Biochemical mechanisms of resistance to p-nitrochloroBenzene of karst caves microorganisms

the biochemical mechanisms of resistance to persistent organic xenobiotic p-nitrochlorobenzene (NCB) of bacterial strains isolated from two cave clays ecosystems Mushkarova yama (Podolia, Ukraine) and kuybyshevskaya (Western Caucasus, abkhazia) have been established. It has been determined that chemoorganotrophic karst caves strains could interact with NCB and transform it reducing the nitro group with formation of p-chloroaniline (Cla) followed by further destruction of NCB aromatic ring. this explained high resistance of caves strains to NCB. the studied strains could potentially be used in wastewater treatment from nitrochloraromatic compounds.


materials and methods
The objects of study were chemoorganotrophic aerobic microbial communities and dominant 1  strains isolated from the cave clays.One clay sample was collected in the farthest point from the entrance of the Mushkarova Yama labyrinth cave laid in the Neogenic gypsum in Podolia, Ukraine.Another sample was collected in the bottom hall of Kuybyshevskaya vertical cave formed in the Jurassic limestones of the Western Caucasus, at the 1 km depth.After collecting both samples were stored in a refrigerator at 4 °C in sealed plastic bags.Microorganisms isolated on agar media: nutrient agar (HiMedia Laboratories Pvt.Ltd.) and oligotrophic agar (OA).Concentration of organic compounds in the medium is determined by permanganate redox titration method [5].For oligotrophic medium (OA) nutrient broth (HiMedia Laboratories Pvt.Ltd.) was diluted 10 times with distilled water and agarized (15 g/l).Colonies morph types obtained on the agar medium from maximum ten-fold dilutions of clays were reisolated three times to check the purity of the culture and in order to get single colonies.Thus 25 dominant strains were isolated from the studied cave clays.
Strains resistance to NCB were determined in a liquid medium (NB, oligotrophic broth) in a concentration gradient of the xenobiotic (50-300 mg/l).Controlled parameter was the biomass growth determined photocolorimetrically by optical density 1 Dominant strains in the microbial community means isolated from maximum ten-fold dilutions of caves clays samples.експериментальні роботи doi: http://dx.doi.org/10.15407/ubj87.04.032 of the liquid culture on KFK-2MP (ZOMZ, Russia) (λ = 540 nm).Inoculated medium without xenobiotic was used as a growth control.
determination of the strains ability to transform xenobiotic.Microorganisms are cultured in NCB concentration gradient (50-300 mg/l) in NB medium during three days.Concentration of NCB and chloroaniline (ClA) was determined by mass spectrometry using gas chromatography-mass-spectrum system Agilent 6890N/5973inert (capillary column HP-5MS (J&W Scientific, USA)).Gas carrier -helium; initial column temperature -150 °C; final column temperature -250 °C; temperature gradient -4 ° C/min; interface temperature -280 °C; type ionization -electron impact; ionization energy -70 eV.For this, the culture liquid was centrifuged (1700 g, 15 min).Hexane (0.5 ml) was added to the supernatant (0.7 ml) and suspended for 3 min.Next, the suspension was centrifuged (360 g, 10 min) to separate phases.Thereafter, hexane was collected (0.4 ml) and analyzed.Data processing of gas chromatography-mass spectrometry analysis was performed using the computer program ChemStation and integrated database of mass spectra NIST 02.
All experiments were run thrice.Experimental data were analyzed by statistical methods in Excel, P ≤ 0.05.

results and Discussion
Mushkarova Yama and Kuybyshevskaya cave clay samples were collected in zones free of anthropogenic load, which provided no influence of an-thropogenic factors on microbial communities.We isolated 25 bacterial strains from cave clay samples.The majority was highly resistant to NCB.Such high resistance could be explained by the fact that they were able to transform xenobiotic.There three most probable ways of nitroclorinearomatic compounds transformation: nitro group reduction, dehalogenation and aromatic ring reduction [6][7][8].
Previously it was shown the non-specific p-nitrochlorobenzene reduction on the example of facultative and obligate anaerobic bacteria museum cultures [9].Microorganisms can degrade xenobiotic in two ways: reduction to p-chloroaniline (p-ClA); reduction of the aromatic ring with subsequent cleava ge of the cyclic compound.We have shown that consistent transformation of NCB by these two ways was possible as well (Fig. 1).At the first stage NCB transformed to ClA.At the second stage the concentration of ClA was reduced and apparently aromatic ring was destroyed.
A consistent transformation of NCB could be observed on the example of two strains P3 and P11 isolated from Mushkarova Yama karst cave clay.Strain P3 transformed NCB to ClA almost completely and halved its concentration.Strain P11 also reduced NCB concentration in comparing with its initial concentration (50 mg/l) and destroyed the ClA almost completely.
The absence of stoichiometric ratio between the initial and final concentrations of NCB and ClA indicated deep xenobiotic degradation, apparently due to the destruction of aromatic ring.

Fig. 2. Reducing of NCB concentration by copio-and oligocarbotrophic cave strains (initial concentration is 100 mg/l NCB). Note: strains with the name "P" -Mushkarova yama, strains "kC" -kuybyshevskaya cave (M ± m, n = 3)
Copio-and oligocarbotrophic strains of both karst caves have shown the ability to interact with NCB (Fig. 2).The average reducing of NCB concentration by studied strains was 30%.The most active strains KC8, KC13 decreased xenobiotic concentration 2 times during three days of cultivation.
Maximum permissible concentrations of NCB were determined for studied strains (Table ).Almost all strains were resistant to very high concentrations of xenobiotic -100-300 mg/l.It should be noted that resistance to NCB did not depend on strains pigmentation.
Taking into account that the concentration of NCB in such wastewater does not exceed 50 mg/l, we could assume high effectiveness of xenobiotic destruction [10,11].