The Extracellular Vesicles Containing Inorganic Polyphosphate of Candida Yeast upon Growth on Hexadecane.

The cell wall of Candida yeast grown on presence of hexadecane as a sole carbon source undergoes structural and functional changes including the formation of specific supramolecular complexes-canals. The canals contain specific polysaccharides and enzymes that provide primary oxidization of alkanes. In addition, inorganic polyphosphate (polyP) was identified in Candida maltosa canals. The aim of the work was a comparative study of the features of cell walls and extracellular structures in yeast C. maltosa, C. albicans and C. tropicalis with special attention to inorganic polyphosphates as possible part of these structures when grown on the widely used xenobiotic hexadecane (diesel fuel). Fluorescence microscopy with DAPI has shown an unusual localization of polyP on the cell surface and in the exovesicles in the three yeast species, when growing on hexadecane. Electron-scanning microscopy showed that the exovesicles were associated with the cell wall and also presented in the external environment probably as biofilm components. Treatment of hexadecane-grown cells with purified Ppx1 polyphosphatase led to the release of phosphate into the incubation medium and the disappearance of polyP in vesicles and cell wall observed using microscopic methods. The results indicate the important role of polyP in the formation of extracellular structures in the Candida yeast when consuming hexadecane and are important for the design of xenobiotic destructors based on yeast or mixed cultures.

Genome Analysis and Physiology of Pseudomonas sp. Strain OVF7 Degrading Naphthalene and n-Dodecane.

The complete genome of the naphthalene- and n-alkane-degrading strain Pseudomonas sp. strain OVF7 was collected and analyzed. Clusters of genes encoding enzymes for the degradation of naphthalene and n-alkanes are localized on the chromosome. Based on the Average Nucleotide Identity and digital DNA-DNA Hybridization compared with type strains of the group of fluorescent pseudomonads, the bacterium studied probably belongs to a new species. Using light, fluorescent, and scanning electron microscopy, the ability of the studied bacterium to form biofilms of different architectures when cultured in liquid mineral medium with different carbon sources, including naphthalene and n-dodecane, was demonstrated. When grown on a mixture of naphthalene and n-dodecane, the strain first consumed naphthalene and then n-dodecane. Cultivation of the strain on n-dodecane was characterized by a long adaptation phase, in contrast to cultivation on naphthalene and a mixture of naphthalene and n-dodecane.

Biocompatible Silica-Polyethylene Glycol-Based Composites for Immobilization of Microbial Cells by Sol-Gel Synthesis.

Biocatalysts based on the methylotrophic yeast Ogataea polymorpha VKM Y-2559 immobilized in polymer-based nanocomposites for the treatment of methanol-containing wastewater were developed. The organosilica composites with different matrix-to-filler ratios derived from TEOS/MTES in the presence of PEG (SPEG-composite) and from silicon-polyethylene glycol (STPEG-composite) differ in the structure of the silicate phase and its distribution in the composite matrix. Methods of fluorescent and scanning microscopy first confirmed the formation of an organosilica shell around living yeast cells during sol-gel bio-STPEG-composite synthesis. Biosensors based on the yeast cells immobilized in STPEG- and SPEG-composites are characterized by effective operation: the coefficient of sensitivity is 0.85 ± 0.07 mgO2 × min-1 × mmol-1 and 0.87 ± 0.05 mgO2 × min-1 × mmol-1, and the long-term stability is 10 and 15 days, respectively. The encapsulated microbial cells are protected from UV radiation and the toxic action of heavy metal ions. Biofilters based on the developed biocatalysts are characterized by high effectiveness in the utilization of methanol-rich wastewater-their oxidative power reached 900 gO2/(m3 × cycle), and their purification degree was up to 60%.

Cell wall canals formed upon growth of Candida maltosa in the presence of hexadecane are associated with polyphosphates.

Canals are supramolecular complexes observed in the cell wall of Candida maltosa grown in the presence of hexadecane as a sole carbon source. Such structures were not observed in glucose-grown cells. Microscopic observations of cells stained with diaminobenzidine revealed the presence of oxidative enzymes in the canals. 4΄,6΄-diamino-2-phenylindole staining revealed that a substantial part of cellular polyphosphate was present in the cell wall of cells grown on hexadecane in condition of phosphate limitation. The content and chain length of polyphosphates were higher in hexadecane-grown cells than in glucose grown ones. The treatment of cells with yeast polyphosphatase PPX1 resulted in the decrease of the canal size. These data clearly indicated that polyphosphates are constituents of canals; they might play an important role in the canal structure and functioning.

Modifications of the cell wall of yeasts grown on hexadecane and under starvation conditions.

Electron-microscopic examinations have demonstrated local modifications in the cell wall of the yeast Candida maltosa grown on hexadecane. In our earlier studies, these modified sites, observed in other yeasts grown on oil hydrocarbons, were conventionally called 'canals'. The biochemical and cytochemical studies of C. maltosa have revealed a correlation between the formation of 'canals' and decrease in the amount of cell wall polysaccharides, glucan and mannan. The ultrathin sections and surface replicas have shown that the 'canals' are destroyed by pronase, thus indicating that a significant proportion of their content is represented by proteins. This finding was compatible with our earlier data on the localization of oxidative enzymes in 'canals' and possible participation of the 'canals' in the primary oxidation of hydrocarbons. A completely unexpected and intriguing phenomenon has been the appearance of 'canals' in the yeast C. maltosa under starvation conditions. Unlike the yeasts grown on hexadecane, mannan almost disappears in starving cells, while the quantity of glucan first decreases and then is restored to its initial level. The role of 'canals' in starving cells is as yet unclear; it is assumed that they acquire exoenzymes involved in the utilization of products of cell lysis in the starving population. In the future, 'canals' of starving cells will be studied in connection with their possible participation in apoptosis.