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A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

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Abstract

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked.

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References

  • Ardern E & Lockett WT (1914) Experiments on the oxidation of sewage without the aid of filters. J. Soc. Chem. Indust. 33: 523–539.

    CAS  Google Scholar 

  • Auling G, Pilz F, Busse H-J, Karrasch S, Streichan M & Schön G (1991) Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp. Appl. Environ. Microbiol. 57: 3585–3592.

    PubMed  CAS  Google Scholar 

  • Barnard JL (1974) Cut P and N without chemicals. Water Wastes Eng. 11: 33–44.

    CAS  Google Scholar 

  • Beacham AM, Seviour RJ & Lindrea KD (1992) Polyphosphate accumulating abilities of Acinetobacter isolates from a biological nutrient removal plant. Wat. Res. 26: 121–122.

    Article  CAS  Google Scholar 

  • Blackall LL, Rossetti S, Christenssen C, Cunningham M, Hartman P, Hugenholtz P & Tandoi V (1997) The characterization and description of representatives of 'G' bacteria from activated sludge plants. Lett. Appl. Microbiol. 25: 63–69.

    Article  PubMed  CAS  Google Scholar 

  • Bond PL, Erhart R, Wagner M, Keller J & Blackall LL (1999a) Identification of some of the major groups of bacteria in efficient and nonefficient biological phosphorus removal activated sludge systems. Appl. Environ. Microbiol. 65: 4077–4084.

    PubMed  CAS  Google Scholar 

  • Bond PL, Hugenholtz P, Keller J & Blackall LL (1995) Bacterial community structures of phosphate-removing and non-phosphate-removing activated sludges from sequencing batch reactors. Appl. Environ. Microbiol. 61: 1910–1916.

    PubMed  CAS  Google Scholar 

  • Bond PL, Keller J & Blackall LL (1998) Characterisation of enhanced biological phosphorus removal activated sludges with dissimilar phosphorus removal performance. Wat. Sci. Tech. 37(4-5): 567–571.

    Article  CAS  Google Scholar 

  • Bond PL, Keller J & Blackall LL (1999b) Bio-P and non-bio-P bacteria identification by a novel microbial approach. Wat. Sci. Tech. 39(6): 13–20.

    Article  CAS  Google Scholar 

  • Bond PL & Rees GN (1999) Microbiological aspects of phosphorus removal in activated sludge systems. In: Seviour RJ & Blackall LL (Eds) The Microbiology of Activated Sludge (pp 227–256). Kluwer Academic Publishers, London.

    Google Scholar 

  • Buchan L (1983) Possible biological mechanism of phosphorus removal. Wat. Sci. Tech. 15: 87–103.

    CAS  Google Scholar 

  • Cech JS & Hartman P (1990) Glucose induced break down of enhanced biological phosphate removal. Environ. Tech. 11: 651–656.

    Article  CAS  Google Scholar 

  • Cech JS & Hartman P (1993) Competition between polyphosphate and polysaccharide accumulating bacteria in enhanced biological phosphate removal systems. Wat. Res. 27: 1219–1225.

    Article  CAS  Google Scholar 

  • Christensson M, Blackall LL & Welander T (1998) Metabolic transformations and characterisation of the sludge community in an enhanced biological phosphorus removal system. Appl. Microbiol. Biotech. 49: 226–234.

    Article  CAS  Google Scholar 

  • Cloete TE & Steyn PL (1987) A combined fluorescent antibody-membrane filter technique for enumerating Acinetobacter in activated sludge. In: Ramadori R (Ed) Biological Phosphate Removal from Wastewaters (pp 335–338). Pergamon Press, Oxford.

    Google Scholar 

  • Crocetti GR, Banfield JF, Keller J, Bond PL & Blackall LL (2001) The identification of glycogen accumulating organisms from a poorly-operating enhanced biological phosphorus removal laboratory-scale SBR. In: Activated Sludge Population Dynamics Committee of IWA (Ed) 3rd International Conference on Microorganisms in Activated Sludge and Biofilm Processes (pp CD-ROM). Rome, Italy.

  • Crocetti GR, Hugenholtz P, Bond PL, Schuler A, Keller J, Jenkins D & Blackall LL (2000) Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation. Appl. Environ. Microbiol. 66: 1175–1182.

    Article  PubMed  CAS  Google Scholar 

  • Cullen P (1994) The link between effluent standards and receiving water guidelines. In: Bayly R & Pilkington N (Ed) Second Australian Conference on Biological Nutrient Removal from Wastewater (pp 25-30). Albury, Victoria.

  • Dabert P, Fleurat-Lessard A, Mounier E, Delgenés J-P, Moletta R & Godon J-J (2001a) Monitoring of the microbial community of a sequencing batch reactor bioaugmented to improve its phosphorus removal capabilities. Wat. Sci. Tech. 43 (3): 1–8.

    CAS  Google Scholar 

  • Dabert P, Sialve B, Delgenès J-P, Moletta R & Godon J-J (2001b) Characterisation of the microbial 16S rDNA diversity of an aerobic phosphorus-removal ecosystem and monitoring of its transition to nitrate respiration. Appl. Microbiol. Biotech. 55: 500–509.

    Article  CAS  Google Scholar 

  • Deinema MH, Habets LHA, Scholten J, Turkstra E & Webers HA (1980) The accumulation of polyphosphates in Acinetobacter spp. FEMS Microbiol. Lett. 9: 275–279.

    Article  CAS  Google Scholar 

  • Deinema MH, van Loosdrecht MCM & Scholten A (1985) Some physiological characteristics of Acinetobacter spp. accumulating large amounts of phosphate. Wat. Sci. Tech. 17(12): 119–125.

    CAS  Google Scholar 

  • Falvo A, Levantesi C, Rossetti S, Seviour RJ & Tandoi V (2001) Synthesis of intracellular storage polymers by Amaricoccus kaplicensis, a tetrad forming bacterium present in activated sludge. J. Appl. Microbiol. 91: 299–305.

    Article  PubMed  CAS  Google Scholar 

  • Fuhs GW & Chen M (1975) Microbiological basis of phosphate removal in the activated sludge process for the treatment of wastewater. Microb. Ecol. 2: 119–138.

    Article  CAS  Google Scholar 

  • Fukase T, Shibata M & Miyaji Y (1985) The role of an anaerobic stage on biological phosphorus removal. Wat. Sci. Tech. 17(2-3): 69–80.

    CAS  Google Scholar 

  • Gieseke A, Purkhold U, Wagner M, Amann R & Schramm A (2001) Community structure and activity dynamics of nitrifying bacteria in a phosphate-removing biofilm. Appl. Environ. Microbiol. 67: 1351–1362.

    Article  PubMed  CAS  Google Scholar 

  • Harremoës P (1994) The rational for demanding nutrient removal from wastewater. The Danish experience. In: Bayly R & Pilkington N (Eds) Second Australian Conference on Biological Nutrient Removal from Wastewater (pp 15-24). Albury, Victoria.

  • Hartley KJ & Sickerdick L (1994) Performance of Australian BNR plants. In: Bayly R & Pilkington N (Eds) Second Australian Conference on Biological Nutrient Removal from Wastewater (pp 65-72). Albury, Victoria.

  • Hesselmann RPX, Werlen C, Hahn D, van der Meer JR & Zehnder AJB (1999) Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge. Syst. Appl. Microbiol. 22: 454–465.

    PubMed  CAS  Google Scholar 

  • Hiraishi A & Morishima Y (1990) Capacity for polyphosphate accumulation of predominant bacteria in activated sludge showing enhanced phosphate removal. J. Ferm. Bioeng. 69: 368–371.

    Article  CAS  Google Scholar 

  • Jenkins D, Richard MG & Daigger GT (1993) Manual on the Causes and Control of Activated Sludge Bulking and Foaming. Lewis Publishers, New York.

    Google Scholar 

  • Jenkins D & Tandoi V (1991) The applied microbiology of enhanced biological phosphate removal-accomplishments and needs. Wat. Res. 25: 1471–1478.

    Article  CAS  Google Scholar 

  • Kämpfer P, Erhart R, Beimfohr C, Bohringer J, Wagner M & 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. Microb. Ecol. 32: 101–121.

    Article  PubMed  Google Scholar 

  • Kawaharasaki M, Manome A, Kanagawa T & Nakamura K (2001) Flow cytometric sorting and RFLP analysis of phosphate accumulating bacteria in an enhanced biological phosphorus removal system. In: Activated Sludge Population Dynamics Committee of IWA (Ed) 3rd International Conference on Microorganisms in Activated Sludge and Biofilm Processes (pp 92-97). Rome, Italy.

  • Kawaharasaki M, Tanaka H, Kanagawa T & 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. Wat. Res. 33: 257–265.

    Article  CAS  Google Scholar 

  • Kortstee GJJ, Appeldoorn KJ, Bonting CFC, van Niel EWJ & van Veen HW (1994) Biology of polyphosphate-accumulating bacteria involved in enhanced biological phosphorus removal. FEMS Microbiol. Rev. 15: 137–153.

    Article  PubMed  CAS  Google Scholar 

  • Lee N, Jansen J, Aspegren H, Kircks K, Henze M, Nielsen PH, Schleifer K-H & Wagner M (2001) Population dynamics and in situ physiology of phosphorus-accumulating bacteria in wastewater treatment plants without enhanced biological phosphorus removal operated with and without nitrogen removal. In: Activated Sludge Population Dynamics Committee of IWA (Ed) 3rd International Conference on Microorganisms in Activated Sludge and Biofilm Processes (pp 114-122). Rome, Italy.

  • Liu W-T, Linning KD, Nakamura K, Mino T, Matsuo T & Forney LJ (2000a) Microbial community changes in biological phosphate-removal systems on altering sludge phosphorus content. Microbiology. 146: 1099–1107.

    PubMed  CAS  Google Scholar 

  • Liu W-T, Mino T, Nakamura K & Matsuo T (1996) Glycogen accumulating population and its anaerobic substrate uptake in anaerobic-aerobic activated sludge without biological phosphorus removal. Wat. Res. 30: 75–82.

    Article  CAS  Google Scholar 

  • Liu W-T, Nielsen AT, Wu J-H, Tsai C-S, Matsuo Y & Molin S (2001) In situ identification of polyphosphate-and polyhydroxyalkanoate-accumulating traits for microbial populations in a biological phosphorus removal process. Environ. Microbiol. 3: 110–122.

    Article  PubMed  CAS  Google Scholar 

  • Liu WT, Marsh TL & Forney LJ (1998) Determination of the microbial diversity of anaerobic-aerobic activated sludge by a novel molecular biological technique. Wat. Sci. Tech. 37(4-5): 417–422.

    Article  CAS  Google Scholar 

  • Liu WT, Mino T, Matsuo T & Nakamura K (2000b) Isolation, characterization and identification of polyhydroxyalkanoate-accumulating bacteria from activated sludge. J. Biosci. Bioeng. 90: 494–500.

    PubMed  CAS  Google Scholar 

  • Lötter LH (1985) The role of bacterial phosphate metabolism in enhanced phosphorus removal from the activated sludge process. Wat. Sci. Tech. 17: 127–138.

    Google Scholar 

  • Manz W, Amann R, Ludwig W, Wagner M & Schleifer K-H (1992) Phylogenetic oligonucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15: 593–600.

    Google Scholar 

  • Maszenan AM, Seviour RJ, Patel BKC, Rees GN & McDougall BM (1997) Amaricoccus gen. nov., a gram negative coccus occurring in regular packages or tetrads isolated from activated sludge and descriptions of Amaricoccus bendigoensis sp. nov., Amaricoccus tamworthensis sp. nov., Amaricoccus macauensis sp.nov.and Amaricoccus kaplicensis sp. nov. Int. J. Syst. Bacteriol. 47: 727–734.

    PubMed  CAS  Google Scholar 

  • Maszenan AM, Seviour RJ, Patel BKC, Schumann P, Burghardt J, Tokiwa Y & Stratton HM (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. Int. J. Syst. Evol. Microbiol. 50: 593–603.

    PubMed  CAS  Google Scholar 

  • Mino T (2000) Microbial selection of polyphosphate-accumulating bacteria in activated sludge wastewater treatment processes for enhanced biological phosphate removal. Biochemistry-Moscow. 65: 341–348.

    PubMed  CAS  Google Scholar 

  • Mino T, Liu WT, Kurisu F & Matsuo T (1995) Modelling glycogen storage and denitrification capability of microorganisms in enhanced biological phosphate removal processes. Wat. Sci. Tech. 31(2): 25–34.

    Article  CAS  Google Scholar 

  • Mino T, van Loosdrecht MCM & Heijnen JJ (1998) Microbiology and biochemistry of the enhanced biological phosphate removal process. Wat. Res. 32: 3193–3207.

    Article  CAS  Google Scholar 

  • Nakamura K, Hiraishi A, Yoshimi Y, Kawaharasaki M, Masuda K & Kamagata Y (1995) Microlunatus phosphovorus gen. nov., sp. nov., a new gram-positive polyphosphate-accumulating bacterium isolated from activated sludge. Int. J. Syst. Bacteriol. 45: 17–22.

    Article  PubMed  CAS  Google Scholar 

  • Nielsen AT, Liu W-T, Filipe C, Grady L, Molin S & Stahl DA (1999) Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor. Appl. Environ. Microbiol. 65: 1251–1258.

    PubMed  CAS  Google Scholar 

  • Olsen GJ, Lane DJ, Giovannoni SJ, Pace NR & Stahl DA (1986) Microbial ecology and evolution: a ribosomal RNA approach. Annu. Rev. Microbiol. 40: 337–365.

    Article  PubMed  CAS  Google Scholar 

  • Onuki M, Satoh H & Mino T (2001) Analysis of microbial com-munity that performs enhanced biological phosphorus removal in activated sludge fed with acetate. In: Activated Sludge Population Dynamics Committee of IWA (Ed) 3rd International Conference on Microorganisms in Activated Sludge and Biofilm Processes (pp 98-105). Rome, Italy.

  • Osborn DW & Nicholls HA (1978) Bacterial stress: a prerequisite for the biological removal of phosphorus. J. Wat. Poll. Cont. Fed. 51: 557–569.

    Google Scholar 

  • Ramadori R (1987) Biological Phosphate Removal from Wastewaters. Pergamon Press, New York.

    Google Scholar 

  • Santos MM, Lemos PC, Reis MAM & Santos H (1999) Glucose metabolism and kinetics of phosphorus removal by the fermentative bacterium Microlunatus phosphovorus. Appl. Environ. Microbiol. 65: 3920–3928.

    PubMed  CAS  Google Scholar 

  • Schlegel HG (1993) General Microbiology. Cambridge University Press, Cambridge.

    Google Scholar 

  • Seviour RJ, Maszenan AM, Soddell JA, Tandoi V, Patel BKC, Kong Y & Schumann P (2000) Microbiology of the 'G-bacteria' in activated sludge. Environ. Microbiol. 2: 581–593.

    Article  PubMed  CAS  Google Scholar 

  • Shintani T, Liu WT, Hanada S, Kamagata Y, Miyaoka S, Suzuki T & Nakamura K (2000) Micropruina glycogenica gen. nov., sp, nov., a new Gram-positive glycogen-accumulating bacterium isolated from activated sludge. Int. J. Syst. Evol. Microbiol. 1: 201–207.

    Google Scholar 

  • Snaidr J, Amann R, Huber I, Ludwig W & Schleifer K-H (1997) Phylogenetic analysis and in situidentification of bacteria in activated sludge. Appl. Environ. Microbiol. 63: 2884–2896.

    PubMed  CAS  Google Scholar 

  • Stante L, Cellamare CM, Malaspina F, Bortone G & Tilche A (1997) Biological phosphorus removal by pure culture of Lampropedia spp. Wat. Res. 31: 1317–1324.

    Article  CAS  Google Scholar 

  • Sudiana IM, Mino T, Satoh H & Matsuo T (1998) Morphology, in-situ characterization with rDNA targetted probes and respiratory quinone profiles of enhanced biological phosphorus removal sludge. Wat. Sci. Tech. 38(8-9): 69–76.

    Article  CAS  Google Scholar 

  • Vallentyne JR (1994) Prevention of eutrophication of the Great Lakes. In: Bayly R & Pilkington N (Eds) Second Australian Conference on Biological Nutrient Removal from Wastewater, Vol (pp 31–38). AWWA, Albury, Victoria.

    Google Scholar 

  • van Loosdrecht MCM, Hooijmans CM, Brdjanovic D & Heijnen JJ (1997a) Biological phosphate removal processes. Appl. Microbiol. Biotech. 48: 289–296.

    Article  CAS  Google Scholar 

  • van Loosdrecht MCM, Smolders GJ, Kuba T & Heijnen JJ (1997b) Metabolism of microorganisms responsible for enhanced biological phosphorus removal from wastewater. Antonie van Leeuwenhoek 71: 109–116.

    Article  PubMed  CAS  Google Scholar 

  • Wagner M, Amann R, Lemmer H & Schleifer K-H (1993) Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbiol. 59: 1520–1525.

    PubMed  CAS  Google Scholar 

  • Wagner M, Erhart R, Manz W, Amann R, Lemmer H, Wedi D & 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. Appl. Environ. Microbiol. 60: 792–800.

    PubMed  CAS  Google Scholar 

  • Wentzel MC, Loewenthal RE, Ekama GA & Marais Gv (1988) Enhanced polyphosphate organism cultures in activated sludge systems - Part 1: Enhanced culture development. Water SA 14: 81–92.

    CAS  Google Scholar 

  • Zilles JL, Hung C-H & Noguera DR (2001) Presence of Rhodocyclus in a full-scale wastewater treatment plant and their participation in enhanced biological phosphorus removal. In: Activated Sludge Population Dynamics Committee of IWA (Ed) 3rd International Conference on Microorganisms in Activated Sludge and Biofilm Processes (pp 75-81). Rome, Italy.

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Authors and Affiliations

  1. Advanced Wastewater Management Centre, The University of Queensland, St. Lucia, 4072, Queensland, Australia

    Linda L. Blackall, Gregory R. Crocetti & Aaron M. Saunders

  2. School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

    Philip L. Bond

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  1. Linda L. Blackall
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  2. Gregory R. Crocetti
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  3. Aaron M. Saunders
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  4. Philip L. Bond
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Correspondence to Linda L. Blackall.

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Blackall, L.L., Crocetti, G.R., Saunders, A.M. et al. A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants. Antonie Van Leeuwenhoek 81, 681–691 (2002). https://doi.org/10.1023/A:1020538429009

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