Identification of Active Microorganisms in Environmental Biofilms Using 16SrRNA Based Approaches: The Challenge of Obtaining Representative Information About the Microbial Community

A protocol for identification of physiologically active microbes within biofilms based on extraction of ribosomal RNA was validated. Cloning of cDNA conserved information about the relative proportion of the three members (Escherichia coli, Burkholderia cepacia and Pseudodomonas aeruginosa) of a community established by mixing their 16SrRNA cDNA. 30 cycle PCR amplification of these cDNA mixtures, however, resulted in considerable distortion of proportionality, a problem fixed by reducing the number of PCR cycles to 10. Application of the optimized protocol to the analysis of a biofilm from a bioreactor treating surface water for biofouling control of reverse osmosis membranes showed good reproducibility of identification of active community members (ARDRA clones present in a frequency of >2% in the clone library) in replicate samples, analysed either directly after sampling or after storage of samples at -20°C for 2 months. Clone libraries obtained after extracting biofilm samples with a commercial kit, however, showed a totally different distribution of clones.