© 2002 Blackwell Science Ltd In situ studies of the phylogeny and physiology of filamentous bacteria with attached growth ferent bacterial phyla were represented in the identi- fied fraction suggesting that the attached microor- ganisms are phylogenetically diverse. The study of the in situ physiology of Type 0041 using MAR-FISH revealed that both the filaments and the attached bac- teria on Type 0041 were versatile in the use of organic substrates and electron acceptors. It was observed that all Type 0041 could consume glucose, but none of the filaments were able to consume acetate under any conditions tested, in contrast to some of the attached bacteria. No significant physiological differences were found between TM7–positive and TM7–negative Type 0041 filaments, and only minor differences were observed between the two treatment plants tested. These are the first data on the phy- siology of the almost entirely uncharacterized TM7 phylum and show that TM7 filamentous bacteria can uptake carbon substrates under aerobic and anaero- bic conditions. Introduction Attached growth of unicellular bacteria to filamentous bac- teria is occasionally observed in the microbial world. A well described example is the filamentous white sulphur bacterium Thiothrix, where single Thiothrix cells (gonidia) attach to the sheath and grow into new filaments, often forming a rosette (Nelson, 1989; Nielsen, 1984). In acti- vated sludge, several filamentous bacteria have been described with attached growth, ranging from a few attached cells to heavy attachment. The majority of these filaments have not been isolated or identified and are only described by their morphotype and simple staining reactions using the classification system of Eikelboom (Eikelboom, 1975; Eikelboom and van Buijsen, 1983). The most commonly observed Eikelboom morphotypes with attached growth in activated sludges are Types 0041, 1851 and 1701 all of which occasionally cause bulking problems in activated sludge plants (Seviour et al., 1994; Wanner et al., 1998). Very limited information is available about the phylogeny and physiology of filamentous bac- teria with epiflora. Furthermore, the identity and function of the attached growth is largely unknown. An improved knowledge of the physiology and phylogeny of these filamentous bacteria is necessary for the development of control strategies to prevent bulking (Seviour et al., 1994). Environmental Microbiology (2002) 4(7), 383–391 Trine Rolighed Thomsen 1 , Birthe Venø Kjellerup 1 , Jeppe Lund Nielsen 1 , Philip Hugenholtz 2† and Per Halkjær Nielsen 1 * 1 Department of Environmental Engineering, Aalborg University, Sohngardsholmsvej 57, DK-9000 Aalborg, Denmark. 2 ComBinE group, Advanced Computational Modelling Centre, The University of Queensland, Brisbane 4072, Australia. Summary Among the filamentous bacteria occasionally causing bulking problems in activated sludge treatment plants, three morphotypes with attached microbial growth are common, Eikelboom Type 0041, Type 1851 and Type 1701. A better knowledge of the phylogeny and physiology of these filamentous bacteria is necessary in order to develop control strategies for bulking. In this study we have used a combination of fluorescence in situ hybridization (FISH) and microau- toradiography (MAR) to investigate the identity and in situ physiology of the Type 0041-morphotype and its attached bacteria in two wastewater treatment plants. Identification and enumeration of Type 0041 using group-specific 16S rRNA-targeted FISH probes revealed that approximately 15% of the filaments hybridized with a gene probe specific for the TM7 group, a recently recognized major lineage in the bacterial domain. All other filaments morphologically identified as Type 0041 only hybridized to the general bacterial EUB338-probe, indicating that they probably do not belong to commonly isolated bacterial phyla such as the Proteobacteria, Firmicutes, Actinoba- cteria and Bacteroidetes, for which group-specific probes were used. The phylogenetic heterogeneity of Type 0041 again highlights the inadequacy of a morphology-based classification system. Like the fil- aments, most of the attached microbial cells were not identified beyond their affiliation to the Bacteria using the group-specific FISH probes. However, several dif- Accepted 1 May, 2002. *For correspondence. E-mail: phn@civil.auc.dk; Tel. +45 96358503; Fax +45 98142555. † Present address: Department of Environmental Science, Policy and Manage- ment, Hilgard Hall, University of California Berkeley, Berkeley CA 94720, USA.