T. G. Volova

Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood

Samples of olyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) and copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with 4 and 18 mol% hydroxyvalerate, synthesized by the bacteria Ralstonia eutropha B5786, were investigated. PHA films in contact with blood did not activate the hemostasis system at the level of cell response, but they did activate the coagulation system and the complement reaction. To detect biologically-active components in the PHAs, a detailed analysis of the composition of the polymers was conducted. Gas chromatography-mass spectrometry revealed long-chain fatty acids (FAs) in the tested PHAs. Their total concentration in the polymer ranged from tenths of mol% to 2-3 mol%, depending on the purification method. C16:0 constituted the largest proportion, up to 70%. Of the long-chain hydroxy acids, only β-OH-C14:0 was detected and it did not exceed 0.06 mol%. The analysis of the hemocompatibility properties of the PHAs purified by a specialized procedure, including the quantitative and morphological estimation of platelets adherent to the surface of polymer films, the plasma recalcification time and complement activation studies, indicated that PHB and PHBV can be used in contact with blood. It has been found out that the lipopolysaccharides of bacteria producing PHAs, which contain mostly long-chain hydroxy acids, can be the factor activating the hemostasis systems. Thus, the technology of PHA purification must satisfy rather stringent specific requirements.

Effect of Nitrogen Limitation on the Growth and Lipid Composition of the Green Alga Botryococcus braunii Kutz IPPAS H-252

The effect of nitrogen limitation in a medium on the composition of intracellular lipids in the alga Botryococcus braunii Kutz IPPAS H-252 in the course of culture development was investigated. Under the conditions of nitrogen limitation, the alga under investigation accumulated lipids as triacylglycerols, and this process was accompanied by substantial changes in the total fatty acid (FA) composition, which were manifested in a decrease in trienoic acids (from 52.8–57.2 to 19.5–24.7% of total FAs) and an increase in the content of oleic (from 1.1–1.2 to 17.1–24.4%) and saturated (from 23.7–26.0 to 32.9–46.1%) acids. In the control culture, the directionality of FA redistribution was less marked, and these changes were noticed at the later stages of culture development. Under nitrogen limitation, marked changes in the FA composition of polar lipids occurred by the 13th day, and they were characterized by an increase in the content of saturated acids (up to 76.8%) and a dramatic decrease in the content of all polyenoic acids (up to 6.8%). The changes in the FA composition of triacylglycerols were noticed as early as by the 7th day; these changes consisted in an increase in the content of oleic acid, and its high content (28.4–38.4%) was maintained up to the end of culturing. In the control culture, triacylglycerols with a high content of oleic acid were found by the 13th day, although, by this time, the content of total lipids and triacylglycerols did not change.

A hybrid PHB–hydroxyapatite composite for biomedical application: production, in vitro and in vivo investigation

Samples of a hybrid composite of polyhydroxybutyrate (PHB), a biodegradable polyester, and hydroxyapatite (HA), with different PHB/HA ratios, have been prepared using mechanical-physical method. Electron microscopy, X-ray structure analysis and differential thermal analysis have been used to investigate the structure and physicochemical properties of the composite, depending on the PHB/HA ratio. The properties of the surface of the HA-loaded composite are significantly different from those of the pure polymer. As the HA percentage in the composite increases, free interface energy, the cohesive force, i.e., the strength of the adhesive bond between the composite surface and the water phase, and surface wettability increase. The HA percentage of the composite does not influence its melting temperature, but affects the temperature for the onset of decomposition: as the HA content increases from 0 to 10% (w/w), T d decreases from 260°C to 225°C. The degree of crystallinity of PHB/HA increases from 77% to 89% with an increase in the HA fraction from 10% to 50%. Functional properties of the composites have been investigated in vitro and in vivo. The best parameters of growth and differentiation of murine marrow osteoblasts are registered on PHB/HA samples containing 10% and 20% HA. In ectopic bone formation assay it has been proven that the hybrid PHB/HA composites can function as scaffolds and that bone tissue develops on their surface and in pores.

A Temperature Dependence of the Intra- and Extracellular Fatty-Acid Composition of Green Algae and Cyanobacterium

The effect of ambient temperature on the composition of intracellular fatty acids and the release of free fatty acids (FFA) into a medium by cyanobacterium Spirulina platensis and eukaryotic microalgae, Chlorella vulgaris and Botryococcus braunii, was studied using their batch cultures. It was found that all the species studied, regardless of their taxonomic status, responded to the temperature regime by similar changes in their intracellular fatty acid composition: the relative content of more unsaturated fatty acids decreased with the elevation of temperature. At the same time, in the prokaryote, this temperature shift blocked, first of all, the elongation of 16:0 to 18:0 and then their further desaturation. In eukaryotes, the change in the desaturation of dienoic to trienoic fatty acids was the most pronounced process. The ratio of dienoic to trienoic fatty acids remained almost unchanged in S. platensis. The relative content of extracellular unsaturated FFA increased in the prokaryotic organism S. platensis at a higher temperature. But no significant changes in the composition of extracellular unsaturated FFA were detected in eukaryotic algae upon temperature elevation.

Influence of nitrogen deficiency on biochemical composition of the green alga Botryococcus

A study has been carried out to investigate the influence of nitrogen deficiency on intracellular lipid composition, including total fatty acid composition of lipids, polar lipids, and triacylglycerols, of the alga Botryococcus braunii Kütz IPPAS H-252 in batch culture. Under nitrogen limitation, the alga accumulates lipids as triacylglycerols and the total fatty acid (FA) composition changes: trienoic acids decrease (from 52.8–57.2 to 19.5–24.7% of the total FAs) and the oleic acid increases (from 1.1–1.2 to 17.1–24.4%) as does the saturated acids (from 23.7–26 to 32.9–46.1%). A similar rearrangement in the FA spectrum occurs at later times in the control culture, but it is less pronounced. Under nitrogen limitation, considerable changes in the polar lipid FAs are registered at day 13: saturated acids increase (from 28.6–35.5 to 76.8%) and all polyenoic acids markedly decrease (from 56.9–64.1 to 6.8%). Changes in the triacylglycerol fatty acid spectrum are seen on day 7: the oleic acid increases (from 14.7 to 34.2%) and remains at a high level till the end of the culture. In the control, triacylglycerols with large contents of oleic acid are detected at day 13, the total lipids and triacylglycerols still remaining unchanged.

Degradation of P(3HB) and P(3HB-co-3HV) in biological media

The biodegradability of oriented fibers made of polyhydroxybutyrate (P(3HB)) and its co-polymer with β-hydroxyvalerate (P(3HB-co-3HV)) was investigated in buffer solutions and in biological media in vitro and in vivo. The fibers of both polymer types demonstrated resistance to hydrolytic degradation in buffer solutions at 38°C and pH from 4.5 to 7.0 (for up to 180 days). It has been found that the biodegradation of the fibers in vitro in blood and serum and in vivo is accompanied by weight losses and minor changes in the microstructure with no significant losses in the tensile strength over a long time (up to 180 days). The biodegradation rate of the less crystalline co-polymer P(3HB-co-3HV) fibers was 1.4–2.0-times higher than that of the homopolymer P(3HB). It has also been shown that the degradation of the fibers in vivo is influenced both by tissue fluid enzymes and cells (macrophages and foreign-body giant cells). The fibers were eroded on the surface only with no gross defects and no dramatic effects on their mechanical performance.

The Effect of Temperature on the Lipid Composition of the Green Alga Botryococcus

The lipid composition of the green alga Botryococcus was studied at three different cultivation temperatures: suboptimal (18°C), optimal (25°C), and supraoptimal (32°C). Cultivation at the supraoptimal temperature was found to considerably inhibit the synthesis of nearly all intracellular lipids, except for triacylglycerides, and to influence their fatty acid composition. In particular, the content of trienoic fatty acids was significantly lower at the supraoptimal than at the optimal cultivation temperature. At the same time, the fatty acid composition of the extracellular lipids of the alga virtually did not depend on cultivation temperature.

Lipid and hydrocarbon compositions of a collection strain and a wild sample of the green microalga Botryococcus

Lipid composition and hydrocarbon structure of two colonial green algae of the genus Botryococcus, i.e., a museum strain and a field sample collected for the first time from Lake Shira (Khakasia, Siberia), have been compared. Polar lipids, diacylglycerols, alcohols, triacylglycerols, sterols, sterol esters, free fatty acids and hydrocarbons have been identified among lipids in the laboratory culture. The dominant fraction in the museum strain was formed by polar lipids (up to 50% of the lipids) made up of fatty acids from C12 to C24. Palmitic, oleic, C16 - C18 dienoic and trienoic acids were the main fatty acids of the museum strain. Aliphatic hydrocarbons were found in the lipid of the museum strain. However, these amounted maximally to about 1% of the dry biomass at the end of exponential growth phase. The qualitative and quantitative compositions of FAs and hydrocarbons of the museum strain of Botryococcus, (registered at the Cambridge collection as Botryococcus braunii Kutz No LB 807/1 Droop 1950 H-252) differed from those of the Botryococcus strain described in the literature as Botryococcus braunii. The Botryococcus sp. found in Lake Shira is characterized by a higher lipid content (< 40% of the dry weight). Polar lipids, sterols, triacylglycerols, free fatty acids and hydrocarbons have been identified among lipids in the field sample. The main lipids in this sample were dienes and trienes (hydrocarbons < 60% of total lipid). Monounsaturated and very long chain monounsaturated fatty acids, including C28:1 and C32:1 acids, were identified in the Botryococcus found in Lake Shira. The chemo-taxonomic criteria allow us to unequivocally characterize the organism collected from Lake Shira as Botryococcus braunii, race A.

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.