Applied Microbiology and Biotechnology

, Volume 99, Issue 13, pp 5749–5761 | Cite as

The effect of storage conditions on microbial community composition and biomethane potential in a biogas starter culture

  • Live Heldal Hagen
  • Vivekanand Vivekanand
  • Phillip B. Pope
  • Vincent G. H. Eijsink
  • Svein J. HornEmail author
Bioenergy and biofuels


A new biogas process is initiated by adding a microbial community, typically in the form of a sample collected from a functional biogas plant. This inoculum has considerable impact on the initial performance of a biogas reactor, affecting parameters such as stability, biogas production yields and the overall efficiency of the anaerobic digestion process. In this study, we have analyzed changes in the microbial composition and performance of an inoculum during storage using barcoded pyrosequencing of bacterial and archaeal 16S ribosomal RNA (rRNA) genes, and determination of the biomethane potential, respectively. The inoculum was stored at room temperature, 4 and −20 °C for up to 11 months and cellulose was used as a standard substrate to test the biomethane potential. Storage up to 1 month resulted in similar final methane yields, but the rate of methane production was reduced by storage at −20 °C. Longer storage times resulted in reduced methane yields and slower production kinetics for all storage conditions, with room temperature and frozen samples consistently giving the best and worst performance, respectively. Both storage time and temperature affected the microbial community composition and methanogenic activity. In particular, fluctuations in the relative abundance of Bacteroidetes were observed. Interestingly, a shift from hydrogenotrophic methanogens to methanogens with the capacity to perform acetoclastic methanogensis was observed upon prolonged storage. In conclusion, this study suggests that biogas inocula may be stored up to 1 month with low loss of methanogenic activity, and identifies bacterial and archaeal species that are affected by the storage.


Inoculum Anaerobic digestion Methane Biogas Bioenergy Microbial community 



This work was financially supported by the ENERGIX-program of the Norwegian Research Council, grants 203402 and 228747.

Conflict of interest

The authors declare that they have no conflict of interest

Supplementary material

253_2015_6623_MOESM1_ESM.pdf (682 kb)
ESM 1 (PDF 681 KB)


  1. Ahring BK, Biswas R, Ahamed A, Teller PJ, Uellendahl H (2014) Making lignin accessible for anaerobic digestion by wet-explosion pretreatment. Bioresour TechnolGoogle Scholar
  2. Angelidaki I, Alves M, Bolzonella D, Borzacconi L, Campos J, Guwy A, Kalyuzhnyi S, Jenicek P, Van Lier J (2009) Defining the biomethane potential (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assaysGoogle Scholar
  3. Aubart C, Bully F (1984) Anaerobic digestion of rabbit wastes and pig manure mixed with rabbit wastes in various experimental conditions. Agric Wastes 10(1):1–13CrossRefGoogle Scholar
  4. Bae B-U, Shin H-S, Paik B-C, Chung J-C (1995) Re-activation characteristics of preserved anaerobic granular sludges. Bioresour Technol 53(3):231–235CrossRefGoogle Scholar
  5. Bowen EJ, Dolfing J, Davenport RJ, Read FL, Curtis TP (2014) Low-temperature limitation of bioreactor sludge in anaerobic treatment of domestic wastewater. Water Sci Technol 69(5):1004–1013PubMedCrossRefGoogle Scholar
  6. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7(5):335–336PubMedCentralPubMedCrossRefGoogle Scholar
  7. Castro H, Queirolo M, Quevedo M, Muxi L (2002) Preservation methods for the storage of anaerobic sludges. Biotechnol Lett 24(4):329–333CrossRefGoogle Scholar
  8. Chouari R, Le Paslier D, Dauga C, Daegelen P, Weissenbach J, Sghir A (2005) Novel major bacterial candidate division within a municipal anaerobic sludge digester. Appl Environ Microbiol 71(4):2145–2153PubMedCentralPubMedCrossRefGoogle Scholar
  9. De Vrieze J, Hennebel T, Boon N, Verstraete W (2012) Methanosarcina: the rediscovered methanogen for heavy duty biomethanation. Bioresour Technol 112:1–9PubMedCrossRefGoogle Scholar
  10. Díaz I, Donoso-Bravo A, Fdz-Polanco M (2011) Effect of microaerobic conditions on the degradation kinetics of cellulose. Bioresour Technol 102(21):10139–10142PubMedCrossRefGoogle Scholar
  11. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19):2460–2461PubMedCrossRefGoogle Scholar
  12. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27(16):2194–2200PubMedCentralPubMedCrossRefGoogle Scholar
  13. Elbeshbishy E, Nakhla G, Hafez H (2012) Biochemical methane potential (BMP) of food waste and primary sludge: influence of inoculum pre-incubation and inoculum source. Bioresour Technol 110:18–25PubMedCrossRefGoogle Scholar
  14. Ferry JG (1993) Methanogenesis: ecology, physiology, biochemistry & genetics. Chapman & Hall, New YorkCrossRefGoogle Scholar
  15. Gantner S, Andersson AF, Alonso-Sáez L, Bertilsson S (2011) Novel primers for 16S rRNA-based archaeal community analyses in environmental samples. J Microbiol Methods 84(1):12–18PubMedCrossRefGoogle Scholar
  16. Hagen LH, Vivekanand V, Linjordet R, Pope PB, Eijsink VG, Horn SJ (2014) Microbial community structure and dynamics during co-digestion of whey permeate and cow manure in continuous stirred tank reactor systems. Bioresour Technol 171:350–359PubMedCrossRefGoogle Scholar
  17. Hahn H, Krautkremer B, Hartmann K, Wachendorf M (2014) Review of concepts for a demand-driven biogas supply for flexible power generation. Renew Sust Energ Rev 29:383–393CrossRefGoogle Scholar
  18. Hamady M, Walker JJ, Harris JK, Gold NJ, Knight R (2008) Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat Methods 5(3):235–237PubMedCentralPubMedCrossRefGoogle Scholar
  19. Hatamoto M, Kaneshige M, Nakamura A, Yamaguchi T (2014) Bacteroides luti sp. nov., an anaerobic, cellulolytic and xylanolytic bacterium isolated from methanogenic sludge. Int J Syst Evol Microbiol 64(Pt 5):1770–1774PubMedCrossRefGoogle Scholar
  20. Horn SJ (2013) A tetra-transition away from fossil fuels. In: Sygna L, O’Brien K, Wolf J (eds) A changing environment for human security: transformative approaches to research, policy and action. Earthscan, London, p 392Google Scholar
  21. Jimenez S, Cartagena MC, Arce A (1989) Influence of operating variables on the anaerobic digestion of crude and treated vine shoots. Biol Wastes 29(3):211–220CrossRefGoogle Scholar
  22. Kafle GK, Kim SH, Sung KI (2013) Ensiling of fish industry waste for biogas production: a lab scale evaluation of biochemical methane potential (BMP) and kinetics. Bioresour Technol 127:326–336PubMedCrossRefGoogle Scholar
  23. Kotsyurbenko OR, Glagolev MV, Nozhevnikova AN, Conrad R (2001) Competition between homoacetogenic bacteria and methanogenic archaea for hydrogen at low temperature. FEMS Microbiol Ecol 38(2‐3):153–159CrossRefGoogle Scholar
  24. Lauber CL, Zhou N, Gordon JI, Knight R, Fierer N (2010) Effect of storage conditions on the assessment of bacterial community structure in soil and human‐associated samples. FEMS Microbiol Lett 307(1):80–86PubMedCentralPubMedCrossRefGoogle Scholar
  25. Lopes WS, Leite VD, Prasad S (2004) Influence of inoculum on performance of anaerobic reactors for treating municipal solid waste. Bioresour Technol 94(3):261–266PubMedCrossRefGoogle Scholar
  26. Lorenz H, Fischer P, Schumacher B, Adler P (2013) Current EU-27 technical potential of organic waste streams for biogas and energy production. Waste Manag 33(11):2434–2448PubMedCrossRefGoogle Scholar
  27. Mauky E, Jacobi HF, Liebetrau J, Nelles M (2015) Flexible biogas production for demand-driven energy supply–feeding strategies and types of substrates. Bioresour Technol 178:262–269PubMedCrossRefGoogle Scholar
  28. McCalley CK, Woodcroft BJ, Hodgkins SB, Wehr RA, Kim E-H, Mondav R, Crill PM, Chanton JP, Rich VI, Tyson GW, Saleska SR (2014) Methane dynamics regulated by microbial community response to permafrost thaw. Nature 514(7523):478–481PubMedCrossRefGoogle Scholar
  29. Meng J, Xu J, Qin D, He Y, Xiao X, Wang F (2013) Genetic and functional properties of uncultivated MCG archaea assessed by metagenome and gene expression analyses. ISME JGoogle Scholar
  30. Naas A, Mackenzie A, Mravec J, Schückel J, Willats W, Eijsink V, Pope P (2014) Do rumen Bacteroidetes utilize an alternative mechanism for cellulose degradation? MBio 5(4):e01401–e01414PubMedCentralPubMedCrossRefGoogle Scholar
  31. Ott SJ, Musfeldt M, Timmis KN, Hampe J, Wenderoth DF, Schreiber S (2004) In vitro alterations of intestinal bacterial microbiota in fecal samples during storage. Diagn Microbiol Infect Dis 50(4):237–245PubMedCrossRefGoogle Scholar
  32. Raposo F, De la Rubia M, Fernández-Cegrí V, Borja R (2012) Anaerobic digestion of solid organic substrates in batch mode: an overview relating to methane yields and experimental procedures. Renew Sust Energ Rev 16(1):861–877CrossRefGoogle Scholar
  33. Risberg K, Sun L, Levén L, Horn SJ, Schnürer A (2013) Biogas production from wheat straw and manure–impact of pretreatment and process operating parameters. Bioresour Technol 149:232–237PubMedCrossRefGoogle Scholar
  34. Rosewarne CP, Pope PB, Denman SE, McSweeney CS, O’Cuiv P, Morrison M (2010) High-yield and phylogenetically robust methods of DNA recovery for analysis of microbial biofilms adherent to plant biomass in the herbivore gut. Microb Ecol 61(2):448–454PubMedCrossRefGoogle Scholar
  35. Rubin BE, Gibbons SM, Kennedy S, Hampton-Marcell J, Owens S, Gilbert JA (2013) Investigating the impact of storage conditions on microbial community composition in soil samples. PLoS One 8(7), e70460PubMedCentralPubMedCrossRefGoogle Scholar
  36. Schmidt O, Horn MA, Kolb S, Drake HL (2014) Temperature impacts differentially on the methanogenic food web of cellulose‐supplemented peatland soil. Environ Microbiol. doi: 10.1111/1462-2920.12507 Google Scholar
  37. Shehu MS, Abdul Manan Z, Wan Alwi SR (2012) Optimization of thermo-alkaline disintegration of sewage sludge for enhanced biogas yield. Bioresour Technol 114:69–74PubMedCrossRefGoogle Scholar
  38. Shelton DR, Tiedje JM (1984) General method for determining anaerobic biodegradation potential. Appl Environ Microbiol 47(4):850–857PubMedCentralPubMedGoogle Scholar
  39. Shin H-S, Bae B-U, Oh S-E (1993) Preservation characteristics of anaerobic granular sludge. Biotechnol Lett 15(5):537–542CrossRefGoogle Scholar
  40. Solli L, Håvelsrud OE, Horn SJ, Rike AG (2014) A metagenomic study of the microbial communities in four parallel biogas reactors. Biotechnol Biofuels 7(1)Google Scholar
  41. Tzeneva VA, Salles JF, Naumova N, de Vos WM, Kuikman PJ, Dolfing J, Smidt H (2009) Effect of soil sample preservation, compared to the effect of other environmental variables, on bacterial and eukaryotic diversity. Res Microbiol 160(2):89–98PubMedCrossRefGoogle Scholar
  42. Vivekanand V, Olsen EF, Eijsink VG, Horn SJ (2013) Effect of different steam explosion conditions on methane potential and enzymatic saccharification of birch. Bioresour Technol 127:343–349PubMedCrossRefGoogle Scholar
  43. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73(16):5261–5267PubMedCentralPubMedCrossRefGoogle Scholar
  44. Weiland P (2010) Biogas production: current state and perspectives. Appl Microbiol Biotechnol 85(4):849–860PubMedCrossRefGoogle Scholar
  45. Westerholm M, Levén L, Schnürer A (2012) Bioaugmentation of syntrophic acetate-oxidizing culture in biogas reactors exposed to increasing levels of ammonia. Appl Environ Microbiol 78(21):7619–7625PubMedCentralPubMedCrossRefGoogle Scholar
  46. Wu W-M, Jain MK, Thiele JH, Zeikus JG (1995) Effect of storage on the performance of methanogenic granules. Water Res 29(6):1445–1452CrossRefGoogle Scholar
  47. Ziganshin AM, Liebetrau J, Pröter J, Kleinsteuber S (2013) Microbial community structure and dynamics during anaerobic digestion of various agricultural waste materials. Appl Microbiol Biotechnol 97(11):5161–5174PubMedCrossRefGoogle Scholar
  48. Zilouei H, Soares A, Murto M, Guieysse B, Mattiasson B (2006) Influence of temperature on process efficiency and microbial community response during the biological removal of chlorophenols in a packed-bed bioreactor. Appl Microbiol Biotechnol 72(3):591–599PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Live Heldal Hagen
    • 1
  • Vivekanand Vivekanand
    • 1
  • Phillip B. Pope
    • 1
  • Vincent G. H. Eijsink
    • 1
  • Svein J. Horn
    • 1
    Email author
  1. 1.Department of Chemistry, Biotechnology and Food ScienceNorwegian University of Life SciencesÅsNorway

Personalised recommendations