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

  • Miao Hu
  • Matthew Lee
  • Ling Zhong
  • Michael J. ManefieldEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2046)


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.

Key words

Dichloromethane DNA Protein Stable isotope probing (SIP) Activated sludge Metaproteomics Metagenomics 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Miao Hu
    • 1
  • Matthew Lee
    • 1
  • Ling Zhong
    • 2
  • Michael J. Manefield
    • 1
    • 3
    Email author
  1. 1.UNSW Water Research Centre, School of Civil and Environmental EngineeringUniversity of New South WalesSydneyAustralia
  2. 2.Bioanalytical Mass Spectrometry FacilityUniversity of New South WalesSydneyAustralia
  3. 3.School of Chemical EngineeringUniversity of New South WalesSydneyAustralia

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