Hu Miao, Lee Matthew, Zhong Ling, Manefield Michael J
UNSW Water Research Centre, School of Civil and Environmental Engineering, University of New South Wales, Sydney, NSW, Australia.
Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, Australia.
Methods Mol Biol. 2019;2046:207-219. doi: 10.1007/978-1-4939-9721-3_16.
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 C-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.
二氯甲烷（DCM）是一种有毒的、密度较大的非水相液体（DNAPL），在所有工业化国家都会污染地下水。幸运的是，DCM可被厌氧微生物用作唯一的能量和有机碳源，并转化为乙酸盐、甲酸盐和碳酸氢盐等良性终产物。然而，关于能够代谢DCM的厌氧微生物的系统发育多样性的知识有限。所涉及的基因和酶以及反应机制的细节尚不清楚。稳定同位素探测（SIP）是一种用于识别参与元素同化的微生物的技术。同位素标记的底物可在DNA和蛋白质中回收（即DNA-SIP和蛋白质-SIP），这使得能够识别参与底物降解的微生物分类群及其各自的蛋白质。因此，通过将SIP技术与分子方法（即Illumina Miseq测序和宏蛋白质组学）相结合，可以以独立于厌氧富集培养的方式鉴定和表征DCM降解生物。在我们的研究中，分别向污水处理厂的活性污泥中加入未标记的和C标记的DCM。在这里，我们提供了从活性污泥中提取DNA和蛋白质的方案和技术说明，并展示了下游分子技术的分析流程。
