| Summary: | The objective of this study was to isolate and identify strains of <i>Acetobacter</i> suitable for use in the development of a complex microbial culture for producing Kombucha and to examine the fermentation characteristics for selection of suitable strains. A medium supplemented with calcium carbonate was used for isolation of acetic acid bacteria from 22 various sources. Colonies observed in the clear zone resulting from decomposition of calcium carbonate by acid produced by microorganisms were collected. Identification of the collected strains was based on biological and morphological characteristics, and the results of base sequence analysis. A total of 37 strains were identified, including six species in the <i>Acetobacter</i> genus: <i>Acetobacter pasteurianus</i>, <i>Acetobacter orientalis</i>, <i>Acetobacter cibinongensis</i>, <i>Acetobacter pomorum</i>, <i>Acetobacter ascendens</i>, and <i>Acetobacter malorum</i>, as well as one species in the <i>Gluconobacter</i> genus, <i>Gluconobacter oxydans</i>. Among thirty-seven strains, seven strains of acetic acid bacteria with exceptional acid and alcohol tolerance were selected, and an evaluation of their fermentation characteristics according to fermentation temperature and period was performed. The results showed a titratable acidity of 1.68% for the <i>Acetobacter pasteurianus</i> SFT-18 strain, and an acetic acid bacteria count of 9.52 log CFU/mL at a fermentation temperature of 35 °C. The glucuronic acid and gluconate contents for the <i>Gluconobacter oxydans</i> SFT-27 strain were 10.32 mg/mL and 25.49 mg/mL, respectively.
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