In methanogenic habitats, volatile fatty acids (VFA), such as propionate and butyrate, are major intermediates in organic matter degradation. VFA are further metabolized to H, acetate and CO by syntrophic fatty acid-degrading bacteria (SFAB) in association with methanogenic archaea. Despite their indispensable role in VFA degradation, little is known about SFAB abundance and their environmental distribution. To facilitate ecological studies, we developed four novel genus-specific quantitative PCR (qPCR) assays, with primer sets targeting known SFAB: and . Primer set specificity was confirmed using and experimental (target controls, clone libraries and melt-curve analysis) approaches. These qPCR assays were applied to quantify SFAB in a variety of mesophilic methanogenic habitats, including a laboratory propionate enrichment culture, pilot- and full-scale anaerobic reactors, cow rumen, horse faeces, an experimental rice paddy soil, a bog stream and swamp sediments. The highest SFAB 16S rRNA gene copy numbers were found in the propionate enrichment culture and anaerobic reactors, followed by the bog stream and swamp sediment samples. In addition, it was observed that SFAB and methanogen abundance varied with reactor configuration and substrate identity. To our knowledge, this research represents the first comprehensive study to quantify SFAB in methanogenic habitats using qPCR-based methods. These molecular tools will help investigators better understand syntrophic microbial communities in engineered and natural environments.


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  1. Ariesyady H. D., Ito T., Okabe S. (2007a). Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester. Water Res 41, 15541568. [View Article][PubMed] [Google Scholar]
  2. Ariesyady H. D., Ito T., Yoshiguchi K., Okabe S. (2007b). Phylogenetic and functional diversity of propionate-oxidizing bacteria in an anaerobic digester sludge. Appl Microbiol Biotechnol 75, 673683. [View Article][PubMed] [Google Scholar]
  3. Bergman E. N. (1990). Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol Rev 70, 567590. [PubMed] [Google Scholar]
  4. Bouvier T., del Giorgio P. A. (2003). Factors influencing the detection of bacterial cells using fluorescence in situ hybridization (FISH): A quantitative review of published reports. FEMS Microbiol Ecol 44, 315. [View Article][PubMed] [Google Scholar]
  5. Bustin S. A., Benes V., Garson J. A., Hellemans J., Huggett J., Kubista M., Mueller R., Nolan T., Pfaffl M. W., other authors. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55, 611622. [View Article][PubMed] [Google Scholar]
  6. Chauhan A., Ogram A. (2006). Fatty acid-oxidizing consortia along a nutrient gradient in the Florida Everglades. Appl Environ Microbiol 72, 24002406. [View Article][PubMed] [Google Scholar]
  7. Chauhan A., Ogram A., Reddy K. R. (2004). Syntrophic-methanogenic associations along a nutrient gradient in the Florida Everglades. Appl Environ Microbiol 70, 34753484. [View Article][PubMed] [Google Scholar]
  8. Cole J. R., Wang Q., Fish J. A., Chai B., McGarrell D. M., Sun Y., Brown C. T., Porras-Alfaro A., Kuske C. R., Tiedje J. M. (2014). Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res (41), D633D642. [View Article][PubMed] [Google Scholar]
  9. de Bok F. A. M., Stams A. J. M., Dijkema C., Boone D. R. (2001). Pathway of propionate oxidation by a syntrophic culture of Smithella propionica and Methanospirillum hungatei . Appl Environ Microbiol 67, 18001804. [View Article][PubMed] [Google Scholar]
  10. Fernandez A. S., Hashsham S. A., Dollhopf S. L., Raskin L., Glagoleva O., Dazzo F. B., Hickey R. F., Criddle C. S., Tiedje J. M. (2000). Flexible community structure correlates with stable community function in methanogenic bioreactor communities perturbed by glucose. Appl Environ Microbiol 66, 40584067. [View Article][PubMed] [Google Scholar]
  11. Gan Y., Qiu Q., Liu P., Rui J., Lu Y. (2012). Syntrophic oxidation of propionate in rice field soil at 15 and 30 °C under methanogenic conditions. Appl Environ Microbiol 78, 49234932. [View Article][PubMed] [Google Scholar]
  12. Glissmann K., Conrad R. (2000). Fermentation pattern of methanogenic degradation of rice straw in anoxic paddy soil. FEMS Microbiol Ecol 31, 117126. [View Article][PubMed] [Google Scholar]
  13. Gujer W., Zehnder A. J. B. (1983). Conversion processes in anaerobic digestion. Water Sci Technol 15, 127167. [Google Scholar]
  14. Hansen K. H., Ahring B. K., Raskin L. (1999). Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization. Appl Environ Microbiol 65, 47674774. [PubMed] [Google Scholar]
  15. Harmsen H. J. M., Kengen H. M. P., Akkermans A. D. L., Stams A. J. M. (1995). Phylogenetic analysis of two syntrophic propionate-oxidizing bacteria in enrichments cultures. Syst Appl Microbiol 18, 6773. [View Article] [Google Scholar]
  16. Harmsen H. J., Akkermans A. D., Stams A. J., de Vos W. M. (1996). Population dynamics of propionate-oxidizing bacteria under methanogenic and sulfidogenic conditions in anaerobic granular sludge. Appl Environ Microbiol 62, 21632168. [PubMed] [Google Scholar]
  17. Hashsham S. A., Fernandez A. S., Dollhopf S. L., Dazzo F. B., Hickey R. F., Tiedje J. M., Criddle C. S. (2000). Parallel processing of substrate correlates with greater functional stability in methanogenic bioreactor communities perturbed by glucose. Appl Environ Microbiol 66, 40504057. [View Article][PubMed] [Google Scholar]
  18. Hatamoto M., Imachi H., Ohashi A., Harada H. (2007). Identification and cultivation of anaerobic, syntrophic long-chain fatty acid-degrading microbes from mesophilic and thermophilic methanogenic sludges. Appl Environ Microbiol 73, 13321340. [View Article][PubMed] [Google Scholar]
  19. Hori T., Haruta S., Ueno Y., Ishii M., Igarashi Y. (2006). Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester. Appl Environ Microbiol 72, 16231630. [View Article][PubMed] [Google Scholar]
  20. Imachi H., Sekiguchi Y., Kamagata Y., Loy A., Qiu Y. L., Hugenholtz P., Kimura N., Wagner M., Ohashi A., Harada H. (2006). Non-sulfate-reducing, syntrophic bacteria affiliated with desulfotomaculum cluster I are widely distributed in methanogenic environments. Appl Environ Microbiol 72, 20802091. [View Article][PubMed] [Google Scholar]
  21. Ito T., Yoshiguchi K., Ariesyady H. D., Okabe S. (2012). Identification and quantification of key microbial trophic groups of methanogenic glucose degradation in an anaerobic digester sludge. Bioresour Technol 123, 599607. [View Article][PubMed] [Google Scholar]
  22. Juottonen H., Galand P. E., Tuittila E. S., Laine J., Fritze H., Yrjälä K. (2005). Methanogen communities and Bacteria along an ecohydrological gradient in a northern raised bog complex. Environ Microbiol 7, 15471557. [View Article][PubMed] [Google Scholar]
  23. Krylova N. I., Janssen P. H., Conrad R. (1997). Turnover of propionate in methanogenic paddy soil. FEMS Microbiol Ecol 23, 107117. [View Article] [Google Scholar]
  24. Larkin M. A., Blackshields G., Brown N. P., Chenna R., McGettigan P. A., McWilliam H., Valentin F., Wallace I. M., Wilm A., other authors. (2007). clustal w and clustal_x version 2.0. Bioinformatics 23, 29472948. [View Article][PubMed] [Google Scholar]
  25. Liu P., Qiu Q., Lu Y. (2011). Syntrophomonadaceae-affiliated species as active butyrate-utilizing syntrophs in paddy field soil. Appl Environ Microbiol 77, 38843887. [View Article][PubMed] [Google Scholar]
  26. Lovley D. R., Klug M. J. (1982). Intermediary metabolism of organic matter in the sediments of a eutrophic lake. Appl Environ Microbiol 43, 552560. [PubMed] [Google Scholar]
  27. Lueders T., Pommerenke B., Friedrich M. W. (2004). Stable-isotope probing of micro-organisms thriving at thermodynamic limits: syntrophic propionate oxidation in flooded soil. Appl Environ Microbiol 70, 57785786. [View Article][PubMed] [Google Scholar]
  28. Mackie R. I., Wilkins C. A. (1988). Enumeration of anaerobic bacterial microflora of the equine gastrointestinal tract. Appl Environ Microbiol 54, 21552160. [PubMed] [Google Scholar]
  29. McCarty P. L., Smith D. P. (1986). Anaerobic wastewater treatment. Environ Sci Technol 20, 12001206. [View Article] [Google Scholar]
  30. McInerney M. J., Mackie R. I., Bryant M. P. (1981). Syntrophic association of a butyrate-degrading bacterium and methanosarcina enriched from bovine rumen fluid. Appl Environ Microbiol 41, 826828. [PubMed] [Google Scholar]
  31. McInerney M. J., Struchtemeyer C. G., Sieber J., Mouttaki H., Stams A. J., Schink B., Rohlin L., Gunsalus R. P. (2008). Physiology, ecology, phylogeny, and genomics of micro-organisms capable of syntrophic metabolism. Ann N Y Acad Sci 1125, 5872. [View Article][PubMed] [Google Scholar]
  32. McMahon K. D., Zheng D., Stams A. J., Mackie R. I., Raskin L. (2004). Microbial population dynamics during start-up and overload conditions of anaerobic digesters treating municipal solid waste and sewage sludge. Biotechnol Bioeng 87, 823834. [View Article][PubMed] [Google Scholar]
  33. Morris R., Schauer-Gimenez A., Bhattad U., Kearney C., Struble C. A., Zitomer D., Maki J. S. (2014). Methyl coenzyme M reductase (mcrA) gene abundance correlates with activity measurements of methanogenic H2/CO2-enriched anaerobic biomass. Microb Biotechnol 7, 7784. [View Article][PubMed] [Google Scholar]
  34. Muyzer G., de Waal E. C., Uitterlinden A. G. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59, 695700. [PubMed] [Google Scholar]
  35. Narihiro T., Terada T., Ohashi A., Kamagata Y., Nakamura K., Sekiguchi Y. (2012). Quantitative detection of previously characterized syntrophic bacteria in anaerobic wastewater treatment systems by sequence-specific rRNA cleavage method. Water Res 46, 21672175. [View Article][PubMed] [Google Scholar]
  36. Narihiro T., Nobu M. K., Kim N. K., Kamagata Y., Liu W. T. (2015). The nexus of syntrophy-associated microbiota in anaerobic digestion revealed by long-term enrichment and community survey. Environ Microbiol 17, 17071720. [View Article][PubMed] [Google Scholar]
  37. Power M. E., Tilman D., Estes J. A., Menge B. A., Bond W. J., Mills L. S., Daily G., Castilla J. C., Lubchenco J., Paine R. (1996). Challenges in the quest for keystones. Bioscience 46, 609620. [View Article] [Google Scholar]
  38. Russell J. B., Hespell R. B. (1981). Microbial rumen fermentation. J Dairy Sci 64, 11531169. [View Article][PubMed] [Google Scholar]
  39. Schauer-Gimenez A. E., Zitomer D. H., Maki J. S., Struble C. A. (2010). Bioaugmentation for improved recovery of anaerobic digesters after toxicant exposure. Water Res 44, 35553564. [View Article][PubMed] [Google Scholar]
  40. Scheid D., Stubner S. (2001). Structure and diversity of Gram-negative sulfate-reducing bacteria on rice roots. FEMS Microbiol Ecol 36, 175183. [View Article][PubMed] [Google Scholar]
  41. Schink B. (1997). Energetics of syntrophic cooperation in methanogenic degradation. Microbiol Mol Biol Rev 61, 262280. [PubMed] [Google Scholar]
  42. Schink B., Stams A. J. M. (2002). Syntrophism among Prokaryotes. The Prokaryotes 2, 309335. [Google Scholar]
  43. Schink B., Thauer R. K. (1988). Energetics of syntrophic methane formation and the influence of aggregation. In Granular Anaerobic Sludge, Microbiology and Technology, pp. 517. Edited by Lettinga G., Zehnder A. J. B., Grotenhuis J. T. C., Hulshoff-Pol L. W.. Wageningen, The Netherlands: Pudoc. [Google Scholar]
  44. Scholten J. C., Stams A. J. (1995). The effect of sulfate and nitrate on methane formation in a freshwater sediment. Antonie van Leeuwenhoek 68, 309315. [View Article][PubMed] [Google Scholar]
  45. Shigematsu T., Era S., Mizuno Y., Ninomiya K., Kamegawa Y., Morimura S., Kida K. (2006). Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes. Appl Microbiol Biotechnol 72, 401415. [View Article][PubMed] [Google Scholar]
  46. Smith C. J., Osborn A. M. (2009). Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol 67, 620. [View Article][PubMed] [Google Scholar]
  47. Smith C. J., Nedwell D. B., Dong L. F., Osborn A. M. (2006). Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples. Environ Microbiol 8, 804815. [View Article][PubMed] [Google Scholar]
  48. Sorensen A. H., Ahring B. K. (1993). Measurements of the specific methanogenic activity of anaerobic digestor biomass. Appl Microbiol Biotechnol 40, 427443. [Google Scholar]
  49. Sousa D. Z., Pereira M. A., Stams A. J., Alves M. M., Smidt H. (2007). Microbial communities involved in anaerobic degradation of unsaturated or saturated long-chain fatty acids. Appl Environ Microbiol 73, 10541064. [View Article][PubMed] [Google Scholar]
  50. Stams A. J., Sousa D. Z., Kleerebezem R., Plugge C. M. (2012). Role of syntrophic microbial communities in high-rate methanogenic bioreactors. Water Sci Technol 66, 352362. [View Article][PubMed] [Google Scholar]
  51. Sundberg C., Al-Soud W. A., Larsson M., Alm E., Yekta S. S., Svensson B. H., Sørensen S. J., Karlsson A. (2013). 454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters. FEMS Microbiol Ecol 85, 612626. [View Article][PubMed] [Google Scholar]
  52. Tale V. P., Maki J. S., Struble C. A., Zitomer D. H. (2011). Methanogen community structure-activity relationship and bioaugmentation of overloaded anaerobic digesters. Water Res 45, 52495256. [View Article][PubMed] [Google Scholar]
  53. Tang Y. Q., Shigematsu T., Morimura S., Kida K. (2007). Effect of dilution rate on the microbial structure of a mesophilic butyrate-degrading methanogenic community during continuous cultivation. Appl Microbiol Biotechnol 75, 451465. [View Article][PubMed] [Google Scholar]
  54. Thauer R. K., Jungermann K., Decker K. (1977). Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41, 100180. [PubMed] [Google Scholar]
  55. Wang Q., Garrity G. M., Tiedje J. M., Cole J. R. (2007). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73, 52615267. [View Article][PubMed] [Google Scholar]
  56. Yu Y., Lee C., Kim J., Hwang S. (2005). Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89, 670679. [View Article][PubMed] [Google Scholar]
  57. Yu Y., Kim J., Hwang S. (2006). Use of real-time PCR for group-specific quantification of aceticlastic methanogens in anaerobic processes: population dynamics and community structures. Biotechnol Bioeng 93, 424433. [View Article][PubMed] [Google Scholar]

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