Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae. The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented. A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria. Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group. Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species. A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.

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.

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