Dimova M, Dankevich L, Yamborko N.

D.K. Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine,

Department of general and soil microbiology,

Department of plant pathogenic bacteria


Hexachlorobenzene (HCB) is a toxic, persistent organochlorine contaminant with a high ability to bioaccumulate in adipose tissue of animals and humans. Its half-life in soil is more than 6 years. HCB is formed as an intermediate and waste in the chemical industry. The search for microorganisms potentially capable of destroying HCB is relevant. From the soil of sites with local HCB contamination the promising microbial isolates of HCB destructors were selected. After screening bacteria for resistance to contamination, bacterial isolates 46 and 47 were selected for further study.

The aim of the research was to study the resistance of this isolates to high HCB doses and to determine their systematic position.

Materials and methods. To determine the resistance to HCB the isolates were cultured in the Menkina’s liquid medium with different HCB doses. Establishing taxonomic position of the species and selection of species markers was performed using an automated system Vitek 2 Bio-Mérieux according to the manufacturer’s instructions using the VITEK® 2: Gram-Negative identification card (GN). Identification of fatty acid methyl esters was performed using a Agilent 6800N/5973 inert. chromato-mass spectrometric system.

Results. The study of the resistance of selected bacteria to different HCB doses showed that both strains are able to grow in a liquid medium at the presence of ultra-high HCB doses of 50 and 100 mg/L, which corresponds to 1650 and 3300 maximum permissible concentrations (MPC). After 168 hours of cultivation in medium at doses of 50 and 100 mg/L, cell titers of isolate 46 were 8.8∙106 and 6.1∙105 CFU/mL, and for isolate 47 1.2∙106 and 1.0∙105 CFU/mL, respectively. The initial inoculation titers for the 46 and 47 isolates were 3.9·108 and 4·106 CFU/mL. By phenotype properties, the bacteria are aerobic gram-negative rods with dimensions of 0.4×2.5 μm and 0.3×2.1 μm, respectively. Isolates showed catalase and oxidase activity. Identification by physiological and biochemical characteristics in the atomized system Vitek 2 revealed the affiliation of isolate 46 to Delftia acidovorans and isolate 47 to Comamonas testosteroni. To confirm the correctness of the obtained results, a comparative study was conducted for typical bacterial strains Comamonas testosteroni UCM B-213 and Delftia acidovorans UCM B-197 from the Ukrainian Collection of Microorganisms. The results showed the identity of newly isolated and typical bacterial strains. The common physiological and biochemical characteristics of these two species were: capability to perform fermentation of lactate and succinate, inability to assimilate glucose, mannose, trehalose, malonate, and sodium citrate, possessing L-pyrrolidone-arylamidase, tyrosin arylamidase activities, inability to produce hydrogen sulfide. D. acidovorans 46 strain exhibited glutamylarylamidase and gamma-glutamyltransferase activity. The spectrum of fatty acids of total lipids is important for determining the taxonomic position. The unsaturated hexadecenoic (C16: 1cis 9) and cis-9 octadecenoic acids (C18: 1 cis 9) and the saturated hexadecanoic (C16: 0) acids are dominant in the fatty acid spectrum (over 70%). The content of markers for typical species of the genera Comamonas and Delftia hydroxy acids 2 hydroxyhexadecane (C16: 0 2OH) and 3 hydroxydecane (C10: 0 3OH) for cell lipids of the studied strains ranged from 2-5% of the total peak area. A number of researchers emphasize the similarity of the qualitative composition of the fatty acid spectrum of studied species.

Conclusions. Thus, our study of the complex of phenotype traits indicates a significant affinity of our studied bacterial isolates to Delftia acidovorans (isolate 46) and Comamonas testosteroni (isolate 47), but to establish the correct affiliation of the strains, we outline to study certain genotypic properties.