Advertisement

Microbial Ecology

, Volume 63, Issue 4, pp 773–786 | Cite as

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

  • Tegan N. EvansEmail author
  • Robert J. Seviour
Article

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.

Keywords

Internal Transcribe Spacer Activate Sludge Clone Library Fungal Community Aerobic Granule 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

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.

References

  1. 1.
    Amaral Zettler LA, Gómez F, Zettler E, Keenan BG, Amils R, Sogin ML (2002) Eukaryotic diversity in Spain’s River of Fire. Nature 417:137PubMedCrossRefGoogle Scholar
  2. 2.
    Anderson IC, Cairney JWG (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environ Microbiol 6:769–779PubMedCrossRefGoogle Scholar
  3. 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 5:36–47PubMedCrossRefGoogle Scholar
  4. 4.
    Baldrian P, Valášková V (2007) Degradation of cellulose by basidiomycetous fungi. FEMS Microbiol Rev 32:501–521CrossRefGoogle Scholar
  5. 5.
    Bärlocher F (2010) Molecular approaches promise a deeper and broader understanding of the evolutionary ecology of aquatic hyphomycetes. J N Am Benthol Soc 29:1027–1041CrossRefGoogle Scholar
  6. 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 274:3069–3077PubMedCrossRefGoogle Scholar
  7. 7.
    Berney, C, Fahrni, J, Pawlowski, J (2004) How many novel eukaryotic ‘kingdoms’? Pitfalls and limitations of environmental DNA surveys. BMC Biol 2Google Scholar
  8. 8.
    Bonito G, Isikhuemhen OS, Vilgalys R (2010) Identification of fungi associated with municipal compost using DNA-based techniques. Bioresour Technol 101:1021–1027PubMedCrossRefGoogle Scholar
  9. 9.
    Booth C (1971) Introduction to general methods. In: Booth C (ed) Methods in microbiology, vol 4. Academic, London, pp 34–35Google Scholar
  10. 10.
    Borneman J, Hartin RJ (2000) PCR primers that amplify fungal rRNA genes from environmental samples. Appl Environ Microbiol 66:4356–4360PubMedCrossRefGoogle Scholar
  11. 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 74:3959–3968PubMedCrossRefGoogle Scholar
  12. 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 9:259–261CrossRefGoogle Scholar
  13. 13.
    Brodie E, Edwards S, Clipson N (2003) Soil fungal community structure in a temperature upland grassland soil. FEMS Microbiol Ecol 45:105–114PubMedCrossRefGoogle Scholar
  14. 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 77:955–964PubMedCrossRefGoogle Scholar
  15. 15.
    Cooke WB, Ludzack FJ (1958) Predacious fungus behavior in activated sludge systems. Sewage and Industrial Wastes 30:1490–1495Google Scholar
  16. 16.
    Cooke WB, Pipes WO (1969) The occurrence of fungi in activated sludge. Mycopathologia 40:249–270Google Scholar
  17. 17.
    Cooke WB (1970) Fungi associated with the activated-sludge process of sewage treatment at the Lebanon, Ohio, sewage-treatment plant. Ohio J Sci 70:129–146Google Scholar
  18. 18.
    Dashtban M, Schraft H, Syed TA, Qin W (2010) Fungal biodegradation and enzymatic modification of lignin. Int J Biochem Mol Biol 1:36–50PubMedGoogle Scholar
  19. 19.
    Dawson SC, Pace NR (2002) Novel kingdom-level eukaryotic diversity in anoxic environments. PNAS 99:8324–8329PubMedCrossRefGoogle Scholar
  20. 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 36:1607–1614CrossRefGoogle Scholar
  21. 21.
    Diener UL, Morgan-Jones G, Hagler WM, Davis ND (1976) Mycoflora of activated sewage sludge. Mycopathologia 58:115–116PubMedCrossRefGoogle Scholar
  22. 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. 23.
    Eikleboom DH (2000) Process control of activated sludge plants by microscopic investigation. IWA, UKGoogle Scholar
  24. 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 37:309–320CrossRefGoogle Scholar
  25. 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 106:18315–18320PubMedCrossRefGoogle Scholar
  26. 26.
    Fröhlich-Nowoisky J, Pickersgill DA, Després VR, Pöschl U (2009) High diversity of fungi in air particulate matter. PNAS 106:12814–12819PubMedCrossRefGoogle Scholar
  27. 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 74:6091–6101PubMedCrossRefGoogle Scholar
  28. 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 104:143–149CrossRefGoogle Scholar
  29. 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 66:5488–5491PubMedCrossRefGoogle Scholar
  30. 30.
    Guindon S, Gascuel O (2003) A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704PubMedCrossRefGoogle Scholar
  31. 31.
    Hall B (2008) Phylogenetic trees made easy: a how-to manual. Sinauer, SunderlandGoogle Scholar
  32. 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 51:209–214PubMedCrossRefGoogle Scholar
  33. 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 25:281–287CrossRefGoogle Scholar
  34. 34.
    Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319PubMedCrossRefGoogle Scholar
  35. 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 47:385–395PubMedCrossRefGoogle Scholar
  36. 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 71:399–412PubMedCrossRefGoogle Scholar
  37. 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 1–15Google Scholar
  38. 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, LondonGoogle Scholar
  39. 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 474:200–203CrossRefGoogle Scholar
  40. 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 53:233–246PubMedCrossRefGoogle Scholar
  41. 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 97:1177–1194PubMedCrossRefGoogle Scholar
  42. 42.
    Kacprzak M, Neczaj E, Okoniewska E (2005) The comparative mycological analysis of wastewater and sewage sludges from selected wastewater treatment plants. Desalination 185:363–370CrossRefGoogle Scholar
  43. 43.
    Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120PubMedCrossRefGoogle Scholar
  44. 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 305–324Google Scholar
  45. 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 1:756–762PubMedCrossRefGoogle Scholar
  46. 46.
    Lara E, Mitchell EAD, Moreira D, López Garía P (2010) Highly diverse and seasonally dynamic protist community in a pristine peat bog. Protist 162:14–32PubMedCrossRefGoogle Scholar
  47. 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 9:61–71PubMedCrossRefGoogle Scholar
  48. 48.
    Letcher PM, Powell MJ, Barr DJS, Churchill PF, Wakefield WS, Picard KT (2008) Rhizophlyctidales—a new order in Chytridiomycota. Mycol Res 112:1031–1048PubMedCrossRefGoogle Scholar
  49. 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 45:155–165PubMedCrossRefGoogle Scholar
  50. 50.
    Malandra L, Wolfaardt G, Zietsman A, Viljoen-Bloom M (2003) Microbiology of a biological contactor for winery wastewater treatment. Water Res 37:4125–4134PubMedCrossRefGoogle Scholar
  51. 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 47:829–841PubMedCrossRefGoogle Scholar
  52. 52.
    McIlroy SJ, Porter K, Seviour RJ, Tillett D (2009) Extracting nucleic acids from activated sludge which reflect community population diversity. Antonie Van Leeuwenhoek 96:593–605PubMedCrossRefGoogle Scholar
  53. 53.
    More TT, Yan S, Tyagi RD, Surampalli RY (2010) Potential use of filamentous fungi for wastewater sludge treatment. Bioresour Technol 101:7691–7700CrossRefGoogle Scholar
  54. 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 82:983–993PubMedCrossRefGoogle Scholar
  55. 55.
    Nocker A, Burr M, Camper AK (2007) Genotypic microbial community profiling: a critical technical review. Microb Ecol 54:276–289PubMedCrossRefGoogle Scholar
  56. 56.
    Peay KG, Kennedy PG, Bruns TD (2008) Fungal community ecology: a hybrid beast with a molecular master. Bioscience 58:799–810CrossRefGoogle Scholar
  57. 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 37:821–829CrossRefGoogle Scholar
  58. 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 75:1922–1931PubMedCrossRefGoogle Scholar
  59. 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, LondonGoogle Scholar
  60. 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 95–126Google Scholar
  61. 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 57:72–82CrossRefGoogle Scholar
  62. 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 271–286Google Scholar
  63. 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 65:2614–2621PubMedGoogle Scholar
  64. 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 126–142Google Scholar
  65. 65.
    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  66. 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 40:1390–1403CrossRefGoogle Scholar
  67. 67.
    Tomlinson TG, Williams IL (1975) Fungi. In: Curds CR, Hawkes HA (eds) Ecological aspects of used-water treatment, vol 1. Academic, London, pp 93–152Google Scholar
  68. 68.
    Vainio EJ, Hantula J (2000) Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycol Res 104:927–936CrossRefGoogle Scholar
  69. 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 88:299–307CrossRefGoogle Scholar
  70. 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 68:246–254PubMedCrossRefGoogle Scholar
  71. 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 315–321Google Scholar
  72. 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 54:139–146PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Biotechnology Research Centre, La Trobe Institute for Molecular SciencesLa Trobe UniversityBendigoAustralia
  2. 2.Department of MicrobiologyLa Trobe UniversityMelbourneAustralia

Personalised recommendations