Isolation and study of the epiphytic yeast strain Rhodotorula mucilaginosa AgIV RCAM05019 (Basidiomycota)

The pigmented yeast Rhodotorula, which belongs to the division Basidiomycota, family Sporidiobolaceae, and class Microbotryomycetes, is found in a wide variety of natural reservoirs, including air, soil, freshwater, seawater, plant substrates, and milk. This yeast is distributed from tropical regions to the permafrost of the Arctic Circle. Various strains of Rhodotorula are considered safe and promising biotechnological candidates for the production of a wide range of biologically active substances, such as proteins, lipids, and vitamins. However, despite its low pathogenicity, an increasing number of studies are reporting on the pathogenic potential of this species, including cases of dermatomycosis in immunocompromised patients. The aim of this study was to obtain and investigate the cultural, morphological, physiological, and biochemical properties of the yeast strain Rhodotorula mucilaginosa AglV RCAM05019, as well as to explore its potential use as a source of feed protein. The objects of this research were the yeast strain Rhodotorula mucilaginosa AglV, which was previously isolated from the epiphytic yeast complex found in the fruiting bodies of the champignon Agaricus sp. This strain was obtained from the microbiological monitoring laboratory within the Department of Applied Biology and Microbiology at Astrakhan State Technical University. Additionally, the collection strain of Candida tropicalis SK-4-1 was provided by the All-Russian Scientific Research Institute of Agriculture (Pushkin) to serve as a control strain for the experimental enhancement of cell biomass under various submerged cultivation modes. Obtaining a pure culture, analyzing cultural-morphological and physiological-biochemical characteristics, assessing growth kinetics, conducting deep cultivation on an orbital shaker, performing periodic cultivation in a fermenter, and carrying out physicochemical studies to determine biomass quality were carried out using standard methods. The yeast strain under investigation was identified through Sanger sequencing, which determined the nucleotide sequence of a fragment of the ITS region at the All-Russian Research Institute of Agricultural Microbiology in Pushkin. This strain has been deposited in the Departmental Collection of Beneficial Microorganisms for Agricultural Purposes under registration number RCAM05019 (FGBNU "VNIISKHM"). The safety of the yeast strain, including its toxicity, toxigenicity, virulence, and dissemination potential, was evaluated in white male Balb/c mice. On solid Sabouraud medium, the studied strain of Rhodotorula mucilaginosa AgIV RCAM05019 exhibited growth in the form of a smooth, shiny, mucus-like streak of bright pink color. It did not penetrate the surface of the nutrient medium (see Fig. 1a) and formed a large pale pink colony on morphological agar (see Fig. 1b). Microscopic examination revealed round-shaped cells measuring 1.5-2.3 pm (see Fig. 1c). The results of the analysis of micro-, macromorphological, and physiological-biochemical characteristics are presented in the block diagram (see Fig. 2). When comparing the analyzed nucleotide sequences with those deposited in BLAST, the species Rhodotorula mucilaginosa was identified as the closest match to the strain under study, with a similarity of 99%. The R. mucilaginosa AglV strain RCAM05019 has been deposited in GenBank under accession number PP531621. No signs of acute toxicity, toxigenicity, virulent properties, or dissemination effects were observed in the identified strain of R. mucilaginosa AglV RCAM05019; this strain did not result in the death of laboratory animals. The mice remained active and mobile, with clean skin and unchanged fur. They exhibited a normal appetite and reactions, did not lose weight, and the surfaces of their internal organs appeared smooth, with no visible pathology, normal coloration, and a dense structure (see Tables 3 and 4). When assessing growth kinetics, the most significant effect was observed in the growth medium containing molasses, which is characterized by a pronounced exponential growth phase of the test strain (see Fig. 3). When the stock culture was established using depth culture on an orbital shaker, intensive growth of R. mucilaginosa AglV RCAM05019 strain cells (107 cells/mL) was detected as early as one day after the initiation of the culture, in contrast to the control strain C. tropicalis CK-4-1 (4.0 x 106 cells/mL) (see Table 1). From the analysis of experimental data obtained during periodic cultivation in a fermenter, it was established that the R. mucilaginosa AglV RCAM05019 strain (6.0 x 107 cells/mL) accumulates biomass in a shorter period compared to the C. tropicalis CK-4-1 strain (5.0 x 107 cells/mL) (see Table 1). The quality indicators of the biomass for the tested strains meet the requirements outlined in regulatory documentation: moisture mass fraction for R. mucilaginosa AglV RCAM05019 is 11.5%, while for C. tropicalis CK-4-1 it is 11.8%; ash mass fraction for R. mucilaginosa AglV RCAM05019 is 7.0%, and for C. tropicalis CK-4-1 it is 7.8%; and the mass fraction of crude protein for R. mucilaginosa AglV RCAM05019 is 67%, whereas for C. tropicalis CK-4-1 it is 48% (see Table 2). The article contains 4 Figures, 4 Tables, 45 References. The authors declare no conflict of interest.

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