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"Candidatus Microthrix parvicella" is a filamentous bacterium that grows with great difficulty in cultures from the mixed liquor of activated sludge sewage treatment plants. It is gram positive, and the ultrastructure of its cell walls has been determined to be of the gram-positive type by electron microscopical examination. Phylogenetically, it is a deep-branching member of the subphylum actinomycetes within the gram-positive phylum of the domain Bacteria. As for phenotypic features, it is known that the organism contains polyphosphate inclusions and that it is catalase positive. In mixed cultures in activated sludge plants and in pure culture in the laboratory, it has a characteristic and distinctive winding filamentous morphology, with filaments hundreds of micrometers long."Microthrix pawicella" is a filamentous bacterium found in activated sludge sewage treatment plants, where it causes solids separation problems known as foaming and bulking (7). It is found in these plants throughout the world, and when surveys of the solids separation problems are carried out, this bacterium features near the top of the list of causative organisms. "M. pawicella" is notorious in the wastewater industry because of its problem-causing properties. Although some researchers isolated it with apparent ease (4,5,15,17), in recent years it has eluded many, as evidenced by active discussion at the International Association on Water Quality conference on Microorganisms in Activated Sludge and Biofilm Processes (Paris, France [12]) and the workshop on Microbial Ecology in Wastewater (Stuttgart, Germany [9]).For a summary of the information on "M. pawicella", the work of Blackall et al. (2) should be consulted. However, much information on the attempts to isolate this organism is not included in that publication. We have attempted to obtain "M. pawicella" in axenic culture for 12 years. During this time it is estimated that more than 700 "M. pawicella" filaments have been micromanipulated. A wide range of media which were incubated in a range of atmospheres and temperatures was evaluated. In our experience, the "M. pawicel1a"-specific, chemically defined medium of Slijkhuis (14) has never supported the growth of any micromanipulated filaments nor that of any isolates of "M. pawicella" obtained from other media. Of all the media reported to support the growth of filamentous bacteria from activated sludge, only freshly prepared R2A (13) and modified NTM (2, 8) have been able to sustain "M. parvicella" growth, and these media are not universally successful. Atmospheres tested with a range of media included 90% CO, plus 10% H, and the microaerophilic conditions generated by so-called candle jars. Neither condition increased isolation success, nor did either accelerate the growth rates of isolates. Filaments of "M. pawicella" were manipulated to other media and incubated at natural atmosphere and 20 to 22°C. These media included (i) liquid and solid (1.5% agar) media based on activated sludge (autoclaved, filtered mixed liquor; autoclaved...

Five strains of ' Candidatus Nostocoida limicola ' were isolated by micromanipulation from two activated sludge plants. Two (Ben17 and Ben18) were from Sunbury, Victoria, Australia, and three (Ben67, Ver1 and Ver2) were from Verona, Italy. The near complete 16S rDNA sequences were determined for five strains and the phylogenetic location of this important bulking filament in the actinomycete subphylum is reported for the first time. Phylogenetically, the Ben strains formed one group with 994 % 16S rDNA similarity, and the Ver strains formed another with 999 % 16S rDNA similarity. The mean similarity between the two groups was 974 %. By 16S rDNA comparison, the closest relative to all strains was Terrabacter sp. strain DPO1361 (950-955 % identical). On R2A medium, all strains generally grew as short filaments or clumps of cocci, whereas on glucose sulfide (GS) medium, all grew as irregular twisting filaments comprising Gram-positive and Gramnegative cells, which is close to their in situ morphology. Polyphosphate was stored either as granules (R2A) or throughout the trichomes (GS). None of the strains could grow without added nitrogen, reduce nitrate to nitrogen gas or grow anaerobically, whereas all could grow at 15-30 SC, produce catalase and reduce nitrate to nitrite. All were inactive in the Hugh & Leifson test. This paper describes ' Candidatus Nostocoida limicola '.

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Clone libraries were prepared from polymerase chain reaction amplified 16S rDNAs from activated sludge community DNAs. Eight different libraries from a range of samples were prepared. From each library, up to 100 clones were examined. In some libraries, the clone inserts were grouped into operational taxonomic units (OTUs) by restriction enzyme analysis (REA). Then, either the clones or representatives of OTUs were partially sequenced using either 27f or 530f conserved bacterial primers. The sequence data was phylogenetically analysed to group the clones and the method currently gives the best insight into the activated sludge microbial community biodiversity. The method for clone library production is described and the pros and cons of the procedure are outlined. In summary, the use of clone libraries has resulted in the discovery of unimagined biodiversity in activated sludge. The abundance of some unpredicted bacterial groups (e.g. beta subclass Proteobacteria) and the paucity of expected ones (e.g. Acinetobacter) highlights the inadequacy of traditional culture dependent methods that rely on sample dilution and spread plate inoculation.