Natalia O. Zhila

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 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.

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.

Manipulation of Ralstonia eutropha Carbon Storage Pathways to Produce Useful Bio-Based Products

Ralstonia eutrophais a Gram-negative betaproteobacterium found natively in soils that can utilize a wide array of carbon sources for growth, and can store carbon intracellularly in the form of polyhydroxyalkanoate. Many aspects of R. eutrophamake it a good candidate for use in biotechnological production of polyhydroxyalkanoate and other bio-based, value added compounds. Manipulation of the organisms carbon flux is a cornerstone to success in developing it as a biotechnologically relevant organism. Here, we examine the methods of controlling and adapting the flow of carbon in R. eutrophametabolism and the wide range of compounds that can be synthesized as a result. The presence of many different carbon utilization pathways and the custom genetic toolkit for manipulation of those pathways gives R. eutrophaa versatility that allows it to be a biotechnologically important organism.

Specificity of Lipid Composition in Two Botryococcus Strains, the Producers of Liquid Hydrocarbons

The structure of liquid hydrocarbons and fatty acids produced by the green alga Botryococcus was identified. Two representatives of this alga, Botryococcus braunii Kütz, strain IPPAS H-252, introduced into culture earlier and an organism isolated for the first time from the Shira Lake, were used for this identification. Fatty acid composition of B. braunii, strain H-252, lipids was characterized by a high content of trienoic acids of C16–C18 series. The hydrocarbon composition of this strain was represented by straight-chain and branched-chain C14–C28 components; long-chain linear aliphatic C20–C27 hydrocarbons (54.4%) and 2,6,10,14-tetramethylhexadecane (20.5%) predominated among them. The strain H-252 differed in its fatty acid and hydrocarbon composition from the strains described earlier as Botryococcus braunii. The fatty acid composition of the Botryococcus isolate was represented mainly by C12–C32 saturated and monoenoic acids. The hydrocarbons formed by this isolate were represented by dienoic and trienoic components. C29 (48.9–56.3%) and C31 (11.1–16.3%) hydrocarbons predominated among the C23–C31 dienoic hydrocarbons, and C27, C29, and C31 trienoic hydrocarbons comprised 2.5–2.6% of total hydrocarbons. This type of hydrocarbons and the lipid fatty acid composition were characteristic for the race A of B. braunii.

Degradation of polyhydroxyalkanoates and the composition of microbial destructors under natural conditions

The degradation dynamics of polyhydroxyalkanoates of different composition has been studied in an eutrophic storage reservoir for two seasons. It has been shown that the biodegradation of polymers under natural conditions depends not only on their structure and physicochemical properties but also, to a great extent, on a complex of weather-climatic conditions affecting the state of the reservoir ecosystem. The molecular genetic analysis of 16S rRNA has revealed bacterial species (clones) probably involved in the degradation of polyhydroxyalkanoates in a model storage reservoir.

Effects of intracellular poly(3-hydroxybutyrate) reserves on physiological–biochemical properties and growth of Ralstonia eutropha

Microbial polyhydroxyalkanoates (PHAs), because of their well studied complex physiology and commercial potential, are vehicles for carbon and potential storage reduction for many microbial species. Even with the wealth of studies about microbial PHAs in the scientific literature, polymer accumulation and degradation are still not comprehensively understood. Poly(3-hydroxybutyrate) (P3HB) granule formation and polymer mobility were studied here in the bacterium Ralstonia eutropha strain B5786 in autotrophic cultures. Electron microscopy studies revealed decreasing cell size concomitant with enlargement of size and number of intracellular granules, and inhibition of cell division during intracellular polymer production. Activities of key P3HB biosynthetic enzymes demonstrated correlations with each other during polymer accumulation, suggesting an intricately regulated P3HB cycle in autotrophically grown R. eutropha cells.