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Method Development for DNA and Proteome SIP Analysis of Activated Sludge for Anaerobic Dichloromethane Biodegradation

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Stable Isotope Probing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2046))

Abstract

Dichloromethane (DCM) is a toxic, dense non-aqueous phase liquid (DNAPL) that pollutes groundwater in all industrialized countries. Fortunately, DCM can be used as the sole source of energy and organic carbon by anaerobic microorganisms and be transformed to benign end products such as acetate, formate, and bicarbonate. However, knowledge around the phylogenetic diversity of anaerobic microorganisms capable of DCM metabolism is limited. The genes and enzymes involved and details of the reaction mechanism are not known. Stable isotope probing (SIP) is a technique used to identify microbes involved in assimilation of elements. The isotopically labeled substrate can be recovered in DNA and protein (i.e., DNA-SIP and protein-SIP) which enables identification of both the microbial taxa and their respective proteins involved in the substrate degradation. Therefore, by applying a combination of SIP techniques with molecular approaches (i.e., Illumina Miseq sequencing and metaproteomics), DCM degrading organisms can be identified and characterized in a manner independent of anaerobic enrichment cultures. In our research, activated sludge from wastewater treatment plant was fed with unlabeled and 13C-labeled DCM, respectively. Here, we provide protocols and technical notes for DNA and protein extraction from activated sludge and present analysis pipelines for downstream molecular techniques.

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Author information

Authors and Affiliations

  1. UNSW Water Research Centre, School of Civil and Environmental Engineering, University of New South Wales, Sydney, NSW, Australia

    Miao Hu, Matthew Lee & Michael J. Manefield

  2. Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, Australia

    Ling Zhong

  3. School of Chemical Engineering, University of New South Wales, Sydney, NSW, Australia

    Michael J. Manefield

Authors
  1. Miao Hu
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  2. Matthew Lee
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  3. Ling Zhong
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  4. Michael J. Manefield
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Corresponding author

Correspondence to Michael J. Manefield .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, University of Southampton, Southampton, UK

    Marc G. Dumont

  2. School of Biological Sciences, University of Southampton, Southampton, UK

    Marcela Hernández García

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Hu, M., Lee, M., Zhong, L., Manefield, M.J. (2019). Method Development for DNA and Proteome SIP Analysis of Activated Sludge for Anaerobic Dichloromethane Biodegradation. In: Dumont, M., Hernández García, M. (eds) Stable Isotope Probing. Methods in Molecular Biology, vol 2046. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9721-3_16

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  • DOI: https://doi.org/10.1007/978-1-4939-9721-3_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9720-6

  • Online ISBN: 978-1-4939-9721-3

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