Biomedical Investigations of Biodegradable PHAs

This work is a review of the results of biomedical studies of polymer devices (films, fibers, microparticles, 3D implants) made from resorbable PHAs synthesized by the bacterium Wautersia (Ralstonia) eutropha B5786, using the technology developed at the Institute of Biophysics of the Siberian Branch of the Russian Academy of Sciences. Two types of PHAs – polyhydroxybutyrate (PHB) and a hydroxybutyrate/hydroxyvalerate copolymer (PHB/PHV) – have been proven to be biocompatible in vitro in cultures of fibroblasts, endothelial cells, hepatocytes, and osteoblasts, and in short- and long-duration experiments on animals. Polymer films and membranes have been found to be usable as scaffolds for functioning cells and monofilament suture fibers – for stitching muscular-fascial wounds and in abdominal surgery. Ectopic bone formation assay and experiments with the model of segmental osteotomy showed that 3D PHB and PHB/HA implants can be used for reparative osteogenesis. The paper reports beneficial results of using polymers to repair bone defects in oral surgery.     

Medical applications of biopolyesters polyhydroxyalkanoates

Microbial polyhydroxyalkanoates(PHAs) are a family of biopolyesters produced by many wild type and engineered bacteria.PHAs have diverse structures accompanied by flexible thermal and mechanical properties.Combined with their in vitro biodegradation,cell and tissue compatibility,PHAs have been studied for medical applications,especially medical implants applications,including heart valve tissue engineering,vascular tissue engineering,bone tissue engineering,cartilage tissue engineering,nerve conduit tissue engineering as well as esophagus tissue engineering.Most studies have been conducted in the authors’ lab in the past 20+ years.Recently,mechanism on PHA promoted tissue regeneration was revealed to relate to cell responses to PHA biodegradation products and cell-material interactions mediated by microRNA.Very importantly,PHA implants were found not to cause carcinogenesis during long-term implantation.Thus,PHAs should have a bright future in biomedical areas.     

Construction of recombinant Bacillus subtilis for production of polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1, and pBE2C1AB, containing one or two PHA synthse, genes, respectively. The two plasmids were inserted into Bacillus subtilis DB104 to generate modified strains, B. subtilis/pBE2C1 and B. subtilis/pBE2C1AB. The two recombinants strains were subjected to fermentation and showed PHA accumulation, the first reported example of mcl-PHA production in B. subtilis. Gas Chromatography analysis identified the compound produced by B. subtilis/pBE2C1 to be a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer whereas that produced by B. subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxyde-canoate-co-hydroxydodecanoate (HB-HD-HDD) polymer.     

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.     

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.     

Expression Profile of pha Gene Cluster of Pseudomonas putida KT2442

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that many bacteria accumulate as carbon and energy storage when growth conditions are unbalanced. Pseudomonas strains belonging to the rRNA homology group I such as P. putida can accumulate medium-chain-length-PHA from monomers in the C8 to C10 range. Regulation of PHA synthesis and degradation in P. putida KT2442 has been studied using different molecular approaches. In this study six promoter regions located upstream of each pha gene were identified. The expression of the pha cluster have been analysed in the presence of octanoic acid versus glucose in the culture medium. Results demonstrated that the system is activated in the presence of octanoic acid as PHA precursor.     

Synthesis and characterization of poly-β-hydroxybutyrate. I. Synthesis of crystalline DL-poly-β-hydroxybutyrate from DL-β-butyrolactone

The polymerization of β-butyrolactone was investigated as a possible monomer for a proposed synthesis of the naturally occurring polyester, D -poly-β-hydroxybutyrate (D -PHB). The racemic DL -monomer was used in this initial study to determine the best conditions and catalyst system for use in a subsequent study of the polymerization of optically active β-butyrolactone. In so doing it was found that certain organometallic catalysts (Et₂Zn and Et₃Al) plus a cocatalyst of water produced highly crystalline samples of polyester from the racemic monomer. This paper describes the synthesis and characterization of the racemic polymer obtained using these catalyst systems, and compares the results obtained with certain other catalysts that were also investigated for this purpose. Examination of the DL -PHB by infrared, NMR, x-ray, and electron microscopy shows that it is possible to synthesize a crystalline racemic polymer that is virtually identical (excepting optical activity) to the naturally occurring polymer, D -PHB.     

Adsorption effects of poly(hydroxybutyric acid) depolymerase on chain-folding surface of polyester single crystals revealed by mutant enzyme and frictional force microscopy

Adsorption effects of poly(hydroxybutyric acid) (PHB) depolymerase from Ralstonia pickettii T1 on various polymer single crystals were studied using a catalytically inactive mutant of PHB depolymerase by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), and frictional force microscopy (FFM). Six types of polymer single crystals, poly[(R)-3-hydroxybutyric acid] (P(3HB)), poly[(R)-3-hydroxybutyric acid-co-6 mol% (R)-3-hydroxyvaleric acid] (P(3HB-co-6 mol% 3HV)), poly[(R)-3-hydroxybutyric acid-co-8 mol% (R)-3-hydroxyhexanoic acid] (P(3HB-co-8 mol% 3HH)), poly(l-lactic acid) (PLLA), poly(d-lactic acid) (PDLA), and polyethylene (PE), were prepared to examine the influence of an ester bond and stereoregularity of a polymer on the enzymatic adsorption. The numbers of PHB depolymerase enzymes adsorbed on P(3HB) and P(3HB-co-6 mol% 3HV) single crystals were determined as 171 and 183 enzymes/μm² by AFM, respectively. AFM observation revealed that the concentration of PHB depolymerase enzymes adsorbed onto PLLA and PDLA single crystals is much higher compared to those on a P(3HB) single crystal, whereas the concentration of enzyme adsorbed onto PE and P(3HB-co-8 mol% 3HH) single crystals is much less. In addition, the single crystals of each polymer were characterized by TEM and FFM before and after enzymatic treatment by mutant for 1 h at 37 °C. The surface properties of P(3HB), P(3HB-co-6 mol% 3HV), and P(3HB-co-8 mol% 3HH) single crystals were changed by the enzymatic adsorption, whereas the internal structures were not affected. On the basis of these results, the properties of the binding domain of PHB depolymerase to polymer chain-folding surfaces have been discussed.     

Production of Polyhydroxyalkanoates in Unsterilized Hyper-Saline Medium by Halophiles Using Waste Silkworm Excrement as Carbon Source

The chlorophyll ethanol-extracted silkworm excrement was hardly biologically reused or fermented by most microorganisms. However, partial extremely environmental halophiles were reported to be able to utilize a variety of inexpensive carbon sources to accumulate polyhydroxyalkanoates. In this study, by using the nile red staining and gas chromatography assays, two endogenous haloarchaea strains: Haloarcula hispanica A85 and Natrinema altunense A112 of silkworm excrement were shown to accumulate poly(3-hydroxybutyrate) up to 0.23 g/L and 0.08 g/L, respectively, when using the silkworm excrement as the sole carbon source. The PHA production of two haloarchaea showed no significant decreases in the silkworm excrement medium without being sterilized compared to that of the sterilized medium. Meanwhile, the CFU experiments revealed that there were more than 60% target PHAs producing haloarchaea cells at the time of the highest PHAs production, and the addition of 0.5% glucose into the open fermentation medium can largely increase both the ratio of target haloarchaea cells (to nearly 100%) and the production of PHAs. In conclusion, our study demonstrated the feasibility of using endogenous haloarchaea to utilize waste silkworm excrement, effectively. The introduce of halophiles could provide a potential way for open fermentation to further lower the cost of the production of PHAs.     

Effects of residual metal compounds and chain-end structure on thermal degradation of poly(3-hydroxybutyric acid)

Thermal degradation behaviours of poly(3-hydroxybutyric acid) (P(3HB); bacterial poly[(R)-3-hydroxybutyric acid] and synthetic poly[(R,S)-3-hydroxybutyric acid] samples, were examined under both isothermal and non-isothermal conditions. The inverse of number-average degree of polymerisation for all P(3HB) samples decreased linearly with degradation time during the initial stage of isothermal degradation at a temperature ranging from 170-190 °C. In addition, crotonyl unit was detected in the residual polymer samples as main ω-chain-end. These results indicate that the dominant thermal degradation reaction for P(3HB) is a random chain scission via cis-elimination reaction of P(3HB) molecules. It was found that the presence of either Ca or Mg ions enhances the depolymerisation of P(3HB) molecules, while that Zn ions hardly catalyse the reaction. As a result, a shift of thermogravimetric curves toward the lower temperature regions was observed for the P(3HB) samples containing high amounts of Ca and Mg compounds.     

Gamma Irradiation Effects on Mechanical and Thermal Properties and Biodegradation of Poly(3-hydroxybutyrate) Based Films

Summary: Their biodegradable properties make polyhydroxyalkanoates (PHAs) ideal candidates for innovative applications. Many studies have been primarily oriented to poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-valerate) (PHBV) and afterwards to blends of PHAs with synthetic biodegradable polymers, such as poly(ε-caprolactone) (PCL). Medical and pharmaceutical devices require sterilization and γ irradiation could provide a proper alternative since it assures storage stability and microbiological safety. This contribution presents the effect of γ irradiation on the mechanical and thermal properties and on the biodegradation of PHB, PHBV and a commercial PHB/PCL blend. Samples, prepared by compression moulding, were irradiated in air at a constant dose rate of 10 kGy/h, from 10 to 179 kGy. Polymer chain scission was assessed by changes in the molecular weight, thermal properties and tensile behaviour. The correlation between absorbed dose and changes in the mechanical properties and biodegradation is discussed in detail. The optimum dose to guarantee microbiological sterilization without damage of the structure or meaningful loss of the mechanical properties is also reported.     

Synthesis,recovery and possible application of medium-chain-length polyhydroxyalkanoates: A short overview

Bacterial polyhydroxyalkanoates (PHAs) are polyesters of 3-hydroxyacids produced as intracellular granules by a large variety of bacteria, currently receiving much attention because of their potential as renewable and biodegradable plastics. The monomer units in these microbial polyesters are all in the R-configuration due to the stereospecificity of biosynthetic enzymes. Pseudomonads synthesise mainly medium-chain-lenght PHAs, formed of monomers of 6 to 14 carbons. The PHA monomer composition is influenced by the substrate added to the growth media and determines the physical properties of the plastic material. The capability of Pseudomonads to incorporate many different functional groups into the PHAs does extend their physical properties and potential applications, and suggests various possibilities to produce tailor-made polymers. The mcl-PHAs are of major interest for specific uses, where chirality and elastomeric property of the polymers are important. In this report we will focus on the biotechnological production, recovery and possible applications of mcl-PHAs.     

Purification and characterization of an extracellular medium-chain length polyhydroxyalkanoate depolymerase from Thermus thermophilus HB8

During growth on medium-chain length (mcl) polyhydroxyalkanoates (PHAs), or on sodium octanoate Thermus thermophilus HB8 produces an extracellular mcl-PHA depolymerase. This enzyme was purified from the culture medium of sodium octanoate-grown cells to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the purified enzyme was approximately 28 kDa. A part of the gene TTHA1605 encoding a 24.17 kDa protein was demonstrated to encode the mcl-PHA depolymerase of T. thermophilus. The primary amino-acid sequence of purified enzyme reveals similarity to all reported so far extracellular mcl-PHA depolymerases. The purified enzyme could hydrolyze mcl - PHAs and p-nitrophenyl (pNP) esters but not short chain length (scl) - PHAs. The optimum pH range was 7.5-9 and the optimum temperature was 70 °C for pNP-octanoate (pNPO) hydrolysis. The Km value for pNPO was 53.2 μM. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and non-ionic detergents (Tween 20, Tween 80 and Triton X-100). The results demonstrated in this study revealed that the mcl-PHA depolymerase from T. thermophilus is a distinct enzyme, which is different from those of other mcl-PHA-degrading bacteria.     

Influence of pH on the Molecular Weight of Poly-3-hydroxybutyric Acid (P3HB) Produced by Recombinant Escherichia coli

The production of ultrahigh molecular weight poly-3-hydroxybutyric acid (P3HB) from carbohydrates by recombinant Escherichia coli harboring genes from Ralstonia eutropha was evaluated. In shaken-flask experiments, E. coli XL1 Blue harboring plasmid pSK::phaCAB produced P3HB corresponding to 40 and 27 % of cell dry weight from glucose and xylose, respectively. Cultures in bioreactor using glucose as the sole carbon source at variable pH values (6.0, 6.5, or 7.0) allowed the production of P3HB with molecular weight varying between 2.0 and 2.5 MDa. These figures are significantly higher than the values often obtained by natural bacterial strains (0.5–1.0 MDa). Contrary to reports of other authors, no influence of pH was observed on the molecular weight of the polymer produced. Using xylose, P3HB with high molecular weight was also produced, indicating the possibility to produce these polymers from lignocellulosic materials.     

Development of a closed-loop control system for production of medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs) from bacteria

Large scale availability of bacterial polyhydroxyalkanoates (PHAs) is still limited to a few types of short-chain-length PHAs, namely poly(3-hydroxybutyrate) (PHB) and its copolymer Biopol™, consisting of 3-hydroxybutyrate and 3-hydroxyvalerate repeating units. In order to increase the number of available medium-chain-length PHA (mcl-PHA) copolymers a flexible high-cell-density fed-batch process was developed. Continuous process monitoring and substrate control were achieved by coupling on-line gaschromatography (on-line GC) to a software-based Proportional Integral (PI) substrate controller. System development time and continuous system upgrading were considerably shortened by using LABView™, a powerful graphical programming environment. The control of octanoic acid and 10-undecenoic acid at 1.5 and 0.5 gL⁻¹ respectively, enabled the production of high levels of biomass (30 gL⁻¹) and mcl-PHA (10.5 gL⁻¹) by avoiding substrate limitations or toxicities. The resulting mcl-PHA was an amorphous copolyester consisting of 37 mol% unsaturated monomers. The present system represents a valuable tool for the production of tailor-made mcl-PHAs, where the desired monomer composition is determined by the ratio of added cosubstrates.     

Production of PHAs from agricultural waste material

Polyhydroxyalkanoates (PHAs) are biodegradable substitutes to fossil fuel plastics that can be produced from renewable raw materials such as saccharides, alcohols and low-molecular-weight fatty acids. They are completely degradable to carbon dioxide and water through natural microbiological mineralization. Consequently, neither their production nor their use or degradation have a negative ecological impact. By keeping closed the cycle of production and re-use, PHAs can enable at least part of the polymer-producing industry to switch from ecologically harmful end-of-the-pipe production methods towards sounder technologies. Up to now such polyesters have been produced biotechnologically from refined raw materials (e.g. glucose and sodium propionate), but they can as well be produced much cheaper from agricultural waste materials (e.g. molasses, maltose, glycerol phase from biodiesel production, whey), as long as these materials have a known composition and are available in appropriate quantities. Yield factors and specific rates for growth and PHA accumulation are shown for 3 strains of Alcaligenes latus for different agricultural waste carbon sources.     

Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production,biocompatibility, biodegradation,physical properties and applications

ABSTRACT

Polyhydroxyalkanoates (PHAs) are intracellular aliphatic polyesters synthesized as energy reserves, in the form of water-insoluble, nano-sized discrete and optically dense granules in cytoplasm by a diverse bacteria and some archae under conditions of limiting nutrients in the presence of excess carbon source. Bacteria synthesize different PHAs from coenzyme A thioesters of respective hydroxyalkanoic acid, and degrade intracellularly for reuse and extracellularly in natural environments by other microorganisms. In vivo, PHAs exist as amorphous mobile liquid and water-insoluble inclusions but in vitro, exhibit material and mechanical properties, ranging from stiff and brittle crystalline to elastomeric and molding, similar to petrochemical thermoplastics. Further, they are hydrophobic, isotactic, biocompatible and exhibit piezoelectric properties. But as commodity plastics their applications are limited by high production cost, low yield, in vivo degradation, complexity of technology and difficulty of extraction. Therefore, to replace the conventional plastic with PHAs, it is prerequisite to standardize the PHA production systems.     

Conversion of industrial food wastes by Alcaligenes latus into polyhydroxyalkanoates

Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. As the first step in our pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesise environmental-friendly bioplastics, we investigated the usage of soya wastes from a soya milk dairy, and malt wastes from a beer brewery plant as the carbon sources for the production of polyhydroxyalkanoates (PHA) by selected strain of microorganism. Bench experiments showed that Alcaligenes latus DSM 1124 used the nutrients from malt and soya wastes to biosynthesise PHAs. The final dried cell mass and specific polymer production of A. latus DSM 1124 were 32g/L and 70% polymer/cells (g/g), 18.42 g/L and 32.57% polymer/cell (g/g), and 28 g/L and 36% polymer/cells (g/g), from malt waste, soya waste, and from sucrose, responctively. These results suggest that many types of food wastes might be used as the carbon source for the production of PHA.     

Marine Diatom, Navicula sp. Strain JPCC DA0580 and Marine Green Alga, Chlorella sp. Strain NKG400014 as Potential Sources for Biodiesel Production

Marine diatom, strain JPCC DA0580, and marine green microalga strain NKG400014 were selected as high neutral lipid-producers from marine microalgal culture collection toward biodiesel production. These strains were tentatively identified as Navicula sp. and Chlorella sp., respectively, by 18S rDNA analysis. Growth and lipid accumulation conditions of both strains were analyzed by changing nutrient concentrations in growth media and initial illuminance intensity. The highest productivity of fatty acid methyl ester (FAME) reached to 154 mg/L/week for NKG400014 and 185 mg/L/week for JPCC DA0580. Gas chromatography/mass spectrometry analysis indicates that FAME fraction from NKG400014 mainly contained 9-12-15-octadecatrienoate (C18:3) and that from JPCC DA0580 mainly contained methyl palmitate (C16:0) and methyl palmitoleate (C16:1). Furthermore, calorimetric analysis revealed that the energy content of strain was 4,233?±?55 kcal/kg (i.e., 15.9?±?0.2 MJ/kg) for NKG400014 and 6,423?±?139 kcal/mg (i.e., 26.9?±?0.6 MJ/kg) for JPCC DA0580, respectively. The value from JPCC DA0580 was equivalent to that of coal. The strains NKG400014 and JPCC DA0580 will become a promising resource that can grow as dominant species in the open ocean toward production of both liquid and solid biofuels.     

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.