Background: The bacterial destructing activity toward pesticides has been the focus of research in the last few decades. Hexachlorobenzene is included in the organochlorine pesticides group that are prohibited for use. However, large hexachlorobenzene amounts are still concentrated in the soil, stressing the relevance of research on hexachlorobenzene-destroying bacteria. Methods: The ability to destroy hexachlorobenzene by Comamonas testosteroni UCM B-400, B-401, B-213 strains was investigated and established. Bacteria were cultivated (7 days at 28 °C) in mineral Luria-Bertrani (LB) medium with three hexachlorobenzene doses: 10, 20, 50 mg/L. The hexachlorobenzene concentrations were recorded by a gas chromatography method. Results: The results showed that C. testosteroni UCM B-400, B-401 have high destructive activity toward hexachlorobenzene. The highest (50 mg/L) initial concentration decreased to 41.5 and 43.8%, respectively, for C. testosteroni UCM B-400, B-401. The unadapted C. testosteroni UCM B-213 was tolerant to hexachlorobenzene (cell titers after cultivating with 10.0, 20.0, 50.0 mg/mL were higher compared to initial titer), but had a low-destructing activity level (two times less than B-400 and B-401). Conclusions: Bacterial strains C. testosteroni UCM B-400, B-401 can be seen as a potential soil bioremediation from hexachlorobenzene pollution.

The species of Comamonas testosteroni is the most common human pathogen of the genus, which can be associated with acute appendicitis, infections of the bloodstream, the peritoneal cavity, cerebrospinal fluid, inflammatory bowel disease, and in general, bacteremia. According to the literature, Comamonas testosteroni has destructive activity to a wide range of toxic chemical compounds, including chlorobenzenes. The specified strains were isolated from the soil of the organochlorine waste landfill, where hexachlorobenzene (HCB) was predominant. These strains were expected to be capable of degrading HCB. Microbiological (bacterial enrichment and cultivating, bacterial biomass obtaining), molecular biology, biochemical (enzymatic activities, malondialdehyde measuring, peroxidation lipid products measuring), and statistical methods were carried out in this research. The reaction of both strains (UCM B-400 and UCM B-401) to the hexachlorobenzene presence differed in the content of diene and triene conjugates and malondialdehyde, as well as different catalase and peroxidase activity levels. In terms of primary peroxidation products, diene conjugates were lower, except conditions with 20 mg/L HCB, where these were higher up to two times, than the pure control. Malondialdehyde in strain B-400 cells decreased up to five times, in B-401, but increased up to two times, compared to the pure control. Schiff bases in strain B-400 cells were 2–3 times lower than the pure control. However, in B-401 cells Schiff bases under higher HCB dose were in the same level with the pure control. Catalase activity was 1.5 times higher in all experimental variants, compared to the pure control (in the strain B-401 cells), but in the B-400 strain, cells were 2 times lower, compared to the pure control. The response of the two strains to hexachlorobenzene was similar only in peroxidase activity terms, which was slightly higher compared to the pure control. The physiological response of Comamonas testosteroni strains to hexachlorobenzene has a typical strain reaction. The physiological response level of these strains to hexachlorobenzene confirms its tolerance, and indirectly, the ability to destroy the specified toxic compound.

Based on the analysing phenotypic and genotypic properties, the taxonomic position of isolates # 46 and # 47 was established and assigned to the species Comamonas testosteronі UCM B-400 and UCM B-401. Isolates were selected from the soil with organochlorine wastes landfill of chemical enterprises in Kalush, Ivano-Frankivsk region, Ukraine. The strains are resistant to high hexachlorobenzene concentrations. According to morphological, cultural and biochemical properties, the studied strains are related to typical members of the Comamonadaceae family, they are motile gram-negative rods, aerobic, chemoorganotrophic. Saturated and unsaturated fatty acids with a carbon chain length from C10 to C18 were found in composition of cellular lipids. The qualitative and quantitative composition of marker hydroxy acids, namely: 2 hydroxyhexadecane (C_{16: 0 2OH}) and 3 hydroxydecane (C_{10: 0 3OH}) in the lipids of the strains ranged from 2-5%, which corresponded to their content in the representatives of the Comamonas genus. Phylogenetic analysing the nucleotide sequences of the 16S rRNA gene revealed the affinity of isolates # 46 (97.77%) and # 47 (98.05%) with a similar nucleotide sequence of a typical strain of Comamonas testosteroni LMG 1800. The 16S rRNA nucleotide sequences of Comamonas testosteroni UCM B-400 and UCM B-401 were placed in GenBank under the numbers SUB9376895 Comamonas_testosteroni_46 MW861636 and SUB9376895 Comamonas_testosteroni_47 MW861637.