Applied Microbiology and Biotechnology

, Volume 99, Issue 20, pp 8777–8792 | Cite as

Effect of organic loading on the microbiota in a temperature-phased anaerobic digestion (TPAD) system co-digesting dairy manure and waste whey

  • Yueh-Fen Li
  • Christopher Abraham
  • Michael C. Nelson
  • Po-Hsu Chen
  • Joerg Graf
  • Zhongtang Yu
Environmental biotechnology


Temperature-phased anaerobic digestion (TPAD) has gained increasing attention because it provides the flexibility to operate digesters under conditions that enhance overall digester performance. However, research on impact of organic overloading rate (OLR) to microbiota of TPAD systems was limited. In this study, we investigated the composition and successions of the microbiota in both the thermophilic and the mesophilic digesters of a laboratory-scale TPAD system co-digesting dairy manure and waste whey before and during organic overloading. The thermophilic and the mesophilic digesters were operated at 50 and 35 °C, respectively, with a hydraulic retention time (HRT) of 10 days for each digester. High OLR (dairy manure with 5 % total solid and waste whey of ≥60.4 g chemical oxygen demand (COD)/l/day) resulted in decrease in pH and in biogas production and accumulation of volatile fatty acids (VFAs) in the thermophilic digester, while the mesophilic digester remained unchanged except a transient increase in biogas production. Both denaturant gradient gel electrophoresis (DGGE) and Illumina sequencing of 16S ribosomal RNA (rRNA) gene amplicons showed dramatic change in microbiota composition and profound successions of both bacterial and methanogenic communities. During the overloading, Thermotogae was replaced by Proteobacteria, while Methanobrevibacter and archaeon classified as WCHD3-02 grew in predominance at the expense of Methanoculleus in the thermophilic digester, whereas Methanosarcina dominated the methanogenic community, while Methanobacterium and Methanobrevibacter became less predominant in the mesophilic digester. Canonical correspondence analysis (CCA) revealed that digester temperature and pH were the most influential environmental factors that explained much of the variations of the microbiota in this TPAD system when it was overloaded.


DGGE Illumina sequencing Microbiota Organic overloading TPAD 

Supplementary material

253_2015_6738_MOESM1_ESM.xlsx (148 kb)
Table S1(XLSX 147 kb)
253_2015_6738_MOESM2_ESM.pdf (34 kb)
Fig. S1(PDF 34 kb)


  1. Alkaya E, Demirer GN (2011) Anaerobic acidification of sugar-beet processing wastes: effect of operational parameters. Biomass Bioenergy 35:32–39CrossRefGoogle Scholar
  2. American Public Health Association, American Water Works Association, Water Environment Federation (2005) Standard methods for the examination of water and wastewater, 21st edn. APHA, Washington, DCGoogle Scholar
  3. Banks C, Chesshire M, Stringfellow A (2008) A pilot-scale comparison of mesophilic and thermophilic digestion of source segregated domestic food waste. Water Sci Technol 58:1475–1481CrossRefPubMedGoogle Scholar
  4. Batstone DJ, Keller J, Angelidaki I, Kalyuzhnyi SV, Pavlostathis SG, Rozzi A, Sanders WTM, Siegrist H, Vavilin VA (2002) Anaerobic digestion model no. 1 (ADM1). IWA Publishing, LondonGoogle Scholar
  5. Bergmann GT, Bates ST, Eilers KG, Lauber CL, Caporaso JG, Walters WA, Knight R, Fierer N (2011) The under-recognized dominance of Verrucomicrobia in soil bacterial communities. Soil Biol Biochem 43(7):1450–1455PubMedCentralCrossRefPubMedGoogle Scholar
  6. Blume F, Bergmann I, Nettmann E, Schelle H, Rehde G, Mundt K, Klocke M (2010) Methanogenic population dynamics during semi-continuous biogas fermentation and acidification by overloading. J Appl Microbiol 109(2):441–450PubMedGoogle Scholar
  7. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7(5):335–336PubMedCentralCrossRefPubMedGoogle Scholar
  8. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6(8):1621–1624PubMedCentralCrossRefPubMedGoogle Scholar
  9. Chen CH (2002) Generalized association plots: information visualization via iteratively generated correlation matrices. Stat Sin 12:7–29.33Google Scholar
  10. Chen Y, Cheng JJ, Creamer KS (2008) Inhibition of anaerobic digestion process: a review. Bioresour Technol 99(10):4044–4064CrossRefPubMedGoogle Scholar
  11. Chen S, Zamudio Cañas EM, Zhang Y, Zhu Z, He Q (2012) Impact of substrate overloading on archaeal populations in anaerobic digestion of animal waste. J Appl Microbiol 113(6):1371–1379CrossRefPubMedGoogle Scholar
  12. Chouari R, Le Paslier D, Daegelen P, Ginestet P, Weissenbach J, Sghir A (2005) Novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester. Environ Microbiol 7(8):1110–1115CrossRefGoogle Scholar
  13. Conklin A, Stensel HD, Ferguson J (2006) Growth kinetics and competition between Methanosarcina and Methanosaeta in mesophilic anaerobic digestion. Water Environ Res 78(5):486–496Google Scholar
  14. Delbes D, Moletta R, Godon JJ (2000) Monitoring of activity dynamics of an anaerobic digester bacterial community using 16S rRNA polymerase chain reaction–single-strand conformation polymorphism analysis. Environ Microbiol 2(5):506–515CrossRefPubMedGoogle Scholar
  15. Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M (2012) Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces. Int J Syst Evol Microbiol 62(8):1902–1907CrossRefPubMedGoogle Scholar
  16. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19):2460–2461CrossRefPubMedGoogle Scholar
  17. Ghanimeh SA, Saikaly PE, Li D, El-Fadel M (2013) Population dynamics during startup of thermophilic anaerobic digesters: the mixing factor. Waste Manag 33(11):2211–2218CrossRefPubMedGoogle Scholar
  18. Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methé B, DeSantis TZ, Human Microbiome Consortium, Petrosino JF, Knight R, Birren BW (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21(3):494–504PubMedCentralCrossRefPubMedGoogle Scholar
  19. 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 50:1601–1609Google Scholar
  20. 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(2):1623–1630PubMedCentralCrossRefPubMedGoogle Scholar
  21. Huber R, Hanning M (2005) Thermotogales. The prokaryotes: an evolving electronic resource for the microbiological community, 3rd edn (DworkinM, ed), Springer-Verlag, New YorkGoogle Scholar
  22. Iino T, Tamaki H, Tamazawa S, Ueno Y, Ohkuma M, Suzuki K, Igarashi Y, Haruta S (2013) Candidatus Methanogranum caenicola: a novel methanogen from the anaerobic digested sludge, and proposal of Methanomassiliicoccaceae fam. nov. and Methanomassiliicoccales ord. nov., for a methanogenic lineage of the class Thermoplasmata. Microbes Environ 28(2):244–250PubMedCentralCrossRefPubMedGoogle Scholar
  23. Jabbour D, Sorger A, Sahm K, Antranikian G (2013) A highly thermoactive and salt-tolerant α-amylase isolated from a pilot-plant biogas reactor. Appl Microbiol Biotechnol 97(7):2971–2978PubMedCentralCrossRefPubMedGoogle Scholar
  24. Janse I, Bok J, Zwart G (2004) A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis. J Microbiol Methods 57(2):279–281CrossRefPubMedGoogle Scholar
  25. Karakashev D, Batstone DJ, Angelidaki I (2005) Influence of environmental conditions on methanogenic compositions in anaerobic biogas reactors. Appl Environ Microbiol 71(1):331–338PubMedCentralCrossRefPubMedGoogle Scholar
  26. Kim HW, Han SK, Shin HS (2004) Anaerobic co-digestion of sewage sludge and food waste using temperature-phased anaerobic digestion process. Water Sci Technol 50(9):107–114PubMedGoogle Scholar
  27. Kim M, Morrison M, Yu Z (2011) Status of the phylogenetic diversity census of ruminal microbiomes. FEMS Microbiol Ecol 76(1):49–63CrossRefPubMedGoogle Scholar
  28. Lee SH, Park JH, Kang HJ, Lee YH, Lee TJ, Park HD (2013) Distribution and abundance of Spirochaetes in full-scale anaerobic digesters. Bioresour Technol 145:25–32CrossRefPubMedGoogle Scholar
  29. Lerm S, Kleyböcker A, Miethling-Graff R, Alawi M, Kasina M, Liebrich M, Würdemann H (2012) Archaeal community composition affects the function of anaerobic co-digesters in response to organic overload. Waste Manag 32(3):389–399CrossRefPubMedGoogle Scholar
  30. Levén L, Eriksson AR, Schnürer A (2007) Effect of process temperature on bacterial and archaeal communities in two methanogenic bioreactors treating organic household waste. FEMS Microbiol Ecol 59(3):683–693CrossRefPubMedGoogle Scholar
  31. Lv W, Schanbacher FL, Yu Z (2010) Putting microbes to work in sequence: recent advances in temperature-phased anaerobic digestion processes. Bioresour Technol 101(24):9409–9414CrossRefPubMedGoogle Scholar
  32. Lv W, Zhang W, Yu Z (2013a) Evaluation of system performance and microbial communities of a temperature-phased anaerobic digestion system treating dairy manure: thermophilic digester operated at acidic pH. Bioresour Technol 142:625–632CrossRefPubMedGoogle Scholar
  33. Lv W, Zhang W, Yu Z (2013b) Evaluation of system performances and microbial communities of two temperature-phased anaerobic digestion systems treating dairy manure. Bioresour Technol 143:431–438CrossRefPubMedGoogle Scholar
  34. Maune MW, Tanner RS (2012a) Description of Anaerobaculum hydrogeniformans sp. nov., an anaerobe that produces hydrogen from glucose, and emended description of the genus Anaerobaculum. Int J Syst Evol Microbiol 62(4):832–838CrossRefPubMedGoogle Scholar
  35. Maune MW, Tanner RS (2012b) Description of Anaerobaculum hydrogeniformans sp. nov., an anaerobe that produces hydrogen from glucose, and emended description of the genus Anaerobaculum. Int J Syst Evol Microbiol 62:832–838CrossRefPubMedGoogle Scholar
  36. McMahon KD, Zheng D, Stams AJ, Mackie RI, 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(7):823–834CrossRefPubMedGoogle Scholar
  37. Menes RJ, Muxí L (2002) Anaerobaculum mobile sp. nov., a novel anaerobic, moderately thermophilic, peptide-fermenting bacterium that uses crotonate as an electron acceptor, and emended description of the genus Anaerobaculum. Int J Syst Evol Microbiol 52(1):157–164CrossRefPubMedGoogle Scholar
  38. Miranda-Tello E, Fardeau ML, Thomas P, Ramirez F, Casalot L, Cayol JL, Garcia JL, Ollivier B (2004) Petrotoga mexicana sp. nov., a novel thermophilic, anaerobic and xylanolytic bacterium isolated from an oil-producing well in the Gulf of Mexico. Int J Syst Evol Microbiol 54(1):169–174CrossRefPubMedGoogle Scholar
  39. Nelson MC, Morrison HG, Benjamino J, Grim SL, Graf J (2014) Analysis, optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys. PLoS One 9(4):e94249PubMedCentralCrossRefPubMedGoogle Scholar
  40. Nesbø CL, Dlutek M, Zhaxybayeva O, Doolittle WF (2006) Evidence for existence of “mesotogas,” members of the order Thermotogales adapted to low-temperature environments. Appl Environ Microbiol 72(7):5061–5068PubMedCentralCrossRefPubMedGoogle Scholar
  41. Price MN, Dehal PS, Arkin AP (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS One 5:e9490PubMedCentralCrossRefPubMedGoogle Scholar
  42. Pycke BF, Etchebehere C, Van de Caveye P, Negroni A, Verstraete W, Boon N (2011) A time-course analysis of four full-scale anaerobic digesters in relation to the dynamics of change of their microbial communities. Water Sci Technol 63(4):769–775CrossRefPubMedGoogle Scholar
  43. Qiao JT, Qiu YL, Yuan XZ, Shi XS, Xu XH, Guo RB (2013) Molecular characterization of bacterial and archaeal communities in a full-scale anaerobic reactor treating corn straw. Bioresour Technol 143:512–518CrossRefPubMedGoogle Scholar
  44. Ramette A (2007) Multivariate analyses in microbial ecology. FEMS Microbiol Ecol 62:142–160PubMedCentralCrossRefPubMedGoogle Scholar
  45. Riau V, De la Rubia MA, Pérez M (2010) Temperature-phased anaerobic digestion (TPAD) to obtain class A biosolids: a semi-continuous study. Bioresour Technol 101(8):2706–2712CrossRefPubMedGoogle Scholar
  46. Sangwan P, Chen X, Hugenholtz P, Janssen PH (2004) Chthoniobacter flavus gen. nov., sp. nov., the first pure-culture representative of subdivision two, Spartobacteria classis nov., of the phylum Verrucomicrobia. Appl Environ Microbiol 70(10):5875–5881PubMedCentralCrossRefPubMedGoogle Scholar
  47. Santha H, Sandino J, Shimp GF, Sung S (2006) Performance evaluation of a ‘sequential-batch’ temperature-phased anaerobic digestion (TPAD) scheme for producing class A biosolids. Water Environ Res 78(3):221–226CrossRefPubMedGoogle Scholar
  48. Sasaki D, Hori T, Haruta S, Ueno Y, Ishii M, Igarashi Y (2011) Methanogenic pathway and community structure in a thermophilic anaerobic digestion process of organic solid waste. J Biosci Bioeng 111:41–46CrossRefPubMedGoogle Scholar
  49. Savant DV, Shouche YS, Prakash S, Ranade DR (2002) Methanobrevibacter acididurans sp. nov., a novel methanogen from a sour anaerobic digester. Int J Syst Evol Microbiol 52(4):1081–1087PubMedGoogle Scholar
  50. Steinberg LM, Regan JM (2011) Response of lab-scale methanogenic reactors inoculated from different sources to organic loading rate shocks. Bioresour Technol 102(19):8790–8798CrossRefPubMedGoogle Scholar
  51. Sundberg C, Al-Soud WA, Larsson M, Alm E, Yekta SS, Svensson BH, Sørensen SJ, Karlsson A (2013) 454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters. FEMS Microbiol Ecol 85(3):612–626CrossRefPubMedGoogle Scholar
  52. Tale VP, Maki JS, Struble CA, Zitomer DH (2011) Methanogen community structure-activity relationship and bioaugmentation of overloaded anaerobic digesters. Water Res 45(16):5249–5256CrossRefPubMedGoogle Scholar
  53. Vandenburgh SR, Ellis TG (2002) Effect of varying solids concentration and organic loading on the performance of temperature phased anaerobic digestion process. Water Environ Res 74(2):142–148CrossRefPubMedGoogle Scholar
  54. 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 73:5261–5267PubMedCentralCrossRefPubMedGoogle Scholar
  55. Weiss A, Jérôme V, Freitag R, Mayer HK (2008) Diversity of the resident microbiota in a thermophilic municipal biogas plant. Appl Microbiol Biotechnol 81(1):163–173CrossRefPubMedGoogle Scholar
  56. Werner JJ, Zhou D, Caporaso JG, Knight R, Angenent LT (2012) Comparison of Illumina paired-end and single-direction sequencing for microbial 16S rRNA gene amplicon surveys. ISME J 6:1273–1276PubMedCentralCrossRefPubMedGoogle Scholar
  57. Wu HM, Tien YJ, Chen C (2010) GAP: a graphical environment for matrix visualization and cluster analysis. Comput Stat Data Anal 54:767–778CrossRefGoogle Scholar
  58. Yan JQ, Lo KV, Pinder KL (1993) Instability caused by high strength of cheese whey in a UASB reactor. Biotechnol Bioeng 41:700–706CrossRefPubMedGoogle Scholar
  59. Yu Z, Morrison M (2004a) Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis. Appl Environ Microbiol 70:4800–4806PubMedCentralCrossRefPubMedGoogle Scholar
  60. Yu Z, Morrison M (2004b) Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques 36:808–812PubMedGoogle Scholar
  61. Yu Z, García-González R, Schanbacher FL, Morrison M (2008) Evaluations of different hypervariable regions of archaeal 16S rRNA genes in profiling of methanogens by Archaea-specific PCR and denaturing gradient gel electrophoresis. Appl Environ Microbiol 74(3):889–893PubMedCentralCrossRefPubMedGoogle Scholar
  62. Zhou Z, Meng Q, Yu Z (2011) Effects of methanogenic inhibitors on methane production and abundances of methanogens and cellulolytic bacteria in in vitro ruminal cultures. Appl Environ Microbiol 77(8):2634–2639PubMedCentralCrossRefPubMedGoogle Scholar
  63. Zuo Z, Wu S, Zhang W, Dong R (2013) Effects of organic loading rate and effluent recirculation on the performance of two-stage anaerobic digestion of vegetable waste. Bioresour Technol 146:556–561CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Yueh-Fen Li
    • 1
  • Christopher Abraham
    • 2
  • Michael C. Nelson
    • 4
  • Po-Hsu Chen
    • 3
  • Joerg Graf
    • 4
  • Zhongtang Yu
    • 1
    • 2
  1. 1.Environmental Science Graduate ProgramThe Ohio State UniversityColumbusUSA
  2. 2.Department of Animal SciencesThe Ohio State UniversityColumbusUSA
  3. 3.Department of StatisticsThe Ohio State UniversityColumbusUSA
  4. 4.Department of Molecular and Cell BiologyUniversity of ConnecticutStorrsUSA

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