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

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.