Two environmental bacterial isolates KCM-R5 and KCM-RG5 were selected from xenobiotic-polluted environment. KCM-R5 was identified as Pseudomonas rhodesiae and KCM-RG5 as Bacillus subtilis. KCM-R5 demonstrated tolerance to heavy metals and KCM-RG5 to heavy metals and phenol derivatives. Both strains were studied for xenobiotic biotransformation

in order to contribute towards bioremediation of polluted environments.

Pseudomonas rhodesiae KCM-R5 and Bacillus subtilis. KCM-RG5 possess unusual ability to utilize ortho-nitrophenol (o-NP) and 2,4- dichlorophenoxyacetic acid (2,4-D). o-NP and 2,4-D were added at concentration 30 mg/l. The possible indictive/inhibiting effect of Pb cations (40mg/l) was also studied. Pseudomonas rhodesiae KCM-R5 removed 86 % of o-NP and below 1% of 2,4-D. Bacillus subtilis KCM-RG5 eliminated 83% of o-NP andunder 1% of 2,4-D. Biotransformation effectiveness of o-NP reached 95-100% in contrast to 2,4-D where the effectiveness was just 15-20%. Cell morphological changes were registered during the biotransformation processes. The obtained results could contribute to manage bioremediation processes in polluted with heavy metals and phenol derivatives environments.

. 1, . 2, . 2, . 

Golovinsky, Evgeny

Ivanov, Ivailo

Satchanska, Galina

Topalova, Yana

English

New Bulgarian University Scholar Electronic Repository

 Biotechnol. & Biotechnol. Eq. 20/2006/1 97XENOBIOTIC BIOTRANSFORMATION POTENTIAL  OF PSEUDOMONAS RHODESIAE KCM-R5 AND  BACILLUS SUBTILIS KCM-RG5, TOLERANT TO  HEAVY METALS AND PHENOL DERIVATIVES  G. Satchanska1, Y. Topalova2, I. Ivanov2, E. Golovinsky1 Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria1 Sofia University "St. Kliment Ohridski", Faculty of Biology, Sofia, Bulgaria2  ABSTRACT Two environmental bacterial isolates KCM-R5 and KCM-RG5 were selected from xenobi-otic-polluted environment. KCM-R5 was identified as Pseudomonas rhodesiae and KCM-RG5 as Bacillus subtilis. KCM-R5 demonstrated tolerance to heavy metals and KCM-RG5 – to heavy metals and phenol derivatives. Both strains were studied for xenobiotic bio-transformation in order to contribute towards bioremediation of polluted environments. Pseudomonas rhodesiae KCM-R5 and Bacillus subtilis. KCM-RG5 possess unusual ability to utilize ortho-nitrophenol (o-NP) and 2,4- dichlorophenoxyacetic acid (2,4-D). o-NP and 2,4-D were added at concentration 30 mg/l. The possible indictive/inhibiting effect of Pb cations (40mg/l) was also studied. Pseudomonas rhodesiae KCM-R5 removed 86 % of o-NP and below 1% of 2,4-D. Bacillus subtilis KCM-RG5 eliminated 83% of o-NP and under 1% of 2,4-D. Biotransformation effectiveness of o-NP reached 95-100% in contrast to 2,4-D where the effectiveness was just 15-20%. Cell morphological changes were regi-stered during the biotransformation processes. The obtained results could contribute to manage bioremediation processes in polluted with heavy metals and phenol derivatives environments.  Introduction During last decades enormous amount in-dustrial pollutants was released in the envi-ronment. The remediation technologies using bacteria appeared to be one of the most effective and environment-friendly techniques. Many investigations on bacte-ria converting metals (1,2) and phenol de-rivatives (5, 6, 7, 8, 12) were performed. But little is known about the bacterial ac-tivity in the environments, simultaneously contaminated with above mentioned pollu-tants. Possible inductive/inhibiting effect of metal cations on the biotransformation of aromatic compounds were reported in lim-ited number of publications (3, 9). The in-vestigation of bacteria isolated from poly-contaminated environments is of special interest. Such autochthonic bacteria showed to be effective in the bioindication of environment contamination, biotrans-formation of xenobiotics as well as in the optimisation the bioremediation processes. Present paper describes biotransforma-tion abilities to nitro-and chlorophenols by two bacterial isolates collected in the re-gion of the Pb-Zn metallurgical factory- “KCM” and the factory for producing of pesticides “AGRIA”, Bulgaria. Additiona-lly was investigated the probable induc-tive/inhibiting effect of Pb cations on the biotransformation processes. The study was performed in order to contribute, using above mentioned bacterial strains, for  Biotechnol. & Biotechnol. Eq. 20/2006/1 98bioremiation of similar environmental pol-lutants.  Materials and Methods Microorganisms  KCM-R5 was isolated from metal-contami-nated industrial soil and KCM-RG5- from a polluted groundwater, collected near the KCM and AGRIA factories. Both isolates were characterized using general and mo-lecular microbiology methods (not pub-lished data). Isolate KCM-R5 was affiliated to Pseudomonas rhodesie and isolate KCM-RG5- to Bacillus subtilis. Isolation procedure was performed on selective me-dia according to standard method (4). Both strains were maintained at 4 ºC and stored at -20 ºC.  2,4-D and o-nitrophenol biotransformation Two aromatic compounds: 2,4-dichloro-phenoxiacetic acid (2,4-D) and o-nitrophe-nol (o-NP) were studied for their biotrans-formation by Pseudomonas rhodesiae KCM-R5 and Bacillus subtilis KCM-RG5. Strains were grown for 144h/28 ºC/220 rpm on 2,4-D and o-NP (30 mg/l) as a sole carbon and energy source. The biomass accumulation, residual amount of xenobi-otics and cell morphology were studied at initial time, 24h, 48h, 72h and 96h. The effectiveness of biotransformation (Eff.) and specific rate of biodegradation (SRB) were calculated according to the following formulas:   Eff. = К0 _ Кt / [x 100]  where К0 - quantity of xenobiotic in the initial moment;  Кt -quantity of xenobiotic in the moment t.   SRB = К0 _ Кt / (t x biomass)      [мМol/h.mg Protein]. Modulative effect of Pb cations Pb cations (40 mg/l) as lead acetate were added in additional parallel experiment in order to investigate their probable induc-tive/inhibiting effect on the biotransformation.  Analytical methods Biomass accumulation was analyzed in the supernatant as protein dry weight. Residual amount of o-NP and 2,4-D was examined by HPLC (Waters, U.K.) on a reverse phase C18 column with methanol-water liquid phase.  Study of cell morphology  Variation of cell morphology was studied using light microscopy at initial time, 24h, 48h, 72h and 96h. Results and Discussion Environmental isolates Pseudomonas rho-desiae KCM-R5 and Bacillus subtilis KCM-RG5 were studied for their abilities to utilize nitro- and chlorophenols as a sole carbon and energy source. These products are hazardous pollutants released by chemical and plant-protection industry (1, 6). They demonstrate toxic, mutagenic and terratogenic effect in humans (13). Bacterial strains were isolated from pol-luted soil and water near the Pb-Zn smelter and factory for producing pesticides in South Bulgaria. As it was published earlier the area was strongly polluted by heavy metals. A moderate pesticides contamina-tion was detected (10, 11).  The isolate KCM-R5 is determined as Gram(-), motile and ovalshaped bacteria while the isolate KCM-RG5 is a Gram(+), motile and rod-shaped one. 16S rDNA of both strains was sequenced and aligned using BLAST program to the sequences available in EMBL database. Sequences were deposited in the Gene Bank under accession numbers AJ830707 and AJ830708. As it was published earlier (10, 11) Pseudomonas rhodesiae KCM-R5 demonstrated tolerance to 2,4- D and Ba-cillus subtilis KCM-RG5- tolerance to 2,4-D as well as Pb, Cu, Zn. Biotransformation of o-NP and 2,4-D at concentration 30 mg/l was studied using batch method. To evaluate the probable indictive/inhibiting effect of metal cations an addition of Pb cations at concentration 40 mg/l were added in parallel experiment. (see Table, in bold).  The  reason to choose   Biotechnol. & Biotechnol. Eq. 20/2006/1 99TABLE Experimental scheme  Microorganisms, phenol derivatives and metal in the experiment 1. Pseudomonas rhodesiae KCM-R5+ 30mg/l o-NP 2. Pseudomonas rhodesiae KCM-R5+ 30mg/l o-NP + 40 mg/l Pb2+ 3. Bacillus subtilis KCM-RG5 + 30mg/l o-NP 4. Bacillus subtilis KCM-RG5 + 30mg/l o-NP + 40 mg/l Pb2+ 5. Pseudomonas rhodesiae KCM-R5 + 30mg/l 2,4-D 6. Pseudomonas rhodesiae KCM-R5 + 30mg/l 2,4-D + 40 mg/l Pb2+ 7. Bacillus subtilis KCM-RG5 + 30mg/l 2,4-D 8. Bacillus subtilis KCM-RG5 + 30mg/l 2,4-D + 40 mg/l Pb2+ in bold- parallel experiment with addition of Pb cations.  Pb as metal modulator of biotransformation is that Pb was the strongest pollutant in the area (1.5 - 2560 ppm) and exceed in times the maximum permission standard (MPL) (10, 11).  The dynamic of biomass accumulation and residual amount of xenobiotic is shown in Fig. 1.  Growing on o-NP Pseudomonas pro-duced more biomass (0.3g dry weight/l) compared то Bacillus (0.8 g dry weight/l) (see Fig.1-a) Slight stimulating effect of Pb cations was found in the parallel experi-ment with Pb (see Fig.1–b). When grew on 2,4-D Pseudomonas again demonstrated higher biomass accumulation (0.7 g dry weight/l) while Bacillus biomass was re-duced to 0.2 g dry weight/l. As seen from the figure Pb cations did not influence the biomass accumulation during the 2,4-D biotransformation (see Fig. 1). In the Fig. 1 is also presented the residual amount of xenobiotics in the water phase. The reduced xenobiotic concentration (0.1 - 0.7 mg/l) in the initial time of experiment possibly resulted from its fast cell uptake. It might be due to mutagenic effect of the aromatic compounds on the cell wall per-meability. Both bacterial strains removed significant amount of o-NP within 96th hour (between 25 and 26 mg/l) (see Fig. 1). In contrast 2,4-D was weakly eliminated (between 0 and 3 mg/l) by both bacteria (see Fig. 1). The possible reason for the slight 2,4-D removal is the difficulty of chlorine atom removal and consecutive break of the aromatic nucleus.  In Fig. 2 is shown the effectiveness of the biotransformation process. For o-NP it reached 95 to 100 % (see Fig. 2-a) while for 2,4-D it was only 15 to 20 % (see Fig. 2-b). As seen from the figure the SRB is significantly high: 5 to 12 мМol/h.mg protein during biotransformation of o-NP in contrast to 2,4-D biotransformation where SRB is just 0.3 to 0.6 мМol/h.mg protein (see Fig. 2).  Morphological changes in bacteria was studied at initial time, 24 h, 48 h, and 96 h. Regarding Pseudomonas rhodesiae KCM-R5 cells became smaller and roundshaped. For Ba-cillus subtilis KCM-RG5 a fragmentation and sporeforming was detected. Conclusions Pseudomonas rhodesiae KCM-R5 и Ba-cillus subtilis KCM-RG5 are able to utilize o-nitrophenol and 2,4-dichlorophenoxya-cetic acid as a sole carbon and energy source. They eliminated between 76 and 83 % of o- NP and just 1 % and lower of 2,4-D. The effectiveness of o-NP biotransfor-mation reached 90 %. Pb cations did not influence significantly the biotransforma-tion processes. This fact proposes the de-velopment of a stability of the biodegrada-tion   apparatus  towards  the  Pb-inhibiting   Biotechnol. & Biotechnol. Eq. 20/2006/1 100a)          Pseudomonas + o-NP  86 % eliminated xenobiotic        Bacillus + o-NP 83 % eliminated xenobiotic           Pseudomonas + 2,4-D   0 % eliminated xenobiotic        Bacillus + 2,4-D 0,1 % eliminated xenobiotic   b)             Pseudomonas + o-NP+Pb  83 % eliminated xenobiotic     Bacillus + o-NP+Pb 76 % eliminated xenobiotic              Pseudomonas + 2,4-D+Pb  0,03 % eliminated xenobiotic        Bacillus + 2,4-D+Pb 0,2 % eliminated xenobiotic     ■  Residual amount of xenobiotic  ▲     Dry weight Fig. 1. Biomass accumulation and elimination of a). o-NP and 2,4-D with Pb as modulator- b). Experiment was performed in triplicate.  Biotechnol. & Biotechnol. Eq. 20/2006/1 101                     Pseudomonas + o-NP          Pseudomonas + o-NP + Pb                              Bacillus + o-NP                              Bacillus + o-NP + Pb a) Effectiveness (Eff): 95-100 %,  Specific rate of biodegradation (SRB): 5-12 [mMol/h.mg Protein]/24 h                               Pseudomonas + 2,4-D               Pseudomonas + 2,4-D + Pb                           Bacillus + 2,4-D                 Bacillus + 2,4-D + Pb b) Effectiveness (Eff): 15-20 %,  Specific rate of biodegradation (SRB): 0.3 - 0.6 [mMol/h.mg Protein]/24 h Fig. 2. Effectiveness and specific rate of biotransformation processes of a). o-NP and b). 2,4-D. Experiment was performed in triplicate.  Biotechnol. & Biotechnol. Eq. 20/2006/1 102effect. Cell morphology changes as size-reducing, roundshaping, fragmentation and sporeforming of bacterial cells were regis-tered during biotransformation. The ob-tained resultes showed that above men-tioned bacterial strains could be applied succesfully in the bioindication of envi-ronmental pollution. Additionally they could contribute for bioremediation of en-vironments, simultaneously contaminated with heavy metals and phenol derivatives.  Acknowledgements Sequence analysis was done in Molecular Microbiology Department of the Instutute of radiochemistry, Forcshungszentrum Rossendorf, Dresden, Germany. The authors also thank G. Aleksieva and L. Bojilova from Environmental Biotechno-logy Lab, Sofia University for their valu-able laboratory assistance. This work was supported by grant MU-B-03 financed by the Ministry of Education and Science, Sofia, Bulgaria. REFERENCES 1.  Ehrlich H. (1997) Appl. Microbiol. Biotechnol., 48, 687-692. 2.  Fowle D., Fein J. (2001) Geomicrobiology Jour-nal, 18, 77-91. 3.  Grekova M., Kachamakova N., Dencheva V., Evtimova V., Harizanova D., Chapanova R., Dim-kov R., Topalova Y. 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Annuare de l,Universite de Sofia ”St.

Bioaccumulation and precipitation of metals by microorganisms. In:

Effect of heavy metals on ortho-Nitrophenol biodegradation by Pseudomonas aeruginosa B8. In: Bioremediation of Recalcitrant Organics,

Xenobiotic biotransformation potential of Pseudomonas rhodesiae KCM-R5 and Bacillus subtilis KCM-RG5, tolerant to heavy metals and phenol derivatives

G. Satchanska

Y. Topalova

I. Ivanov

E. Golovinsky

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Xenobiotic biotransformation potential of Pseudomonas rhodesiae KCM-R5 and Bacillus subtilis KCM-RG5, tolerant to heavy metals and phenol derivatives

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