The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acidcontaining actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata, a protocol to permeabilize mycolata, and a statistically robust quantification method. Statistical analyses showed that a lipase-based permeabilization method was quantitatively superior to previously described methods (P << 0.05). When mixed liquor and foam samples were examined, most of the mycolata present were rods or cocci, although filamentous mycolata were also observed. A nested analysis of variance showed that virtually all of the measured variance occurred between fields of view and not between samples. On this basis we determined that as few as five fields of view could be used to give a statistically meaningful sample. Quantitative fluorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m 3 completely mixed activated sludge plant. Foaming occurred when the number of mycolata exceeded a certain threshold value. Baffling of the plant affected foaming without affecting the number of mycolata. We tentatively estimated that the threshold foaming concentration of mycolata was about 2 ؋ 10 6 cells ml ؊1 or 4 ؋ 10 12 cells m ؊2. We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigation of foaming in activated sludge.Microbe-based treatment problems occur in the plants of even the most sophisticated and experienced water utilities. One of the most intractable and widespread problems is the formation of viscous foams in activated sludge plants. Such foams appear as bacterial biomass floating on the surfaces of aeration basins and secondary sedimentation tanks. The presence of foam may lead to severe operational problems and may cause plants to fail effluent standards. The occurrence of foam is unpredictable, and there is uncertainty about the cause and mechanism of foaming; the control mechanisms used currently are empirical and frequently ineffective (45). It is thought that foams may form when gas bubbles are stabilized by the presence of hydrophobic particles and surfactants; the bubbles then rise to the surface and accumulate (45). The hydrophobic particles are assumed to be bacteria.Microscopic examination of foams usually reveals the presence of large numbers of one or two filamentous bacterial morphotypes. It is assumed that the morphotypes that are branched are mycolic acid-containing actinomycetes (mycolata) (13, 48) and that unbranched filaments are Microthrix parvicella (45). Putative mycolata morphotypes are the most widely reported morphotypes (39,43) and are often referred to as "nocardia." However, a wide range of mycolata are now associated with foaming. Plate cou...

Isolates from activated sludge foam were provisionally assigned to the genera Gordona and Tsukamurella on the basis of colony morphology and pigmentation. Representatives of the first group were compared with marker strains of validly described species of Gordona by using Curie-point pyrolysis mass spectrometry. Most of the isolates fell into a number of taxa which were equated with groups of marker strains which corresponded to the known Gordona species. In a corresponding experiment the activated sludge isolates, which were provisionally labelled “Tsukamurella spumae” formed a group which was well separated from the marker strains of the genus. Representatives of the isolates were found to have chemical properties consistent with their classification in the genus Tsukamurella. 16S rDNA sequence data, when taken with previous DNA:DNA relatedness results, suggest that Tsukamurella paurometabola is heterogeneous. Similarly, the sequence data provide some evidence that “Tsukamurella spumae” may merit recognition as a novel species.

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...

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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: