Effects of freezing storage on the DNA extraction and microbial evaluation from anaerobic digested sludges.

Background: The anaerobic digestion is one of the most spread renewable energy technology. The input biomasses included various environmental problematic wastes such as sludge coming from wastewater treatment plant (WWTP) and organic fraction of municipal solid waste (OFMSW). As biomolecular procedures have become important tools for the microbial characterisation of anaerobic samples coming from the reactors, it is crucial sampling and extracting properly DNA in order to employ such types of techniques. The current study is aimed to evaluate how freezing temperature and length of storage at −20 °C influence both the extracted DNA yield and microbial community quantifications from digested sludge samples collected at full-scale plants. Results: From WWTP sludge samples, we observed a reduction of DNA concentration comparing fresh and stored samples for 10 days at −20 °C (ANOVA test p < 0.0001), with an estimated DNA loss of approximately 65 % for such types of samples, however the methanogen communities can be assessed respecting the fresh conditions. From OFMSW sludge samples, we observed a reduction in extracted DNA (−90 %), after 120 frozen days, while microbial communities are determined respecting the fresh conditions within 2 months of frozen storage. Conclusions: The remarkable effect of frozen storage on sludge samples suggests as the better procedure to perform the DNA extraction from fresh sample. On the other hand it is not generally possible, so approximately 2 months of storage at −20 °C appears to be suitable time at which DNA concentrations remain sufficient to perform coherent microbial characterization through quantitative qRT-PCR.