A. N. Boyandin

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.

Biodegradation of polyhydroxyalkanoates by soil microbial communities of different structures and detection of PHA degrading microorganisms

Biodegradation of microbial linear polymers of hydroxyalkanoic acids (polyhydroxyalkanoates, PHAs) by soil microbial communities of different structures has been studied during two field seasons in different weather conditions. This process was shown to be influenced by the polymer chemical composition, temperature, humidity, and the microbial soil component. The PHA degradation was accompanied by a decrease in the polymer molecular weight and an increase in the degree of crystallinity, indicating the preferential destruction of the amorphous phase compared to the crystalline one. The quantity of the true PHA destructors developing at the surface of the polymer samples was lower than the quantity of accompanying bacteria. The dominant PHA degrading microorganisms under the test conditions were identified as bacteria of the genera Variovorax, Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Xanthomonas and as micromycetes from Penicillium, Paecilomyces, Acremonium, Verticillium, and Zygosporium.

Degradable Polyhydroxyalkanoates as Herbicide Carriers

The biodegradable polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) has been used to design experimental sustained-release formulations of the herbicide Zellek Super in the form of films and microgranules. The kinetics of polymer degradation and the dynamics of herbicide release show that the rate and extent of herbicide release from the polymer matrix into the soil depends on the geometry of the carrier and the proportion of the pesticide loaded into it (polymer/pesticide mass ratio). Experiments with the creeping bentgrass (Agrostis stolonifera L.) show that the formulations of the herbicide Zellek Super constructed as microgranules and films can be successfully used to suppress the growth of grasses. This study is the first to demonstrate that biodegradable polyhydroxyalkanoates can be used effectively to construct environmentally friendly sustained-release PHA-herbicide systems that can be placed into the soil together with seeds.

Surface wettability and energy effects on the biological performance of poly-3-hydroxybutyrate films treated with RF plasma.

The surface properties of poly-3-hydroxybutyrate (P3HB) membranes were modified using oxygen and an ammonia radio-frequency (RF, 13.56 MHz) plasma. The plasma treatment procedures used in the study only affected the surface properties, including surface topography, without inducing any significant changes in the crystalline structure of the polymer, with the exception being a power level of 250 W. The wettability of the modified P3HB surfaces was significantly increased after the plasma treatment, irrespective of the treatment procedure used. It was revealed that both surface chemistry and surface roughness changes caused by the plasma treatment affected surface wettability. A treatment-induced surface aging effect was observed and resulted in an increase in the water contact angle and a decrease in the surface free energy. However, the difference in the water contact angle between the polymers that had been treated for 4 weeks and the untreated polymer surfaces was still significant. A dependence between cell adhesion and proliferation and the polar component of the surface energy was revealed. The increase in the polar component after the ammonia plasma modification significantly increased cell adhesion and proliferation on biodegradable polymer surfaces compared to the untreated P3HB and the P3HB modified using an oxygen plasma.

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.

Biodegradable poly-3-hydroxybutyrate as a fertiliser carrier.

BACKGROUND Increasing use of mineral fertilisers can lead to accumulation of fertilisers in soil, water and foodstuffs. One of the approaches to preventing these problems is to develop controlled release forms of fertilisers. RESULTS Experimental formulations of the nitrogen fertiliser urea loaded in a degradable matrix of the natural polymer poly-3-hydroxybutyrate (P3HB) in the form of films, pellets and coated granules were constructed and investigated. Nitrogen release into soil occurred as the polymer was degraded, and it was dependent on the geometry of the carrier and the amount of nitrogen loaded in it, showing that nitrogen release can last for 30 days or longer and that release rates can be controlled by varying the fabrication technique employed. P3HB/urea formulations have a favourable effect on the soil microbial community. The use of embedded urea has a beneficial influence on the growth of creeping bentgrass (Agrostis stolonifera) and lettuce (Latuca sativa) and reduces removal of nitrogen with drain water. CONCLUSION The slow-release nitrogen formulations developed in this study can be buried in soil together with seeds preventing nitrogen deficiency. The use of such slow-release formulations can decrease the amounts of chemicals in the environment and prevent their adverse effects on the biosphere.

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.

Synthesis of reserve polyhydroxyalkanoates by luminescent bacteria

The ability of marine luminescent bacteria to synthesize polyesters of hydroxycarboxylic acids (polyhydroxyalkanoates, PHA) as reserve macromolecules was studied. Twenty strains from the collection of the luminescent bacteria CCIBSO (WDCM839) of the Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, assigned to different taxa (Photobacterium leiognathi, Ph. phosphoreum, Vibrio harveyi, and V. fischeri) were analyzed. The most productive strains were identified, and the conditions ensuring high polymer yields in batch culture (40–70% of the cell dry mass weight) were determined. The capacity for synthesizing two-and three-component polymers containing hydroxybutyric acid as the main monomer and hydroxyvaleric and hydroxyhexanoic acids was revealed in Ph. leiognathi and V. harveyi strains. The results allow luminescent microorganisms to be regarded as new producers of multicomponent polyhydroxyalkanoates.

Experimental evaluation of the processes resulting from the introduction of the transgenic microorganism Escherichia coli Z905/pPHL7 (lux+) into aquatic microcosms

The processes resulting from the introduction of the tranagenic microorganism (TM) E. coli Z905/pPHL7 into aquatic microcosms have been modeled experimentally. It has been shown that the TM E. coli is able to adapt to a long co-existence with indigenous heterotrophic microflora in variously structured microcosms. In more complex microcosms the numerical dynamics of the introduced E. coli Z905/pPHL7 population is more stable. In the TM populations staying in the microcosms for a prolonged time, changes are recorded in the phenotypic expression of plasmid genes (ampicillin resistance and the luminescence level) and chromosome genes (morphological and physiological traits). However, in our study microcosms, the recombinant plasmid persisted in the TM cells for 6 years after the introduction, and as the population adapts to the conditions of the microcosms, the efficiency of the cloned gene expression in the cells is restored. In the microcosms with high microalgal counts (10(7) cells/ml), cells with a high threshold of sensitivity to ampicillin dominate in the population of the TM E. coli Z905/pPHL7.

Population dynamics of transgenic microorganisms in the different microecosystem conditions

The role of key environmental factors in adaptation of spore-forming and non-spore-forming transgenic microorganisms (TM) have been studied in model ecosystems. Model TM Escherichia coli Z905 (bearing plasmid genes of bacterial luminescence Ap (r) Lux+) has been found to have a higher adaptation potential than TM Bacillus subtilis 2335/105 (bearing genes of human alpha 2-interferon Km (r) Inf+), planned for employment as a living vaccine under varying environmental conditions. Effects of abiotic factors on migration of natural and recombinant plasmids between microorganisms under model ecosystem conditions has been estimated. The transgenic microorganisms with low copy number survived better under introduction conditions in the microcosms studied. This trend has been shown to be independent of the microcosm type and its complexity. Grant numbers: 99-04-96017, 25, 00-07-9011.