غربالگری و شناسایی سویه‌های جدید باکتریایی اکسید کننده متان از خاک‌های آلوده به نفت خوزستان
محورهای موضوعی : میکروب شناسی محیطینازنین صانعی 1 , محمد رعایایی اردکانی 2 , محمدرضا صعودی 3
1 - گروه بیولوژی، دانشکده علوم، دانشگاه شهید چمران، اهواز، ایرا
2 - گروه بیولوژی، دانشکده علوم، دانشگاه شهید چمران، اهواز، ایران
3 - گروه بیولوژی، دانشکده علوم، دانشگاه الزهرا، تهران، ایران
کلید واژه: باکتری‌های اکسید‌کننده متان, خاک‌های آلوده به نفت, اسفنگوموناس, کروموباکتر,
چکیده مقاله :
سابقه و هدف: با استفاده از اطلاعات به دست آمده از نوع و پراکندگی میکروارگانیسم ها در نمونه های خاک های سطحی می توان به میزان توزیع مخازن نفت و گاز پی برد. هدف از این پژوهش، جداسازی و شناسایی باکتری های اکسید کننده متان از خاک های آلوده به نفت در خوزستان و بهینه سازی دما و اسیدیته در حضور متان بود.مواد و روش ها: باکتری ها در محیط نمک های معدنی و نیترات (NMS) در حضور 50 درصد هوا و 50 درصد متان جداسازی شدند. محیط های کشت به مدت 10 روز بر روی شیکر در دمای °C 30 در تاریکی گرمخانه گذاری شدند و هر 2 روز یک بار مخلوط گازها تجدیدکشت شد. جدایه ها با استفاده از ویژگی های بیوشیمیایی و مولکولی شناسایی شدند. همچنین بهینه سازی رشد باکتری ها در دما و اسیدیته های گوناگون در محیط کشت NMS در حضور متان به عنوان منبع کربن انجام شد.یافته ها: در شرایط مورد بررسی، سه باسیل گرم منفی از نمونه های خاک جداسازی گردید. جدایه ها متعلق به گونه هایی از اکروموباکتر و اسفنگوموناس بودند. تمامی سویه ها قادر به رشد در محیطNMS دارای غلظت بالای متانول (3%) بودند. بهترین شرایط pH و دمای بهینه رشد باکتری های جداسازی شده به ترتیب 4/7 و °C 30 به دست آمد.نتیجه گیری: سویه هایمتانوتروف جداسازی شده در بررسی حاضر قادر به رشد و اکسیداسیون متان در محدوده وسیع اسیدیته و دما بودند و می توانند برای حذف ترکیبات تک کربنه مانند متان و به عنوان شناساگر بیولوژیک مخازن نفت و گاز پیشنهاد شوند.
Background & objectives:Based on information about the types and numbers of microorganisms in the surface soil samples, the distribution range of underlying oil and gas reservoirs can be judged. In the present study, methane oxidizing bacteria were isolated and characterized from oil contaminated soils in Khuzestan, Iran and their growth was optimized in the presence of methane as sole carbon and energy sources. Materials & Methods: Bacteria were isolated in nitrate mineral salt (NMS) medium in the presence of 50% air and 50% methane. The cultures were incubated on a shaker for 10 days at 30°C in dark place, and every two days, the gas mixture was replaced. The isolated bacteria were characterized based on biochemical and molecular identification tests. Then the optimum growth conditions was detected in different pH values and incubation temperatures in NMS medium containing methane. Results:Three Gram-negative rods were isolated from soil samples that were able to grow in isolation condition. The isolates were characterized as Achromobacter and Sphingomonas spp. The strains could also grow in the NMS medium with a high methanol concentration (3%). The optimal pH and temperature for the isolates were 7.4 and 30°C respectively. Conclusion: Methanotrophic strains that were isolated in the present study were able to grow and oxidize methane in high ranges of temperature and pH and can be proposed for the removal of mono-carbon compounds such as methane and as biological detectors for prospecting for oil and gas reservoirs
hydrocarbons: realities, challenges and prospects. Biotechnol J Int. 2018; 14:1-0.
2. Chan JB. 2011. PCR primers for the detection of propane and butane-oxidizing
microorganisms. MSc, California Polytechnic State University.
3. Liu YC, He Z, Zhang Sh, Yin MY, Ning Zh, Zhang CY. Abundance and diversity of
methanotrophs and propanotrophs in soils above Yangxin oil reservoir, China. Geomicrobiol
J. 2016; 33(8):661-670.
4. Hanson RS, Hanson TE. Methanotrophic bacteria. Microbiol Rev. 1996; 60(2):439-471.
1. Rahalkar M. 2007. Aerobic methanotrophic bacterial communities in sediments of Lake
Constance. MSc, University of Konstanz.
1. Abushammala MF, Basri NEA, Younes MK Methane oxidation in landfill cover soils: A
Review. Asian J. Atmos Environ. 2014; 8(1):1-14.
7. Jhala YK, Vyas RV, Shelat HN, Patel HK, Patel KT. Isolation and characterization of methane
utilizing bacteria from wetland paddy ecosystem. World JMicrobiol Biotechnol. 2014; 30
(6):1845-1860.
1. Ghashghavi M, Jetten MSM, Lüke C. Survey of methanotrophic diversity in various
ecosystems by degenerate methane monooxygenase gene primers. AMB Express. 2017; 7
(1):162.
1. Stepniewska Z, Goraj W, Kuzniar A, Lopacka N, Malysza M. Enrichment culture and
identification of endophytic methanotrophs isolated from peatland plants. Folia Microbiol.
2017; 62(5):381-391.
10. Baesman SM, Miller LG, Wei JH, Cho Y, Matys ED, Summons RE, Welander PV, Oremland
RS. Methane oxidation and molecular characterization of methanotrophs from a former
mercury mine impoundment. Microorganisms 2015; 3(2):290-309.
11. Henard CA, Smith H, Dowe N, Kalyuzhnaya MG, Pienkos PT, Guarnieri MT. Bioconversion
of methane to lactate by an obligate methanotrophic bacterium. Sci Rep. 2016; 6:21585.
12. Lakshmi M, Rasheed MA, Madhavi T, Kalpana MS, Patil DJ, Dayal AM. Characterization of
light gaseous hydrocarbons of the surface soils of Krishna-Godavari basin, India. J Environ
Biol. 2012; 33(1):67-79.
13. Dianou D, Espiritu BM, Adachi K, Senboku T. Isolation and some properties of
methane-oxidizing bacteria from a subtropical paddy field. Soil Sci Plant Nutr. 1997; 43
(3):735-740.
14. Dianou D, Adachi K. Characterization of methanotrophic bacteria isolated from a subtropical
paddy field. FEMS Microbiol Lett. 1999; 173(1):163-173.
11. Rusmana I, Akhdiya A. Isolation and characterizatin of methanotrophic bacteria from rice
fields. Biotropia 2009; 16(2):71-78.
11. Kim HG, Han GH, Eom CY, Kim SW. Isolation and taxonomic characterization of a novel
type Ι methanotrophic bacterium. J Microbiol. 2008; 46(1):45-50.
17. Kip N, Ouyang W, Van Winden J, Raghoebarsing A, Van Niftrik L, Pol A, Pan Y, Bodrossy
L, Van Donselaar EG, Reichart GJ, Jetten MSM, Sinninghe Damste JS, Op Den Camp HJM.
Detection, isolation and characterization of acidophilic methanotrophs from sphagnum
mosses. Appl Environ Microbiol. 2011; 77(16):5643-5654.
11. Prescott LM, Harley JP (2002) Laboratory exercise in microbiology, 5th edn. New York,
MCGraw hill; 2002.
11. McDonald IR, Kenna EM, Murrell JC. Detection of methanotrophic bacteria in environmental
samples with the PCR. Appl Environ Microbiol. 1995; 61(1):116-121.
20. Cheng YS, Halsey JL, Fode KA, Remsen CC, Collins MLP. Detection of methanotrophs in
ground water by PCR. Appl Environ Microbiol. 1999; 65(2):648-651.
21. Kalyuzhnaya MG, Makutina VA, Rusakova TG, Nikitin DV, Khmelenina VN, Dmitriev VV,
Trotsenko YA. Methanotrophic communities in the soils of the Russian northern taiga and
subarctic tundra. Microbiology 2002; 71(2):227-233.
22. Anthony C. The Biochemistry of methylotrophs. London, Academic Press; 1982.
23. Qiang K, Zhang G, Zhang L, Lu B, Zhong G. Application of microbiological method to
hydrocarbon exploration in Eastern Pearl River Mouth Basin. Acta Geol Sin. 2019; 93(S2):
99-102.
24. Rasheed MA, Patil DJ, Dayal AM. Microbial techniques for hydrocarbon exploration. In:
Kutcherov V, Kolesnikov A editors. Hydrocarbon, 1st ed. In Tech, Croatia, 2013; 195-200.
21. Peltoniemi K, Laiho R, Juottonen H, Bodrossy L, Kell DK, Minkkinen K, Mäkiranta P,
Mehtätalo L, Penttilä T, Siljanen HM, Tuittila ES. Responses of methanogenic and
methanotrophic communities to warming in varying moisture regimes of two boreal fens. Soil
Biol Biochem. 2016; 97:144-56.
21. Heyer J, Galchenko VF, Dunfield PF. Molecular phylogeny of type II methane-oxidizing
bacteria isolated from various environments. Microbiology 2002; 148(9):2831-46.
27. Cho KS, Lee JH, Moon KE, Kim TG, Lee SH, inventors; Ewha University-industry
collaboration foundation, assignee. Sphingomonas sp. microorganism and method for
decomposing methane or odor-producing compounds using the same. US patent US 2014;
8,748,154.
21. Tambekar DH, Patil RV, Pawar AL. Studies on methanotrophs from Lonar Lake. J Res Biol.
2011; 3:230-236.
21. Rodrigues ADS, Valdman B, Salgado AM. Analysis of methane biodegradation by
Methylosinus trichosporium OB3b. Brazilian Microbiol. 2009; 40(2):301-307.
30. Saari A, Rinnan R, Martikainen PJ. Methane oxidation in boreal forest soils: kinetics and
sensitivity to pH and ammonium. Soil Biol Biochem. 2004; 36(7):1037-1046.
31. Abazari M, Owlia P, Zarrini G, Aghdasinia H. Isolation of Methylococcus strain resistant to
abnormal climate change to reduce methane emissions from the Iranian salt Lake.
Geomicrobiol J. 2020; 16:1-8.
_||_
hydrocarbons: realities, challenges and prospects. Biotechnol J Int. 2018; 14:1-0.
2. Chan JB. 2011. PCR primers for the detection of propane and butane-oxidizing
microorganisms. MSc, California Polytechnic State University.
3. Liu YC, He Z, Zhang Sh, Yin MY, Ning Zh, Zhang CY. Abundance and diversity of
methanotrophs and propanotrophs in soils above Yangxin oil reservoir, China. Geomicrobiol
J. 2016; 33(8):661-670.
4. Hanson RS, Hanson TE. Methanotrophic bacteria. Microbiol Rev. 1996; 60(2):439-471.
1. Rahalkar M. 2007. Aerobic methanotrophic bacterial communities in sediments of Lake
Constance. MSc, University of Konstanz.
1. Abushammala MF, Basri NEA, Younes MK Methane oxidation in landfill cover soils: A
Review. Asian J. Atmos Environ. 2014; 8(1):1-14.
7. Jhala YK, Vyas RV, Shelat HN, Patel HK, Patel KT. Isolation and characterization of methane
utilizing bacteria from wetland paddy ecosystem. World JMicrobiol Biotechnol. 2014; 30
(6):1845-1860.
1. Ghashghavi M, Jetten MSM, Lüke C. Survey of methanotrophic diversity in various
ecosystems by degenerate methane monooxygenase gene primers. AMB Express. 2017; 7
(1):162.
1. Stepniewska Z, Goraj W, Kuzniar A, Lopacka N, Malysza M. Enrichment culture and
identification of endophytic methanotrophs isolated from peatland plants. Folia Microbiol.
2017; 62(5):381-391.
10. Baesman SM, Miller LG, Wei JH, Cho Y, Matys ED, Summons RE, Welander PV, Oremland
RS. Methane oxidation and molecular characterization of methanotrophs from a former
mercury mine impoundment. Microorganisms 2015; 3(2):290-309.
11. Henard CA, Smith H, Dowe N, Kalyuzhnaya MG, Pienkos PT, Guarnieri MT. Bioconversion
of methane to lactate by an obligate methanotrophic bacterium. Sci Rep. 2016; 6:21585.
12. Lakshmi M, Rasheed MA, Madhavi T, Kalpana MS, Patil DJ, Dayal AM. Characterization of
light gaseous hydrocarbons of the surface soils of Krishna-Godavari basin, India. J Environ
Biol. 2012; 33(1):67-79.
13. Dianou D, Espiritu BM, Adachi K, Senboku T. Isolation and some properties of
methane-oxidizing bacteria from a subtropical paddy field. Soil Sci Plant Nutr. 1997; 43
(3):735-740.
14. Dianou D, Adachi K. Characterization of methanotrophic bacteria isolated from a subtropical
paddy field. FEMS Microbiol Lett. 1999; 173(1):163-173.
11. Rusmana I, Akhdiya A. Isolation and characterizatin of methanotrophic bacteria from rice
fields. Biotropia 2009; 16(2):71-78.
11. Kim HG, Han GH, Eom CY, Kim SW. Isolation and taxonomic characterization of a novel
type Ι methanotrophic bacterium. J Microbiol. 2008; 46(1):45-50.
17. Kip N, Ouyang W, Van Winden J, Raghoebarsing A, Van Niftrik L, Pol A, Pan Y, Bodrossy
L, Van Donselaar EG, Reichart GJ, Jetten MSM, Sinninghe Damste JS, Op Den Camp HJM.
Detection, isolation and characterization of acidophilic methanotrophs from sphagnum
mosses. Appl Environ Microbiol. 2011; 77(16):5643-5654.
11. Prescott LM, Harley JP (2002) Laboratory exercise in microbiology, 5th edn. New York,
MCGraw hill; 2002.
11. McDonald IR, Kenna EM, Murrell JC. Detection of methanotrophic bacteria in environmental
samples with the PCR. Appl Environ Microbiol. 1995; 61(1):116-121.
20. Cheng YS, Halsey JL, Fode KA, Remsen CC, Collins MLP. Detection of methanotrophs in
ground water by PCR. Appl Environ Microbiol. 1999; 65(2):648-651.
21. Kalyuzhnaya MG, Makutina VA, Rusakova TG, Nikitin DV, Khmelenina VN, Dmitriev VV,
Trotsenko YA. Methanotrophic communities in the soils of the Russian northern taiga and
subarctic tundra. Microbiology 2002; 71(2):227-233.
22. Anthony C. The Biochemistry of methylotrophs. London, Academic Press; 1982.
23. Qiang K, Zhang G, Zhang L, Lu B, Zhong G. Application of microbiological method to
hydrocarbon exploration in Eastern Pearl River Mouth Basin. Acta Geol Sin. 2019; 93(S2):
99-102.
24. Rasheed MA, Patil DJ, Dayal AM. Microbial techniques for hydrocarbon exploration. In:
Kutcherov V, Kolesnikov A editors. Hydrocarbon, 1st ed. In Tech, Croatia, 2013; 195-200.
21. Peltoniemi K, Laiho R, Juottonen H, Bodrossy L, Kell DK, Minkkinen K, Mäkiranta P,
Mehtätalo L, Penttilä T, Siljanen HM, Tuittila ES. Responses of methanogenic and
methanotrophic communities to warming in varying moisture regimes of two boreal fens. Soil
Biol Biochem. 2016; 97:144-56.
21. Heyer J, Galchenko VF, Dunfield PF. Molecular phylogeny of type II methane-oxidizing
bacteria isolated from various environments. Microbiology 2002; 148(9):2831-46.
27. Cho KS, Lee JH, Moon KE, Kim TG, Lee SH, inventors; Ewha University-industry
collaboration foundation, assignee. Sphingomonas sp. microorganism and method for
decomposing methane or odor-producing compounds using the same. US patent US 2014;
8,748,154.
21. Tambekar DH, Patil RV, Pawar AL. Studies on methanotrophs from Lonar Lake. J Res Biol.
2011; 3:230-236.
21. Rodrigues ADS, Valdman B, Salgado AM. Analysis of methane biodegradation by
Methylosinus trichosporium OB3b. Brazilian Microbiol. 2009; 40(2):301-307.
30. Saari A, Rinnan R, Martikainen PJ. Methane oxidation in boreal forest soils: kinetics and
sensitivity to pH and ammonium. Soil Biol Biochem. 2004; 36(7):1037-1046.
31. Abazari M, Owlia P, Zarrini G, Aghdasinia H. Isolation of Methylococcus strain resistant to
abnormal climate change to reduce methane emissions from the Iranian salt Lake.
Geomicrobiol J. 2020; 16:1-8.