A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli andHerpetosiphon aurantiacus under aerobic incubation conditions with added [3H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33Pi) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.

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A joint EU research project aimed at solving activated sludge bulking in nutrient removal plants was initiated in 1993. The project started with a survey of the size and composition of the filamentous population in nutrient removal plants in Denmark, Germany, Greece and the Netherlands. The results show that biological nutrient removal process conditions indeed favour filamentous microorganisms in their competition with floc forming organisms. An increase in the size of the filamentous population resulted in a deterioration of the settling properties of the biomass, except for plants with Bio-P removal conditions. It is assumed that in the latter case the dense clusters of Bio-P bacteria increase the weight of the flocs, and compensate for the effect of the larger number of filaments. Although exceptions frequently occur, the following sequence in decreasing filamentous organism population size was observed for the process conditions indicated: - completely mixed + simultaneous denitrification; - completely mixed + intermittent aeration/denitrification; - alternating anoxic/oxic process conditions, with an anaerobic tank for biological phosphate removal (Bio-Denipho); - alternating anoxic/oxic process conditions (Bio-Denitro); - predenitrification The surveys provided little information about the effect of nutrient removal in plants with plug flow aeration basins. Simultaneous precipitation with aluminium salts nearly always resulted in a low number of filaments and a good settling sludge. The size of the filamentous organism population showed a seasonal pattern with a maximum in winter/early spring and a minimum during summer (in Greece: during autumn). This seasonal variation is primarily caused by the effect of the season on the population sizes of M. parvicella, N. limicola and Type 0092. M. parvicella is by far the most important filamentous species in nutrient removal plants. In Denmark only, Type 0041 also frequently dominates the filamentous population, but seldom causes severe bulking. Considering their frequency of occurrence, approx. 10 other filamentous micro-organisms are of minor importance. Growth of some of these species, viz. those which use soluble substrate, can be prevented by the introduction of Bio-P process conditions. M. parvicella and Type 0041 (and probably also Actinomycetes and the Types 1851 and 0092) seem to compete for the same substrates i.e. the influent particulate fraction. Most of the differences in composition of the filamentous microorganism population can be explained by whether or not premixing of influent and recycled sludge is used. In general, premixing for a short period of time followed by anoxic conditions favours Type 0041. M. parvicella seems to proliferate if the particulate fraction is first hydrolysed or if it enters the plant via an oxic zone. It is concluded that bulking in nutrient removal plants is mainly caused by filamentous species requiring the particulate fraction for their growth.

Excessive growth of filamentous microorganisms in activated-sludge treatment plants is a major operational problem which causes poor settlement of activated sludge. An enhanced understanding of the factors controlling growth of different filamentous microorganisms is necessary in order to establish more successful control strategies. In the present study, the in situ substrate uptake was investigated by means of microautoradiography. It was demonstrated that the uptake of labeled organic substrates by the filamentous microorganisms, during short-term incubation, could be detected by microautoradiography. Viability and respiratory activity of the filaments were also detected by reduction of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and by incorporation of [ 3 H]thymidine. Gram, Neisser, and fluorescence staining techniques were used for the localization and identification of the filaments. Activated-sludge samples from five wastewater treatment plants with bulking problems due to filamentous microorganisms were investigated. Microthrix parvicella, Nostocoida limicola, and Eikelboom's type 0041 and type 021N were investigated for their ability to take up organic substrates. A panel of six substrates, i.e., [ 14 C]acetate, [ 3 H]glucose, [ 14 C]ethanol, [ 3 H]glycine, [ 3 H]leucine, and [ 3 H]oleic acid, was tested. The uptake response was found to be very specific not only between the different filamentous types but also among filaments of the same type from different treatment plants. Interestingly, M. parvicella consistently took up only oleic acid among the tested substrates. It is concluded that microautoradiography is a useful method for investigation of in situ substrate uptake by filamentous microorganisms in activated sludge.

Microthrix parvicella is a filamentous microorganism responsible for bulking and foaming problems in many activated sludge treatment plants. The problems have increased with the introduction of nutrient removal in many countries, and presently, there is no reliable control strategy for M. parvicella. Little is known about the physiology of M. parvicella, and conflicting data exist about its preferred organic substrates, and whether it is able to be physiologically active under anaerobic and anoxic conditions. In this study, the ability of M. parvicella to take up various radioactively labeled organic compounds was investigated in situ at three nutrient removal plants using a microautoradiographic technique. Of 12 compounds tested under aerobic conditions only the long chain fatty acids (LCFA), oleic acid and palmitic acid, and to some extent a lipid, trioleic acid, were assimilated. None of the simple substrates such as acetate, propionate, butyrate, glucose, ethanol, glycine and leucine were taken up. Furthermore, the uptake of oleic acid was compared under anaerobic, anoxic and aerobic conditions, and it was demonstrated that in addition to aerobic conditions M. parvicella was also able to take up oleic acid under anaerobic and anoxic conditions. No difference in substrate uptake pattern for M. parvicella was found among the tested activated sludge plants. The results strongly indicate that a better control strategy against M. parvicella must rely on a better understanding of presence and availability of triglycerides and LCFA, and an improved knowledge of the physiology of M. parvicella under anaerobic and anoxic conditions.