Applied Microbiology and Biotechnology

, Volume 100, Issue 2, pp 1019–1026 | Cite as

Biogas process parameters—energetics and kinetics of secondary fermentations in methanogenic biomass degradation

  • Dominik Montag
  • Bernhard SchinkEmail author
Bioenergy and Biofuels


Pool sizes of short-chain fatty acids (formate, acetate, propionate, and butyrate), hydrogen, and carbon monoxide were assayed in digesting sludge from four different methanogenic reactors degrading either sewage sludge or agricultural products and wastes at pH 8.0 and 40 or 47 °C. Free reaction energies were calculated for the respective degradation reactions involved, indicating that acetate, propionate, and butyrate degradation all supplied sufficient energy (−10 to −30 kJ per mol reaction) to sustain the microbial communities involved in the respective processes. Pools of formate and hydrogen were energetically equivalent as electron carriers. In the sewage sludge reactor, homoacetogenic acetate formation from H2 and CO2 was energetically feasible whereas syntrophic acetate oxidation appeared to be possible in two biogas reactors, one operating at enhanced ammonia content (4.5 g NH4 +-N per l) and the other one at enhanced temperature (47 °C). Maximum capacities for production of methanogenic substrates did not exceed the consumption capacities by hydrogenotrophic and aceticlastic methanogens. Nonetheless, the capacity for acetate degradation appeared to be a limiting factor especially in the reactor operating at enhanced ammonia concentration.


Methanogenesis Energetics Pool sizes Fatty acids Syntrophy Secondary fermentations 



The authors are grateful to Melanie Hecht, Thomas Dickhaus, and Sarah Refai for organizing the sampling campaigns at the biogas reactors at Troisdorf and to Erich Kronenthaler and Martin Kaspar for the supply of sewage sludge and operation parameters of the reactor at the wastewater treatment plant in Konstanz. The technical help with specific experiments in the lab by Antje Wiese, Ye Schmidt, and Stefan Bieletzki is highly appreciated.

Compliance with ethical standards


This study was funded by the German Federal Ministry for Education and Research, project BioPara, project number 03SF0421E.

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2015_7069_MOESM1_ESM.pdf (438 kb)
ESM 1 (PDF 437 kb)


  1. Adams CJ, Redmond MC, Valentine DL (2006) Pure-culture growth of fermentative bacteria, facilitated by H2 removal: bioenergetics and H2 production. Appl Environ Microbiol 72(2):1079–1085PubMedPubMedCentralCrossRefGoogle Scholar
  2. Ahring BK, Sandberg M, Angelidaki I (1995) Volatile fatty acids as indicators of process imbalance in anaerobic digestors. Appl Microbiol Biotechnol 43(3):559–565CrossRefGoogle Scholar
  3. Amend JP, Shock EL (2001) Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and bacteria. FEMS Microbiol Rev 25(2):175–243PubMedCrossRefGoogle Scholar
  4. Batstone DJ, Pind PF, Angelidaki I (2003) Kinetics of thermophilic, anaerobic oxidation of straight and branched chain butyrate and valerate. Biotechnol Bioeng 84(2):195–204PubMedCrossRefGoogle Scholar
  5. Bryant M, Wolin E, Wolin M, Wolfe R (1967) Methanobacillus omelianskii, a symbiotic association of two species of bacteria. Arch Mikrobiol 59(1–3):20–31PubMedCrossRefGoogle Scholar
  6. Buswell A, Mueller H (1952) Mechanism of methane fermentation. Industrial & Engineering Chemistry 44(3):550–552CrossRefGoogle Scholar
  7. Can M, Armstrong FA, Ragsdale SW (2014) Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and acetyl-CoA synthase. Chem Rev 114(8):4149–4174PubMedPubMedCentralCrossRefGoogle Scholar
  8. Conrad R, Wetter B (1990) Influence of temperature on energetics of hydrogen metabolism in homoacetogenic, methanogenic, and other anaerobic bacteria. Arch Microbiol 155(1):94–98CrossRefGoogle Scholar
  9. Dwyer DF, Weeg-Aerssens E, Shelton DR, Tiedje JM (1988) Bioenergetic conditions of butyrate metabolism by a syntrophic, anaerobic bacterium in coculture with hydrogen-oxidizing methanogenic and sulfidogenic bacteria. Appl Environ Microbiol 54(6):1354–1359PubMedPubMedCentralGoogle Scholar
  10. Fey A, Conrad R (2000) Effect of temperature on carbon and electron flow and on the archaeal community in methanogenic rice field soil. Appl Environ Microbiol 66(11):4790–4797PubMedPubMedCentralCrossRefGoogle Scholar
  11. Gujer W, Zehnder A (1983) Conversion processes in anaerobic digestion. Water Science & Technology 15(8–9):127–167Google Scholar
  12. Hao L-P, Lü F, He P-J, Li L, Shao L-M (2010) Predominant contribution of syntrophic acetate oxidation to thermophilic methane formation at high acetate concentrations. Environmental Science & Technology 45(2):508–513CrossRefGoogle Scholar
  13. Ho DP, Jensen PD, Batstone DJ (2013) Methanosarcinaceae and acetate-oxidizing pathways dominate in high-rate thermophilic anaerobic digestion of waste-activated sludge. Appl Environ Microbiol 79(20):6491–6500PubMedPubMedCentralCrossRefGoogle Scholar
  14. Hoehler TM, Alperin MJ, Albert DB, Martens CS (2001) Apparent minimum free energy requirements for methanogenic Archaea and sulfate-reducing bacteria in an anoxic marine sediment. FEMS Microbiol Ecol 38(1):33–41CrossRefGoogle Scholar
  15. Kaspar HF, Wuhrmann K (1978) Kinetic parameters and relative turnovers of some important catabolic reactions in digesting sludge. Appl Environ Microbiol 36(1):1–7PubMedPubMedCentralGoogle Scholar
  16. Krylova NI, Conrad R (1998) Thermodynamics of propionate degradation in methanogenic paddy soil. FEMS Microbiol Ecol 26(4):281–288CrossRefGoogle Scholar
  17. Lim JK, Mayer F, Kang SG, Müller V (2014) Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon. Proc Natl Acad Sci 111(31):11497–11502PubMedPubMedCentralCrossRefGoogle Scholar
  18. Lü F, Hao L, Guan D, Qi Y, Shao L, He P (2013) Synergetic stress of acids and ammonium on the shift in the methanogenic pathways during thermophilic anaerobic digestion of organics. Water Res 47(7):2297–2306PubMedCrossRefGoogle Scholar
  19. McCarty PL, Smith DP (1986) Anaerobic Waste-Water Treatment. 4. Environmental Science & Technology 20(12):1200–1206. doi: 10.1021/es00154a002 CrossRefGoogle Scholar
  20. McInerney MJ, Bryant MP, Pfennig N (1979) Anaerobic bacterium that degrades fatty acids in syntrophic association with methanogens. Arch Microbiol 122(2):129–135CrossRefGoogle Scholar
  21. Moestedt J, Nordell E, Schnürer A (2014) Comparison of operating strategies for increased biogas production from thin stillage. J Biotechnol 175:22–30PubMedCrossRefGoogle Scholar
  22. Müller N, Schleheck D, Schink B (2009) Involvement of NADH: acceptor oxidoreductase and butyryl coenzyme A dehydrogenase in reversed electron transport during syntrophic butyrate oxidation by Syntrophomonas wolfei. J Bacteriol 191(19):6167–6177PubMedPubMedCentralCrossRefGoogle Scholar
  23. Müller N, Worm P, Schink B, Stams AJ, Plugge CM (2010) Syntrophic butyrate and propionate oxidation processes: from genomes to reaction mechanisms. Environ Microbiol Rep 2(4):489–499PubMedCrossRefGoogle Scholar
  24. Müller V (2015) Microbial life at the thermodynamic limit: how much energy is required to sustain life? Environ Microbiol Rep 7(1):31–32PubMedCrossRefGoogle Scholar
  25. Nationale Akademie der Wissenschaften Leopoldina (2012) Bioenergie: Möglichkeiten und Grenzen. Halle (Saale) Online unter: http://wwwleopoldinaorg/uploads/tx_leopublication/201207_Empfehlungen_Bioenergie_02pdfGoogle Scholar
  26. Noike T, Endo G, Chang JE, Yaguchi JI, Matsumoto JI (1985) Characteristics of carbohydrate degradation and the rate-limiting step in anaerobic digestion. Biotechnol Bioeng 27(10):1482–1489PubMedCrossRefGoogle Scholar
  27. Palatsi J, Viñas M, Guivernau M, Fernandez B, Flotats X (2011) Anaerobic digestion of slaughterhouse waste: main process limitations and microbial community interactions. Bioresour Technol 102(3):2219–2227PubMedCrossRefGoogle Scholar
  28. Pauss A, Samson R, Guiot S (1990) Thermodynamic evidence of trophic microniches in methanogenic granular sludge-bed reactors. Appl Microbiol Biotechnol 33(1):88–92PubMedGoogle Scholar
  29. Penning H, Conrad R (2006) Effect of inhibition of acetoclastic methanogenesis on growth of archaeal populations in an anoxic model environment. Appl Environ Microbiol 72(1):178–184PubMedPubMedCentralCrossRefGoogle Scholar
  30. Ragsdale SW, Pierce E (2008) Acetogenesis and the Wood–Ljungdahl pathway of CO2 fixation. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1784(12):1873–1898CrossRefGoogle Scholar
  31. Schink B (1997) Energetics of syntrophic cooperation in methanogenic degradation. Microbiol Mol Biol Rev 61(2):262–280PubMedPubMedCentralGoogle Scholar
  32. Schink B, Stams AJ (2013) Syntrophism among prokaryotes. In: Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (eds) The prokaryotes - prokaryotic communitites and ecophysiology, 4th edn. Springer, Berlin Heidelberg, pp 471–493Google Scholar
  33. Schmidt A, Müller N, Schink B, Schleheck D (2013) A proteomic view at the biochemistry of syntrophic butyrate oxidation in Syntrophomonas wolfei. PLoS One 8(2):e56905PubMedPubMedCentralCrossRefGoogle Scholar
  34. Schnürer A, Houwen FP, Svensson BH (1994) Mesophilic syntrophic acetate oxidation during methane formation by a triculture at high ammonium concentration. Arch Microbiol 162(1–2):70–74CrossRefGoogle Scholar
  35. Schnürer A, Schink B, Svensson BH (1996) Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium. Int J Syst Bacteriol 46(4):1145–1152PubMedCrossRefGoogle Scholar
  36. Schnürer A, Svensson BH, Schink B (1997) Enzyme activities in and energetics of acetate metabolism by the mesophilic syntrophically acetate-oxidizing anaerobe Clostridium ultunense. FEMS Microbiol Lett 154(2):331–336CrossRefGoogle Scholar
  37. Scholten JC, Conrad R (2000) Energetics of syntrophic propionate oxidation in defined batch and chemostat cocultures. Appl Environ Microbiol 66(7):2934–2942PubMedPubMedCentralCrossRefGoogle Scholar
  38. Schuchmann K, Müller V (2014) Autotrophy at the thermodynamic limit of life: a model for energy conservation in acetogenic bacteria. Nat Rev Microbiol 12:809–821Google Scholar
  39. Seiler W, Giehl H, Roggendorf P (1980) Detection of carbon monoxide and hydrogen by conversion of mercury oxide to mercury vapor. Atmos Technol, United States, p. 12Google Scholar
  40. Seitz H-J, Schink B, Pfennig N, Conrad R (1990) Energetics of syntrophic ethanol oxidation in defined chemostat cocultures. Arch Microbiol 155(1):82–88CrossRefGoogle Scholar
  41. Spahn S, Brandt K, Müller V (2015) A low phosphorylation potential in the acetogen Acetobacterium woodii reflects its lifestyle at the thermodynamic edge of life. Arch Microbiol 197:745–751Google Scholar
  42. Thauer RK, Jungermann K, Decker K (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41(1):100PubMedPubMedCentralGoogle Scholar
  43. Wang J, Liu H, Fu B, Xu K, Chen J (2013) Trophic link between syntrophic acetogens and homoacetogens during the anaerobic acidogenic fermentation of sewage sludge. Biochem Eng J 70:1–8CrossRefGoogle Scholar
  44. Worm P, Fermoso FG, Stams AJ, Lens PN, Plugge CM (2011) Transcription of fdh and hyd in Syntrophobacter spp. and Methanospirillum spp. as a diagnostic tool for monitoring anaerobic sludge deprived of molybdenum, tungsten and selenium. Environ Microbiol 13(5):1228–1235PubMedCrossRefGoogle Scholar
  45. Zinder SH, Koch M (1984) Non-aceticlastic methanogenesis from acetate: acetate oxidation by a thermophilic syntrophic coculture. Arch Microbiol 138(3):263–272CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Department of BiologyUniversity of KonstanzKonstanzGermany

Personalised recommendations