Optimizing the extraction of extracellular DNA of aerobic granular biofilms

Formation and properties of aerobic granules for enhanced biological phosphorus removal (EBPR) from sequencing batch reactor (SBR) strongly depend on the extracellular polymeric substances (EPS) in which the microorganisms are embedded. The composition of EPS varies between different species but mostly it is made up of proteins, polysaccharides and extracellular DNA (eDNA), that has been recently recognized as an important structural component. Although great amount of literature on the EPS extraction is available, till now, there is no standardized protocol for EPS extraction. Extraction methods involving multi step processes seem to be the most promising, although too harsh extraction procedures can lyse the cells and release intracellular compounds, which contaminate the EPS samples creating bias. Commonly employed intracellular markers of cell lysis i.e. Glucose-6-phosphate dehydrogenase (G6PDH) or 2-keto-3deoxyoctonate (KDO) are not always applicable, especially in systems where glycolysis does not occur and in mixed communities with gram-positive bacteria, respectively. Therefore a new method to measure cell lysis is needed. The activity of isocitrate dehydrogenase (ICDH), an ubiquitous enzyme found in all living organisms utilzing the Krebs (citric acid) cycle, has been assessed. Initial assays using this enzyme activity during EPS extraction revealed to be very sensitive to detect cell lysis in samples from mixed microbial communities as granular sludge biofilms cultivated in SBR. Preliminary results of EPS extraction using multi-step method and enzymatic extraction as well as selection of appropriate dyes to visualize and distinguish intracellular and extracellular DNA in granular samples from the reactor are discussed.