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Micromanipulated filamentous bacteria from bulking and foaming activated sludge morphologically identified as Eikelboom type 0803 were shown to be affiliated to the genus Caldilinea within the phylum Chloroflexi. Specific FISH probes were designed for their in situ detection and quantification in seven Danish wastewater treatment plants with biological nutrient removal. The survey applied all species-specific probes for Chloroflexi of relevance in activated sludge treatment plants as well as the phylum-specific probes. Type 0803 filaments constituted around 20% of the total Chloroflexi population. In four of the treatment plants, type 0803 and type 0092 co-occurred and were the dominating fraction of the Chloroflexi population. In the other plants, most Chloroflexi could not be identified beyond the phylum level, suggesting a yet far larger diversity. On average, for all plants, the total Chloroflexi population constituted 12% of the entire microbial population and seems to play an important structural role in the sludge floc formation. Ecophysiological characterization of type 0803 showed their potential role in macromolecule conversion as evident by high levels of exoenzyme expression. Acetate was not consumed. Glucose was consumed with oxygen, nitrite and nitrite as electron acceptors, suggesting that type 0803 may be a denitrifier. Their surfaces were hydrophobic, explaining their occasional occurrence in foaming incidents., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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.

The abundance of potential denitrifiers in full-scale wastewater treatment plants with biological nitrogen and phosphorus removal was investigated by FISH and various oligonucleotide probes. The potential denitrifiers were characterized as probe-defined populations that were able to consume radiolabelled substrate with oxygen, nitrate and nitrite as electron acceptor as determined by microautoradiography. The most abundant potential denitrifiers were related to the genera Aquaspirillum, Azoarcus, Thauera and Rhodocyclus, all within the Betaproteobacteria. They made up 20-49% of all bacteria in most of the 17 nitrogen removal plants investigated and were hardly present in four plants without denitrification. The ecophysiology of Aquaspirillum, Azoarcus and Thauera-related bacteria was consistent within each probe-defined group in the plants investigated. These three groups showed distinct physiological differences, with the Aquaspirillum-related bacteria appearing as the most specialized one, consuming only amino acids among the substrates tested, and Thauera as the most versatile consuming some volatile fatty acids, ethanol and amino acids. The coexistence of Aquaspirillum, Azoarcus and Thauera-related bacteria in a range of treatment plants with differences in wastewater, design and operation suggest that the populations ensure a functional stability of the plants by occupying different ecological niches related to the carbon transformation.

The diversity of sulfate-reducing bacteria (SRB) inhabiting the extreme hypersaline sediment (270 g L(-1) NaCl) of the northern arm of Great Salt Lake was studied by integrating cultivation and genotypic identification approaches involving PCR-based retrieval of 16S rRNA and dsrAB genes, the latter encoding major subunits of dissimilatory (bi) sulfite reductase. The majority (85%) of dsrAB sequences retrieved directly from the sediment formed a lineage of high (micro) diversity affiliated with the genus Desulfohalobium, while others represented novel lineages within the families Desulfohalobiaceae and Desulfobacteraceae or among Gram-positive SRB. Using the same sediment, SRB enrichment cultures were established in parallel at 100 and at 190 g L(-1) NaCl using different electron donors. After 5-6 transfers, dsrAB and 16S rRNA gene-based profiling of these enrichment cultures recovered a SRB community composition congruent with the cultivation-independent profiling of the sediment. Pure culture representatives of the predominant Desulfohalobium-related lineage and of one of the Desulfobacteraceae-affilated lineages were successfully obtained. The growth performance of these isolates and of the enrichment cultures suggests that the sediment SRB community of the northern arm of Great Salt Lake consists of moderate halophiles, which are salt-stressed at the in situ salinity of 27%.

Filamentous Chloroflexi species are often present in activated sludge wastewater treatment plants in relatively low numbers, although bulking incidences caused by Chloroflexi filaments have been observed. A new species-specific gene probe for FISH was designed and using phylum-, subdivision-, morphotype 1851- and species-specific gene probes, the abundance of Chloroflexi filaments were monitored in samples from 126 industrial wastewater treatment plants from five European countries. Chloroflexi filaments were present in 50% of the samples, although in low quantities. In most treatment plants the filaments could only be identified with phylum or subdivision probes, indicating the presence of great undescribed biodiversity. The ecophysiology of various Chloroflexi filaments was investigated by a suite of in situ methods. The experiments revealed that Chloroflexi constituted a specialized group of filamentous bacteria only active under aerobic conditions consuming primarily carbohydrates. Many exo-enzymes were excreted, e.g. chitinase, glucuronidase and galactosidase, suggesting growth on complex polysaccharides. The surface of Chloroflexi filaments appeared to be hydrophilic compared to other filaments present. These results are generally supported by physiological studies of two new isolates. Based on the results obtained in this study, the potential role of filamentous Chloroflexi species in activated sludge is discussed.

A comprehensive study of the ecophysiology of the filamentous Meganema perideroedes affiliated to the Alphaproteobacteria, possessing a "Nostocoida limicola Type II" filamentous morphology was conducted. This morphotype often causes serious bulking problems in activated sludge wastewater treatment plants, and hardly anything is known about its physiology. The study was carried out by applying a suite of in situ methods in an industrial activated sludge treatment plant with excessive growth of this species. The experiments revealed a very versatile organism able to take up a large variety of organic substrates under aerobic conditions. It had a remarkably high storage capacity forming polyhydroxyalkanoates from most substrates tested. When nitrate was present as e-acceptor, the number of substrates to be consumed by M. perideroedes was more restricted compared to aerobic conditions. With nitrite as e-acceptor, only acetate and glucose among the substrates tested could be assimilated and used for storage and possibly growth. This indicated that M. perideroedes might be able to denitrify under certain conditions, which is unusual for filamentous bacteria in activated sludge. No substrate uptake or storage was seen under anaerobic conditions. M. perideroedes was relatively hydrophobic, compared to other filamentous bacteria and microcolonies present in the sludge, indicating the presence of a hydrophobic sheath. Several excreted surface-associated exoenzymes were detected in the sludge, but M. perideroedes never showed any activity, except once after a breakdown in the production facility. This confirmed that M. perideroedes mainly grows on soluble substrates. Based on the studies of the ecophysiology of M. perideroedes, potential control strategies are suggested.