جداسازی و شناسایی باکتری های تجزیه کننده تولوئن از مناطق آلوده به نفت رودخانه قره سو در کرمانشاه
محورهای موضوعی : میکروب شناسی محیطی
1 - کارشناس ارشد، گروه زیست شناسی، دانشکده علوم پایه، دانشگاه آزاد اسلامی واحد سنندج
2 - استادیار، گروه زیست شناسی، دانشکده علوم پایه، دانشگاه آزاد اسلامی واحد سنندج
کلید واژه: تجزیه زیستی, تولوئن, رودخانه قره سو, سودوموناس پوتیدا, پساب پالایشگاه نفت,
چکیده مقاله :
سابقه و هدف: اصلاح زیستی یکی از روش های پاکسازی آلودگی های نفتی است که به دلیل مزایایی مانند هزینه اندک، کارایی بالا و سازگاری با محیط زیست، در سال های اخیر بسیار مورد توجه بوده است. رودخانه قره سو نیز یکی از مناطقی است که به دلیل مجاورت با پالایشگاه نفت کرمانشاه، در سال های اخیر دچار آلودگی نفتی شده است. این مطالعه با هدف جداسازی و شناسایی باکتری های تجزیه کننده تولوئن از مناطق آلوده به نفت رودخانه قره سو در کرمانشاه انجام شد. مواد و روش ها: این مطالعه به صورت تجربی بر روی نمونه های آب، خاک و لجن فعال مناطق آلوده انجام شد. دو جدایه تجزیه کننده تولوئن با استفاده از غنی سازی بر روی محیط کشت انتخابی حاوی تولوئن به دست آمدند. این جدایه ها با روش های ریخت شناسی کلنی، رنگ آمیزی گرم، آزمون های بیوشیمیایی و توالی یابی ژن 16S rRNA شناسایی شدند. همچنین میزان حذف تولوئن توسط جدایه ها با روش کروماتوگرافی گازی ارزیابی گردید. یافته ها: هر دو جدایه متعلق به گونه باکتریایی سودوموناس پوتیدا بودند. با استفاده از روش کروماتوگرافی گازی ثابت شد که جدایه ها توانستند تولوئن موجود در محیط کشت (با غلظت v/v 0.5 درصد) را در مدت 72 ساعت به ترتیب به میزان 89% و 87% تجزیه نمایند. همچنین این جدایه ها قادر بودند شرایط نامساعد دمایی، pH و اسمولاریته را تحمل نمایند. همچنین اثبات گردید که این جدایه ها قادرند در حضور دیگر آلاینده های نفتی (بنزن، اتیل بنزن، زایلن) نیز به طور کارآمد به فعالیت و رشد خود ادامه دهند. نتیجه گیری: نتایج نشان داد که این جدایه ها به دلیل توانایی رشد در غلظت بالای تولوئن و تجزیه طیف وسیعی از آلاینده ها می توانند کارآیی بالایی برای حذف آلاینده های نفتی از محیط زیست داشته باشند.
Background & Objectives: Biodegradation is one of the most useful methods for elimination of oil spills and is recently considered as a promising approaches due to numbers of advantages, including low costs, high efficiency and being environment friendly. Gharehsoo river is one of those regions which have been contaminated by oil spills during recent years due to its vicinity to Kermanshah Oil Refining Company. This study was aimed to isolate and identify the toluene-degrading bacteria from oil spills in Gharehsoo river located at Kermanshah city. Materials & Methods: In this experimental study, the samples were collected from water, soil and active sludge of the contaminated areas. Two isolates were achieved by enrichment of the samples into a selective medium containing toluene. Then, the isolates were identified using morphology, Gram staining, biochemical methods and 16S rRNA sequencing. Also, the ability of isolates to eliminate toluene was testes based on Gas chromatography. Results: Both isolates were identified as Pseudomonas putida strains. Gas chromatography tests showed that the isolates 1 and 2 were able to degrade toluene into the selective medium (0.5% v/v) 89% and 87%, at 72 C, respectively. The isolates were also able to resist and grow under harsh conditions of temperature, pH and osmolality. It was proved that the isolates were able to continue their activity and growth in the presence of other crude oil pollutants (benzene, xylene, ethyl-benzene). Conclusion: Our results showed that these isolates were very efficient for elimination of oil pollutants due to their high growth rate in the presence of relatively high toluene concentration and to the ability to degrade a wide range of oil toxic compounds.
1. Atlas RM. Petroleum biodegradation and oil spill bioremediation. Marine Pollution Bull. 1995; 31(4): 178-182.
2. Hinchee RE, Kittel JA, Reisinger HJ. Applied bioremediation of petroleum hydrocarbons. Battelle Press, Columbus, OH (United States), 1995.
3. Harayama S, Kishira H, Kasai Y, Shutsubo K. Petroleum biodegradation in marine environments. J Mol Microbiol Biotechnol. 1999; 1(1): 63-70.
4. Mohajeri L, Aziz HA, Isa MH, Zahed MA. A statistical experiment design approach for optimizing biodegradation of weathered crude oil in coastal sediments. Bioresource Technol. 2010;101(3):893-900.
5. Zhou J, Yu X, Ding C, Wang Z, Zhou Q, Pao H. Optimization of phenol degradation by Candida tropicalis Z-04 using Plackett-Burman design and response surface methodology. J Environ Sci. 2011; 23(1): 22-30.
6. Jin HM, Choi EJ, Jeon CO. Isolation of a BTEX-degrading bacterium, Janibacter sp. SB2, from a sea-tidal flat and optimization of biodegradation conditions. Bioresource Technol. 2013;145: 57-64.
7. Weelink SB, van Eekert MA, Stams AM. Degradation of BTEX by anaerobic bacteria: physiology and application. Rev Environ Sci Biotechnol. 2010; 9(4): 359-385.
8. Margesin R, Schinner F. Biodegradation and bioremediation of hydrocarbons in extreme environments. Appl Microbiol Biotechnol. 2001; 56(5-6): 650-663.
9. Al-Wasify RS, Hamed SR. Bacterial biodegradation of crude oil using local isolates. Int J Bacteriol. 2014. Doi: 10.1155/2014/863272
10. Wang L, Shao ZZ. Isolation and characterization of 4 benzene/toluene-degrading bacterial strains and detection of related degradation genes. Acta Microbiologica Sinica. 2006; 46(5): 753-757.
11. Mukherjee S, Bardolui NK, Karim S, Patnaik VV, Nandy RK, Bag PK. Isolation and characterization of a monoaromatic hydrocarbon-degrading bacterium, Pseudomonas aeruginosa from crude oil. Journal of environmental science and health Part A, Toxic/hazardous substances & environmental engineering. 2010; 45(9): 1048-1053.
12. Wang L, Qiao N, Sun F, Shao Z. Isolation, gene detection and solvent tolerance of benzene, toluene and xylene degrading bacteria from nearshore surface water and Pacific Ocean sediment. Extremophiles : life under extreme conditions. 2008; 12(3): 335-342.
13. Kim JM, Jeon CO. Isolation and characterization of a new benzene, toluene, and ethylbenzene degrading bacterium, Acinetobacter sp. B113. Current microbiology. 2009; 58(1): 70-75.
14. Afrouzossadat HA, Giti E, SeyedMahdi G. The role of exopolysaccharide, biosurfactant and peroxidase enzymes on toluene degradation by bacteria isolated from marine and wastewater environments. Jundishapur J Microbiol. 2012; 3: 479-485.
15. Slepecky RA, Hemphill HE. The genus Bacillus- nonmedical. The prokaryotes: Springer; 2006; pp: 530-562.
16. Abari AH, Emtiazi G, Ghasemi SM, Roghanian R. Isolation and characterization of a novel toluene-degrading bacterium exhibiting potential application in bioremediation. Jundishapur J Microbiol. 2013; 6(3): 256-261.
17. Eghtesadi-Araghi P, Haffner P, Drouillard K, Maghsoudlou W. Polycyclic aromatic hydrocarbons contaminants in Black-lip (Pearl) Oyster Pinctada margaritifera from Kish Island (Persian Gulf). Iran J Fisheries Sci. 2011; 10(1): 25-34.
18. Hassanshahian M, Emtiazi G, Cappello S. Isolation and characterization of crude-oil-degrading bacteria from the Persian Gulf and the Caspian Sea. Marine Pollution Bull. 2012; 64(1): 7-12.
19. Inoue A, Yamamoto M, Horikoshi K. Pseudomonas putida which can grow in the presence of toluene. Appl Environ Microbiol. 1991; 57(5): 1560-1562.
20. Robledo-Ortíz JR, Ramírez-Arreola DE, Pérez-Fonseca AA, Gómez C, González-Reynoso O, Ramos-Quirarte J. Benzene, toluene, and o-xylene degradation by free and immobilized P. putida F1 of postconsumer agave-fiber/polymer foamed composites. Int Biodeterioration Biodegradation. 2011; 65(3): 539-546.
21. Worsey MJ, Williams PA. Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol. 1975; 124(1): 7-13.
22. Rahman RN, Mahamad S, Salleh AB, Basri M. A new organic solvent tolerant protease from Bacillus pumilus 115b. J Indust Microbiol Biotechnol. 2007; 34(7): 509-517.
23. Zylstra GJ, McCombie WR, Gibson DT, Finette BA. Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon. Appl Environ Microbiol. 1988; 54(6): 1498-1503.
24. Hemalatha S, Veera Manikandan P. Characterization of aromatic hydrocarbon rading bacteria from petroleum contaminated sites. J Environ Protection. 2011; 2(03): 243.
25. Reva ON, Weinel C, Weinel M, Böhm K, Stjepandic D, Hoheisel JD. Functional genomics of stress response in Pseudomonas putida KT2440. J Bacteriol. 2006; 188(11): 4079-4092.
26. You Y, Shim J, Cho CH, Ryu MH, Shea PJ, Kamala-Kannan S. Biodegradation of BTEX mixture by Pseudomonas putida YNS1 isolated from oil-contaminated soil. J Basic Microbiol. 2013; 53(5): 469-475.
_||_1. Atlas RM. Petroleum biodegradation and oil spill bioremediation. Marine Pollution Bull. 1995; 31(4): 178-182.
2. Hinchee RE, Kittel JA, Reisinger HJ. Applied bioremediation of petroleum hydrocarbons. Battelle Press, Columbus, OH (United States), 1995.
3. Harayama S, Kishira H, Kasai Y, Shutsubo K. Petroleum biodegradation in marine environments. J Mol Microbiol Biotechnol. 1999; 1(1): 63-70.
4. Mohajeri L, Aziz HA, Isa MH, Zahed MA. A statistical experiment design approach for optimizing biodegradation of weathered crude oil in coastal sediments. Bioresource Technol. 2010;101(3):893-900.
5. Zhou J, Yu X, Ding C, Wang Z, Zhou Q, Pao H. Optimization of phenol degradation by Candida tropicalis Z-04 using Plackett-Burman design and response surface methodology. J Environ Sci. 2011; 23(1): 22-30.
6. Jin HM, Choi EJ, Jeon CO. Isolation of a BTEX-degrading bacterium, Janibacter sp. SB2, from a sea-tidal flat and optimization of biodegradation conditions. Bioresource Technol. 2013;145: 57-64.
7. Weelink SB, van Eekert MA, Stams AM. Degradation of BTEX by anaerobic bacteria: physiology and application. Rev Environ Sci Biotechnol. 2010; 9(4): 359-385.
8. Margesin R, Schinner F. Biodegradation and bioremediation of hydrocarbons in extreme environments. Appl Microbiol Biotechnol. 2001; 56(5-6): 650-663.
9. Al-Wasify RS, Hamed SR. Bacterial biodegradation of crude oil using local isolates. Int J Bacteriol. 2014. Doi: 10.1155/2014/863272
10. Wang L, Shao ZZ. Isolation and characterization of 4 benzene/toluene-degrading bacterial strains and detection of related degradation genes. Acta Microbiologica Sinica. 2006; 46(5): 753-757.
11. Mukherjee S, Bardolui NK, Karim S, Patnaik VV, Nandy RK, Bag PK. Isolation and characterization of a monoaromatic hydrocarbon-degrading bacterium, Pseudomonas aeruginosa from crude oil. Journal of environmental science and health Part A, Toxic/hazardous substances & environmental engineering. 2010; 45(9): 1048-1053.
12. Wang L, Qiao N, Sun F, Shao Z. Isolation, gene detection and solvent tolerance of benzene, toluene and xylene degrading bacteria from nearshore surface water and Pacific Ocean sediment. Extremophiles : life under extreme conditions. 2008; 12(3): 335-342.
13. Kim JM, Jeon CO. Isolation and characterization of a new benzene, toluene, and ethylbenzene degrading bacterium, Acinetobacter sp. B113. Current microbiology. 2009; 58(1): 70-75.
14. Afrouzossadat HA, Giti E, SeyedMahdi G. The role of exopolysaccharide, biosurfactant and peroxidase enzymes on toluene degradation by bacteria isolated from marine and wastewater environments. Jundishapur J Microbiol. 2012; 3: 479-485.
15. Slepecky RA, Hemphill HE. The genus Bacillus- nonmedical. The prokaryotes: Springer; 2006; pp: 530-562.
16. Abari AH, Emtiazi G, Ghasemi SM, Roghanian R. Isolation and characterization of a novel toluene-degrading bacterium exhibiting potential application in bioremediation. Jundishapur J Microbiol. 2013; 6(3): 256-261.
17. Eghtesadi-Araghi P, Haffner P, Drouillard K, Maghsoudlou W. Polycyclic aromatic hydrocarbons contaminants in Black-lip (Pearl) Oyster Pinctada margaritifera from Kish Island (Persian Gulf). Iran J Fisheries Sci. 2011; 10(1): 25-34.
18. Hassanshahian M, Emtiazi G, Cappello S. Isolation and characterization of crude-oil-degrading bacteria from the Persian Gulf and the Caspian Sea. Marine Pollution Bull. 2012; 64(1): 7-12.
19. Inoue A, Yamamoto M, Horikoshi K. Pseudomonas putida which can grow in the presence of toluene. Appl Environ Microbiol. 1991; 57(5): 1560-1562.
20. Robledo-Ortíz JR, Ramírez-Arreola DE, Pérez-Fonseca AA, Gómez C, González-Reynoso O, Ramos-Quirarte J. Benzene, toluene, and o-xylene degradation by free and immobilized P. putida F1 of postconsumer agave-fiber/polymer foamed composites. Int Biodeterioration Biodegradation. 2011; 65(3): 539-546.
21. Worsey MJ, Williams PA. Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol. 1975; 124(1): 7-13.
22. Rahman RN, Mahamad S, Salleh AB, Basri M. A new organic solvent tolerant protease from Bacillus pumilus 115b. J Indust Microbiol Biotechnol. 2007; 34(7): 509-517.
23. Zylstra GJ, McCombie WR, Gibson DT, Finette BA. Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon. Appl Environ Microbiol. 1988; 54(6): 1498-1503.
24. Hemalatha S, Veera Manikandan P. Characterization of aromatic hydrocarbon rading bacteria from petroleum contaminated sites. J Environ Protection. 2011; 2(03): 243.
25. Reva ON, Weinel C, Weinel M, Böhm K, Stjepandic D, Hoheisel JD. Functional genomics of stress response in Pseudomonas putida KT2440. J Bacteriol. 2006; 188(11): 4079-4092.
26. You Y, Shim J, Cho CH, Ryu MH, Shea PJ, Kamala-Kannan S. Biodegradation of BTEX mixture by Pseudomonas putida YNS1 isolated from oil-contaminated soil. J Basic Microbiol. 2013; 53(5): 469-475.