Skip to main content
SpringerLink
Log in

Estimating Biodiversity of Fungi in Activated Sludge Communities Using Culture-Independent Methods

  • Published:
Microbial Ecology Aims and scope Submit manuscript

Abstract

Fungal diversity of communities in several activated sludge plants treating different influent wastes was determined by comparative sequence analyses of their 18S rRNA genes. Methods for DNA extraction and choice of primers for PCR amplification were both optimised using denaturing gradient gel electrophoresis profile patterns. Phylogenetic analysis revealed that the levels of fungal biodiversity in some communities, like those treating paper pulp wastes, were low, and most of the fungi detected in all communities examined were novel uncultured representatives of the major fungal subdivisions, in particular, the newly described clade Cryptomycota. The fungal populations in activated sludge revealed by these culture-independent methods were markedly different to those based on culture-dependent data. Members of the genera Penicillium, Cladosporium, Aspergillus and Mucor, which have been commonly identified in mixed liquor, were not identified in any of these plant communities. Non-fungal eukaryotic 18S rRNA genes were also amplified with the primer sets used. This is the first report where culture-independent methods have been applied to flocculated activated sludge biomass samples to estimate fungal community composition and, as expected, the data obtained gave a markedly different view of their population biodiversity compared to that based on culture-dependent methods.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Amaral Zettler LA, Gómez F, Zettler E, Keenan BG, Amils R, Sogin ML (2002) Eukaryotic diversity in Spain’s River of Fire. Nature 417:137

    Article  PubMed  CAS  Google Scholar 

  2. Anderson IC, Cairney JWG (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environ Microbiol 6:769–779

    Article  PubMed  CAS  Google Scholar 

  3. Anderson IC, Campbell CD, Prosser JI (2003) Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil. Environ Microbiol 5:36–47

    Article  PubMed  CAS  Google Scholar 

  4. Baldrian P, Valášková V (2007) Degradation of cellulose by basidiomycetous fungi. FEMS Microbiol Rev 32:501–521

    Article  Google Scholar 

  5. Bärlocher F (2010) Molecular approaches promise a deeper and broader understanding of the evolutionary ecology of aquatic hyphomycetes. J N Am Benthol Soc 29:1027–1041

    Article  Google Scholar 

  6. Bass D, Howe A, Brown N, Barton H, Demidova M, Michelle H, Li L, Sanders H, Watkinson SC, Willcock S, Richards TA (2007) Yeast forms dominate fungal diversity in the deep oceans. Proc R Soc B 274:3069–3077

    Article  PubMed  CAS  Google Scholar 

  7. Berney, C, Fahrni, J, Pawlowski, J (2004) How many novel eukaryotic ‘kingdoms’? Pitfalls and limitations of environmental DNA surveys. BMC Biol 2

  8. Bonito G, Isikhuemhen OS, Vilgalys R (2010) Identification of fungi associated with municipal compost using DNA-based techniques. Bioresour Technol 101:1021–1027

    Article  PubMed  CAS  Google Scholar 

  9. Booth C (1971) Introduction to general methods. In: Booth C (ed) Methods in microbiology, vol 4. Academic, London, pp 34–35

    Google Scholar 

  10. Borneman J, Hartin RJ (2000) PCR primers that amplify fungal rRNA genes from environmental samples. Appl Environ Microbiol 66:4356–4360

    Article  PubMed  CAS  Google Scholar 

  11. Brad T, Braster M, van Breukelen BM, van Straalen NM, Röling WFM (2008) Eukaryotic diversity in an anaerobic aquifer polluted with landfill leachate. Appl Environ Microbiol 74:3959–3968

    Article  PubMed  CAS  Google Scholar 

  12. Brinkhoff T, van Hannen EJ (2001) Use of silicone grease to avoid ‘smiling effect’ in denaturing gradient gel electrophoresis. J Rapid Methods Automat Micro 9:259–261

    Article  Google Scholar 

  13. Brodie E, Edwards S, Clipson N (2003) Soil fungal community structure in a temperature upland grassland soil. FEMS Microbiol Ecol 45:105–114

    Article  PubMed  CAS  Google Scholar 

  14. Carrigg C, Rice O, Kavanagh S, Collins G, O’Flaherty V (2007) DNA extraction method affects microbial community profiles from soils and sediment. Appl Microbiol Biotechnol 77:955–964

    Article  PubMed  CAS  Google Scholar 

  15. Cooke WB, Ludzack FJ (1958) Predacious fungus behavior in activated sludge systems. Sewage and Industrial Wastes 30:1490–1495

    Google Scholar 

  16. Cooke WB, Pipes WO (1969) The occurrence of fungi in activated sludge. Mycopathologia 40:249–270

    Google Scholar 

  17. Cooke WB (1970) Fungi associated with the activated-sludge process of sewage treatment at the Lebanon, Ohio, sewage-treatment plant. Ohio J Sci 70:129–146

    Google Scholar 

  18. Dashtban M, Schraft H, Syed TA, Qin W (2010) Fungal biodegradation and enzymatic modification of lignin. Int J Biochem Mol Biol 1:36–50

    PubMed  CAS  Google Scholar 

  19. Dawson SC, Pace NR (2002) Novel kingdom-level eukaryotic diversity in anoxic environments. PNAS 99:8324–8329

    Article  PubMed  CAS  Google Scholar 

  20. de Lipthay JR, Enzinger C, Johnsen K, Aamand J, Sørensen SJ (2004) Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis. Soil Biol Biochem 36:1607–1614

    Article  Google Scholar 

  21. Diener UL, Morgan-Jones G, Hagler WM, Davis ND (1976) Mycoflora of activated sewage sludge. Mycopathologia 58:115–116

    Article  PubMed  CAS  Google Scholar 

  22. Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2010) Geneious. Available from http://www.geneious.com

  23. Eikleboom DH (2000) Process control of activated sludge plants by microscopic investigation. IWA, UK

    Google Scholar 

  24. Fakhru’l-Razi A, Alam MZ, Idris A, Abd-Aziz S, Molla AH (2002) Filamentous fungi in Indah Water Konsortium (IWK) sewage treatment plant for biological treatment of domestic wastewater sludge. J Environ Sci Health, Pt A: Toxic/Hazard Subst Environ Eng 37:309–320

    Article  Google Scholar 

  25. Freeman KR, Martin AP, Karki D, Lynch RC, Mitter MS, Meyer AF, Longcore JE, Simmons DR, Schmidt SK (2009) Evidence that chytrids dominate fungal communities in high-elevation soils. PNAS 106:18315–18320

    Article  PubMed  CAS  Google Scholar 

  26. Fröhlich-Nowoisky J, Pickersgill DA, Després VR, Pöschl U (2009) High diversity of fungi in air particulate matter. PNAS 106:12814–12819

    Article  PubMed  Google Scholar 

  27. Gao Z, Li B, Zheng C, Wang G (2008) Molecular detection of fungal communities in the Hawaiian marine sponges Suberites zeteki and Mycale armata. Appl Environ Microbiol 74:6091–6101

    Article  PubMed  CAS  Google Scholar 

  28. Gray NF (1984) The effect of fungal parasitism and predation on the population dynamics of nematodes in the activated sludge process. Ann Appl Biol 104:143–149

    Article  Google Scholar 

  29. Griffiths RI, Whiteley AS, O’Donnell AG, Bailey MJ (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA and rRNA-based microbial community composition. Appl Environ Microbiol 66:5488–5491

    Article  PubMed  CAS  Google Scholar 

  30. Guindon S, Gascuel O (2003) A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704

    Article  PubMed  Google Scholar 

  31. Hall B (2008) Phylogenetic trees made easy: a how-to manual. Sinauer, Sunderland

    Google Scholar 

  32. Hansgate AM, Schloss PD, Hay AG, Walker LP (2005) Molecular characterization of fungal community dynamics in the initial stages of composting. FEMS Microbiol Ecol 51:209–214

    Article  PubMed  CAS  Google Scholar 

  33. Hoshino YT, Matsumoto S (2010) Soil clone library analyses to evaluate specificity and selectivity of PCR primers targeting fungal 18S rDNA for denaturing-gradient gel electrophoresis (DGGE). Microbes Environ 25:281–287

    Article  Google Scholar 

  34. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319

    Article  PubMed  CAS  Google Scholar 

  35. Hunt J, Boddy L, Randerson PF, Rogers HJ (2004) An evaluation of 18S rDNA approaches for the study of fungal diversity in grassland soils. Microb Ecol 47:385–395

    Article  PubMed  CAS  Google Scholar 

  36. Jebaraj CS, Raghukumar C, Behnke A, Stoeck T (2010) Fungal diversity in oxygen-depleted regions of the Arabian Sea revealed by targeted environmental sequencing combined with cultivation. FEMS Microbiol Ecol 71:399–412

    Article  PubMed  CAS  Google Scholar 

  37. Jeewon R, Hyde KD (2007) Detection and diversity of fungi from environmental samples: traditional versus molecular approaches. In: Varma A, Oelmüller R (eds) Soil biology, vol 11. Springer, Berlin, pp 1–15

    Google Scholar 

  38. Jenkins D, Richard MG, Daigger GT (2004) Manual of the causes and control of activated sludge bulking, foaming and other solids separation problems. CRC, London

    Google Scholar 

  39. Jones MDM, Forn I, Gadelha C, Egan MJ, Bass D, Massana R, Richards TA (2011) Discovery of novel intermediate forms redefines the fungal tree of life. Nature Letter 474:200–203

    Article  CAS  Google Scholar 

  40. Jumpponen A (2007) Soil fungal communities underneath willow canopies on a primary successional glacier forefront: rDNA sequence results can be affected by primer selection and chimeric data. Microb Ecol 53:233–246

    Article  PubMed  CAS  Google Scholar 

  41. Jumpponen A, Johnson LC (2005) Can rDNA analyses of diverse fungal communities in soil and roots detect effects of environmental manipulations—a case study from tallgrass prairie. Mycologia 97:1177–1194

    Article  PubMed  CAS  Google Scholar 

  42. Kacprzak M, Neczaj E, Okoniewska E (2005) The comparative mycological analysis of wastewater and sewage sludges from selected wastewater treatment plants. Desalination 185:363–370

    Article  CAS  Google Scholar 

  43. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120

    Article  PubMed  CAS  Google Scholar 

  44. Kjøller AH, Struwe S (2002) Fungal communities, succession, enzymes and decomposition. In: Burns RG, Dick RP (eds) Enzymes in the environment: activity, ecology, and applications. Marcel Dekker, New York, pp 305–324

    Google Scholar 

  45. Lai X, Cao L, Tan H, Fang S, Huang Y, Zhou S (2007) Fungal communities from methane hydrate-bearing deep-sea marine sediments in South China Sea. ISME J 1:756–762

    Article  PubMed  CAS  Google Scholar 

  46. Lara E, Mitchell EAD, Moreira D, López Garía P (2010) Highly diverse and seasonally dynamic protist community in a pristine peat bog. Protist 162:14–32

    Article  PubMed  Google Scholar 

  47. Lefèvre E, Bardot C, Noёl C, Carrias J, Viscogliosi E, Amblard C, Sime-Ngando T (2007) Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake. Environ Microbiol 9:61–71

    Article  PubMed  Google Scholar 

  48. Letcher PM, Powell MJ, Barr DJS, Churchill PF, Wakefield WS, Picard KT (2008) Rhizophlyctidales—a new order in Chytridiomycota. Mycol Res 112:1031–1048

    Article  PubMed  Google Scholar 

  49. Maarit-Niemi R, Heiskanen I, Wallenius K, Lindström K (2001) Extraction and purification of DNA in rhizosphere soil samples for PCR–DGGE analysis of bacterial consortia. J Microbiol Methods 45:155–165

    Article  PubMed  CAS  Google Scholar 

  50. Malandra L, Wolfaardt G, Zietsman A, Viljoen-Bloom M (2003) Microbiology of a biological contactor for winery wastewater treatment. Water Res 37:4125–4134

    Article  PubMed  CAS  Google Scholar 

  51. May LA, Smiley B, Schmidt MG (2001) Comparative denaturing gradient gel electrophoresis analysis of fungal communities associated with whole plant corn silage. Can J Microbiol 47:829–841

    Article  PubMed  CAS  Google Scholar 

  52. McIlroy SJ, Porter K, Seviour RJ, Tillett D (2009) Extracting nucleic acids from activated sludge which reflect community population diversity. Antonie Van Leeuwenhoek 96:593–605

    Article  PubMed  CAS  Google Scholar 

  53. More TT, Yan S, Tyagi RD, Surampalli RY (2010) Potential use of filamentous fungi for wastewater sludge treatment. Bioresour Technol 101:7691–7700

    Article  CAS  Google Scholar 

  54. Ning J, Liebich J, Kästner M, Zhou J, Schäffer A, Burauel P (2009) Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study. Appl Microbiol Biotechnol 82:983–993

    Article  PubMed  CAS  Google Scholar 

  55. Nocker A, Burr M, Camper AK (2007) Genotypic microbial community profiling: a critical technical review. Microb Ecol 54:276–289

    Article  PubMed  CAS  Google Scholar 

  56. Peay KG, Kennedy PG, Bruns TD (2008) Fungal community ecology: a hybrid beast with a molecular master. Bioscience 58:799–810

    Article  Google Scholar 

  57. Petruccioli M, Cardoso Duarte J, Eusébio A, Federici F (2002) Aerobic treatment of winery wastewater using a jet-loop activated sludge reactor. Process Biochem 37:821–829

    Article  CAS  Google Scholar 

  58. Prat C, Ruiz-Rueda O, Trias R, Anticó E, Capone D, Sefton M, Bañeras L (2009) Molecular fingerprinting by PCR-denaturing gradient gel electrophoresis reveals differences in the levels of microbial diversity for musty-earthy tainted corks. Appl Environ Microbiol 75:1922–1931

    Article  PubMed  CAS  Google Scholar 

  59. Schäfer H, Muzyer G (2001) Denaturing gradient gel electrophoresis in marine microbial ecology. In: Paul JH (ed) Marine microbiology: methods in microbiology, vol 30. Elsevier, London

    Google Scholar 

  60. Seviour R, Nielson PH (2010) Microbial communities in activated sludge. In: Seviour R, Nielson PH (eds) Microbial ecology of activated sludge. IWA, London, pp 95–126

    Google Scholar 

  61. Singh BK, Munro S, Reid E, Ord B, Potts JM, Paterson E, Millard P (2006) Investigating microbial community structure in soils by physiological, biochemical and molecular fingerprinting. Eur J Soil Sci 57:72–82

    Article  CAS  Google Scholar 

  62. Smit E, de Souza F, Landeweert R (2005) Molecular detection of fungal communities in soil. In: Olson MA, Smith CT (eds) Molecular microbial ecology. Taylor and Francis, New York, pp 271–286

    Google Scholar 

  63. Smit E, Leeflang P, Glandorf B, van Elsas J, Wernars K (1999) Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18 s rRNA and temperature gradient gel electrophoresis. Appl Environ Microbiol 65:2614–2621

    PubMed  CAS  Google Scholar 

  64. Stackebrandt E (2011) Pitfalls of PCR-based rRNA gene sequence analysis: an update on some parameters. In: de Bruijin FJ (ed) Handbook of molecular microbial ecology I: metagenomics and complementary approaches. Wiley, Hoboken, pp 126–142

    Google Scholar 

  65. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  PubMed  CAS  Google Scholar 

  66. Thakuria D, Schmidt O, Siúrtáin MM, Egan D, Doohan FM (2008) Importance of DNA quality in comparative soil microbial community structure analyses. Soil Biol Biochem 40:1390–1403

    Article  CAS  Google Scholar 

  67. Tomlinson TG, Williams IL (1975) Fungi. In: Curds CR, Hawkes HA (eds) Ecological aspects of used-water treatment, vol 1. Academic, London, pp 93–152

    Google Scholar 

  68. Vainio EJ, Hantula J (2000) Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycol Res 104:927–936

    Article  CAS  Google Scholar 

  69. Vanyasker L, Declerck SAJ, Hellemans B, De Meester L, Vankelecom I, Declerck P (2010) Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods. Appl Microbiol Biotechnol 88:299–307

    Article  Google Scholar 

  70. Weber SD, Hofmann A, Pilhofer M, Wanner G, Agerer R, Ludwig W, Schliefer K, Fried J (2009) The diversity of fungi in aerobic sewage granules assessed by 18S rRNA gene and ITS sequence analyses. FEMS Microbiol Ecol 68:246–254

    Article  PubMed  CAS  Google Scholar 

  71. White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, New York, pp 315–321

    Google Scholar 

  72. Williams JC, de los Reyes FL III (2006) Microbial community structure of activated sludge during aerobic granulation in an annular gap bioreactor. Water Sci Technol 54:139–146

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We wish to thank Jian Rong Liu, Robert Morris, Kate Pauley, Catherine Watts and Michael Machin for providing biomass samples and information on the treatment plants. Tegan Evans was a recipient of a La Trobe University Pharmacy and Applied Sciences Department postgraduate scholarship.

Author information

Authors and Affiliations

  1. Biotechnology Research Centre, La Trobe Institute for Molecular Sciences, La Trobe University, P.O. Box 199, Bendigo, Victoria, 3552, Australia

    Tegan N. Evans

  2. Department of Microbiology, La Trobe University, Bundoora, Melbourne, Victoria, 3086, Australia

    Robert J. Seviour

Authors
  1. Tegan N. Evans
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert J. Seviour
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tegan N. Evans.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Evans, T.N., Seviour, R.J. Estimating Biodiversity of Fungi in Activated Sludge Communities Using Culture-Independent Methods. Microb Ecol 63, 773–786 (2012). https://doi.org/10.1007/s00248-011-9984-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00248-011-9984-7

Keywords

Search

Navigation