Evgeniy G. Kiselev

Cell growth and accumulation of polyhydroxyalkanoates from CO2 and H2 of a hydrogen-oxidizing bacterium, Cupriavidus eutrophus B-10646.

Synthesis of polyhydroxyalkanoates (PHAs) by a new strain of Cupriavidus - Cupriavidus eutrophus B-10646 - was investigated under autotrophic growth conditions. Under chemostat, at the specific flow rate D=0.1h(-1), on sole carbon substrate (CO2), with nitrogen, sulfur, phosphorus, potassium, and manganese used as growth limiting elements, the highest poly(3-hydroxybutyrate) [P(3HB)] yields were obtained under nitrogen deficiency. In batch autotrophic culture, in the fermenter with oxygen mass transfer coefficient 0.460 h(-1), P(3HB) yields reached 85% of dry cell weight (DCW) and DCW reached 50 g/l. Concentrations of supplementary PHA precursor substrates (valerate, hexanoate, γ-butyrolactone) and culture conditions were varied to produce, for the first time under autotrophic growth conditions, PHA ter- and tetra-polymers with widely varying major fractions of 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate, and 3-hydroxyhexanoate monomer units. Investigation of the high-purity PHA specimens showed significant differences in their physicochemical and physicomechanical properties.

A Glucose-Utilizing Strain, Cupriavidus euthrophus B-10646: Growth Kinetics, Characterization and Synthesis of Multicomponent PHAs

This study investigates kinetic and production parameters of a glucose-utilizing bacterial strain, C. eutrophus B-10646, and its ability to synthesize PHA terpolymers. Optimization of a number of parameters of bacterial culture (cell concentration in the inoculum, physiological activity of the inoculum, determined by the initial intracellular polymer content, and glucose concentration in the culture medium during cultivation) provided cell concentrations and PHA yields reaching 110 g/L and 80%, respectively, under two-stage batch culture conditions. Addition of precursor substrates (valerate, hexanoate, propionate, γ-butyrolactone) to the culture medium enabled synthesis of PHA terpolymers, P(3HB/3HV/4HB) and P(3HB/3HV/3HHx), with different composition and different molar fractions of 3HB, 3HV, 4HB, and 3HHx. Different types of PHA terpolymers synthesized by C. eutrophus B-10646 were used to prepare films, whose physicochemical and physical-mechanical properties were investigated. The properties of PHA terpolymers were significantly different from those of the P3HB homopolymer: they had much lower degrees of crystallinity and lower melting points and thermal decomposition temperatures, with the difference between these temperatures remaining practically unchanged. Films prepared from all PHA terpolymers had higher mechanical strength and elasticity than P3HB films. In spite of dissimilar surface structures, all films prepared from PHA terpolymers facilitated attachment and proliferation of mouse fibroblast NIH 3T3 cells more effectively than polystyrene and the highly crystalline P3HB.

Constructing herbicide metribuzin sustained-release formulations based on the natural polymer poly-3-hydroxybutyrate as a degradable matrix.

ABSTRACT Polymer poly(3-hydroxybutyrate) [P(3HB)] has been used as a matrix in slow-release formulations of the herbicide metribuzin (MET). Physical P(3HB)/MET mixtures in the form of solutions, powders, and emulsions were used to construct different metribuzin formulations (films, granules, pellets, and microparticles). SEM, X-Ray, and DSC proved the stability of these formulations incubated in sterile water in vitro for long periods of time (up to 49 days). Metribuzin release from the polymer matrix has been also studied. By varying the shape of formulations (microparticles, granules, films, and pellets), we were able to control the release time of metribuzin, increasing or decreasing it.

Constructing Slow-Release Formulations of Metribuzin Based on Degradable Poly(3-hydroxybutyrate)

Experimental formulations of herbicide metribuzin embedded in matrices of degradable natural polymer poly(3-hydroxybutyrate) (P3HB) and its composites with poly(ethylene glycol) (PEG), poly-ε-caprolactone (PCL), and wood powder have been prepared in the form of pressed pellets containing 75% polymeric basis (pure P3HB or its composite with a second component at a ratio of 7:3) and 25% metribuzin. Incubation of formulations in soil laboratory systems led to the degradation of the matrix and herbicide release. The most active release of metribuzin (about 60% of the embedded herbicide over 35 days) was detected for the P3HB/PEG carrier compared to the P3HB, P3HB/wood, and P3HB/PCL forms (30-40%). Thus, the study shows that herbicide release can be controlled by the matrix formulation. Metribuzin formulations exerted a significant herbicidal effect on the plant Agrostis stolonifera, used as a weed plant model. Application of these long-term formulations will make it possible to reduce environmental release of chemicals, which will restrict the rate of their accumulation in trophic chains of ecosystems and abate their adverse effects on the biosphere.

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.

Efficacy of tebuconazole embedded in biodegradable poly-3-hydroxybutyrate to inhibit the development of Fusarium moniliforme in soil microecosystems.

BACKGROUND An important line of research is the development of a new generation of formulations with targeted and controlled release of the pesticide, using matrices made from biodegradable materials. In this study, slow-release formulations of the fungicide tebuconazole (TEB) have been prepared by embedding it into the matrix of poly-3-hydroxybutyrate (P3HB) in the form of films, microgranules and pellets. RESULTS The average rates of P3HB degradation were determined by the geometry of the formulation, reaching, for 63 days, 0.095-0.116, 0.081-0.083 and 0.030-0.055 mg day-1 for films, microgranules and pellets respectively. The fungicidal activity of P3HB/TEB against the plant pathogen Fusarium moniliforme was compared with that of the commercial formulation Raxil Ultra. A pronounced fungicidal effect of the experimental P3HB/TEB formulations was observed in 2-4 weeks after application, and it was retained for 8 weeks, without affecting significantly the development of soil aboriginal microflora. CONCLUSION TEB release can be regulated by the process employed to fabricate the formulation and the fungicide loading, and the TEB accumulates in the soil gradually, as the polymer is degraded. The experimental forms of TEB embedded in the slowly degraded P3HB can be used as a basis for developing slow-release fungicide formulations.

A study of synthesis and properties of poly-3-hydroxybutyrate/diethylene glycol copolymers

This study investigates synthesis of poly(3‐hydroxybutyrate)/diethylene glycol copolymers (P3HB/DEG) by Cupriavidus eutrophus B‐10646 cells as related to DEG concentration in the medium and the time when it is added to the culture of cells synthesizing P3HB. The study determines the limits of physiological effect of DEG on C. eutrophus cells, showing that at DEG concentrations above 30 g/L, it inhibits cell growth, decreasing cell concentration and total P3HB/DEG yield and inducing an increase in the degree of saturation of fatty acids in lipids of cell cytoplasmic membrane. A series of copolymers containing different molar fractions of DEG (between 0.13 and 3.0 mol%) have been synthesized and their physicochemical, physical/mechanical, and biological properties have been investigated as related to the chemical composition and proportions of DEG monomers of the polymers.

The Kinetics of Fungicide and Herbicide Release from Slow-Release Formulations Prepared from Degradable Poly-3- Hydroxybutyrate

Release kinetics of the fungicide tebuconazole and the herbicide metribuzin from polymeric carriers was investigated. Carriers were prepared from poly-3- hydroxybutyrate as granules, films and 3D forms. The main mechanisms of pesticide release were discovered; kinetic constants for the first order model and Higuchi model were calculated.

Effect of Drying Mode of Bacterial Biomass on the Effectiveness of Extraction and Physicochemical Properties of the Product (Polymer)

Comparative study of different drying modes of bacterial biomass of Cupriavidus eutrophus В10646 was conducted. Different modes differently affect the yield of the product (polymer) and its physicochemical properties (molecular weight distribution and temperature characteristics). High temperatures caused the polymer degradation and decrease of its molecular weight. Using freeze drying allows to obtain friable biomass with an extended surface which is favorable for extraction process and to preserve the molecular weight characteristics of the polymer.