Molecular biological methods to detect “Microthrix parvicella” and to determine its abundance in activated sludge

Abstract

Molecular biological methods were evaluated in attempts to detect and quantitate levels of “Microthrix parvicella” in activated sludges. Approximately 66% of the 23S rRNA gene sequence of a strain (Ben43) of the Gram positive bulking and foaming organism “Microthrix parvicella” was determined, while a lesser amount was determined for “M. parvicella” strain RN1. The high mol%G+C Gram positive bacteria (HGCGPBs) possess two powerfully diagnostic regions in the 23S rDNA and these were investigated in both strains. Firstly, the 18 nucleotide HGCGPB probe sequence (HGC69a) varied in at least two nucleotides with the sequence from both strains of “M. parvicella”. Secondly, an approximately 100 nucleotide stable insert between helices 54 and 55 in the 23S rRNA of HGCGPBs was discovered to be present in “M. parvicella”, but in both strains it was unique in length (79 nucleotides) and sequence. The region of the 23S rDNA with the stable insert was exploited to develop a polymerase chain reaction assay in which amplicons from “M. parvicella” were larger than those from nonHGCGPBs (i.e. all Bacteria except the HGCGPBs), and smaller than those from HGCGPBs. This assay was evaluated with DNAs extracted from activated sludges but although “M. parvicella” was morphologically identified, and was a dominant filament in at least one of the samples, no “M. parvicella” specific sized amplicons could be recovered from it. Amplicons of sizes generated by nonHGCGPBs and HGCGPBs were routinely produced in the stable insert PCR with DNAs from activated sludges where the highest yield was of amplicons from nonHGCGPBs. A second series of experiments were undertaken with the objective of evaluating the use of a non-radioactive hybridization method, based on extraction of bacterial RNA, for quantifying “M. parvicella” in activated sludge samples. Total nucleic acids were extracted from activated sludge samples and immobilized on nylon membranes. Probing with 16S rRNA-directed DIG-labelled oligonucleotide probes, detection of chemiluminescent signals on membranes and densitometry allowed hybridization signals to be quantified. The relationship between the amount of nucleic acid hybridized and the hybridization signal intensity observed was found to be linear over a specified range of signal intensities. A range of activated sludge samples were analysed for “M. parvicella” and variations in levels could be distinguished.