Investigating the structure-property relationship of bacterial PHA block copolymers

Mechanical testing of solvent cast films consisting of short-chain-length (SCL) polyhydroxyalkanoate (PHA) films suggested that films consisting of block copolymers retained more elasticity over time with respect to films of similar random copolymers of comparable composition. Two experimental techniques, wide angle X-ray scattering (WAXS) and uniaxial extension, were used to quantitatively investigate the structure-property relationship of bacterially synthesized PHA block copolymers of poly(3-hydroxybutyrate) (PHB) homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) random copolymer (PHBV) segments. Uniaxial testing experiments yielded the Young's modulus, ultimate tensile strength, and the elongation until fracture of the films. Percent crystallinity was determined by deconvolution of amorphous and crystalline scattering peaks obtained from WAXS. Two PHBV films containing either 8% 3-hydroxyvalerate monomer (3HV) or 29% 3HV exhibited a quick transition to brittle behavior, decreasing to less than 20% percent elongation at fracture within a few days after annealing. Conversely, the block copolymer samples remained higher than 100% elongation at fracture a full 3 months after annealing. Because block copolymers covalently link polymers that would otherwise form thermodynamically separate phases, the rates and degrees of crystallization of the block copolymers are less than the random copolymer samples. These differences translate into materials that extend the property space of biologically synthesized SCL PHA.     

Microbial production of polyhydroxyalkanoate block copolymer by recombinant Pseudomonas putida

Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ _Ac cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC (phaPCJ _A.c ) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC studies showed the polymer to possess two glass transition temperatures (T _g), one melting temperature (T _m) and one cool crystallization temperature (T _c). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young’s modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio. Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length block copolymer PHB-b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community.     

Biosynthesis and native granule characteristics of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Delftia acidovorans

The ability of Delftia acidovorans to incorporate a broad range of 3-hydroxyvalerate (3HV) monomers into polyhydroxyalkanoate (PHA) copolymers was evaluated in this study. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] containing 0–90 mol% of 3HV was obtained when a mixture of sodium 3-hydroxybutyrate and sodium valerate was used as the carbon sources. Transmission electron microscopy analysis revealed an interesting aspect of the P(3HB-co-3HV) granules containing high molar ratios of 3HV whereby, the copolymer granules were generally larger than those of poly(3-hydroxybutyrate) [P(3HB)] granules, despite having almost the same cellular PHA contents. The large number of P(3HB-co-3HV) granules occupying almost the entire cell volume did not correspond to a higher amount of polymer by weight. This indicated that the granules of P(3HB-co-3HV) contain polymer chains that are loosely packed and therefore have lower density than P(3HB) granules. It was also interesting to note that a decrease in the length of the side chain from 3HV to 4-hydroxybutyrate (4HB) corresponded to an increase in the density of the respective PHA granules. The presence of longer side chain monomers (3HV) in the PHA structure seem to exhibit steric effects that prevent the polymer chains in the granules from being closely packed. The results reported here have important implications on the maximum ability of bacterial cells to accumulate PHA containing monomers with longer side chain length.     

Polyhydroxybutyrate: An intriguing biopolymer

The microbial polymer poly-3-hydroxybutyrate (PHB) and related poly-hydroxyalkanoates, such as poly-3-hydroxyvalerate and poly-3-hydroxyoctanoate, are unique biodegradable thermoplastics of considerable commercial importance. The structure, properties and regulation of synthesis and degradation of PHB are reviewed and the microbial production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, with properties varying according to copolymer composition, is discussed.     

Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126

A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources. One of these, Rhodococcus sp. NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source. Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates. With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced. N.m.r. analysis confirmed the composition of these polyesters. The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.     

Recent Progress in Polyhydroxyalkanoates‐Based Copolymers for Biomedical Applications

In recent years, naturally biodegradable polyhydroxyalkanoate (PHA) monopolymers have become focus of public attentions due to their good biocompatibility. However, due to its poor mechanical properties, high production costs, and limited functionality, its applications in materials, energy, and biomedical applications are greatly limited. In recent years, researchers have found that PHA copolymers have better thermal properties, mechanical processability, and physicochemical properties relative to their homopolymers. This review summarizes the synthesis of PHA copolymers by the latest biosynthetic and chemical modification methods. The modified PHA copolymer could greatly reduce the production cost with elevated mechanical or physicochemical properties, which can further meet the practical needs of various fields. This review further summarizes the broad applications of modified PHA copolymers in biomedical applications, which might shred lights on their commercial applications.     

The synthesis of hydroxybutyrate and hydroxyvalerate copolymers by the bacterium Ralstonia eutropha

The paper deals with the study of the synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymers by the bacterium Ralstonia eutropha B-5786 grown under different carbon nutrition conditions (growth on carbon dioxide, fructose, and CO2-valerate and fructose-valerate mixtures). The parameters to be analyzed included the yield of biomass, the yield, synthesis rate, and composition of copolymers, the activity of the key enzymes of polyhydroxyalkanoate (PHA) synthesis (beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase), the maximum tolerable concentration of valerate to the bacterium, and the conditions that govern the incorporation of hydroxyvalerate to copolymers. This allowed the relationship between cultivation conditions and the proportion of monomers in the copolymers to be deduced. We were able to synthesize a range of 3HB/3HV copolymers and found that the thermal characteristics and the degree of crystallinity of these copolymers depend on the molar fraction of 3HV.     

Scanning electron microscopy of polyhydroxyalkanoate degradation by bacteria

Bacterial degradation of sheets of selected polyhydroxyalkanoates by Comamonas sp., Pseudomonas lemoignei and Pseudomonas fluorescens GK13 is reported. Five natural polyhydroxyalkanoates were used, namely poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate, a copolymer of mainly 3-hydroxyoctanoate and minor amounts of 3-hydroxyhexanoate, and two rubber-like copolymers of saturated and unsaturated hydroxyalkanoic acids that had been modified by electron-beam-induced cross-linking. Each of these polymers was degraded by at least one bacterial strain, the rate of hydrolysis being dependent on the surface area of the polymer exposed to attack. Scanning electron microscopy of partially degraded samples showed that hydrolysis started at the surface and at physical lesions in the polymer and proceeded to the inner part of the material. No evidence for areas of non-degradable polymer was found for any of the polymers analysed, even if the polymer contained chemical cross-links. Received: 24 July 1996 / Accepted: 29 August 1996     

Synthesis of 3-hydroxybutyrate-CO-4-hydroxybutyrate copolymers by hydrogen-oxidizing bacteria

Synthesis of 3- and 4-hydroxybutyrate copolymer (3HB-co-4HB), the most promising member of the biodegradable polyhydroxyalkanoate (PHA) family, has been studied. Cultivation conditions of naturally occurring strains of hydrogen-oxidizing bacteria Ralstonia eutropha B5786 and Cupriavidus eutrophus B10646 have been optimized to ensure efficient synthesis of the 3HB-co-4HB copolymer. A set of highly pure samples of the 3HB-co-4HB copolymer with 4HB content varying from 8.7 to 24.3 mol% has been obtained. Incorporation of 4HB into the copolymer was shown to cause a more pronounced decrease in polymer crystallinity than the incorporation of 3-hydroxyvalerate or 3-hydroxyhexanoate; samples with a degree of crystallinity below 30% have been obtained. The weight average molecular mass of the 3HB-co-4HB copolymers was shown to be independent on the monomer ratio and to vary broadly (from 540 to 1110 kDa).     

Local sequence dependence of polyhydroxyalkanoic acid degradation in Hydrogenophaga pseudoflava

The first order intracellular degradation of various polyhydroxyalkanoic acid (PHA) inclusions in Hydrogenophaga pseudoflava cells was investigated by analyzing the compositional and microstructural changes of the PHA using gas chromatography, (13)C NMR spectroscopy, and differential scanning calorimetry. Two types of PHA, copolymers and blend-type polymers, were separately accumulated in cells for comparison. The constituent monomers were 3-hydroxybutyric acid (3HB), 4-hydroxybutyric acid (4HB), and 3-hydroxyvaleric acid (3HV). It was found that the 3HB-4HB copolymer was degraded only when the polymer contained a minimal level of 3HB units. With the cells containing a 3HB/4HB blend-type polymer, only poly(3HB) was degraded, whereas poly(4HB) was not degraded, indicating the totally inactive nature of the intracellular depolymerase against poly(4HB). On the basis of the magnitude of the first order degradation rate constants, the relative substrate specificity of the depolymerase toward the constituting monomer units was determined to decrease in the order 3HB > 3HV > 4HB. (13)C NMR resonances of the tetrad, triad, and dyad sequences were analyzed for the samples isolated before and after degradation experiments. The results showed that the intracellular degradation depended on the local monomer sequence of the copolymers. The relative substrate specificity of the depolymerase determined from the NMR local sequence analysis agreed well with that obtained from the kinetics analysis. It is suggested that, without isolation and purification of the intracellular PHA depolymerase and "native" PHA substrates, the relative specificity of the enzyme as well as the microstructural heterogeneity of the PHA could be determined by measuring in situ the first order degradation rate constants of the PHA in cells.     

Biosynthesis and composition of bacterial poly(hydroxyalkanoates)

It is well established that Alcaligenes eutrophus can accumulate a copolymer containing 3-hydroxybutyrate and 3-hydroxyvalerate, but longer 3-hydroxyacid monomers have not been reported to occur in this organism. The properties of the enzymes of poly(hydroxyalkanoate) (PHA) biosynthesis are discussed and it is proposed that the substrate specificity of the polymerizing enzyme restricts the range of monomer units incorporated into PHA. Various other bacteria produce similar copolymers from propionic acid and/or valeric acid. A number of Pseudomonas species accumulate PHAs containing longer-chain monomer units from linear alkanoic acids, alkanes and alcohols.     

Factors affecting the freeze-fracture morphology of in vivo polyhydroxyalkanoate granules

Interesting morphologies were observed when Comamonas acidovorans containing polyhydroxyalkanoates (PHA) of various compositions was freeze-fractured at temperatures far below the glass transition temperatures of PHA. In vivo granules of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comparatively showed the most ductility, and could be stretched extensively. Contrary to the uniform needle-type deformation shown by the poly(3-hydroxybutyrate) homopolymer when fractured at -110 degrees C, copolymers containing 3-hydroxyvalerate units showed various deformation structures. Similar observations were made when in vivo granules of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) were freeze-fractured, although the ductility of the latter was much reduced. In addition, it was found that fracturing at -160 degrees C resulted in decreased ductility of the PHA granules with the concomitant increase in the number of mushroom-type deformation structures. Our results suggest that PHA granules with higher resistance to freeze-fracture deformation show less ductility, and therefore produce the mushroom-type morphology. This is the first report on the freeze-fracture morphology of PHA copolymers containing short-chain-length monomers.     

Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae

In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.     

Biodegradation of polyhydroxyalkanoates (PHAs) in the South China Sea and identification of PHA-degrading bacteria

The biodegradation patterns of two types of PHA, a 3-hydroxybutyrate (3-PHB) polymer and a 3-hydroxybutyrate and 3-hydroxyvalerate (3-PHB/3-PHV) copolymer, were studied in tropical marine environments (Dam Bay, South China Sea, Nha Trang, Vietnam). No reliable differences in the degradation of 3-PHB and 3-PHB/3-PHV were revealed. It was shown that the degradation process depended mainly on the shape of a polymer product and its production method: the degradation of polymer films was found to be more active than that of molded solids. A decrease in the molecular mass of both types of PHA was detected in the course of the degradation of PHA samples. However, the degree of PHA crystallinity did not change; that is, the levels of degradation of both the amorphous and crystalline phases of PHA were almost the same. Among microbial PHA degraders, three bacterial strains, Bacillus sp. IBP-V002, Enterobacter cloacae sp. IBP-V001, and Gracilibacillus sp. IBP-V003, were identified based on the results of morphological, biochemical, and molecular phylogenetic analyses. The ability of the representatives of the genera Gracilibacillus and Enterobacter to degrade PHA was revealed for the first time.     

Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by fourier transform infrared spectroscopy and scanning electron microscopy

Rhizobium meliloti produced a copolymer of short chain length polyhydroxyalkanoate (scl-PHA) on sucrose and rice bran oil as carbon substrates. Recombinant Escherichia coli (JC7623ABC1J4), bearing PHA synthesis genes, was used to synthesize short chain length-co-medium chain length PHA (scl-co-mcl-PHA) on glucose and decanoic acid. Fourier transform infrared spectroscopy (FTIR) spectra of the PHAs indicated strong characteristic bands at 1282, 1723, and 2934 cm⁻¹ for scl-PHA and at 2933 and 2976 cm⁻¹ for scl-co-mcl-PHA polymer. Differentiation of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate-P(HB-co-HV) copolymer was obseverd using FTIR, with absorption bands at 1723 and 1281 for PHB, and at 1738, 1134, 1215 cm⁻¹ for HV-copolymer. The copolymers were analyzed by GC and ¹H NMR spectroscopy. Films of polymer blends of PHA produced by R. meliloti and recombinant E. coli were prepared using glycerol, polyethylene glycol, polyvinyl acetate, individually (1:1 ratio), to modify the mechanical properties of the films and these films were evaluated by FTIR and scanning electron microscopy.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator from waste rapeseed oil using propanol as a precursor of 3-hydroxyvalerate

Waste rapeseed oil is a useful substrate for polyhydroxyalkanoates (PHA) production employing Cupriavidus necator H16. In fed-batch mode, we obtained biomass and PHA yields of 138 and 105 g l⁻¹, respectively. Yield coefficient and volumetric productivity were 0.83 g PHA per g oil and 1.46 g l⁻¹ h⁻¹, respectively. Propanol at 1% (v/v) enhanced both PHA and biomass formation significantly and, furthermore, resulted in incorporation of 3-hydroxyvalerate units into PHA structure. Thus, propanol can be used as an effective precursor of 3-hydroxyvalarete for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. During the fed-batch cultivation, propanol concentration was maintained at 1% which resulted in 8% content of 3-hydroxyvalerate in copolymer.     

Biosynthesis and characterization of polyhydroxyalkanoate block copolymer P3HB-b-P4HB

Polyhydroxyalkanoates (PHA) synthesis genes phbC and orfZ cloned from Ralstonia eutropha H16 were transformed into beta-oxidation weakened Pseudomonas putida KTOY08ΔGC, a mutant of P. putida KT2442. The recombinant P. putida strain termed KTHH06 was able to produce a short-chain-length PHA block copolymer consisting of poly(3-hydroxybutyrate) (P3HB) as one block and poly(4-hydroxybutyrate) (P4HB) as another block. One-dimensional and two-dimensional nuclear magnetic resonance (NMR) clearly indicated the polymer was a diblock copolymer consisting of 20 mol % P3HB as one block and 80 mol % P4HB as another one. Differential scanning calorimetric (DSC) showed that P3HB block melting temperatures (T(m)) in the block copolymer P3HB-b-P4HB was shift to low temperature compared with homopolymer P3HB and a blend of P3HB and P4HB. The block copolymer with a number average molecular weight of 50000 Da and a polydispersity of 3.1 demonstrated a better yield and tensile strength compared with that of its related random copolymer and blend of homopolymers of P3HB and P4HB.     

Well-controlled cationic water-soluble phospholipid polymer-DNA nanocomplexes for gene delivery

The facile synthesis of biocompatible and nontoxic gene delivery vectors has been the focus of research in recent years due to the high potential in treating genetic diseases. 2-Methacryloxyethyl phosphorylcholine (MPC) copolymers were recently studied for their ability to produce nontoxic and biocompatible materials. The synthesis of well-defined and water-soluble MPC polymer based cationic vectors for gene delivery purposes was therefore attractive, due to the potential excellent biocompatibility of the resulting copolymers. Herein, cationic MPC copolymers of varying architectures (block versus random) were produced by the reversible addition--fragmentation chain transfer (RAFT) polymerization technique. The copolymers produced were evaluated for their gene delivery efficacy in the presence and absence of serum. It was found that copolymer architectures and molecular weights do affect their gene delivery efficacy. The statistical copolymers produced larger particles, and showed poor gene transfection efficiency as compared to the diblock copolymers. The diblock copolymers served as efficient gene delivery vectors, in both the presence and absence of serum in vitro. To the best of our knowledge, this is the first report where the effect of architecture of MPC based copolymer on gene delivery efficacy has been studied.     

Polyhydroxyalkanoate production in recombinant Escherichia coli.

The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-beta-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-3HV).     

Thermoreversible hydrogels from RAFT-synthesized BAB triblock copolymers: steps toward biomimetic matrices for tissue regeneration

Narrowly dispersed, temperature-responsive BAB block copolymers capable of forming physical gels under physiological conditions were synthesized via aqueous reversible addition fragmentation chain transfer (RAFT) polymerization. The use of a difunctional trithiocarbonate facilitates the two-step synthesis of BAB copolymers with symmetrical outer blocks. The outer B blocks of the triblock copolymers consist of poly(N-isopropylacrylamide) (PNIPAM) and the inner A block consists of poly(N,N-dimethylacrylamide). The copolymers form reversible physical gels above the phase transition temperature of PNIPAM at concentrations as low as 7.5 wt % copolymer. Mechanical properties similar to collagen, a naturally occurring polypeptide used as a three-dimensional in vitro cell growth scaffold, have been achieved. Herein, we report the mechanical properties of the gels as a function of solvent, polymer concentration, and inner block length. Structural information about the gels was obtained through pulsed field gradient NMR experiments and confocal microscopy.