جداسازی و شناسایی فیلوژنتیکی باکتری های بومی نفت خوار از خاک منطقه کارون اهواز
محورهای موضوعی : زیست فناوری میکروبیمحمد حسین آرش اسدی راد 1 , مهناز مظاهری اسدی 2 , حمید راشدی 3 , طاهر نژاد ستاری 4
1 - دکتری، دانشگاه آزاد اسلامی، واحد علوم و تحقیقات، گروه زیست شناسی، تهران
2 - استاد، سازمان پژوهش های علمی و صنعتی ایران، پژوهشکده زیست فناوری، تهران
3 - دانشیار، گروه مهندسی شیمی، دانشگاه تهران، تهران
4 - دانشیار، دانشگاه آزاد اسلامی، واحد علوم و تحقیقات، گروه زیست شناسی، تهران
کلید واژه: کروماتوگرافی گازی, نفت خام, زیست پالایی, باکتری های خاک,
چکیده مقاله :
سابقه و هدف: زیست پالایی روشی مقرون به صرفه و پاک به منظور تیمار مکان های آلوده است و می تواند منجر به معدنی شدن هیدروکربن ها گردد. میکروب های بومی موجود در منطقه می توانند نقش مؤثری در این رابطه ایفا نمایند. این مطالعه با هدف جداسازی و شناسایی فیلوژنتیکی باکتری های بومی نفت خوار از خاک منطقه کارون اهواز انجام شد. مواد و روش ها: خاک آلوده به نفت منطقه کارون اهواز به صورت تصادفی و در شرایط استریل نمونه برداری شد. مقدار فسفر قابل جذب با استفاده از روش اولسن و مقادیر کربن، هیدروژن و نیتروژن توسط دستگاه CHN meter تعیین گردید. محیط کشت نمک های معدنی حاوی 2 درصد نفت خام به منظور جداسازی باکتری های نفت خوار استفاده شد. سویه نفت خوار با استفاده از روش چاهک گذاری غربال گردید. نمونه خاک با استفاده از روش سوکسله عصاره گیری و میزان کربن کل توسط روش کروماتوگرافی گازی مورد سنجش قرار گرفت. به منظور شناسایی سویه باکتریایی برتر از آزمون های بیوشیمیایی و روش مولکولی PCR استفاده گردید. یافته ها: در این مطالعه تعداد 44 سویه باکتریایی جداسازی گردید. در غربالگری اولیه 20 و در غربالگری ثانویه یک جدایه انتخاب و S31 نام گرفت. جدایه مورد نظر باسیلوس لیکنی فورمیس گزارش شد. جدایه منتخب نسبت به سویه استاندارد در محیط حاوی 2 درصد نفت خام رشد بهتری داشت. به طوری که نفت خام را در مدت زمان 30 روز گرماگذاری در دمای تقریبی 30 درجه سلیسیوس به میزان 84 درصد تجزیه نمود. نتیجه گیری: باسیلوس منتخب می تواند از 2 درصد نفت خام به عنوان منبع کربن و انرژی استفاده نماید. مطالعه فراتر آن در اجتماع باکتریایی پیشنهاد می گردد.
Background & Objectives: Bioremediation is the promising technology for the treatment of the contaminated sites since it is cost-effective and will lead to complete mineralization. This study was aimed to isolation and phylogenetic identification of indigenous oil-degrading bacteria from soil of Karoon area in Ahvaz. Materials & Methods: The crude oil contaminated soil of Karoon area in Ahvaz was sampled accidentally and under sterile condition. The amount of absorbable phosphorus was determined using Olson method and also, carbon, hydrogen and nitrogen by CHN meter device. Mineral salt medium containing 2% crude oil was used for isolation of oil eating bacteria. Following sieving the soil samples, the total carbon content of the soils were analysed by gas chromatography. Biochemical tests and PCR method were used to identify the dominant bacteria. Results: In this study, 44 bacterial strains were isolated, among them 20 isolates in the first and one in the second screening methods were selected, which was nominated as S31.This strain belonged to Bacillus licheniformis. The growth of the selected isolate in the media with 2% crude oil was better than the standard strain and remediated 84% of the crude oil in 30 days incubation time at about 30o C. Conclusion: The selected Bacillus could use 2% of crude oil as source of carbon and energy and we suggest further studies on this bacterium in bacterial consortia.
1. Tabari K, Tabari M. Biodegradation potential of hydrocarbons by bacterial diversity in soil. Iran J Environ Health Sci Eng. 2010; 7(4): 319-326.
2. Tanee FBG, Albert E. Reconnaissance assessment of long-term effects of crude oil spill on soil chemical properties and plant composition at Kwawa, Ogoni, Nigeria. J Environ Sci Technol. 2015; 8(6):320-329.
3. Peng R, Xiong A, Sue Y, Fu X, Gao F, Zhao W, Tian Y, Yao Q. Microbial biodegradation of polyaromatic hydrocarbons. FEMS Microbiol Rev. 2008; 32:927–955.
4. Xiong S, Li X, Chen J, Zhao L, Zhang H, Zhang X. Crude oil degradation by bacterial consortia under four different redox and temperature conditions. Appl Microbiol Biotechnol. 2015; 99(3):1451-1461.
5. Rodríguez DM, Andrade RFS, Ribeiro DLR, Rubio-Ribeaux D, Lima RA, Araújo HWC, Campos-Takaki GM. Bioremediation of petroleum derivative using biosurfactant produced by Serratia marcescens UCP/WFCC 1549 in low-cost medium. Int J Curr Microbiol App Sci. 2015; 4(7): 550-562.
6. Elmahdi AM, Abdul Aziz H, El-Gendy NS, Abu Amr SS, Nassar HN. Optimization of Libyan crude oil biodegradation by using solid waste date as a natural low-cost material. J Biorem Biodeg. 2014; 5:252-262.
7. Das N, Chandran P. Microbial degradation of petroleum hydrocarbon contaminants. Biotech Res Int. 2011; 6:1-13.
8. Wang Q, Zhang S, Li Y, Klassen W. Potential approaches to improving biodegradation of hydrocarbons for bioremediation of crude oil pollution. J Env Protec. 2011; 2:47-55.
9. Brooijmans RJW, Pastink MI, Siezen RJ. Hydrocarbon-degrading bacteria: the oil-spill clean-up crew. Microbial Biotech. 2009; 2(6):587–594.
10. Chikere CB, Okpokwasili GC. Bioreactor-based bioremediation of hydrocarbon-polluted Niger Delta marine sediment. Nigeria. Biotech. 2012; 2:53–66.
11. Yu S, Li S, Tang Y, Wu X. Succession of bacterial community along with the removal of heavy crude oil pollutants by multiple biostimulation treatments in the Yellow River Delta, China. J Env Sci. 2011; 23(9):1533–1543.
12. Mishra S, Jyot J, Kuhad RC, Lal B. Evaluation of inoculums addition to stimulate in situ bioremediation of oily-sludge-contaminated soil. Appl Environ Microbiol.2001; 67(4): 1675-1681.
13. Santisi S, Cappello S, Catalfamo M, Mancini G, Hassanshahian M, Genovese L, Giuliano L, Yakimov MM. Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium. Braz J Microbiol. 2015; 46(2):377-387.
14. Agarry SE, Aremu MO, Aworanti OA. Kinetic modeling and half-life study on enhanced soil bioremediation of Bonny light crude oil amended with crop and animal-derived organic wastes. J Pet Environ Biotech. 2013; 4(2):137-147.
15. Atlas RM. Hand book of Media for Environmental Microbiology. 2nd ed. New York. Taylor & Francis; 2005.
16. Prescott LM, Harley JP. Laboratory exercises in Microbiology. 5th ed. McGraw-Hill Publishers; 2001.
17. Arutchelvi M, Arkatkar A, Doble M, Bhaduri S, Uppara P. Biodegradation of polyethylene and polypropylene. India J Biotech. 2008; 8: 9-22.
18. CameronG, Rood D. Total petroleum hydrocarbon analysis by capillary gas chromatography: decreasing analysis times using small-diameter columns. J Chromato Sci. 1999; 37: 1-28.
19. Korenblum E, Seldin L. Shifts in alkaline-degrading bacteria genotypes during bioremediation of a vegetated coastal soil. World J Microbiol Biotech. 2009; 25:1667-1675.
20. Korenblum E, Seldin L. Shifts in alkane-degrading bacteria genotypes during bioremediation of a vegetated coastal soil. World J Microbiol Biotech. 2009; 25: 1667–1675.
21. Tabatabaee MS, Mazaheri Assadi M. Vacuum distillation residue upgrading by an indigenous Bacillus cereus. J Environ Health Sci Eng. 2013; 11:18-29.
22. Sathishkumar M, Binupriya AR, Baik SH, Yun, SE. Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium isolated from hydrocarbon contaminated areas. Clean.2008; 36(1): 92-96.
23. Wang Q, Zhang S, Li Y, Klassen W. Potential approaches to improving biodegradation of hydrocarbons for bioremediation of crude oil pollution. J Environ Protec. 2011; 2:47-55.
24. Brooijmans RJW, Pastink MI, SiezenRJ. Hydrocarbon-degrading bacteria: the oil-spill clean-up crew. Micro Biotech. 2009; 2(6):587–594.
25. Chikere CB, Okpokwasili, GC. Bioreactor-based bioremediation of hydrocarbon-polluted Niger Delta marine sediment, Nigeria. Biotech. 2012; 2:53–66.
26. Adebusoye SA, Ilori MO, Amund OO, Teniola OD, Olatope SO. Microbial degradation of petroleum hydrocarbons in a polluted tropical stream. World J Microbiol Biotech. 2007; 23(8):1149–1159.
27. Kebria Y, Khodadadi A, Ganjidoust H, Badkoubi A, Amoozegar MA. Isolation and characterization of a novel native Bacillus strain capable of degrading diesel fuel. Int J Environ Sci Tech. 2009; 6(3): 435-442.
28. Singh C, Lin J. Bioaugmentation efficiency of diesel degradation by Bacillus pumilus JLB and Acinetobacter calcoaceticus LT1 in contaminated soils. Afr J Biotech. 2010; 9(41):6881-6888.
29. Kumar A, Bisht BS, Joshi VD, DhewaT. Review on bioremediation of polluted environment: amanagement tool. Int J Environ Sci. 2011; 1(6):13-27.
30. Wongsa P, Tanaka M, Ueno A, Hasanuzzaman M, Yumoto I, Okuyama H. Isolation and characterization of novel strains of Pseudomonas aeruginosa and Serratia marcescens possessing high Efficiency to degrade gasoline, kerosene, diesel oil and lubricating oil. Curr Microbiol. 2004; 49: 415-422.
31. Chorom M, Sharifi HS, Motamedi H. Bioremediation of a crude oil-polluted soil by application of fertilizers. Iran J Environ Health Sci Eng. 2010; 7(4): 319-326.
32. Moghadam MS, Ebrahimipour G, Abtahi B, Ghassempour A, Seyed Hashtroudi M. Biodegradation of polycyclic aromatic hydrocarbons by a bacterial consortium enriched from mangrove sediments. J Environ Health Sci Eng. 2014;12:114-124.
_||_1. Tabari K, Tabari M. Biodegradation potential of hydrocarbons by bacterial diversity in soil. Iran J Environ Health Sci Eng. 2010; 7(4): 319-326.
2. Tanee FBG, Albert E. Reconnaissance assessment of long-term effects of crude oil spill on soil chemical properties and plant composition at Kwawa, Ogoni, Nigeria. J Environ Sci Technol. 2015; 8(6):320-329.
3. Peng R, Xiong A, Sue Y, Fu X, Gao F, Zhao W, Tian Y, Yao Q. Microbial biodegradation of polyaromatic hydrocarbons. FEMS Microbiol Rev. 2008; 32:927–955.
4. Xiong S, Li X, Chen J, Zhao L, Zhang H, Zhang X. Crude oil degradation by bacterial consortia under four different redox and temperature conditions. Appl Microbiol Biotechnol. 2015; 99(3):1451-1461.
5. Rodríguez DM, Andrade RFS, Ribeiro DLR, Rubio-Ribeaux D, Lima RA, Araújo HWC, Campos-Takaki GM. Bioremediation of petroleum derivative using biosurfactant produced by Serratia marcescens UCP/WFCC 1549 in low-cost medium. Int J Curr Microbiol App Sci. 2015; 4(7): 550-562.
6. Elmahdi AM, Abdul Aziz H, El-Gendy NS, Abu Amr SS, Nassar HN. Optimization of Libyan crude oil biodegradation by using solid waste date as a natural low-cost material. J Biorem Biodeg. 2014; 5:252-262.
7. Das N, Chandran P. Microbial degradation of petroleum hydrocarbon contaminants. Biotech Res Int. 2011; 6:1-13.
8. Wang Q, Zhang S, Li Y, Klassen W. Potential approaches to improving biodegradation of hydrocarbons for bioremediation of crude oil pollution. J Env Protec. 2011; 2:47-55.
9. Brooijmans RJW, Pastink MI, Siezen RJ. Hydrocarbon-degrading bacteria: the oil-spill clean-up crew. Microbial Biotech. 2009; 2(6):587–594.
10. Chikere CB, Okpokwasili GC. Bioreactor-based bioremediation of hydrocarbon-polluted Niger Delta marine sediment. Nigeria. Biotech. 2012; 2:53–66.
11. Yu S, Li S, Tang Y, Wu X. Succession of bacterial community along with the removal of heavy crude oil pollutants by multiple biostimulation treatments in the Yellow River Delta, China. J Env Sci. 2011; 23(9):1533–1543.
12. Mishra S, Jyot J, Kuhad RC, Lal B. Evaluation of inoculums addition to stimulate in situ bioremediation of oily-sludge-contaminated soil. Appl Environ Microbiol.2001; 67(4): 1675-1681.
13. Santisi S, Cappello S, Catalfamo M, Mancini G, Hassanshahian M, Genovese L, Giuliano L, Yakimov MM. Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium. Braz J Microbiol. 2015; 46(2):377-387.
14. Agarry SE, Aremu MO, Aworanti OA. Kinetic modeling and half-life study on enhanced soil bioremediation of Bonny light crude oil amended with crop and animal-derived organic wastes. J Pet Environ Biotech. 2013; 4(2):137-147.
15. Atlas RM. Hand book of Media for Environmental Microbiology. 2nd ed. New York. Taylor & Francis; 2005.
16. Prescott LM, Harley JP. Laboratory exercises in Microbiology. 5th ed. McGraw-Hill Publishers; 2001.
17. Arutchelvi M, Arkatkar A, Doble M, Bhaduri S, Uppara P. Biodegradation of polyethylene and polypropylene. India J Biotech. 2008; 8: 9-22.
18. CameronG, Rood D. Total petroleum hydrocarbon analysis by capillary gas chromatography: decreasing analysis times using small-diameter columns. J Chromato Sci. 1999; 37: 1-28.
19. Korenblum E, Seldin L. Shifts in alkaline-degrading bacteria genotypes during bioremediation of a vegetated coastal soil. World J Microbiol Biotech. 2009; 25:1667-1675.
20. Korenblum E, Seldin L. Shifts in alkane-degrading bacteria genotypes during bioremediation of a vegetated coastal soil. World J Microbiol Biotech. 2009; 25: 1667–1675.
21. Tabatabaee MS, Mazaheri Assadi M. Vacuum distillation residue upgrading by an indigenous Bacillus cereus. J Environ Health Sci Eng. 2013; 11:18-29.
22. Sathishkumar M, Binupriya AR, Baik SH, Yun, SE. Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium isolated from hydrocarbon contaminated areas. Clean.2008; 36(1): 92-96.
23. Wang Q, Zhang S, Li Y, Klassen W. Potential approaches to improving biodegradation of hydrocarbons for bioremediation of crude oil pollution. J Environ Protec. 2011; 2:47-55.
24. Brooijmans RJW, Pastink MI, SiezenRJ. Hydrocarbon-degrading bacteria: the oil-spill clean-up crew. Micro Biotech. 2009; 2(6):587–594.
25. Chikere CB, Okpokwasili, GC. Bioreactor-based bioremediation of hydrocarbon-polluted Niger Delta marine sediment, Nigeria. Biotech. 2012; 2:53–66.
26. Adebusoye SA, Ilori MO, Amund OO, Teniola OD, Olatope SO. Microbial degradation of petroleum hydrocarbons in a polluted tropical stream. World J Microbiol Biotech. 2007; 23(8):1149–1159.
27. Kebria Y, Khodadadi A, Ganjidoust H, Badkoubi A, Amoozegar MA. Isolation and characterization of a novel native Bacillus strain capable of degrading diesel fuel. Int J Environ Sci Tech. 2009; 6(3): 435-442.
28. Singh C, Lin J. Bioaugmentation efficiency of diesel degradation by Bacillus pumilus JLB and Acinetobacter calcoaceticus LT1 in contaminated soils. Afr J Biotech. 2010; 9(41):6881-6888.
29. Kumar A, Bisht BS, Joshi VD, DhewaT. Review on bioremediation of polluted environment: amanagement tool. Int J Environ Sci. 2011; 1(6):13-27.
30. Wongsa P, Tanaka M, Ueno A, Hasanuzzaman M, Yumoto I, Okuyama H. Isolation and characterization of novel strains of Pseudomonas aeruginosa and Serratia marcescens possessing high Efficiency to degrade gasoline, kerosene, diesel oil and lubricating oil. Curr Microbiol. 2004; 49: 415-422.
31. Chorom M, Sharifi HS, Motamedi H. Bioremediation of a crude oil-polluted soil by application of fertilizers. Iran J Environ Health Sci Eng. 2010; 7(4): 319-326.
32. Moghadam MS, Ebrahimipour G, Abtahi B, Ghassempour A, Seyed Hashtroudi M. Biodegradation of polycyclic aromatic hydrocarbons by a bacterial consortium enriched from mangrove sediments. J Environ Health Sci Eng. 2014;12:114-124.