Australasian Biotechnology (backfiles) AusBiotech ISSN: 1036-7128 Vol. 10, Num. 3, 2000, pp. 29-32

Australasian Biotechnology, Vol. 10 No. 3, 2000, pp. 29-32

Linda L. Blackall

Advanced Wastewater Management Centre, Department of Microbiology & Parasitology, The University of Queensland, St. Lucia, 4072, Queensland

Code Number: au00034

Abstract

A number of recent studies has focussed on discovering the microorganisms responsible for various nutrient removal processes in the wastewater treatment industry. This is despite the fact that many wastewater personnel would think that these microorganisms were already known. The sample dilution and plating methods, previously the major ones used by microbiologists, have lead to the isolation of various microorganisms with specific phenotypes of interest. Consequently, Nitrosomonas and Nitrobacter have been described as the major ammonia and nitrite oxidisers, respectively. Acinetobacter has been attributed with the enhanced biological phosphorus removal (EBPR) phenotype due to its predominance in isolations from EBPR plants. However, the true situation is not so clear and even worse, the above organisms may not even play any role in the transformations for which they have achieved such acclaim. The introduction of methods that allow microbiologists to investigate the microbial composition in mixed cultures has begun to resolve the questions on the true identity of microorganisms responsible for nutrient transformations. These methods also allow the ready in situ confirmation of such hypotheses on organism identity and allow this to be linked with organism phenotype. To answer the question on organism identity, enrichment culture around specific phenotypes can still be employed, however, instead of the enrichment of one microorganism over all others, a consortium is obtained. The enriched consortium is investigated by in situ identification methods like fluorescence in situ hybridisation (FISH) with domain, division, and sub-division level probes. A fairly clear picture of the “global” microbial community structure is thus obtained. Evolutionarily conserved genes like those for the 16S rRNA are extracted from the enriched culture and analysed to place them in the phylogenetic tree. Sequences from groups known to dominate the enrichment culture from FISH, can be focussed upon and more intensively studied, including the design of novel probes for FISH on the enrichment culture. The same approach using FISH and analysis of conserved genes from full-scale operations has also been employed to identify the organisms of relevance to specific nutrient transformations. The final product is a suite of FISH probes that can be used with any mixed culture biomass for enumeration of organisms of interest and correlation of their abundance with process performance. Once the identity of the organisms truly responsible for nutrient transformations is known, their physiological parameters can be studied.

Using the above summarised methods, bacteria closely related to the genus Rhodocyclus in the beta-2 Proteobacteria were found to be one representative of polyphosphate accumulating organisms (PAOs). Nitrospira-like bacteria were the dominant nitrite oxidisers in both enriched and full scale nitrifying systems. Both these discoveries are novel and both contradict previous opinions as to the organisms thought responsible for these phenotypes. The value of molecular biological methods in this work is highlighted.

biological nutrient removal, molecular ecology, 16S rDNA, clone libraries, sequencing batch reactors, EBPR, nitrification

Activated sludge plants are commonly used as the process of choice for treating wastewater from domestic and industrial sources. There are myriad designs and operations of the activated sludge process but all of them rely on the growth of a complex microbial community on the pollutants in the wastewater (Seviour and Blackall, 1999). Thus, the pollutants are transformed by the microbial ecosystem into innocuous compounds that can be discharged into the atmosphere if they are volatile or into a receiving water body with the effluent from the process. In some cases when transformations have not occurred, the pollutant may be complexed with or contained within the microbial biomass and the harvesting of this removes the pollutant from the environment. The major constituent of the microbial community in the activated sludge process is the Bacteria which comprise one domain of biological life on earth (Hugenholtz et al., 1998). There are two other domains of life on Earth - the Archaea which include methogenic organisms found in anaerobic treatment processes and the most well known domain, the Eucarya containing the protozoa and metazoa.

Nitrogen is removed microbiologically from wastewater by the sequential processes of nitrification followed by denitrification (Shin et al., 1992) which is almost exclusively promoted by highly specialised microbes, the nitrifiers and the less specialised denitrifiers present in activated sludge. In wastewater treatment systems, Nitrosomonas (an ammonia oxidiser) and Nitrobacter (a nitrite oxidiser) are the two autotrophs presumed to be collectively responsible for nitrification. Although ammonia oxidisers have been intensively studied by the use of molecular methods (Wagner et al., 1995; Wagner et al., 1996), the nitrite oxidisers had not been similarly studied. In one study of activated sludge flocs (Mobarry et al., 1996), clusters of Nitrosomonas and Nitrobacter were adjacent to each other as revealed by FISH. However, in other studies, Nitrobacter could not be detected and it was speculated that other bacteria were likely responsible for nitrite oxidation (Hovanec and DeLong, 1996; Wagner et al., 1996).

Denitrification is the process where nitrate (NO3-) and/or nitrite (NO2-) are reduced to NO, N2O or N2, and represents one of the key processes in the nitrogen cycle (Robertson and Kuenen, 1991). Both assimilatory and dissimilatory nitrate reduction can occur, but it is now clear that dissimilatory nitrate reduction is commonly found in bacteria, and more than 130 species of chemoheterotrophic bacteria are now known which can denitrify. These organisms can employ NO3- instead of O2 as a terminal electron acceptor to reoxidise NADH generated from the oxidation of organic carbon sources by a process often referred to as “anaerobic respiration”.

The removal of phosphorus from wastewaters is known as enhanced biological phosphorus removal (EBPR). A significant effort has been expended in modelling, designing, building and operating EBPR activated sludge plants. There have been many investigations attempting to match the metabolic performance of bacterial isolates (obtained by sample dilution and spread plate for isolation) with the biochemical model suggested for EBPR. These have concentrated mostly on isolates of the genus Acinetobacter because members of this genus are easily isolated from EBPR sludges (Fuhs and Chen, 1975; Wentzel et al., 1988) and some isolates show some characteristics that may be important to EBPR (Deinema et al., 1985; Streichan et al., 1990). However, evidence indicating that Acinetobacter may not be responsible for EBPR includes pure culture performances not correlating with biological models (Bond, 1997; Tandoi et al., 1998), and analyses of EBPR bacterial communities indicating that Acinetobacter are not present in high enough numbers to account for EBPR (Bond, 1997; Cloete and Steyn, 1987; Kämpfer et al., 1996; Melasniemi et al., 1999; Wagner et al., 1994). Investigations of other EBPR-associated microorganisms have been undertaken with interest in Gram positive bacteria such as Microlunatus phosphovorus (Nakamura et al., 1995; Santos et al., 1999; Ubukata, 1994), the Gram negative Lampropedia (Stante et al., 1997), the Actinobacteria and a-Proteobacteria (Kawaharasaki et al., 1999) and Tetrasphaera spp. which accumulates polyphosphate (Maszenan et al., 2000). However, there is no general consensus that these bacteria are examples of polyphosphate-accumulating organisms (PAOs), and indeed Mino et al. (1998) concluded that rather than being a single dominant PAO several different bacterial groups may be important. The isolation of putative PAOs is hampered by the lack of an easy method to use the P removal phenotype in isolation strategies.

Several studies into activated sludge microbial communities using cloning of 16S rDNA have been reported (Bond et al., 1995; Burrell et al., 1998; Juretschko et al., 1998; Schuppler et al., 1995). The method has allowed the description of candidates for nitrification (Burrell et al., 1998; Mobarry et al., 1996), for competition with PAOs in EBPR (Bond et al., 1999; Nielsen et al., 1999), and for description of candidate PAOs (Bond et al., 1999; Bond et al., 1995; Crocetti et al., 2000; Hesselmann et al., 1999).

The first description of gene probes from 16S rDNA data for in situ hybridisation experiments was by Giovannoni et al. (1988). The detection was by autoradiography from radioactively labelled probes but the method was quickly superseded by one employing fluorescently-labelled oligonucleotides and called “phylogenetic stains” (DeLong et al., 1989). Today we talk about fluorescence in situ hybridisation or FISH probing and the methods are routinely employed by many laboratories. Oligonucleotides complimentary to the rRNA at varying levels of specificity from species to domain are labelled with fluorochromes and they can enter bacterial cells, bind to the ribosomes via base pairing and the probe-target interaction can be observed by viewing the sample by epifluorescence microscopy or confocal laser scanning microscopy (CLSM). Whole cells which have bound probes can be visualised and their morphology and spatial arrangement in the mixed culture determined. The CLSM makes the interpretation and documentation of results more convincing.

FISH has clearly demonstrated the role of Nitrospira as a major member of the nitrite oxidising microbial community in flocs (Juretschko et al., 1998; Schramm et al., 1998). Previously, Nitrobacter was thought to be the major nitrite oxidiser and culture dependent methods were unable to clarify the situation. Juretschko et al. (1998) demonstrated that the nitrifiers appear clustered and that the ammonia oxidiser clusters are closely juxtaposed to the nitrite oxidiser clusters.

The identity of the PAOs focussed on b-Proteobacteria and the Actinobacteria since EBPR process performance was linked with their dominance in the sludge as determined by FISH (Bond et al., 1999; Bond and Rees, 1999; Kämpfer et al., 1996; Wagner et al., 1994). A combination of molecular ecology methods such as FISH with published division and sub-division probes, analysis of 16S rRNA genes from EBPR processes including design of FISH probes from the gene sequence data, and use of the newly designed probes, were used to demonstrate that bacteria related to Rhodocyclus are the dominant PAOs in lab-scale EBPR processes (Crocetti et al., 2000; Hesselmann et al., 1999). These organisms were proposed to be called Candidatus Accumulibacter phosphatis (Hesselmann et al., 1999).

The activated sludge process comprises a complex enrichment culture of a mixture of generalist and specialist microorganisms. Problems in and lack of fundamental understanding of EBPR processes and aspects of processes optimised to remove nitrogen from the influent have compelled researchers to examine and attempt to optimise the biological component of the mixed liquor of activated sludge plants. Since it is unlikely dilution and spread plate inoculation will allow us great insight into the true microbial diversity of activated sludge, molecular biological methods (e.g. Crocetti et al., 2000) in concert with community function methods like microelectrodes (Schramm et al., 1998) and in situ microautoradiography (Andreasen and Nielsen, 1997) will likely lead to understanding and consequently productively manipulating the microbial communities. It is paramount that if one is going to investigate the microbial community structure of an activated sludge plant, as much detail about the operation of the plant including its operating data prior to and including the sampling time is available. In the past, this has not been done rigorously enough and elegant molecular studies of the microbial community structure and function cannot be linked with the operation of the plant.

• Andreasen, K., and Nielsen, P.H. (1997). Application of microautoradiography to study substrate uptake by filamentous microorganisms in activated sludge. Applied and Environmental Microbiology 63, 3662-3668.
• Bond, P.L. (1997). Investigations of the microbial ecology of enhanced biological phosphorus removal in the activated sludge process. PhD. The University of Queensland.Bond, P.L., Erhart, R., Wagner, M., Keller, J., and Blackall, L.L. (1999). The identification of some of the major groups of bacteria in efficient and non-efficient biological phosphorus removal activated sludge systems. Applied and Environmental Microbiology 65, 4077-4084.
• Bond, P.L., Hugenholtz, P., Keller, J., and Blackall, L.L. (1995). Bacterial community structures of phosphate-removing and non-phosphate-removing activated sludges from sequencing batch reactors. Applied and Environmental Microbiology 61, 1910-1916.
• Bond, P.L., and Rees, G.N. (1999). Microbiological aspects of phosphorus removal in activated sludge systems. In The Microbiology of Activated Sludge, pp. 227-256. Edited by R. J. Seviour & L. L. Blackall. London: Kluwer Academic Publishers.
• Burrell, P.C., Keller, J., and Blackall, L.L. (1998). Microbiology of a nitrite-oxidizing bioreactor. Applied and Environmental Microbiology 64, 1878-1883.
• Cloete, T.E., and Steyn, P.L. (1987). A combined fluorescent antibody-membrane filter technique for enumerating Acinetobacter in activated sludge. In Biological Phosphate Removal from Wastewaters, pp. 335-338. Edited by R. Ramadori. Oxford: Pergamon Press.
• Crocetti, G.R., Hugenholtz, P., Bond, P.L., Schuler, A., Keller, J., Jenkins, D., and Blackall, L.L. (2000). Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation. Applied and Environmental Microbiology 66, 1175-1182.
• Deinema, M.H., van Loosdrecht, M.C.M., and Scholten, A. (1985). Some physiological characteristics of Acinetobacter spp. accumulating large amounts of phosphate. Water Science and Technology 17 (12), 119-125.
• DeLong, E.F., Wickham, G.S., and Pace, N.R. (1989). Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243, 1360-1363.
• Fuhs, G.W., and Chen, M. (1975). Microbiological basis of phosphate removal in the activated sludge process for the treatment of wastewater. Microbial Ecology 2, 119-138.
• Giovannoni, S.J., DeLong, E.F., Olsen, G.J., and Pace, N.R. (1988). Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. Journal of Bacteriology 170, 720-726.
• Hesselmann, R.P.X., Werlen, C., Hahn, D., van der Meer, J.R., and Zehnder, A.J.B. (1999). Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge. Systematic and Applied Microbiology 22, 454-465.
• Hovanec, T.A., and DeLong, E.F. (1996). Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria. Applied and Environmental Microbiology 62, 2888-2896.
• Hugenholtz, P., Goebel, B.M., and Pace, N.R. (1998). Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. Journal of Bacteriology 180, 4765-4774.
• Juretschko, S., Timmermann, G., Schmid, M., Schleifer, K.-H., Pommerening-Rîser, A., Koops, H.-P., and Wagner, M. (1998). Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrococcus mobilis and Nitrospira-like bacteria as dominant populations. Applied and Environmental Microbiology 64, 3042-3051.
• Kämpfer, P., Erhart, R., Beimfohr, C., Bohringer, J., Wagner, M., and Amann, R. (1996). Characterization of bacterial communities from activated sludge - culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes. Microbial Ecology 32, 101-121.
• Kawaharasaki, M., Tanaka, H., Kanagawa, T., and Nakamura, K. (1999). In situ identification of polyphosphate-accumulating bacteria in activated sludge by dual staining with rRNA-targeted oligonucleotide probes and 4 ‘,6-diamidino-2-phenylindol (DAPI) at a polyphosphate-probing concentration. Water Research 33, 257-265.
• Maszenan, A.M., Seviour, R.J., Patel, B.K.C., Schumann, P., Burghardt, J., Tokiwa, Y., and Stratton, H.M. (2000). Three isolates of novel polyphosphate-accumulating Gram-positive cocci, obtained from activated sludge, belong to a new genus, Tetrasphaera gen. nov., and description of two new species, Tetrasphaera japonica sp. nov. and Tetrasphaera australiensis sp. nov. International Journal of Systematic and Evolutionary Microbiology 50, 593-603.
• Melasniemi, H., Hernesmaa, A., Pauli, A.S.-L., Rantanen, P., and Salkinoja-Salonen, M. (1999). Comparative analysis of biological phosphate removal (BPR) and non-BPR activated sludge bacterial communities with particular reference to Acinetobacter. Journal of Industrial Microbiology 21, 300-306.
• Mino, T., van Loosdrecht, M.C.M., and Heijnen, J.J. (1998). Microbiology and biochemistry of the enhanced biological phosphate removal process. Water Research 32, 3193-3207.
• Mobarry, B.K., Wagner, M., Urbain, V., Rittmann, B.E., and Stahl, D.A. (1996). Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Applied and Environmental Microbiology 62, 2156-2162.
• Nakamura, K., Hiraishi, A., Yoshimi, Y., Kawaharasaki, M., Masuda, K., and Kamagata, Y. (1995). Microlunatus phosphovorus gen. nov., sp. nov., a new gram-positive polyphosphate-accumulating bacterium isolated from activated sludge. International Journal of Systematic Bacteriology 45, 17-22.
• Nielsen, A.T., Liu, W.-T., Filipe, C., Grady, L., Molin, S., and Stahl, D.A. (1999). Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor. Applied and Environmental Microbiology 65, 1251-1258.
• Robertson, L.A., and Kuenen, J.G. (1991). Physiology of nitrifying and denitrifying bacteria. In Microbial Production and Consumption of Greenhouse Gases: Methane, Nitrogen Oxides and Halomethanes, pp. 189-199. Edited by J. E. Rogers & W. B. Whitman. Washington D.C.: American Society for Microbiology.
• Santos, M.M., Lemos, P.C., Reis, M.A.M., and Santos, H. (1999). Glucose metabolism and kinetics of phosphorus removal by the fermentative bacterium Microlunatus phosphovorus. Applied and Environmental Microbiology 65, 3920-3928.
• Schramm, A., de Beer, D., Wagner, M., and Amann, R. (1998). Identification and activities in situ of Nitrosospira and Nitrospira spp. as dominant populations in an nitrifying fluidized bed reactor. Applied and Environmental Microbiology 64, 3480-3485.Schuppler, M., Mertens, F., Schön, G., and Göbel, U.B. (1995). Molecular characterization of nocardioform actinomycetes in activated sludge by 16S rRNA analysis. Microbiology 141, 513-521.
• Seviour, R.J., and Blackall, L.L. (1999). The Microbiology of Activated Sludge. London: Kluwer Academic Publishers.
• Shin, H.S., Jun, H.B., and Park, H.S. (1992). Simultaneous removal of phosphorus and nitrogen in sequencing batch reactor. Biodegradation 3, 105-111.
• Stante, L., Cellamare, C.M., Malaspina, F., Bortone, G., and Tilche, A. (1997). Biological phosphorus removal by pure culture of Lampropedia spp. Water Research 31, 1317-1324.
• Streichan, M., Golecki, J.R., and Schon, G. (1990). Polyphosphate-accumulating bacteria from sewage treatment plants with different processes for biological phosphorus removal. FEMS Microbiology Ecology 73, 113-124.
• Tandoi, V., Majone, M., May, J., and Ramadori, R. (1998). The behaviour of polyphosphate accumulating Acinetobacter isolates in an anaerobic-aerobic chemostat. Water Research 32, 2903-2912.
• Ubukata, Y. (1994). Some physiological characteristics of a phosphate removing bacterium isolated from anaerobic/ aerobic activated sludge. Water Science and Technology 30-6, 229-235.
• Wagner, M., Erhart, R., Manz, W., Amann, R., Lemmer, H., Wedi, D., and Schleifer, K.-H. (1994). Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Applied and Environmental Microbiology 60, 792-800.
• Wagner, M., Rath, G., Amann, R., Koops, H.P., and Schleifer, K.H. (1995). In situ identification of ammonia-oxidizing bacteria. Systematic and Applied Microbiology 18, 251-264.
• Wagner, M., Rath, G., Koops, H.-P., Flood, J., and Amann, R. (1996). In situ analysis of nitrifying bacteria in sewage treatment plants. Water Science and Technology 34 (1-2), 237-244.
• Wentzel, M.C., Loewenthal, R.E., Ekama, G.A., and Marais, G.v. (1988). Enhanced polyphosphate organism cultures in activated sludge systems - Part 1: Enhanced culture development. Water SA 14, 81-92.

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