopy (GC/MS). PHAs contained C6 to C14 3-hydroxy acids, with a strong presence of 3-hydroxyoctanoate when coconut oil, mustard oil, cotton seed oil and groundnut oil were supplied. 3-hydroxydecanoate was incorporated at higher concentrations when castor seed oil, olive oil and sesame oil were the substrates. Purified PHAs samples were characterized by Fourier Transform Infrared (FTIR) and 13C NMR analysis. During cultivation on various vegetable oils, C. testosteroni accumulated PHAs up to 78.5-87.5% of the cellular dry material (CDM). The efficiency of the culture to convert oil to PHAs ranged from 53.1% to 58.3% for different vegetable oils. Further more, the composition of the PHAs formed was not found to be substrate dependent as PHAs obtained from C. testosteroni during growth on variety of vegetable oils showed similar compositions; 3-hydroxyoctanoic acid and/or 3-hydroxydecanoic acid being always predominant. The polymerizing system of C. testosteroni showed higher preference for C8 and C10 monomers as longer and smaller monomers were incorporated less efficiently.