جداسازی و شناسایی باکتری تجزیه کننده هیدروکربن های نفتی از مخازن نفتی آسماری اهواز
محورهای موضوعی : میکروب شناسی صنعتیراحیل کیانپور برجوئی 1 , حسین معتمدی 2 , زهرا بم زاده 3
1 - کارشناس ارشد، گروه زیست شناسی، دانشگاه آزاد اسلامی، واحد شهرکرد، شهرکرد
2 - استاد، گروه زیست شناسی، دانشکده علوم، دانشگاه شهید چمران اهواز، اهواز
3 - استادیار، گروه زیست شناسی، دانشگاه آزاد اسلامی واحد شهرکرد، شهرکرد
کلید واژه: تجزیه زیستی, باکتری تجزیه کننده نفت خام, آلاینده های هیدروکربنی,
چکیده مقاله :
سابقه و هدف: زیست پالایی فناوری حذف آلودگی های نفتی می باشد. از میان تمامی روش های حذف آلودگی، زیست پالایی به کمک فرآیند های میکروبی با کمترین مقدار انرژی، ماده شیمیایی و زمان قادر به تبدیل آلاینده ها به مواد غیر سمی می باشند. این مطالعه با هدف جداسازی و شناسایی باکتری تجزیه کننده هیدروکربن های نفتی از مخازن نفتی آسماری اهواز انجام شد. مواد و روش ها: این پژوهش به صورت میدانی در مخازن نفتی آسمار اهواز انجام شد. با استفاده از محیط پایه نمکی، به وسیله غنی سازی چند مرحله ای جدایه ها جداسازی و میزان تحمل نمک آن ها بررسی شد. در نهایت با استفاده از دستگاه کروماتوگرافی گازی، میزان تجزیه وحذف ترکیبات هیدروکربنی توسط باکتری جداسازی شده مورد بررسی قرار گرفت. سپس سویه مورد نظر بر اساس آزمونهای بیوشیمیایی و توالی یابی ژن 16S rRNA در حد جنس شناسایی گردید. یافته ها: در این مطالعه یک سویه باکتریایی گرم مثبت هالوتولرانت از جنس استرپتومایسس جداسازی شد. این جدایه رشد قابل قبولی را در غلظت 7.5 درصد نمک نشان داد و قادر به استفاده از نفت به عنوان تنها منبع کربن بود. همچنین این جدایه قادر به کاهش 71.58 درصد هیدروکربنهای موجود در محیط پایه نمکی در مدت 10 روز بود. نتیجه گیری: با توجه به نتایج به دست آمده می توان بیان کرد این جدایه قادر به تحمل غلظت بالای نمک و حذف مطلوب ترکیبات هیدروکربنی می باشد. این امر به دلیل تفاوت غلظت نمک مناطق آلوده یک مزیت محسوب می شود. بنابراین با این جدایه میتوان ورود آلاینده ها به محیط زیست و عوارض حاصل از آن را کاهش داد.
Background & Objectives: Bioremediation is a technology to remove oil pollution. Microbial bioremediation id the best technology to remove contaminants in which pollutants are converted to non-toxic chemicals in expenses of lowest amount of energy, chemicals and time. This study aimed to isolate and identify the petroleum hydrocarbons degrading bacteria from oil reservoirs located at Asmari Ahwaz. Materials & Methods: This field study was performed in the oil reservoirs located at Asmar, Ahvaz. The primary isolation was performed using a salt containing base medium through a multistep process, and the salt tolerance of isolates were tested by this medium. The elimination of the hydrocarbons by isolated bacteria were studied using gas chromatography. The identity of bacteria was determined based on biochemical tests and sequencing of 16S rRNA gene. Results: In this study, a halotolerant Gram-positive bacteria, belonged to Streptomyces, was isolated from the field. This isolate showed an acceptable growth into 7.5% salt concentration and was able to use oil as the sole source of carbon. Also, this strain was able to reduce the level of hydrocarbons to 71.58 % through incubation in the saline medium for 10 days. Conclusion: According to the results, the isolated strain is capable to tolerate high concentrations of salts and is desirable to remove the hydrocarbons, which is beneficiary due to the difference in salt concentration in contaminated areas. As a result, this isolate can be useful for removal of pollutants from the environment and reduction of their side effects on life.
1. Narimani S, Bazigar A, Mirzaee Najafgholi H. Identification of oil degrading bacteria from Poldokhtar polluted areas and investigation of factors affecting their degradation performance. Biotechnol Agri. 2015; 2(13): 11-19. [In Persian]
2. Mirsal Ibrahim A. Soil pollution: origin, monitoring and remediation, IstEd. Germany, Springer. 2004; 1: 5-11.
3. 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.
4. Maki HS, Haramaya S. Photo-oxidation of biodegradable crude oil and toxicity of the photooxidized products. Chemosphere. 2005; 44: 1145- 1151.
5. Taghvaei GS, Nahri NB, Khosravi M. Photooxidation of crude petroleum maltenic fraction in natural simulated conditions and structural elucidation of photo products. Iran J Environ Health Sci Eng. 2007; 4(1): 37-42.
6. Saleha H. Microbial metabolism of high molecular weight polycyclic aromatic hydrocarbons. J Chem Technol Biotechnol. 2008; 80: 723-736.
7. Shen T, Pi Y, Bao M, Xu N, LiN, Lu J. Biodegradation of different petroleum hydrocarbons by free and immobilized microbial consortia. Environ Sci Processes Impacts. 2015; 17: 2022-2033.
8. Jain PK, Gupta VK, Gaur RK, Lowry M, Jaroli DP, Chauhan UK. Bioremediation of petroleum oil contaminated soil and water. Res J Environ Toxicol. 2011; 5(6): 1-26.
9. Margesin R, Schinner F. Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles. 2001; 5: 73-83.
10. Dastgheib MM, Amoozegar MA, Khajeh Kh, Ventosa A. A halotolerant Alcanivorax sp. strain with potential application in saline soil remediation. Appl Microbial Cell Physiol. 2011; 90(1): 305-312.
11. Cheema S, Lavania M, Lal B. Impact of petroleum hydrocarbon contamination on the indigenous soil microbial community. Ann Microbiol. 2015; 65: 359-369.
12. Mohsenzadeh F. The study of microbial removal efficiency of crude oil in contaminated soils of cold climates areas (case study: Tabriz refinery). Mol Biol Res Comm. 2014; 3(27): 406-417.[In Persian]
13. Brakstad OG, Bonaunet K. Biodegradation of petroleum hydrocarbons in seawater at low temperatures (0–5°C) and bacterial communities associated with degradation. Biodeg. 2006; 17: 71- 82.
14. Chaillan F, Fleche AL, Bury E, Phantavong Y, Grimont P, saliot A, Oudot J. Identification and biodegradation potential of tropical aerobic hydrocarbon-degrading microorganisms. Res Microbiol. 2004; 155: 587-595.
15. Okerentugba PO, Ezerony OU. Petroleum degrading potentials of single and mixed microbial cultures isolated from rivers and refinery effluent in Nigeria. Afr J Biotech. 2003; 2(9): 288-292.
16. Hasanshahian M, Emtiazi G. Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation. Int Biodeterior Biodegrad. 2008; 62: 170-178.
17. Hajizadeh N, Sefidi Heris Y, Zununi Vahed S, Vallipour J, Hejazi MA, Golabi SM, Asadpour-Zeynali K, Hejazi MS. Biodegradation of para amino acetanilide by Halomonas sp. TBZ3. Jundishapur J Microbiol. 2015; 8(9): e18622.
18. Ferguson SH, Franzmann PD, Revill AT, Snape I, Rayner JL. The effects of nitrogen and water on mineralization of hydrocarbons in diesel-contaminated terrestrial Antarctic soils. Cold Region Sci Technol. 2003; 37: 197-212.
19. Mittal A, Singh P. Isolation of hydrocarbon degrading bacteria from soils contaminated with crude oil spills. Indian J Exp Biol. 2009; 47: 760-765.
20. Hunter RD, EKunwe SIN, Dodor DE, Hwang H M, Ekunwe L. Bacillus subtilis is a potential degrader of pyrene and benzo[a] pyrene. Int J Environ Res Public Health. 2005; 2(2): 267-271.
21. Burton GA. High incidence of selenite-resistant bacteria from a site polluted with selenium. Appl Environ Microbiol. 1987; 53: 185-188.
22. Smibert RM, Krieg NR. Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR, editors. Methods for general and molecular bacteriology. Washington, DC: American Society for Microbiology; 1994: 607-654.
23. Weisburg WG, Barns SM, Selletier DA, Lane DJ. 16S Ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991; 173(2): 697-703.
24. Azizi niko P, Hadadi A, Shavandi M, Soleimani M, Tabib A. Isolation of bacteria long-chain alkane’s parser of oil-contaminated soils. New J Cell Biotechnol Mol. 2014; 3(12): 99-105.
25. Holt SG, Kriey NR, Sneath PHA, Staley JT, Williams ST. Bergy’s manual of determinative for bacteriology, Williams and Wilkins, New York; 1998.
26. Onwurah INE, Ogugua VN, Onyike NB, Ochonogor AE, Otitoju OF. Crude oil spills in the environment, effects and some innovative cleanup biotechnologies. Int J Environ Res. 2007; 1(4): 307-320.
27. Vennila R, Kannan V. Bioremediation of petroleum refinery effluent by Planococcus halophilus. Afr J Biotechnol. 2011; 10(44): 8829-8833.
28. Lazar I, Doborta S, Voicu A, Stefanescu M, Sandulescu L, Petrisor IG. Microbial degradation of waste hydrocarbons in oily sludge from some Romanian oil fields. J Petroleum Sci Eng. 1997; 22: 151-160.
29. Gibbs CF. Quantitative studies on marine oil biodegradation of crude oil. I. Nutrient limitation at 14ºC. Proc Roy Soc. 1975; 188: 61-82.
30. Atlas RM. Stimulated petroleum biodegradation. Crit Rev Microbiol. 1977; 5(4): 86-371.
31. Solanas M, Marc V, Espuny M. Bacterial community dynamics and polycyclic aromatic hydrocarbons degradation during bioremediation of heavily creosote- contaminated soil. Appl Environ Microbiol. 2005; 11(71): 7008-7018.
32. Rozlyn FC, Stephane M, Fedroak M. Aerobic biodegradation of 2-2-dithiodibenzoic acid produced from dibenzothiophene methabolite. Appl Environ Microbiol. 2006; 1(72): 491-496.
33. Mohana S, Chirayu D, Madamwar D. Biodegradation and decolourization of anaerobically treated Distillery spent wash by a novel bacterial consortium. Bioresource Technol. 2006; 2 (98): 333-339.
34. Qin X, Tang JC, Li DS, Zhang QM. Effect of salinity on the bioremediation of petroleum hydrocarbons in a saline-alkaline soil. Lett Appl Microb. 2011; 55: 210-217.
35. Amozegar MA, Akhavansepahi A, Akhavansepahi F. Isolation and identification of soil bacteria halotolerant parser benzene oil wells Qom. Quarterly J Microbiol Knowledge. 2009; 3 (1): 33-39. [In Persian]
36. Ryu A, Wook H, Yang H, Jooan Y, Sukcho K. Isolation and characterization of psychrophilic and halotolerant Rhodococcus Sp. YHLT-M. Microbiol Biotechnol. 2006; 16(4): 605- 612.
37. Safari M, Ahmadi Asbchin S, Soltani N. The ability of Cyanobacteria Schizothrix vaginata ISC 108 for crude oil biodegradation. Iran J Health Environ. 2014; 3(7): 363-373. [In Persian]
38. Amini A, Tahmorspour A, Abdolahi A, Dodi M. Isolation, characterization and evaluation of bio-molecular anthracene by Nocardia cyriacigeorgica isolated from soil of the refinery. J Microbial World. 2013; 3: 228-236.
_||_1. Narimani S, Bazigar A, Mirzaee Najafgholi H. Identification of oil degrading bacteria from Poldokhtar polluted areas and investigation of factors affecting their degradation performance. Biotechnol Agri. 2015; 2(13): 11-19. [In Persian]
2. Mirsal Ibrahim A. Soil pollution: origin, monitoring and remediation, IstEd. Germany, Springer. 2004; 1: 5-11.
3. 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.
4. Maki HS, Haramaya S. Photo-oxidation of biodegradable crude oil and toxicity of the photooxidized products. Chemosphere. 2005; 44: 1145- 1151.
5. Taghvaei GS, Nahri NB, Khosravi M. Photooxidation of crude petroleum maltenic fraction in natural simulated conditions and structural elucidation of photo products. Iran J Environ Health Sci Eng. 2007; 4(1): 37-42.
6. Saleha H. Microbial metabolism of high molecular weight polycyclic aromatic hydrocarbons. J Chem Technol Biotechnol. 2008; 80: 723-736.
7. Shen T, Pi Y, Bao M, Xu N, LiN, Lu J. Biodegradation of different petroleum hydrocarbons by free and immobilized microbial consortia. Environ Sci Processes Impacts. 2015; 17: 2022-2033.
8. Jain PK, Gupta VK, Gaur RK, Lowry M, Jaroli DP, Chauhan UK. Bioremediation of petroleum oil contaminated soil and water. Res J Environ Toxicol. 2011; 5(6): 1-26.
9. Margesin R, Schinner F. Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles. 2001; 5: 73-83.
10. Dastgheib MM, Amoozegar MA, Khajeh Kh, Ventosa A. A halotolerant Alcanivorax sp. strain with potential application in saline soil remediation. Appl Microbial Cell Physiol. 2011; 90(1): 305-312.
11. Cheema S, Lavania M, Lal B. Impact of petroleum hydrocarbon contamination on the indigenous soil microbial community. Ann Microbiol. 2015; 65: 359-369.
12. Mohsenzadeh F. The study of microbial removal efficiency of crude oil in contaminated soils of cold climates areas (case study: Tabriz refinery). Mol Biol Res Comm. 2014; 3(27): 406-417.[In Persian]
13. Brakstad OG, Bonaunet K. Biodegradation of petroleum hydrocarbons in seawater at low temperatures (0–5°C) and bacterial communities associated with degradation. Biodeg. 2006; 17: 71- 82.
14. Chaillan F, Fleche AL, Bury E, Phantavong Y, Grimont P, saliot A, Oudot J. Identification and biodegradation potential of tropical aerobic hydrocarbon-degrading microorganisms. Res Microbiol. 2004; 155: 587-595.
15. Okerentugba PO, Ezerony OU. Petroleum degrading potentials of single and mixed microbial cultures isolated from rivers and refinery effluent in Nigeria. Afr J Biotech. 2003; 2(9): 288-292.
16. Hasanshahian M, Emtiazi G. Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation. Int Biodeterior Biodegrad. 2008; 62: 170-178.
17. Hajizadeh N, Sefidi Heris Y, Zununi Vahed S, Vallipour J, Hejazi MA, Golabi SM, Asadpour-Zeynali K, Hejazi MS. Biodegradation of para amino acetanilide by Halomonas sp. TBZ3. Jundishapur J Microbiol. 2015; 8(9): e18622.
18. Ferguson SH, Franzmann PD, Revill AT, Snape I, Rayner JL. The effects of nitrogen and water on mineralization of hydrocarbons in diesel-contaminated terrestrial Antarctic soils. Cold Region Sci Technol. 2003; 37: 197-212.
19. Mittal A, Singh P. Isolation of hydrocarbon degrading bacteria from soils contaminated with crude oil spills. Indian J Exp Biol. 2009; 47: 760-765.
20. Hunter RD, EKunwe SIN, Dodor DE, Hwang H M, Ekunwe L. Bacillus subtilis is a potential degrader of pyrene and benzo[a] pyrene. Int J Environ Res Public Health. 2005; 2(2): 267-271.
21. Burton GA. High incidence of selenite-resistant bacteria from a site polluted with selenium. Appl Environ Microbiol. 1987; 53: 185-188.
22. Smibert RM, Krieg NR. Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR, editors. Methods for general and molecular bacteriology. Washington, DC: American Society for Microbiology; 1994: 607-654.
23. Weisburg WG, Barns SM, Selletier DA, Lane DJ. 16S Ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991; 173(2): 697-703.
24. Azizi niko P, Hadadi A, Shavandi M, Soleimani M, Tabib A. Isolation of bacteria long-chain alkane’s parser of oil-contaminated soils. New J Cell Biotechnol Mol. 2014; 3(12): 99-105.
25. Holt SG, Kriey NR, Sneath PHA, Staley JT, Williams ST. Bergy’s manual of determinative for bacteriology, Williams and Wilkins, New York; 1998.
26. Onwurah INE, Ogugua VN, Onyike NB, Ochonogor AE, Otitoju OF. Crude oil spills in the environment, effects and some innovative cleanup biotechnologies. Int J Environ Res. 2007; 1(4): 307-320.
27. Vennila R, Kannan V. Bioremediation of petroleum refinery effluent by Planococcus halophilus. Afr J Biotechnol. 2011; 10(44): 8829-8833.
28. Lazar I, Doborta S, Voicu A, Stefanescu M, Sandulescu L, Petrisor IG. Microbial degradation of waste hydrocarbons in oily sludge from some Romanian oil fields. J Petroleum Sci Eng. 1997; 22: 151-160.
29. Gibbs CF. Quantitative studies on marine oil biodegradation of crude oil. I. Nutrient limitation at 14ºC. Proc Roy Soc. 1975; 188: 61-82.
30. Atlas RM. Stimulated petroleum biodegradation. Crit Rev Microbiol. 1977; 5(4): 86-371.
31. Solanas M, Marc V, Espuny M. Bacterial community dynamics and polycyclic aromatic hydrocarbons degradation during bioremediation of heavily creosote- contaminated soil. Appl Environ Microbiol. 2005; 11(71): 7008-7018.
32. Rozlyn FC, Stephane M, Fedroak M. Aerobic biodegradation of 2-2-dithiodibenzoic acid produced from dibenzothiophene methabolite. Appl Environ Microbiol. 2006; 1(72): 491-496.
33. Mohana S, Chirayu D, Madamwar D. Biodegradation and decolourization of anaerobically treated Distillery spent wash by a novel bacterial consortium. Bioresource Technol. 2006; 2 (98): 333-339.
34. Qin X, Tang JC, Li DS, Zhang QM. Effect of salinity on the bioremediation of petroleum hydrocarbons in a saline-alkaline soil. Lett Appl Microb. 2011; 55: 210-217.
35. Amozegar MA, Akhavansepahi A, Akhavansepahi F. Isolation and identification of soil bacteria halotolerant parser benzene oil wells Qom. Quarterly J Microbiol Knowledge. 2009; 3 (1): 33-39. [In Persian]
36. Ryu A, Wook H, Yang H, Jooan Y, Sukcho K. Isolation and characterization of psychrophilic and halotolerant Rhodococcus Sp. YHLT-M. Microbiol Biotechnol. 2006; 16(4): 605- 612.
37. Safari M, Ahmadi Asbchin S, Soltani N. The ability of Cyanobacteria Schizothrix vaginata ISC 108 for crude oil biodegradation. Iran J Health Environ. 2014; 3(7): 363-373. [In Persian]
38. Amini A, Tahmorspour A, Abdolahi A, Dodi M. Isolation, characterization and evaluation of bio-molecular anthracene by Nocardia cyriacigeorgica isolated from soil of the refinery. J Microbial World. 2013; 3: 228-236.