1887

Abstract

Syntrophic oxidation of acetate, so-called reversed reductive acetogenesis, is one of the most important degradation steps in anaerobic digesters. However, little is known about the genetic diversity of the micro-organisms involved. Here we investigated the activity and composition of potentially acetate-oxidizing syntrophs using a combinatorial approach of flux measurement and transcriptional profiling of the formyltetrahydrofolate synthetase (FTHFS) gene, an ecological biomarker for reductive acetogenesis. During the operation of a thermophilic anaerobic digester, volatile fatty acids were mostly depleted, suggesting a high turnover rate for dissolved H, and hydrogenotrophic methanogens were the dominant archaeal members. Batch cultivation of the digester microbiota with C-labelled acetate indicated that syntrophic oxidation accounted for 13.1–21.3 % of methane production from acetate. FTHFS genes were transcribed in the absence of carbon monoxide, methoxylated compounds and inorganic electron acceptors other than CO, which is implicated in the activity of reversed reductive acetogenesis; however, expression itself does not distinguish whether biosynthesis or biodegradation is functioning. The mRNA- and DNA-based terminal RFLP and clone library analyses indicated that, out of nine FTHFS phylotypes detected, the FTHFS genes from the novel phylotypes I–IV in addition to the known syntroph (i.e. phylotype V) were specifically expressed. These transcripts arose from phylogenetically presumed homoacetogens. The results of this study demonstrate that hitherto unidentified phylotypes of homoacetogens are responsible for syntrophic acetate oxidation in an anaerobic digester.

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2011-07-01
2024-03-28
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References

  1. Ahring B. K. ( 1995). Methanogenesis in thermophilic biogas reactors. Antonie van Leeuwenhoek 67:91–102 [View Article][PubMed]
    [Google Scholar]
  2. Akutsu Y., Li Y.-Y., Harada H., Yu H.-Q. ( 2009). Effects of temperature and substrate concentration on biological hydrogen production from starch. Int J Hydrogen Energy 34:2558–2566 [View Article]
    [Google Scholar]
  3. Akuzawa M., Hori T., Haruta S., Ueno Y., Ishii M., Igarashi Y. ( 2011). Distinctive responses of metabolically active microbiota to acidification in a thermophilic anaerobic digester. Microb Ecol 61:595–605 [View Article][PubMed]
    [Google Scholar]
  4. Balk M., Weijma J., Stams A. J. ( 2002). Thermotoga lettingae sp. nov., a novel thermophilic, methanol-degrading bacterium isolated from a thermophilic anaerobic reactor. Int J Syst Evol Microbiol 52:1361–1368 [View Article][PubMed]
    [Google Scholar]
  5. Cord-Ruwisch R., Mercz T. I., Hoh C.-Y., Strong G. E. ( 1997). Dissolved hydrogen concentration as an on-line control parameter for the automated operation and optimization of anaerobic digesters. Biotechnol Bioeng 56:626–634 [View Article][PubMed]
    [Google Scholar]
  6. Drake H. L. ( 1994). Acetogenesis New York: Chapman & Hall;
    [Google Scholar]
  7. Drake H. L., Daniel S. L. ( 2004). Physiology of the thermophilic acetogen Moorella thermoacetica . Res Microbiol 155:869–883 [View Article][PubMed]
    [Google Scholar]
  8. Dunbar J., Ticknor L. O., Kuske C. R. ( 2001). Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities. Appl Environ Microbiol 67:190–197 [View Article][PubMed]
    [Google Scholar]
  9. Felsenstein J. ( 2005). phylip (phylogeny inference package). Distributed by the author. Department of Genome Sciences, University of Washington; Seattle, WA:
  10. Ferry J. G. ( 1993). Fermentation of acetate. Methanogenesis – Ecology, Physiology, Biochemistry & Genetics304–334 Ferry J. G. New York: Chapman & Hall;
    [Google Scholar]
  11. Gebhardt A., Linder D., Thauer R. K. ( 1983). Anaerobic acetate oxidation to CO2 by Desulfobacter postgatei 2. Evidence from 14C-labelling studies for the operation of the citric acid cycle. Arch Microbiol 136:230–233 [View Article]
    [Google Scholar]
  12. Goberna M., Insam H., Franke-Whittle I. H. ( 2009). Effect of biowaste sludge maturation on the diversity of thermophilic bacteria and archaea in an anaerobic reactor. Appl Environ Microbiol 75:2566–2572 [View Article][PubMed]
    [Google Scholar]
  13. Hattori S. ( 2008). Syntrophic acetate-oxidizing microbes in methanogenic environments. Microbes Environ 23:118–127 [View Article]
    [Google Scholar]
  14. Hattori S., Kamagata Y., Hanada S., Shoun H. ( 2000). Thermacetogenium phaeum gen. nov., sp. nov., a strictly anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium. Int J Syst Evol Microbiol 50:1601–1609[PubMed] [CrossRef]
    [Google Scholar]
  15. Hattori S., Galushko A. S., Kamagata Y., Schink B. ( 2005). Operation of the CO dehydrogenase/acetyl coenzyme A pathway in both acetate oxidation and acetate formation by the syntrophically acetate-oxidizing bacterium Thermacetogenium phaeum . J Bacteriol 187:3471–3476 [View Article][PubMed]
    [Google Scholar]
  16. Henderson G., Naylor G. E., Leahy S. C., Janssen P. H. ( 2010). Presence of novel, potentially homoacetogenic bacteria in the rumen as determined by analysis of formyltetrahydrofolate synthetase sequences from ruminants. Appl Environ Microbiol 76:2058–2066 [View Article][PubMed]
    [Google Scholar]
  17. Hori T., Haruta S., Ueno Y., Ishii M., Igarashi Y. ( 2006a). Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester. Appl Environ Microbiol 72:1623–1630 [View Article][PubMed]
    [Google Scholar]
  18. Hori T., Haruta S., Ueno Y., Ishii M., Igarashi Y. ( 2006b). Direct comparison of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) to characterize a microbial community on the basis of 16S rRNA gene fragments. J Microbiol Methods 66:165–169 [View Article][PubMed]
    [Google Scholar]
  19. Hori T., Noll M., Igarashi Y., Friedrich M. W., Conrad R. ( 2007). Identification of acetate-assimilating microorganisms under methanogenic conditions in anoxic rice field soil by comparative stable isotope probing of RNA. Appl Environ Microbiol 73:101–109 [View Article][PubMed]
    [Google Scholar]
  20. Hori T., Müller A., Igarashi Y., Conrad R., Friedrich M. W. ( 2010). Identification of iron-reducing microorganisms in anoxic rice paddy soil by 13C-acetate probing. ISME J 4:267–278 [View Article][PubMed]
    [Google Scholar]
  21. Jetten M. S. M., Stams A. J. M., Zehnder A. J. B. ( 1992). Methanogenesis from acetate: a comparison of acetate metabolism in Methanothrix soehngenii and Methanosarcina spp. FEMS Microbiol Rev 88:181–198 [View Article]
    [Google Scholar]
  22. Karakashev D., Batstone D. J., Trably E., Angelidaki I. ( 2006). Acetate oxidation is the dominant methanogenic pathway from acetate in the absence of Methanosaetaceae . Appl Environ Microbiol 72:5138–5141 [View Article][PubMed]
    [Google Scholar]
  23. Krakat N., Westphal A., Schmidt S., Scherer P. ( 2010). Anaerobic digestion of renewable biomass: thermophilic temperature governs methanogen population dynamics. Appl Environ Microbiol 76:1842–1850 [View Article][PubMed]
    [Google Scholar]
  24. Krumholz L. R., Bryant M. P. ( 1985). Clostridium pfennigii sp. nov. uses methoxyl groups of monobenzenoids and produces butyrate. Int J Syst Bacteriol 35:454–456 [View Article]
    [Google Scholar]
  25. Kumar S., Nei M., Dudley J., Tamura K. ( 2008). mega: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9:299–306 [View Article][PubMed]
    [Google Scholar]
  26. Leaphart A. B., Lovell C. R. ( 2001). Recovery and analysis of formyltetrahydrofolate synthetase gene sequences from natural populations of acetogenic bacteria. Appl Environ Microbiol 67:1392–1395 [View Article][PubMed]
    [Google Scholar]
  27. Leaphart A. B., Friez M. J., Lovell C. R. ( 2003). Formyltetrahydrofolate synthetase sequences from salt marsh plant roots reveal a diversity of acetogenic bacteria and other bacterial functional groups. Appl Environ Microbiol 69:693–696 [View Article][PubMed]
    [Google Scholar]
  28. Lee M. J., Zinder S. H. ( 1988a). Hydrogen partial pressures in a thermophilic acetate-oxidizing methanogenic coculture. Appl Environ Microbiol 54:1457–1461[PubMed]
    [Google Scholar]
  29. Lee M. J., Zinder S. H. ( 1988b). Isolation and characterization of a thermophilic bacterium which oxidizes acetate in syntrophic association with a methanogen and which grows acetogenically on H2–CO2 . Appl Environ Microbiol 54:124–129[PubMed]
    [Google Scholar]
  30. Lee M. J., Zinder S. H. ( 1988c). Carbon monoxide pathway enzyme activities in a thermophilic anaerobic bacterium grown acetogenically and in a syntrophic acetate-oxidizing coculture. Arch Microbiol 150:513–518 [View Article]
    [Google Scholar]
  31. Lettinga G. ( 1995). Anaerobic digestion and wastewater treatment systems. Antonie van Leeuwenhoek 67:3–28 [View Article][PubMed]
    [Google Scholar]
  32. Liu W. T., Marsh T. L., Cheng H., Forney L. J. ( 1997). Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63:4516–4522[PubMed]
    [Google Scholar]
  33. Lovell C. R., Leaphart A. B. ( 2005). Community-level analysis: key genes of CO2-reductive acetogenesis. Methods Enzymol 397:454–469 [View Article][PubMed]
    [Google Scholar]
  34. Lovley D. R., Klug M. J. ( 1982). Intermediary metabolism of organic matter in the sediments of a eutrophic lake. Appl Environ Microbiol 43:552–560[PubMed]
    [Google Scholar]
  35. Ludwig W., Strunk O., Klugbauer S., Klugbauer N., Weizenegger M., Neumaier J., Bachleitner M., Schleifer K. H. ( 1998). Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19:554–568 [View Article][PubMed]
    [Google Scholar]
  36. Ludwig W., Strunk O., Westram R., Richter L., Meier H., Yadhukumar, Buchner A., Lai T., Steppi S. et al. ( 2004). arb: a software environment for sequence data. Nucleic Acids Res 32:1363–1371 [View Article][PubMed]
    [Google Scholar]
  37. Lueders T., Friedrich M. W. ( 2002). Effects of amendment with ferrihydrite and gypsum on the structure and activity of methanogenic populations in rice field soil. Appl Environ Microbiol 68:2484–2494 [View Article][PubMed]
    [Google Scholar]
  38. Mountfort D. O., Asher R. A. ( 1978). Changes in proportions of acetate and carbon dioxide used as methane precursors during the anaerobic digestion of bovine waste. Appl Environ Microbiol 35:648–654[PubMed]
    [Google Scholar]
  39. Narihiro T., Sekiguchi Y. ( 2007). Microbial communities in anaerobic digestion processes for waste and wastewater treatment: a microbiological update. Curr Opin Biotechnol 18:273–278 [View Article][PubMed]
    [Google Scholar]
  40. Noll M., Matthies D., Frenzel P., Derakshani M., Liesack W. ( 2005). Succession of bacterial community structure and diversity in a paddy soil oxygen gradient. Environ Microbiol 7:382–395 [View Article][PubMed]
    [Google Scholar]
  41. Nüsslein B., Chin K. J., Eckert W., Conrad R. ( 2001). Evidence for anaerobic syntrophic acetate oxidation during methane production in the profundal sediment of subtropical Lake Kinneret (Israel). Environ Microbiol 3:460–470 [View Article][PubMed]
    [Google Scholar]
  42. Pester M., Brune A. ( 2006). Expression profiles of fhs (FTHFS) genes support the hypothesis that spirochaetes dominate reductive acetogenesis in the hindgut of lower termites. Environ Microbiol 8:1261–1270 [View Article][PubMed]
    [Google Scholar]
  43. Petersen S. P., Ahring B. K. ( 1991). Acetate oxidation in a thermophilic anaerobic sewage-sludge digestor: the importance of non-aceticlastic methanogenesis from acetate. FEMS Microbiol Ecol 86:149–158 [View Article]
    [Google Scholar]
  44. Rothfuss F., Conrad R. ( 1993). Thermodynamics of methanogenic intermediary metabolism in littoral sediment of Lake Constance. FEMS Microbiol Ecol 12:265–276 [View Article]
    [Google Scholar]
  45. Ryan P., Forbes C., McHugh S., O'Reilly C., Fleming G. T., Colleran E. ( 2010). Enrichment of acetogenic bacteria in high rate anaerobic reactors under mesophilic and thermophilic conditions. Water Res 44:4261–4269 [View Article][PubMed]
    [Google Scholar]
  46. Salmassi T. M., Leadbetter J. R. ( 2003). Analysis of genes of tetrahydrofolate-dependent metabolism from cultivated spirochaetes and the gut community of the termite Zootermopsis angusticollis . Microbiology 149:2529–2537 [View Article][PubMed]
    [Google Scholar]
  47. Sansone F. J., Martens C. S. ( 1981). Methane production from acetate and associated methane fluxes from anoxic coastal sediments. Science 211:707–709 [View Article][PubMed]
    [Google Scholar]
  48. Schauder R., Widdel F., Fuchs G. ( 1987). Carbon assimilation pathways in sulfate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus . Arch Microbiol 148:218–225 [View Article]
    [Google Scholar]
  49. Schink B. ( 1997). Energetics of syntrophic cooperation in methanogenic degradation. Microbiol Mol Biol Rev 61:262–280[PubMed]
    [Google Scholar]
  50. Schnürer A., Schink B., Svensson B. H. ( 1996). Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium. Int J Syst Bacteriol 46:1145–1152 [View Article][PubMed]
    [Google Scholar]
  51. Schnürer A., Svensson B. H., Schink B. ( 1997). Enzyme activities in and energetics of acetate metabolism by the mesophilic syntrophically acetate-oxidizing anaerobe Clostridium ultunense . FEMS Microbiol Lett 154:331–336 [View Article]
    [Google Scholar]
  52. Schnürer A., Zellner G., Svensson B. H. ( 1999). Mesophilic syntrophic acetate oxidation during methane formation in biogas reactors. FEMS Microbiol Ecol 29:249–261 [CrossRef]
    [Google Scholar]
  53. Shigematsu T., Tang Y., Kobayashi T., Kawaguchi H., Morimura S., Kida K. ( 2004). Effect of dilution rate on metabolic pathway shift between aceticlastic and nonaceticlastic methanogenesis in chemostat cultivation. Appl Environ Microbiol 70:4048–4052 [View Article][PubMed]
    [Google Scholar]
  54. Speece R. E. ( 1996). Anaerobic Biotechnology for Industrial Wastewaters Nashville, TN: Archae Press;
    [Google Scholar]
  55. Talbot G., Topp E., Palin M. F., Massé D. I. ( 2008). Evaluation of molecular methods used for establishing the interactions and functions of microorganisms in anaerobic bioreactors. Water Res 42:513–537 [View Article][PubMed]
    [Google Scholar]
  56. 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 [View Article][PubMed]
    [Google Scholar]
  57. Thauer R. K. ( 1988). Citric-acid cycle, 50 years on. Modifications and an alternative pathway in anaerobic bacteria. Eur J Biochem 176:497–508 [View Article][PubMed]
    [Google Scholar]
  58. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. ( 1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 [View Article][PubMed]
    [Google Scholar]
  59. Ueno Y., Haruta S., Ishii M., Igarashi Y. ( 2001). Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor. Appl Microbiol Biotechnol 57:65–73 [View Article][PubMed]
    [Google Scholar]
  60. Westerholm M., Roos S., Schnürer A. ( 2010). Syntrophaceticus schinkii gen. nov., sp. nov., an anaerobic, syntrophic acetate-oxidizing bacterium isolated from a mesophilic anaerobic filter. FEMS Microbiol Lett 309:100–104[PubMed]
    [Google Scholar]
  61. Zehnder A. J. B., Stumm W. ( 1988). Geochemistry and biochemistry of anaerobic habitats. Biology of Anaerobic Microorganisms1–38 Zehnder A. J. B. New York: Wiley Interscience;
    [Google Scholar]
  62. Zinder S. H. ( 1994). Syntrophic acetate oxidation and “reversible acetogenesis”. Acetogenesis387–415 Drake H. L. New York: Chapman & Hall;
    [Google Scholar]
  63. Zinder S. H., Koch M. ( 1984). Non-aceticlastic methanogenesis from acetate: acetate oxidation by a thermophilic syntrophic coculture. Arch Microbiol 138:263–272 [View Article]
    [Google Scholar]
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