1887

Abstract

Microbiology in wastewater treatment has mainly been focused on problem-causing filamentous bacteria or bacteria directly involved in nitrogen and phosphorus removal, and to a lesser degree on flanking groups, such as hydrolysing and fermenting bacteria. However, these groups constitute important suppliers of readily degradable substrates for the overall processes in the plant. This study aimed to identify glucose-fermenting bacteria in a full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plant (WWTP), and to determine their abundance in similar WWTPs. Glucose-fermenting micro-organisms were identified by an approach using RNA-based stable isotope probing. Activated sludge was incubated anaerobically with C-labelled glucose, and C-enriched rRNA was subsequently reverse-transcribed and used to construct a 16S rRNA gene clone library. Phylogenetic analysis of the library revealed the presence of two major phylogenetic groups of Gram-positive bacteria affiliating with the genera , (Actinobacteria), and and (Firmicutes). Specific oligonucleotide probes were designed for fluorescence hybridization (FISH) to specifically target the glucose-fermenting bacteria identified in this study. The combination of FISH with microautoradiography confirmed that , and were the dominant glucose fermenters. The probe-defined fermenters were quantified in 10 full-scale EBPR plants and averaged 39 % of the total biovolume. and were the most abundant glucose fermenters (average 33 and 4 %, respectively), while and were present only in some WWTPs (average 1 and 0.4 %, respectively). Thus the population of actively metabolizing glucose fermenters seems to occupy a relatively large component of the total biovolume.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.058818-0
2012-07-01
2021-07-30
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/7/1818.html?itemId=/content/journal/micro/10.1099/mic.0.058818-0&mimeType=html&fmt=ahah

References

  1. Akasaka H., Ueki A., Hanada S., Kamagata Y., Ueki K. ( 2003). Propionicimonas paludicola gen. nov., sp. nov., a novel facultatively anaerobic, Gram-positive, propionate-producing bacterium isolated from plant residue in irrigated rice-field soil. Int J Syst Evol Microbiol 53:1991–1998 [View Article][PubMed]
    [Google Scholar]
  2. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. ( 1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [View Article][PubMed]
    [Google Scholar]
  3. Amann R. I., Binder B. J., Olson R. J., Chisholm S. W., Devereux R., Stahl D. A. ( 1990). Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925[PubMed]
    [Google Scholar]
  4. Daims H., Brühl A., Amann R. I., Schleifer K.-H., Wagner M. ( 1999). The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22:434–444 [View Article][PubMed]
    [Google Scholar]
  5. Egert M., de Graaf A. A., Maathuis A., de Waard P., Plugge C. M., Smidt H., Deutz N. E., Dijkema C., de Vos W. M., Venema K. ( 2007). Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing. FEMS Microbiol Ecol 60:126–135 [View Article][PubMed]
    [Google Scholar]
  6. Fischer-Romero C., Tindall B. J., Jüttner F. ( 1996). Tolumonas auensis gen. nov., sp. nov., a toluene-producing bacterium from anoxic sediments of a freshwater lake. Int J Syst Bacteriol 46:183–188 [View Article][PubMed]
    [Google Scholar]
  7. Ginige M. P., Hugenholtz P., Daims H., Wagner M., Keller J., Blackall L. L. ( 2004). Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community. Appl Environ Microbiol 70:588–596 [View Article][PubMed]
    [Google Scholar]
  8. Hallin S., Throbäck I. N., Dicksved J., Pell M. ( 2006). Metabolic profiles and genetic diversity of denitrifying communities in activated sludge after addition of methanol or ethanol. Appl Environ Microbiol 72:5445–5452 [View Article][PubMed]
    [Google Scholar]
  9. Hatamoto M., Imachi H., Yashiro Y., Ohashi A., Harada H. ( 2007). Diversity of anaerobic microorganisms involved in long-chain fatty acid degradation in methanogenic sludges as revealed by RNA-based stable isotope probing. Appl Environ Microbiol 73:4119–4127 [View Article][PubMed]
    [Google Scholar]
  10. Huber T., Faulkner G., Hugenholtz P. ( 2004). Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319 [View Article][PubMed]
    [Google Scholar]
  11. Kong Y., Nielsen J. L., Nielsen P. H. ( 2005). Identity and ecophysiology of uncultured actinobacterial polyphosphate-accumulating organisms in full-scale enhanced biological phosphorus removal plants. Appl Environ Microbiol 71:4076–4085 [View Article][PubMed]
    [Google Scholar]
  12. Kong Y., Xia Y., Nielsen P. H. ( 2008). Activity and identity of fermenting microorganisms in full-scale biological nutrient removing wastewater treatment plants. Environ Microbiol 10:2008–2019 [View Article][PubMed]
    [Google Scholar]
  13. Kovatcheva-Datchary P., Egert M., Maathuis A., Rajilić-Stojanović M., de Graaf A. A., Smidt H., de Vos W. M., Venema K. ( 2009). Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing. Environ Microbiol 11:914–926 [View Article][PubMed]
    [Google Scholar]
  14. Lane D. J. ( 1991). 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial Systematics113–175 Stackebrandt E., Goodfellow M. Chichester, UK: Wiley Publications;
    [Google Scholar]
  15. Ludwig W., Strunk O., Westram R., Richter L., Meier H., Yadhukumar A., Buchner A., Lai T., Steppi S. & other authors ( 2004). arb: a software environment for sequence data. Nucleic Acids Res 32:1363–1371 [View Article][PubMed]
    [Google Scholar]
  16. Maidak B. L., Cole J. R., Lilburn T. G., Parker C. T. Jr, Saxman P. R., Farris R. J., Garrity G. M., Olsen G. J., Schmidt T. M., Tiedje J. M. ( 2001). The RDP-II (Ribosomal Database Project). Nucleic Acids Res 29:173–174 [View Article][PubMed]
    [Google Scholar]
  17. Manefield M., Griffiths R., McNamara N. P., Sleep D., Ostle N., Whiteley A. ( 2007). Insights into the fate of a 13C labelled phenol pulse for stable isotope probing (SIP) experiments. J Microbiol Methods 69:340–344 [View Article][PubMed]
    [Google Scholar]
  18. Meier H., Amann R. I., Ludwig W., Schleifer K.-H. ( 1999). Specific oligonucleotide probes for in situ detection of a major group of Gram-positive bacteria with low DNA G+C content. Syst Appl Microbiol 22:186–196 [View Article][PubMed]
    [Google Scholar]
  19. Meyer R. L., Saunders A. M., Blackall L. L. ( 2006). Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing. Microbiology 152:419–429 [View Article][PubMed]
    [Google Scholar]
  20. Muyzer G., Teske A., Wirsen C. O., Jannasch H. W. ( 1995). Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol 164:165–172 [View Article][PubMed]
    [Google Scholar]
  21. 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 [View Article][PubMed]
    [Google Scholar]
  22. Nguyen H. T., Le V. Q., Hansen A. A., Nielsen J. L., Nielsen P. H. ( 2011). High diversity and abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated sludge systems. FEMS Microbiol Ecol 76:256–267 [View Article][PubMed]
    [Google Scholar]
  23. Nielsen J. L. ( 2009). Protocol for fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotides. FISH Handbook for Biological Wastewater Treatment73–84 Nielsen P. H., Daims H., Lemmer H. London: IWA Publishing;
    [Google Scholar]
  24. Nielsen J. L., Nielsen P. H. ( 2005). Advances in microscopy, microautoradiography of single cells. Environmental Microbiology vol. 397237–256 Leadbetter J. R. San Diego: Elsevier Academic Press Inc;
    [Google Scholar]
  25. Nielsen P. H., Mielczarek A. T., Kragelund C., Nielsen J. L., Saunders A. M., Kong Y., Hansen A. A., Vollertsen J. ( 2010). A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants. Water Res 44:5070–5088 [View Article][PubMed]
    [Google Scholar]
  26. Nollet L., Demeyer D., Verstraete W. ( 1997). Effect of 2-bromoethanesulfonic acid and Peptostreptococcus productus ATCC 35244 addition on stimulation of reductive acetogenesis in the ruminal ecosystem by selective inhibition of methanogenesis. Appl Environ Microbiol 63:194–200[PubMed]
    [Google Scholar]
  27. Oremland R. S., Capone D. G. ( 1988). Use of specific inhibitors in biogeochemistry and microbial ecology. Adv Microb Ecol 10:285–383 [View Article]
    [Google Scholar]
  28. Pitman A. R., Lötter L. H., Alexander W. V., Deacon S. L. ( 1992). Fermentation of raw sludge and elutriation of resultant fatty acids to promote excess biological phosphorus removal. Water Sci Technol 25:185–194
    [Google Scholar]
  29. Pruesse E., Quast C., Knittel K., Fuchs B. M., Ludwig W. G., Peplies J., Glöckner F. O. ( 2007). SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with arb . Nucleic Acids Res 35:7188–7196 [View Article][PubMed]
    [Google Scholar]
  30. Roller C., Wagner M., Amann R. I., Ludwig W., Schleifer K.-H. ( 1994). In situ probing of Gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides. Microbiology 140:2849–2858 [View Article][PubMed]
    [Google Scholar]
  31. Sambrook J., Russell D. ( 2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  32. Smith E. A., Macfarlane G. T. ( 1998). Enumeration of amino acid fermenting bacteria in the human large intestine, effects of pH and starch on peptide metabolism and dissimilation of amino acids. FEMS Microbiol Ecol 25:355–368 [View Article]
    [Google Scholar]
  33. Trebesius K., Leitritz L., Adler K., Schubert S., Autenrieth I. B., Heesemann J. ( 2000). Culture independent and rapid identification of bacterial pathogens in necrotising fasciitis and streptococcal toxic shock syndrome by fluorescence in situ hybridisation. Med Microbiol Immunol (Berl) 188:169–175[PubMed] [CrossRef]
    [Google Scholar]
  34. Vollertsen J., Petersen G., Borregaard V. R. ( 2006). Hydrolysis and fermentation of activated sludge to enhance biological phosphorus removal. Water Sci Technol 53:55–64 [View Article][PubMed]
    [Google Scholar]
  35. Wallner G., Amann R. I., Beisker W. ( 1993). Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14:136–143 [View Article][PubMed]
    [Google Scholar]
  36. Zhang T., Zhang M., Zhang X., Fang H. H. ( 2009). Tetracycline resistance genes and tetracycline resistant lactose-fermenting Enterobacteriaceae in activated sludge of sewage treatment plants. Environ Sci Technol 43:3455–3460 [View Article][PubMed]
    [Google Scholar]
  37. Zheng D., Alm E. W., Stahl D. A., Raskin L. ( 1996). Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies. Appl Environ Microbiol 62:4504–4513[PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.058818-0
Loading
/content/journal/micro/10.1099/mic.0.058818-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error