ISOLATION AND CHARACTERISTICS OF BACTERIA CITROBACTER SP. SR35 FROM A COAL MINE WASTE DUMP

Abstract

SummaryBacteria of the genus Citrobacter are found in soil, water, the intestinal tract of animals, human clinical samples (urine, sputum, blood, wound drainage, etc.), also in wastewaters and waste dumps of mines. Gram-negative bacteria SR35, capable of reducing sulfur and sulfate ions, were isolated from the waste dump of the Nadiya coal mine, Chervonograd Mining and Industrial district (Lviv region, Ukraine). The work aimed to identify and study morphological, physical and chemical properties (cell shape, size, Gram staining, sporulation, motility, oxygen requirements, ability to form H2S, utilization of carbon sources, catalase activity, oxidase activity) of isolate SR35. Methods. We used standard microbiological and biochemical research methods (microbial culture, microscopy methods, determination of amilase, lipase activity). Chromosomal DNA was isolated by the method of soft lysis. The 16S rRNA gene was amplified using universal primers 27F and 1492R. It was sequenced by the Sanger method. Phylogenetic reconstruction was performed using the MEGA X program. Identification of isolates was carried out based on both the sequence of the 16S rRNA gene and physiological and biochemical properties. Results. The studied bacteria are rods (0.5–0.8×1.5–2.0μm), which form chains and are capable of reducing nitrate and sulfate ions and assimilating several carbon sources. Glucose, lactose, mannose, mannitol, and inositol are fermented with the formation of acid. Motile. Catalase positive, oxidase negative. Form H2S during growth in Kligler’s medium. According to the results of pairwise alignment of the isolate’s 16S rRNA gene sequence, the highest percentage of identity with representatives of the genus Citrobacter was established (99.23–99.86% identity, coverage 98%) and it was confirmed by phylogenetic reconstruction. Citrobacter sp. SR35 is resistant to 2 μM cadmium (II), 5 mM iron (II), 0.25 mM cobalt (II), 10 mM manganese (II), 0.5 mM copper (II), and 0.1 mM chromium (VI). Conclusions. According to the results of sequencing of the 16S rRNA (accession number GenBank OP279754) gene, and physiological, and biochemical characteristics (oxidase, catalase, metabolism of carbon sources, production of H2S, etc.), it was established that isolate SR35 belongs to the genus Citrobacter.