Abstract
Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.
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References
Chauhan NS, Ranjan R, Purohit HJ, Kalia VC, Sharma R (2009) Identification of genes conferring arsenic resistance to Escherichia coli from an effluent treatment plant sludge metagenomic library. FEMS Microbiol Ecol 67:130–139
Chu X, He H, Guo C, Sun B (2008) Identification of two novel esterases from a marine metagenomic library derived from South China Sea. Appl Microbiol Biotechnol 80:615–625
Cowan D, Meyer Q, Stafford W, Muyanga S, Cameron R, Wittwer P (2005) Metagenomic gene discovery: past, present and future. Trends Biotechnol 23:321–329
Daniel R (2004) The soil metagenome—a rich resource for the discovery of novel natural products. Curr Opin Biotechnol 15:199–204
Ewis HE, Abdelal AT, Lu CD (2004) Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus. Gene 329:187–195
Gabor EM, de Vries EJ, Janssen DB (2003) Efficient recovery of environmental DNA for expression cloning by indirect extraction methods. FEMS Microbiol Ecol 44:153–163
Hafez EE, Elbestawy E (2009) Molecular characterization of soil microorganisms: effect of industrial pollution on distribution and biodiversity. World J Microbiol Biotechnol 25:215–224
Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5:245–249
Hardeman F, Sjöling S (2007) Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol 59:524–534
Hong KS, Lim HK, Chung EJ, Park EJ, Lee MH, Kim JC, Choi GJ, Cho KY, Lee SW (2007) Selection and characterization of forest soil metagenome genes encoding lipolytic enzymes. J Microbiol Biotechnol 17:1655–1660
Hu Y, Fu C, Huang Y, Yin Y, Cheng G, Lei F, Lu N, Li J, Ashforth EJ, Zhang L, Zhu B (2010) Novel lipolytic genes from the microbial metagenomic library of the South China Sea marine sediment. FEMS Microbiol Ecol 72:228–237
Jeon JH, Kim JT, Kang SG, Lee JH, Kim SJ (2009) Characterization and its potential application of two esterases derived from the arctic sediment metagenome. Mar Biotechnol 11:307–316
JunGang L, KeGui Z, WenJun H (2010) Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product. Microb Cell Fact 9:83
Kakirde KS, Parsley LC, Liles MR (2010) Size does matter: application-driven approaches for soil metagenomics. Soil Biol Biochem 42:1911–1923
Kapley A, Siddiqui S, Misra K, Ahmad SM, Purohit HJ (2007) Preliminary analysis of bacterial diversity associated with the Porites coral from the Arabian sea. World J Microbiol Biotechnol 23:923–930
Kapley A, Thierry B, Purohit HJ (2007) Eubacterial diversity of activated biomass from a common effluent treatment plant. Res Microbiol 158:494–500
Kapley A, Purohit HJ (2009) Diagnosis of treatment efficiency in industrial wastewater treatment plants: a case study at a refinery ETP. Environ Sci Technol 43:3789–3795
Lammle K, Zipper H, Breuer M, Hauer B, Buta C, Brunner H, Rupp S (2007) Identification of novel enzymes with different hydrolytic activities by metagenome expression cloning. J Biotechnol 127:575–592
Liaw RB, Cheng MP, Wu MC, Lee CY (2010) Use of metagenomic approaches to isolate lipolytic genes from activated sludge. Bioresour Technol 101:8323–8329
Miller DN (2001) Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments. J Microbiol Methods 44:49–58
Purohit HJ, Kapley A, Moharikar AA, Narde G (2003) A novel approach for extraction of PCR-compatible DNA from activated sludge samples collected from different biological effluent treatment plants. J Microbiol Methods 52:315–323
Rajendhran J, Gunasekaran P (2008) Strategies for accessing soil metagenome for desired applications. Biotechnol Adv 26:576–590
Rani A, Porwal S, Sharma R, Kapley A, Purohit HJ, Kalia VC (2008) Assessment of microbial diversity in effluent treatment plants by culture dependent and culture independent approaches. Bioresour Technol 99:7098–7107
Ranjan R, Grover A, Kapardar RK, Sharma R (2005) Isolation of novel lipolytic genes from uncultured bacteria of pond water. Biochem Biophys Res Commun 335:57–65
Roh C, Villatte F (2008) Isolation of a low-temperature adapted lipolytic enzyme from uncultivated microorganism. J Appl Microbiol 105:116–123
Shan G, Jin W, Lam EKH, Xing X (2008) Purification of total DNA extracted from activated sludge. J Environ Sci 20:80–87
Sharma P, Kumari H, Kumar M, Verma M, Kumari K, Malhotra S, Khurana J, Lal R (2008) From bacterial genomics to metagenomics: concept, tools and recent advances. Indian J Microbiol 48:173–194
Sharma PK, Capalash N, Kaur J (2007) An improved method for single step purification of metagenomic DNA. Mol Biotechnol 36:61–63
Streit WR, Daniel R, Jaeger KE (2004) Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms. Curr Opin Biotechnol 15:285–290
Thangaraj K, Kapley A, Purohit HJ (2008) Characterization of diverse Acinetobacter isolates for utilization of multiple aromatic compounds. Bioresour Technol 99:2488–2494
Uchiyama T, Miyazaki K (2009) Functional metagenomics for enzyme discovery: challenges to efficient screening. Curr Opin Biotechnol 20:616–622
Voget S, Steele HL, Streit WR (2006) Characterization of a metagenome-derived halotolerant cellulase. J Biotechnol 126:26–36
Wechter P, Williamson J, Robertson A, Kluepfel D (2003) A rapid, cost-effective procedure for the extraction of microbial DNA from soil. World J Microbiol Biotechnol 19:85–91
Williamson KE, Kan J, Polson SW, Williamson SJ (2010) Optimizing the indirect extraction of prokaryotic DNA from soils. Soil Biol Biochem 43:736–748
Zhang T, Han WJ, Liu ZP (2009) Gene cloning and characterization of a novel esterase from activated sludge metagenome. Microb Cell Fact 8:67
Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322
Acknowledgments
This study was supported by a grant from the Department of Biotechnology (DBT) Ministry of Science and Technology, New Delhi, India. The authors thank Director, CSIR- National Environmental Engineering Research Institute (NEERI), Nagpur, for providing facilities for this work.
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Sharma, N., Tanksale, H., Kapley, A. et al. Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method. Indian J Microbiol 52, 538–543 (2012). https://doi.org/10.1007/s12088-012-0263-1
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DOI: https://doi.org/10.1007/s12088-012-0263-1