Elizabeth M. Seviour

A simple HPLC method for analysing diaminopimelic acid diastereomers in cell walls of Gram-positive bacteria

A simple and sensitive method for separating and detecting the LL, DD and meso diastereomers of the dibasic amino acid diaminopimelic acid (DAP) in the peptidoglycan of Gram‐positive bacteria is described. This method is based on reverse phase HPLC separation of chiral derivatives of DAP followed by fluorescence detection of the o‐phthaldehyde derivatives. Its application to the analyses of cell walls of several Gram‐positive bacteria is described, where 10 mg or less of dry cells is required.

“Microthrix parvicella” is a Novel, Deep Branching Member of the Actinomycetes Subphylum

Summary We have isolated the Gram-positive, straight filamentous bacterium “ Microthrix parvicella ” from an activated sludge sewage treatment plant in Australia by using micromanipulation techniques. The 16S rDNA was amplified directly from cell biomass by the polymerase chain reaction and sequenced. This data has allowed us to phylogenetically place M. parvicella as a deep branching member of the actinomycetes subphylum. It is most closely related to the iron-oxidising strain TH3, members of the order Actinomycetales and to the genus Atopobium . Further phenotypic data for “M. parvicella” are required for the valid naming of this organism.

16S rRNA Analysis of Isolates Obtained from Gram-Negative, Filamentous Bacteria Micromanipulated from Activated Sludge

Summary Individual filaments of the Gram negative, bulking filamentous morphotypes Eikelboom Type 0092, Type 0411, Type 0803 and Herpetosiphon sp. were identified in activated sludge mixed liquors and specifically isolated by micromanipulation. Although their isolation and partial phenotypic description have previously been reported, we sought them to determine their phylogenetic position and to compare our data with the previous descriptions. Direct cell lysis procedures and the polymerase chain reaction were used to obtain their 16S rRNA genes. These were sequenced and the data were analysed to phylogenetically place the filaments into their respective lines of descent in the domain Bacteria . Type 0803 is a member of the Rubrivivax subgroup of the beta proteobacterial subclass; Herpetosiphon sp. belongs in the Chloroflexus subdivision of the green non-sulfur lineage and its sequence is most similar to that of H. aurantiacus ; while Type 0092 and Type 0411 belong in the Flex ibacter-Cytophaga-Bacteroides phylum. The contemporary “identification” of these organisms is ambiguous and relies on subjective morphological criteria and staining reactions. However, the sequence data reported here are being examined for morphotype-specific regions to be exploited for RNA-directed, DNA probes for rapid, unequivocal, in situ identification of each filament type.

The filamentous morphotype Eikelboom Type 1863 is not a single genetic entity

Five isolates of a filamentous bacterial morphotype with the distinctive diagnostic microscopic features of Eikelboom Type 1863 were obtained from activated sludge sewage treatment plants in Victoria, Australia. On the basis of phenotypic evidence and 16S rDNA sequence data, these isolates proved to be polyphyletic. Two (Ben 06 and Ben 06C) are from the Chryseobacterium subgroup which is in the Cytophaga group, subdivision I of the Flexibacter–Cytophaga–Bacteroides phylum. Two (Ben 56 and Ben 59) belong to the genus Acinetobacter, and one (Ben 58) is a Moraxella sp., closest to Mor. osloensis. The significance of these findings to the reliance on microscopic features for identification of these filamentous bacteria in activated sludge is discussed.

Towards understanding the taxonomy of some of the filamentous bacteria causing bulking and foaming in activated sludge plants

Several filamentous bacteria obtained in pure culture from activated sludge plants were characterised using their 16S rDNA sequences to determine their phylogenetic relationship to other bacteria. “ Microthrix parvicella ” was shown to be an unusual actinomycete, while the Gram negative bacteria Type 0092, Type 0411 and Type 1863 all belong to the Flexibacter-Cytophaga-Bacteroides phylum, and Type 0803 is a member of the beta subclass of the Proteobacteria. The practical value of obtaining this information is discussed.

Filamentous members of cluster III Defluviicoccus have the in situ phenotype expected of a glycogen-accumulating organism in activated sludge.

The in situ ecophysiology of alphaproteobacterial filamentous Cluster III Defluviicoccus present in enhanced biological phosphorus removal (EBPR)-activated sludge systems was evaluated using FISH-MAR and histochemical staining methods. These organisms, sharing the Nostocoida limicola morphotype, are known to be responsible for serious episodes of activated sludge bulking. The data presented here also demonstrate an ability to assimilate short-chain fatty acids and synthesize poly-β-hydroxyalkanoates (PHA) anaerobically, and then utilize this stored PHA under aerobic conditions, but with no corresponding synthesis of polyphosphate. These features are consistent with an in situ phenotype of glycogen-accumulating organisms (GAO), populations thought to lower the efficiency of EBPR systems by outcompeting polyphosphate-accumulating organisms (PAO) for substrates in their anaerobic feed phase. Survey data indicate that these GAO are as commonly seen as the known PAO in full-scale EBPR-activated sludge systems, which suggest that they might play important roles there, and therefore should not be viewed just as laboratory curiosities.

Ultrastructure of Microthrix parvicella from activated sludge

H.M. STRATTON, R. WEBB, E.M. SEVIOUR, L.L. BLACKALL AND R.J. SEVIOUR. 1996. Filaments of Microthrix parvicella grow very poorly in pure culture and have a characteristic uneven appearance containing large numbers of distinctive spherical swollen cells. This feature was only rarely seen with filaments of this organism in activated sludge biomass samples. Ultrastructurally, these spherical swollen cells do not appear to be bacterial endospores or cysts, but do show features that are consistent with them being some form of resting structure. Their production could be a response of M. parvicella to environmental stress, an explanation proposed because of their relative predominance in pure cultures of M. parvicella compared to their infrequent occurrence in filaments of this bacterium observed in activated sludge biomass.