Improved rRNA extraction from biofouling and bioreactor samples

Abstract

Mixed microbial communities are major drivers of biofouling phenomena that lead to deterioration of materials or compromise process efficiency and are also extensively employed in environmental biotechnology. The metabolically active organisms in these communities are central to further development of environmental bioprocesses or of countermeasures for biofouling control. These organisms can be targeted selectively by analysis of ribosomal RNA because the number of ribosomes in a cell is proportional to the rate of protein synthesis. Comparative analysis of the deficiencies of 6 different rRNA extraction protocols using activated sludge as model system led to the formulation of a new protocol, that consistently produced cleaner extracts from fresh or frozen samples of activated sludge with better rRNA quality and better preservation of the proportions between signatures from different community members after 10 cycles PCR. PCR amplification was best with pfu buffer + 4 mM MgCl2 at a lower annealing temperature of 60 °C. Inhibition of PCR occurred in solutions with 2% formamide. rRNA of good quality was extracted with the new protocol from samples of corrosion deposits, of both the solid and liquid fraction of chalcopyrite bioleaching experiments, of biofilms on slow sand filter media, from walls of water reservoirs and of water pipes, as well as of fouling deposits on reverse osmosis membranes.