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New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches

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Abstract

The significance of ANME-2d in methane sink in the environment has been overlooked, and there was no any study evaluating the distribution of ANME-2d in the environment. New primers were thus needed to be designed for following research. In this paper, a pair of primers (DP397F and DP569R) was designed to quantify ANME-2d. The specificity and amplification efficiency of this primer pair were acceptable. PCR amplification of another pair of primers (DP142F and DP779R) generated a single, bright targeted band from the enrichment sample, but yielded faint, multiple bands from the environmental samples. Nested PCR was conducted using the primers DP142F/DP779R in the first round and DP142F/DP569R in the second round, which generated a bright targeted band. Further phylogenetic analysis showed that these targeted bands were ANME-2d-related sequences. Real-time PCR showed that the copies of the 16s ribosomal RNA gene of ANME-2d in these samples ranged from 3.72 × 104 to 2.30 × 105 copies μg−1 DNA, indicating that the percentage of ANME-2d was greatest in a polluted river sample and least in a rice paddy sample. These results demonstrate that the newly developed real-time PCR primers could sufficiently quantify ANME-2d and that nested PCR with an appropriate combination of the new primers could successfully detect ANME-2d in environmental samples; the latter finding suggests that ANME-2d may spread in environments.

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References

  • Beal EJ, House CH, Orphan VJ (2009) Manganese- and iron-dependent marine methane oxidation. Science 325(5937):184–187

    Article  CAS  PubMed  Google Scholar 

  • Boetius A, Ravenschlag K, Schubert CJ, Rickert D, Widdel F, Gieseke A, Amann R, Jorgensen BB, Witte U, Pfannkuche O (2000) A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407(6804):623–626

    Article  CAS  PubMed  Google Scholar 

  • Brankatschk R, Bodenhausen N, Zeyer J, Burgmann H (2012) Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples. Appl Environ Microbiol 78(12):4481–4489

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ding ZW, Ding J, Fu L, Zhang F, Zeng RJ (2014) Simultaneous enrichment of denitrifying methanotrophs and anammox bacteria. Appl Microbiol Biotechnol 98(24):10211–10221

    Article  CAS  PubMed  Google Scholar 

  • Du JK, Xiao K, Li L, Ding X, Liu HL, Lu YJ, Zhou SN (2013) Temporal and spatial diversity of bacterial communities in coastal waters of the South China Sea. Plos One 8(6)

  • Ettwig KF, Butler MK, Le Paslier D, Pelletier E, Mangenot S, Kuypers MMM, Schreiber F, Dutilh BE, Zedelius J, de Beer D, Gloerich J, Wessels HJCT, van Alen T, Luesken F, Wu ML, van de Pas-Schoonen KT, den Camp HJMO, Janssen-Megens EM, Francoijs KJ, Stunnenberg H, Weissenbach J, Jetten MSM, Strous M (2010) Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464(7288):543–548

    Article  CAS  PubMed  Google Scholar 

  • Ettwig KF, Shima S, van de Pas-Schoonen KT, Kahnt J, Medema MH, op den HJM C, MSM J, Strous M (2008) Denitrifying bacteria anaerobically oxidize methane in the absence of Archaea. Environmental Microbiology 10(11):3164–3173

    Article  CAS  PubMed  Google Scholar 

  • Ettwig KF, van Alen T, van de Pas-Schoonen KT, Jetten MSM, Strous M (2009) Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum. Appl Environ Microbiol 75(11):3656–3662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hallam SJ, Putnam N, Preston CM, Detter JC, Rokhsar D, Richardson PM, DeLong EF (2004) Reverse methanogenesis: testing the hypothesis with environmental genomics. Science 305(5689):1457–1462

    Article  CAS  PubMed  Google Scholar 

  • Han P, Gu JD (2013) A newly designed degenerate PCR primer based on pmoA gene for detection of nitrite-dependent anaerobic methane-oxidizing bacteria from different ecological niches. Appl Microbiol Biotechnol 97(23):10155–10162

    Article  CAS  PubMed  Google Scholar 

  • Haroon MF, Hu S, Shi Y, Imelfort M, Keller J, Hugenholtz P, Yuan Z, Tyson GW (2013) Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage. Nature 500(7464):567–570

    Article  CAS  PubMed  Google Scholar 

  • Hedrick DB, Peacock A, Stephen JR, Macnaughton SJ, Bruggemann J, White DC (2000) Measuring soil microbial community diversity using polar lipid fatty acid and denaturing gradient gel electrophoresis data. Journal of Microbiological Methods 41(3):235–248

    Article  CAS  PubMed  Google Scholar 

  • Hinrichs KU, Hayes JM, Sylva SP, Brewer PG, DeLong EF (1999) Methane-consuming archaebacteria in marine sediments. Nature 398(6730):802–805

    Article  CAS  PubMed  Google Scholar 

  • Hu BL, Shen LD, Lian X, Zhu Q, Liu S, Huang Q, He ZF, Geng S, Cheng DQ, Lou LP, Xu XY, Zheng P, He YF (2014) Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands. Proc Natl Acad Sci U S A 111(12):4495–4500

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hu BL, Shen LD, Zheng P, Hu AH, Chen TT, Cai C, Liu S, Lou LP (2012) Distribution and diversity of anaerobic ammonium-oxidizing bacteria in the sediments of the Qiantang River. Environ Microbiol Rep 4(5):540–547

    Article  CAS  PubMed  Google Scholar 

  • Hu SH, Zeng RJ, Burow LC, Lant P, Keller J, Yuan ZG (2009) Enrichment of denitrifying anaerobic methane oxidizing microorganisms. Environ Microbiol Rep 1(5):377–384

    Article  CAS  PubMed  Google Scholar 

  • Joye SB, Boetius A, Orcutt BN, Montoya JP, Schulz HN, Erickson MJ, Lugo SK (2004) The anaerobic oxidation of methane and sulfate reduction in sediments from Gulf of Mexico cold seeps. Chem Geol 205(3–4):219–238

    Article  CAS  Google Scholar 

  • Kojima H, Tsutsumi M, Ishikawa K, Iwata T, Mussmann M, Fukui M (2012) Distribution of putative denitrifying methane oxidizing bacteria in sediment of a freshwater lake, Lake Biwa. Syst Appl Microbiol 35(4):233–238

    Article  CAS  PubMed  Google Scholar 

  • Kruger M, Meyerdierks A, Glockner FO, Amann R, Widdel F, Kube M, Reinhardt R, Kahnt R, Bocher R, Thauer RK, Shima S (2003) A conspicuous nickel protein in microbial mats that oxidize methane anaerobically. Nature 426(6968):878–881

    Article  PubMed  Google Scholar 

  • Lim J, Do H, Shin SG, Hwang S (2008) Primer and probe sets for group-specific quantification of the genera Nitrosomonas and Nitrosospira using real-time PCR. Biotechnol Bioeng 99(6):1374–1383

    Article  CAS  PubMed  Google Scholar 

  • Losekann T, Knittel K, Nadalig T, Fuchs B, Niemann H, Boetius A, Amann R (2007) Diversity and abundance of aerobic and anaerobic methane oxidizers at the Haakon Mosby mud volcano, Barents Sea. Appl Environ Microbiol 73(10):3348–3362

    Article  PubMed  PubMed Central  Google Scholar 

  • Luesken FA, van Alen TA, van der Biezen E, Frijters C, Toonen G, Kampman C, Hendrickx TLG, Zeeman G, Temmink H, Strous M, den Camp HJMO, Jetten MSM (2011a) Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge. Applied Microbiology and Biotechnology 92(4):845–854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Luesken FA, Zhu BL, van Alen TA, Butler MK, Diaz MR, Song B, den Camp HJMO, Jetten MSM, Ettwig KF (2011b) pmoA primers for detection of anaerobic methanotrophs. Appl Environ Microbiol 77(11):3877–3880

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Michaelis W, Seifert R, Nauhaus K, Treude T, Thiel V, Blumenberg M, Knittel K, Gieseke A, Peterknecht K, Pape T, Boetius A, Amann R, Jorgensen BB, Widdel F, Peckmann J, Pimenov NV, Gulin MB (2002) Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane. Science 297(5583):1013–1015

    Article  CAS  PubMed  Google Scholar 

  • Niemann H, Losekann T, de Beer D, Elvert M, Nadalig T, Knittel K, Amann R, Sauter EJ, Schluter M, Klages M, Foucher JP, Boetius A (2006) Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink. Nature 443(7113):854–858

    Article  CAS  PubMed  Google Scholar 

  • Orphan VJ, House CH, Hinrichs KU, McKeegan KD, DeLong EF (2002) Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments. Proc Natl Acad Sci U S A 99(11):7663–7668

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qin PZ, Niu CG, Zeng GM, Ruan M, Tang L, Gong JL (2009) Time-resolved fluorescence based DNA detection using novel europium ternary complex doped silica nanoparticles. Talanta 80(2):991–995

    Article  CAS  PubMed  Google Scholar 

  • Raghoebarsing AA, Pol A, van de Pas-Schoonen KT, Smolders AJP, Ettwig KF, Rijpstra WIC, Schouten S, Damste JSS, Op den Camp HJM, Jetten MSM, Strous M (2006) A microbial consortium couples anaerobic methane oxidation to denitrification. Nature 440(7086):918–921

    Article  CAS  PubMed  Google Scholar 

  • Shen LD, Liu S, Zhu Q, Li XY, Cai C, Cheng DQ, Lou LP, Xu XY, Zheng P, Hu BL (2014a) Distribution and diversity of nitrite-dependent anaerobic methane-oxidising bacteria in the sediments of the Qiantang River. Microbial Ecol 67(2):341–349

    Article  CAS  Google Scholar 

  • Shen LD, Zhu Q, Liu S, Du P, Zeng JN, Cheng DQ, Xu XY, Zheng P, Hu BL (2014b) Molecular evidence for nitrite-dependent anaerobic methane-oxidising bacteria in the Jiaojiang Estuary of the East Sea (China). Appl Microbiol Biotechnol 98(11):5029–5038

    Article  CAS  Google Scholar 

  • Wang Y, Zhu G, Harhangi HR, Zhu B, Jetten MSM, Yin C, Op den Camp HJM (2012) Co-occurrence and distribution of nitrite-dependent anaerobic ammonium and methane-oxidizing bacteria in a paddy soil. Fems Microbiol Lett 336(2):79–88

    Article  CAS  PubMed  Google Scholar 

  • Wrede C, Brady S, Rockstroh S, Dreier A, Kokoschka S, Heinzelmann SM, Heller C, Reitner J, Taviani M, Daniel R, Hoppert M (2012) Aerobic and anaerobic methane oxidation in terrestrial mud volcanoes in the Northern Apennines. Sediment Geol 263:210–219

    Article  Google Scholar 

  • Zhu BL, van Dijk G, Fritz C, Smolders AJP, Pol A, Jetten MSM, Ettwig KF (2012) Anaerobic oxidization of methane in a minerotrophic peatland: enrichment of nitrite-dependent methane-oxidizing bacteria. Appl Environ Microbiol 78(24):8657–8665

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors would like to acknowledge the financial support from the National Natural Science Foundation of China (51178444), the National Hi-Technology Development 863 Program of China (2011AA060901), the Hundred-Talent Program of the Chinese Academy of Science (CAS), the Collaborative Innovation Center of Suzhou Nano Science and Technology, the Program for Changjiang Scholars and Innovative Research Team in University, the Fundamental Research Funds for the Central Universities (wk2060190040), and the Research Grants Council of Hong Kong Special Administrative Region, China (project no. CityU 160110).

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Correspondence to Raymond J. Zeng.

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New primers design for DAMO archaea. (DOCX 3.95 mb)

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Ding, J., Ding, ZW., Fu, L. et al. New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches. Appl Microbiol Biotechnol 99, 9805–9812 (2015). https://doi.org/10.1007/s00253-015-6893-6

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  • DOI: https://doi.org/10.1007/s00253-015-6893-6

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