Increasing incidences of activated sludge foaming have been reported in the last decade in Danish plants treating both municipal and industrial wastewaters. In most cases, foaming is caused by the presence of Actinobacteria; branched mycolic acid-containing filaments (the Mycolata) and the unbranched Candidatus'Microthix parvicella'. Surveys from wastewater treatment plants revealed that the Mycolata were the dominant filamentous bacteria in the foam. Gordonia amarae-like organisms and those with the morphology of Skermania piniformis were frequently observed, and they often coexisted. Their identity was confirmed by FISH, using a new permeabilization procedure. It was not possible to identify all abundant Mycolata using existing FISH probes, which suggests the presence of currently undetectable and potentially undescribed populations. Furthermore, some Mycolata failed to give any FISH signal, although substrate uptake experiments with microautoradiography revealed that they were physiologically active. Ecophysiological studies were performed on the Mycolata identified by their morphology or FISH in both foams and mixed liquors. Large differences were seen among the Mycolata in levels of substrate assimilation and substrate uptake abilities in the presence of different electron acceptors. These differences were ascribed mainly to the presence of currently undescribed Mycolata species and/or differences in foam age.

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The taxonomic position of two mycolic-acid-producing actinomycetes, isolates J81 T and J82, which were recovered from activated sludge foam, was clarified. Comparative 16S rRNA gene sequence studies indicated that the organisms formed a distinct lineage within the Corynebacterineae 16S rRNA gene tree. The taxonomic integrity of this group was underpinned by a wealth of phenotypic data, notably characteristic rudimentary right-angled branching. In addition, isolate J81 T contained the following: meso-diaminopimelic acid, arabinose and galactose; N-glycolated muramic acid residues; a dihydrogenated menaquinone with eight isoprene units as the predominant isoprenologue; a fatty acid profile rich in oleic and palmitoleic acids and with relatively small proportions of myristic, stearic and tuberculostearic acids; mycolic acids with 44-52 carbons; and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides as major polar lipids. Strain J81 T was found to have a chemotaxonomic profile that serves to distinguish it from representatives of all of the other taxa classified as belonging to the suborder Corynebacterineae. In the light of these data, it is proposed that the two isolates be classified in a novel monospecific genus. The name proposed for this taxon is Millisia brevis gen. nov., sp. nov.; strain J81 T (=DSM 44463 T =NRRL B-24424 T ) is the type strain of Millisia brevis.The taxonomy of mycolic-acid-containing actinomycetes has been clarified and extended by the application of genotypic and phenotypic procedures to representatives of established and novel taxa (Goodfellow et al., 1998a(Goodfellow et al., , 1999 Gürtler et al., 2004). Actinomycetes characterized by the presence of mycolic acids are classified in the suborder Corynebacterineae Stackebrandt et al. 1997, which encompasses the genera Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocardia, Rhodococcus, Segniliparus, Skermania, Tsukamurella and Williamsia (Butler et al., 2005;Goodfellow & Maldonado, 2006). Members of these taxa can be distinguished from one another using a combination of chemotaxonomic and morphological properties, and form distinct lineages in the Corynebacterineae 16S rRNA gene tree. Activated sludge wastewater-treatment plants with foaming problems (Soddell, 1999) are a rich source of mycolic-acid-containing actinomycetes (Lemmer & Kroppenstedt, 1984;Soddell & Seviour, 1990, 1995Goodfellow et al., 1998b;Stainsby et al., 2002) Two actinomycetes that showed rudimentary right-angled branching and which produced salmon-pink filamentous colonies were isolated from activated sludge foam by using a micromanipulator (Soddell & Seviour, 1994). Subsequent studies based on numerical taxonomic and preliminary 16S rRNA gene sequence data indicated that the two strains, isolates J81 T and J82, might represent a novel genus in the suborder Corynebacterineae , a proposition underpinned by the results of the present investigation.DNA from strains J81 T and J82 was extract...

Three strains of non-motile, Gram-positive, filamentous actinomycetes, isolates J4 T , J5 and J59, initially recognized microscopically in activated sludge foam by their distinctive branching patterns, were isolated by micromanipulation. The taxonomic positions of the isolates were determined using a polyphasic approach. Almost-complete 16S rRNA gene sequences of the isolates were aligned with corresponding sequences of representatives of the suborder Corynebacterineae and phylogenetic trees were inferred using three tree-making algorithms. The organisms formed a distinct phyletic line in the Gordonia 16S rRNA gene tree. The three isolates showed 16S rRNA gene sequence similarities within the range 96?9-97?2 % with their nearest phylogenetic neighbours, namely Gordonia bronchialis DSM 43247 T and Gordonia terrae DSM 43249T was shown to have a chemotaxonomic profile typical of the genus Gordonia and was readily distinguished from representatives of the genus on the basis of Curie-point pyrolysis mass spectrometric data. The isolates shared nearly identical phenotypic profiles that distinguished them from representatives of the most closely related Gordonia species. It is evident from the genotypic and phenotypic data that the three isolates belong to a novel Gordonia species. The name proposed for this taxon is Gordonia defluvii sp. nov.; the type strain is J4Most activated sludge systems suffer intermittently from the serious operational disorder known as foaming or scumming, whereby a stable foam or scum develops on the surface of aeration tanks (Soddell, 1999 et al., 2003, 2004). However, it is clear that many additional mycolic acidcontaining taxa associated with foam need to be formally described (Soddell & Seviour, 1998;Stainsby et al., 2002). Such studies are important, as attempts to control the appearance and persistence of foams are unlikely to succeed until the taxonomic diversity and functional roles of the causal organisms are understood (Goodfellow et al., 1996(Goodfellow et al., , 1998Stainsby et al., 2002).Three isolates with a cellular morphology intermediate between the right-angled branching pattern typical of G. amarae Klatte et al. 1994 and the 'pine-tree-like' morphotype of Skermania piniformis Chun et al. 1997 were isolated, by micromanipulation, in Australia from activated sludge foams at Brimbank Park, Victoria (isolates J4 T and J5) and Craigieburn, Victoria (isolate J59), as described by Soddell & Seviour (1998). These authors considered that the strains might form a novel species on the basis of an extensive numerical taxonomic survey of Skermania and related strains, a proposition underpinned by the results of the present investigation.3Present address:

The in situ physiology of the filamentous bacterium Skermania piniformis frequently seen in activated sludge foams in Australia was investigated. An oligonucleotide probe, Spin1449, targeting the 16S rRNA of S. piniformis was designed for its identification by fluorescence in situ hybridization (FISH), validated with pure cultures and applied successfully to foam samples from two geographically distant Australian plants. While filaments of this bacterium appeared to be comparatively hydrophobic, the organism had no clear preference for hydrophobic or hydrophilic substrates. In both foams examined using microautoradiography (MAR), filaments selectively took up substrates under aerobic and anoxic (NO(3) (-)) but not anaerobic or anoxic (NO(2) (-)) conditions. Skermania piniformis assimilated oleic acid, palmitic acid, glycerol and glycine. Ectoenzyme activities detected suggest that S. piniformis has an ability to assimilate a greater range of substrates than might be concluded from the MAR data obtained here. Based on the substrate uptake data presented here, an anaerobic selector may work for controlling S. piniformis in activated sludge systems.