Applied Microbiology and Biotechnology

, Volume 97, Issue 11, pp 5161–5174 | Cite as

Microbial community structure and dynamics during anaerobic digestion of various agricultural waste materials

  • Ayrat M. Ziganshin
  • Jan Liebetrau
  • Jürgen Pröter
  • Sabine KleinsteuberEmail author
Bioenergy and biofuels


The influence of the feedstock type on the microbial communities involved in anaerobic digestion was investigated in laboratory-scale biogas reactors fed with different agricultural waste materials. Community composition and dynamics over 2 months of reactors’ operation were investigated by amplicon sequencing and profiling terminal restriction fragment length polymorphisms of 16S rRNA genes. Major bacterial taxa belonged to the Clostridia and Bacteroidetes, whereas the archaeal community was dominated by methanogenic archaea of the orders Methanomicrobiales and Methanosarcinales. Correlation analysis revealed that the community composition was mainly influenced by the feedstock type with the exception of a temperature shift from 38 to 55 °C which caused the most pronounced community shifts. Bacterial communities involved in the anaerobic digestion of conventional substrates such as maize silage combined with cattle manure were relatively stable and similar to each other. In contrast, special waste materials such as chicken manure or Jatropha press cake were digested by very distinct and less diverse communities, indicating partial ammonia inhibition or the influence of other inhibiting factors. Anaerobic digestion of chicken manure relied on syntrophic acetate oxidation as the dominant acetate-consuming process due to the inhibition of aceticlastic methanogenesis. Jatropha as substrate led to the enrichment of fiber-degrading specialists belonging to the genera Actinomyces and Fibrobacter.


Biogas Co-digestion 16S rRNA genes T-RFLP Pyrosequencing 



We gratefully acknowledge the technicians and students from the Department of Biochemical Conversion (DBFZ) for running the reactors and performing chemical analyses. We also thank Ute Lohse from the Department of Environmental Microbiology (UFZ) for technical assistance.

Supplementary material

253_2013_4867_MOESM1_ESM.pdf (182 kb)
ESM 1 PDF 182 kb


  1. Abdo Z, Schütte UM, Bent SJ, Williams CJ, Forney LJ, Joyce P (2006) Statistical methods for characterizing diversity of microbial communities by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes. Environ Microbiol 8:929–938CrossRefGoogle Scholar
  2. Ariesyady HD, Ito T, Okabe S (2007) Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic digester. Water Res 41:1554–1568CrossRefGoogle Scholar
  3. Asakawa S, Nagaoka K (2003) Methanoculleus bourgensis, Methanoculleus olentangyi and Methanoculleus oldenburgensis are subjective synonyms. Int J Syst Evol Microbiol 53:1551–1552CrossRefGoogle Scholar
  4. Béra-Maillet C, Ribot Y, Forano E (2004) Fiber-degrading system of different strains of the genus Fibrobacter. Appl Environ Microbiol 70:2172–2179CrossRefGoogle Scholar
  5. Chen Y, Cheng JJ, Creamer KS (2008) Inhibition of anaerobic digestion process: a review. Bioresour Technol 99:4044–4064CrossRefGoogle Scholar
  6. Cheng L, Qiu TL, Li X, Wang WD, Deng Y, Yin XB, Zhang H (2008) Isolation and characterization of Methanoculleus receptaculi sp. nov. from Shengli oil field, China. FEMS Microbiol Lett 285:65–71CrossRefGoogle Scholar
  7. Das D, Veziroglu TN (2001) Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 26:13–28CrossRefGoogle Scholar
  8. Demirel B, Scherer P (2008) The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a review. Rev. Environ. Sci Biotechnol 7:173–190CrossRefGoogle Scholar
  9. Dianou D, Miyaki T, Asakawa S, Morii H, Nagaoka K, Oyaizu H, Matsumoto S (2001) Methanoculleus chikugoensis sp. nov., a novel methanogenic archaeon isolated from paddy field soil in Japan, and DNA-DNA hybridization among Methanoculleus species. Int J Syst Evol Microbiol 51:1663–1669CrossRefGoogle Scholar
  10. Elberson MA, Sowers KR (1997) Isolation of an aceticlastic strain of Methanosarcina siciliae from marine canyon sediments and emendation of the species description for Methanosarcina siciliae. Int J Syst Bacteriol 47:1258–1261CrossRefGoogle Scholar
  11. El-Mashad HM, Zhang R (2010) Biogas production from co-digestion of dairy manure and food waste. Bioresour Technol 101:4021–4028CrossRefGoogle Scholar
  12. Fontes CM, Gilbert HJ (2010) Cellulosomes: highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates. Annu Rev Biochem 79:655–681CrossRefGoogle Scholar
  13. Gerardi MH (2003) The microbiology of anaerobic digesters. Wiley, HobokenCrossRefGoogle Scholar
  14. Goberna M, Insam H, Franke-Whittle IH (2009) Effect of biowaste sludge maturation on the diversity of thermophilic bacteria and archaea in an anaerobic reactor. Appl Environ Microbiol 75:2566–2572CrossRefGoogle Scholar
  15. Hattori S (2008) Syntrophic acetate-oxidizing microbes in methanogenic environments. Microbes Environ 2:118–127CrossRefGoogle Scholar
  16. 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 50:1601–1609CrossRefGoogle Scholar
  17. Holm-Nielsen JB, Al Seadi T, Oleskowicz-Popiel P (2009) The future of anaerobic digestion and biogas utilization. Bioresour Technol 100:5478–5484CrossRefGoogle Scholar
  18. Karlsson A, Einarsson P, Schnürer A, Sundberg C, Ejlertsson J, Svensson B (2012) Impact of trace element addition on degradation efficiency of volatile fatty acids, oleic acid and phenyl acetate and on microbial populations in a biogas digester. J Biosci Bioeng 114:446–452CrossRefGoogle Scholar
  19. Kendall MM, Boone DR (2006) The order Methanosarcinales. Prokaryotes 3:244–256CrossRefGoogle Scholar
  20. Kim MD, Song M, Jo M, Shin SG, Khim JH, Hwang S (2010) Growth condition and bacterial community for maximum hydrolysis of suspended organic materials in anaerobic digestion of food waste-recycling wastewater. Appl Microbiol Biotechnol 85:1611–1618CrossRefGoogle Scholar
  21. Klocke M, Mähnert P, Mundt K, Souidi K, Linke B (2007) Microbial community analysis of a biogas-producing completely stirred tank reactor fed continuously with fodder beet silage as mono-substrate. Syst Appl Microbiol 30:139–151CrossRefGoogle Scholar
  22. Krause L, Diaz NN, Edwards RA, Gartemann KH, Krömeke H, Neuweger H, Pühler A, Runte KJ, Schlüter A, Stoye J, Szczepanowski R, Tauch A, Goesmann A (2008) Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor. J Biotechnol 136:91–101CrossRefGoogle Scholar
  23. Kröber M, Bekel T, Diaz NN, Goesmann A, Jaenicke S, Krause L, Miller D, Runte KJ, Viehöver P, Pühler A, Schlüter A (2009) Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing. J Biotechnol 142:38–49CrossRefGoogle Scholar
  24. Lee C, Kim J, Hwang K, O’Flaherty V, Hwang S (2009) Quantitative analysis of methanogenic community dynamics in three anaerobic batch digesters treating different wastewaters. Water Res 43:157–165CrossRefGoogle Scholar
  25. Lomans BP, Maas R, Luderer R, Op den Camp HJM, Pol A, van der Drift C, Vogels GD (1999) Isolation and characterization of Methanomethylovorans hollandica gen. nov., sp. nov., isolated from freshwater sediment, a methylotrophic methanogen able to grow on dimethyl sulfide and methanethiol. Appl Environ Microbiol 65:3641–3650Google Scholar
  26. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577CrossRefGoogle Scholar
  27. Maestrojuan GM, Boone DR (1991) Characterization of Methanosarcina barkeri MST and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T. Int J Syst Bacteriol 41:267–274CrossRefGoogle Scholar
  28. Morrison M, Miron J (2000) Adhesion to cellulose by Ruminococcus albus: a combination of cellulosomes and Pil-proteins? FEMS Microbiol Lett 185:109–115Google Scholar
  29. Narihiro T, Sekiguchi Y (2007) Microbial communities in anaerobic digestion processes for waste and wastewater treatment: a microbiological update. Curr Opin Biotechnol 18:273–278CrossRefGoogle Scholar
  30. Ntaikou I, Gavala HN, Kornaros M, Lyberatos G (2008) Hydrogen production from sugars and sweet sorghum biomass using Ruminococcus albus. Int J Hydrog Energy 33:1153–1163Google Scholar
  31. O’Reilly J, Lee C, Collins G, Chinalia F, Mahony T, O’Flaherty V (2009) Quantitative and qualitative analysis of methanogenic communities in mesophilically and psychrophilically cultivated anaerobic granular biofilms. Water Res 43:3365–3374CrossRefGoogle Scholar
  32. Oksanen J (2011) Multivariate analysis of ecological communities in R: vegan tutorial. Publisher Univ Oulu Comput Serv Cent 83:1–43Google Scholar
  33. Ollivier BM, Mah RA, Garcia JL, Boone DR (1986) Isolation and characterization of Methanogenium bourgense sp. nov. Int J Syst Bacteriol 36:297–301CrossRefGoogle Scholar
  34. Patel GB, Sprott GD (1990) Methanosaeta concilii gen. nov., sp. nov. (“Methanothrix concilii”) and Methanosaeta thermoacetophila nom. rev., comb. nov. Int J Syst Bacteriol 40:79–82CrossRefGoogle Scholar
  35. Rademacher A, Zakrzewski M, Schlüter A, Schönberg M, Szczepanowski R, Goesmann A, Pühler A, Klocke M (2012) Characterization of microbial biofilms in a thermophilic biogas system by high-throughput metagenome sequencing. FEMS Microbiol Ecol 79:785–799CrossRefGoogle Scholar
  36. Rivière D, Desvignes V, Pelletier E, Chaussonnerie S, Guermazi S, Weissenbach J, Li T, Camacho P, Sghir A (2009) Towards the definition of a core of microorganisms involved in anaerobic digestion of sludge. ISME J 3:700–714CrossRefGoogle Scholar
  37. Sasaki K, Morita M, Hirano S, Ohmura N, Igarashi Y (2011) Decreasing ammonia inhibition in thermophilic methanogenic bioreactors using carbon fiber textiles. Appl Microbiol Biotechnol 90:1555–1561CrossRefGoogle Scholar
  38. Schnürer A, Nordberg A (2008) Ammonia, a selective agent for methane production by syntrophic acetate oxidation at mesophilic temperature. Water Sci Technol 57:735–740CrossRefGoogle Scholar
  39. 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 46:1145–1152CrossRefGoogle Scholar
  40. Schnürer A, Zellner G, Svensson BH (1999) Mesophilic syntrophic acetate oxidation during methane formation in biogas reactors. FEMS Microbiol Ecol 29:249–261CrossRefGoogle Scholar
  41. Sowers KR, Baron SF, Ferry JG (1984) Methanosarcina acetivorans sp. nov., an acetotrophic methane-producing bacterium isolated from marine sediments. Appl Environ Microbiol 47:971–978Google Scholar
  42. Steinberg LM, Regan JM (2009) mcrA-targeted real-time quantitative PCR method to examine methanogen communities. Appl Environ Microbiol 75:4435–4442CrossRefGoogle Scholar
  43. Weiland P (2010) Biogas production: current state and perspectives. Appl Microbiol Biotechnol 85:849–860CrossRefGoogle Scholar
  44. 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 309:100–104Google Scholar
  45. Westerholm M, Roos S, Schnürer A (2011) Tepidanaerobacter acetatoxydans sp. nov., an anaerobic, syntrophic acetate-oxidizing bacterium isolated from two ammonia-enriched mesophilic methanogenic processes. Syst Appl Microbiol 34:260–266CrossRefGoogle Scholar
  46. Ziganshin AM, Schmidt T, Scholwin F, Il’inskaya ON, Harms H, Kleinsteuber S (2011) Bacteria and archaea involved in anaerobic digestion of distillers grains with solubles. Appl Microbiol Biotechnol 89:2039–2052CrossRefGoogle Scholar
  47. Ziganshin AM, Ziganshina EE, Kleinsteuber S, Pröter J, Ilinskaya ON (2012) Methanogenic community dynamics during anaerobic utilization of agricultural wastes. Acta Naturae 4:91–97Google Scholar
  48. Zinder SH, Sowers KR, Ferry JG (1985) Methanosarcina thermophila sp. nov., a thermophilic, acetotrophic, methane-producing bacterium. Int J Syst Bacteriol 35:522–523CrossRefGoogle Scholar
  49. Zverlov VV, Hiegl W, Köck DE, Kellermann J, Köllmeier T, Schwarz WH (2010) Hydrolytic bacteria in mesophilic and thermophilic degradation of plant biomass. Eng Life Sci 10:528–536CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Ayrat M. Ziganshin
    • 1
  • Jan Liebetrau
    • 2
  • Jürgen Pröter
    • 2
  • Sabine Kleinsteuber
    • 3
    Email author
  1. 1.Department of MicrobiologyKazan (Volga Region) Federal UniversityKazanRussia
  2. 2.Department of Biochemical ConversionDeutsches Biomasseforschungszentrum (DBFZ)LeipzigGermany
  3. 3.Department of Environmental MicrobiologyHelmholtz Centre for Environmental Research (UFZ)LeipzigGermany

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