G. A. Bonartseva

Effect of growth conditions on the molecular weight of poly-3-hydroxybutyrate produced by Azotobacter chroococcum 7B

It has been shown that poly-3-hydroxybutyrate (PHB) of predetermined molecular weight can be obtained by varying the growth conditions of the producer strain, Azotobacter chroococcum 7B: pH, temperature, aeration, presence of sodium acetate as an additional carbon source, or growth on crude complex carbon sources (molasses, vinasse, or starch). High-molecular-weight polymer can be obtained at pH 7.0, optimal for the culture (1485 kDa), temperature 30–37°C (1600–1450 kDa, respectively), and low aeration (2215 kDa). The following factors decrease PHB MW: pH deviation to the acidic (pH 6.0, 476 kDa) or alkaline (pH 8.0, 354 kDa) range or lower temperature (20°C, 897 kDa). Introduction of additional carbon source (sodium acetate) at concentrations in the medium varying from 0 to 5 g/l provides an original method of production of PHB with predetermined MW in a wide range, from 270 to 1515 kDa, with high PHB content in the cell.

Cell attachment on poly(3-hydroxybutyrate)-poly (ethylene glycol) copolymer produced by Azotobacter chroococcum 7B

BackgroundThe improvement of biomedical properties, e.g. biocompatibility, of poly(3-hydroxyalkanoates) (PHAs) by copolymerization is a promising trend in bioengineering. We used strain Azotobacter chroococcum 7B, an effective producer of PHAs, for biosynthesis of not only poly(3-hydroxybutyrate) (PHB) and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also alternative copolymer, poly(3-hydroxybutyrate)-poly(ethylene glycol) (PHB-PEG).ResultsIn biosynthesis we used sucrose as the primary carbon source and valeric acid or poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-PEG and PHB-HV was confirmed by 1H nuclear-magnetic resonance (1H NMR) analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) and surface morphology of films from PHB copolymers were studied. To study copolymers biocompatibility in vitro the protein adsorption and COS-1 fibroblasts growth on biopolymer films by XTT assay were analyzed. Both copolymers had changed physico-chemical properties compared to PHB homopolymer: PHB-HV and PHB-PEG had less crystallinity than PHB; PHB-HV was more hydrophobic than PHB in contrast to PHB-PEG appeared to have greater hydrophilicity than PHB; whereas the morphology of polymer films did not differ significantly. The protein adsorption to PHB-PEG was greater and more uniform than to PHB and PHB-PEG copolymer promoted better growth of COS-1 fibroblasts compared with PHB homopolymer.ConclusionsThus, despite low EG-monomers content in bacterial origin PHB-PEG copolymer, this polymer demonstrated significant improvement in biocompatibility in contrast to PHB and PHB-HV copolymers, which may be coupled with increased protein adsorption and hydrophilicity of PEG-containing copolymer.

Hydrolytic Degradation of Poly(3-hydroxybutyrate), Polylactide and their Derivatives: Kinetics, Crystallinity, and Surface Morphology

Hydrolytic degradations of biodegradable poly(3-hydroxybutyrate) (PHB), polylactide (PLA) and their derivatives were explored by kinetic and structure methods at 37 and 70°C in phosphate buffer. It was revealed the kinetic profiles for copolymer PHBV (20% of 3-hydroxyvalerate) and the blend PHB-PLA (1:1 wt. ratio). The intensity of biopolymer hydrolysis depending on temperature is characterized by total weight loss and the viscosity-averaged molecular weight decrement (ΔMW) as well as by WAXS and AMF techniques. Characterization of PHB and PHBV includes both ΔMW and crystallinity evolution (x-ray diffraction) as well as the AFM analysis of PHB film surfaces before and after aggressive medium exposition. The degradation is enhanced in the series PHBV < PHB < PHB-PLA blend < PLA. The impact of MW on the biopolymer hydrolysis is shown.

Biodegradation and Medical Application of Microbial Poly(3-Hydroxybutyrate)

This review is designed to be a comprehensive source for biodegradable polymer: poly(3-hydroxybutyrate) research, including fundamental structure/property relationships and biodegradation kinetics for samples of different geometry. In addition to presenting the scientific framework for the advances in PHB research, this review focuses on applications of PHB in biomedicine and environment with a discussion on commercial applications and health/safety concerns for biodegradable materials.

The Terpolymer Produced by Azotobacter Chroococcum 7B: Effect of Surface Properties on Cell Attachment

The copolymerization of poly(3-hydroxybutyrate) (PHB) is a promising trend in bioengineering to improve biomedical properties, e.g. biocompatibility, of this biodegradable polymer. We used strain Azotobacter chroococcum 7B, an effective producer of PHB, for biosynthesis of not only homopolymer and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also novel terpolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-poly(ethylene glycol) (PHB-HV-PEG), using sucrose as the primary carbon source and valeric acid and poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-HV-PEG was confirmed by 1H nuclear-magnetic resonance analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) of produced biopolymer, the protein adsorption to the terpolymer, and cell growth on biopolymer films were studied. Despite of low EG-monomers content in bacterial-origin PHB-HV-PEG polymer, the terpolymer demonstrated significant improvement in biocompatibility in vitro in contrast to PHB and PHB-HV polymers, which may be coupled with increased protein adsorption, hydrophilicity and surface roughness of PEG-containing copolymer.

Aerobic and Anaerobic Microbial Degradation of Poly-β-Hydroxybutyrate Produced by Azotobacter chroococcum

Food industry wastewater served as a carbon source for the synthesis of poly-β-hydroxybutyrate (PHB) by Azotobacter chroococcum. The content of polymer in bacterial cells grown on the raw materials reached 75%. PHB films were degraded under aerobic, microaerobic, and anaerobic conditions in the presence and absence of nitrate by microbial populations of soil, sludges from anaerobic and nitrifying/denitrifying reactors, and sediment from a sludge deposit site. Changes in molecular mass, crystallinity, and mechanical properties of PHB were studied. Anaerobic degradation was accompanied by acetate formation, which was the main intermediate utilized by denitrifying bacteria or methanogenic archaea. On a decrease in temperature from 20 to 5° C in the presence of nitrate, the rate of PHB degradation was 7.3 times lower. Under anaerobic conditions and in the absence of nitrate, no PHB degradation was observed, even at 11°C. The enrichment cultures of denitrifying bacteria obtained from soil and anaerobic sludge degraded PHB films for a short time (3–7 d). The dominant species in the enrichment culture from soil were Pseudomonas fluorescens and Pseudomonas stutzeri. The rate of PHB degradation by the enrichment cultures depended on the polymer molecular weight, which reduced with time during biodegradation.

New poly(3-hydroxybutyrate)-based systems for controlled release of dipyridamole and indomethacin

New poly(3-hydroxybutyrate)-based systems for controlled release of anti-inflammatory and anti-thrombogenic drugs have been studied. The release occurs via two mechanisms (diffusion and degradation) operating simultaneously. Dipyridamole and indomethacin diffusion processes determine the rate of the release at the early stages of the contact of the system with the environment (the first 6–8 h). The coefficient of the release diffusion of a drug depends on its nature, the thickness of the poly(3-hydroxybutyrate) films containing the drug, the concentrations of dipyridamole and indomethacin, and the molecular weight of the poly(3-hydroxybutyrate). The results obtained are critical for developing systems of release of diverse drugs, thus, enabling the attainment of the requisite physiological effects on tissues and organs of humans.

Biodegradation kinetics of poly(3-hydroxybutyrate)-based biopolymer systems

The aim of this study was to evaluate and to compare the long-term kinetics curves of biodegradation of poly(3-hydroxybutyrate) (PHB), its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and a PHB/polylactic acid composite. The total weight loss and the change of average viscosity molecular weight were used as the parameters reflecting the biodegradation degree. The rate of biodegradation was analyzed in vitro in the presence of lipase and in vivo after film implantation in animal tissues. The morphology of the PHB film surface was studied by the atomic force microscopy technique. It was shown that PHB biodegradation involves both polymer hydrolysis and its enzymatic biodegradation. The results obtained in this study can be used for the development of various PHB-based medical devices.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer by Azotobacter chroococcum strain 7B

The ability of Azotobacter chroococcum strain 7B, producer of poly(3-hydroxybutyrate) (PHB), to synthesize its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was studied. It was demonstrated, for the first time, that A. chroococcum strain 7B was able to synthesize P(3HB-co-3HV) with various molar rates of HV in the polymer chain when cultivated on medium with sucrose and carboxylic acids as precursors of HV elements in the PHB chain, namely, valeric (13.1–21.6 mol %), propanoic (3.1 mol %), and hexanoic (2.1 mol %) acids. Qualitative and functional differences between PHB and P(3HB-co-3HV) were demonstrated by example of the release kinetic of methyl red from films made of synthesized polymers. Maximal HV incorporation into the polymer chain (28.8mol %) was recorded when the nutrient medium was supplemented with 0.1% peptone on the background of 20 mM valerate. These results suggest that that the studied strain can be regarded as a potential producer of not only PHB but also P(3HB-co-3HV).

The Biodegradation of Poly-β-Hydroxybutyrate (PHB) by a Model Soil Community: The Effect of Cultivation Conditions on the Degradation Rate and the Physicochemical Characteristics of PHB

The biodegradation of films made of poly-β-hydroxybutyrate (PHB) with a molecular mass of 1500 kDa was studied using a model soil community in the presence and absence of nitrate and at different concentrations of oxygen in the gas phase. The biodegradation of PHB was investigated with respect to changes in its molecular mass, crystallinity, and some mechanical properties.