Bioprocess and Biosystems Engineering

, Volume 39, Issue 7, pp 1115–1127 | Cite as

Predictive functional profiling using marker gene sequences and community diversity analyses of microbes in full-scale anaerobic sludge digesters

Original Paper


Anaerobic digestion (AD) is widely used in treating the sewage sludge, as it can reduce the amount of sludge, eliminate pathogens and produce biofuel. To enhance the operational performance and stability of anaerobic bioreactors, operational and conventional chemical data from full-scale sludge anaerobic digesters were collected over a 2-year period and summarized, and the microbial community diversity of the sludge sample was investigated at various stages of the AD process. For the purpose of distinguishing between the functional and community diversity of the microbes, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) software was used to impute the prevalence of 16S rDNA marker gene sequences in the difference in various sludge samples. Meanwhile, a taxa analysis was also carried out to investigate the different sludge samples. The microbial community diversity analysis of one AD sludge sample showed that the most dominant bacterial genera were Saccharicrinis, Syntrophus, Anaerotruncus and Thermanaerothrix. Among archaea, acetoclastic Methanosaeta represented 56.0 %, and hydrogenotrophic Methanospirillum, Methanoculleus, Methanothermus and Methanolinea accounted for 41.3 % of all methanogens. The taxa, genetic and functional prediction analyses of the feedstock and AD sludge samples suggested great community diversity differences between them. The taxa of bacteria in two AD sludge samples were considerably different, but the abundances of the functional KEGG pathways took on similar levels. The numbers of identified pathogens were significantly lower in the digested sludge than in the feedstock, but the PICRUSt results showed the difference in “human diseases” abundances in the level-1 pathway between the two sludge samples was small.


Anaerobic digestion Bacteria Function PICRUSt Sludge 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

449_2016_1588_MOESM1_ESM.doc (1 mb)
Supplementary material 1 (DOC 1038 kb)


  1. 1.
    Shu D, He Y, Yue H, Wang Q (2015) Microbial structures and community functions of anaerobic sludge in six full-scale wastewater treatment plants as revealed by 454 high-throughput pyrosequencing. Bioresour Technol 186:163–172CrossRefGoogle Scholar
  2. 2.
    Yang Y, Yu K, Xia Y, Lau FT, Tang DT, Fung WC, Fang HH, Zhang T (2014) Metagenomic analysis of sludge from full-scale anaerobic digesters operated in municipal wastewater treatment plants. Appl Microbiol Biotechnol 98:5709–5718CrossRefGoogle Scholar
  3. 3.
    Kim J, Kim W, Lee C (2013) Absolute dominance of hydrogenotrophic methanogens in full-scale anaerobic sewage sludge digesters. J Environ Sci-China 25:2272–2280CrossRefGoogle Scholar
  4. 4.
    Guo J, Peng Y, Ni B-J, Han X, Fan L, Yuan Z (2015) Dissecting microbial community structure and methane-producing pathways of a full-scale anaerobic reactor digesting activated sludge from wastewater treatment by metagenomic sequencing. Microb Cell Fact 14:33-33Google Scholar
  5. 5.
    Amani T, Nosrati M, Sreekrishnan TR (2010) Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects—a review. Environ Rev 18:255–278CrossRefGoogle Scholar
  6. 6.
    Shen Y, Linville JL, Urgun-Demirtas M, Mintz MM, Snyder SW (2015) An overview of biogas production and utilization at full-scale wastewater treatment plants (WWTPs) in the United States: challenges and opportunities towards energy-neutral WWTPs. Renew Sust Energy Rev 50:346–362CrossRefGoogle Scholar
  7. 7.
    Sundberg C, Al-Soud WA, Larsson M, Alm E, Yekta SS, Svensson BH, Sorensen SJ, Karlsson A (2013) 454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters. FEMS Microbiol Ecol 85:612–626CrossRefGoogle Scholar
  8. 8.
    Ariesyady HD, Ito T, Okabe S (2007) Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester. Water Res 41:1554–1568CrossRefGoogle Scholar
  9. 9.
    Akutsu Y, Li Y-Y, Tandukar M, Kubota K, Harada H (2008) Effects of seed sludge on fermentative characteristics and microbial community structures in thermophilic hydrogen fermentation of starch. Int Hydrogen Energy 33:6541–6548CrossRefGoogle Scholar
  10. 10.
    Solli L, Havelsrud OE, Horn SJ, Rike AG (2014) A metagenomic study of the microbial communities in four parallel biogas reactors. Biotechnol Biofuels 7:146CrossRefGoogle Scholar
  11. 11.
    Werner JJ (2011) Bacterial community structures are unique and resilient in full-scale bioenergy systems. PNAS 108:4158–4163CrossRefGoogle Scholar
  12. 12.
    Lee S-H, Park J-H, Kim S-H, Yu BJ, Yoon J-J, Park H-D (2015) Evidence of syntrophic acetate oxidation by Spirochaetes during anaerobic methane production. Bioresour Technol 190:543–549CrossRefGoogle Scholar
  13. 13.
    Wagner AM, Cloete ET (2002) 16S rRNA sequence analysis of bacteria present in foaming activated sludge. Syst Appl Microbiol 25:434–439CrossRefGoogle Scholar
  14. 14.
    Riviere 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
  15. 15.
    Langille MGI, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes JA, Clemente JC, Burkepile DE, Thurber RLV, Knight R, Beiko RG, Huttenhower C (2013) Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol 31:814–821CrossRefGoogle Scholar
  16. 16.
    Magoc T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963CrossRefGoogle Scholar
  17. 17.
    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena 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 7:335–336CrossRefGoogle Scholar
  18. 18.
    Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microb 75:7537–7541CrossRefGoogle Scholar
  19. 19.
    Huson DH, Mitra S, Ruscheweyh H-J, Weber N, Schuster SC (2011) Integrative analysis of environmental sequences using MEGAN4. Genome Res 21:1552–1560CrossRefGoogle Scholar
  20. 20.
    Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M (2012) KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 40:D109–D114CrossRefGoogle Scholar
  21. 21.
    Razaviarani V, Buchanan ID (2015) Calibration of the Anaerobic Digestion Model No. 1 (ADM1) for steady-state anaerobic co-digestion of municipal wastewater sludge with restaurant grease trap waste. Chem Eng J 266:91–99CrossRefGoogle Scholar
  22. 22.
    Lee SH, Kang HJ, Lee YH, Lee TJ, Han K, Choi Y, Park HD (2012) Monitoring bacterial community structure and variability in time scale in full-scale anaerobic digesters. J Environ Monit 14:1893–1905CrossRefGoogle Scholar
  23. 23.
    DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microb 72:5069–5072CrossRefGoogle Scholar
  24. 24.
    Hagen LH, Vivekanand V, Linjordet R, Pope PB, Eijsink VGH, 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 171:350–359CrossRefGoogle Scholar
  25. 25.
    Engel AS, Porter ML, Stern LA, Quinlan S, Bennett PC (2004) Bacterial diversity and ecosystem function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by chemolithoautotrophic “Epsilonproteobacteria”. FEMS Microbiol Ecol 51:31–53CrossRefGoogle Scholar
  26. 26.
    Wang X, Zhang Y, Zhang T, Zhou J, Chen M (2016) Waste activated sludge fermentation liquid as carbon source for biological treatment of sulfide and nitrate in microaerobic conditions. Chem Eng J 283:167–17427CrossRefGoogle Scholar
  27. 27.
    Kern M, Simon J (2009) Electron transport chains and bioenergetics of respiratory nitrogen metabolism in Wolinella succinogenes and other Epsilonproteobacteria. Biochim Biophys Acta 1787:646–656CrossRefGoogle Scholar
  28. 28.
    Zhou Z, Qiao WM, Xing C, Shen XL, Hu DL, Wang LC (2014) A micro-aerobic hydrolysis process for sludge in situ reduction: performance and microbial community structure. Bioresour Technol 173:452–456CrossRefGoogle Scholar
  29. 29.
    Goud RK, Raghavulu SV, Mohanakrishna G, Naresh K, Mohan SV (2012) Predominance of Bacilli and Clostridia in microbial community of biohydrogen producing biofilm sustained under diverse acidogenic operating conditions. Int J Hydrogen Energy 37:4068–4076CrossRefGoogle Scholar
  30. 30.
    Zhou Z, Qiao W, Xing C, An Y, Shen X, Ren W, L-m Jiang, Wang L (2015) Microbial community structure of anoxic–oxic-settling-anaerobic sludge reduction process revealed by 454-pyrosequencing. Chem Eng J 266:249–257CrossRefGoogle Scholar
  31. 31.
    Shin SG, Lee S, Lee C, Hwang K, Hwang S (2010) Qualitative and quantitative assessment of microbial community in batch anaerobic digestion of secondary sludge. Bioresour Technol 101:9461–9470CrossRefGoogle Scholar
  32. 32.
    Castello E, Santos CGY, Iglesias T, Paolino G, Wenzel J, Borzacconi L, Etchebehere C (2009) Feasibility of biohydrogen production from cheese whey using a UASB reactor: links between microbial community and reactor performance. Int J Hydrogen Energ 34:5674–5682CrossRefGoogle Scholar
  33. 33.
    Gregoire P, Fardeau ML, Joseph M, Guasco S, Hamaide F, Biasutti S, Michotey V, Bonin P, Ollivier B (2011) Isolation and characterization of Thermanaerothrix daxensis gen. nov., sp. nov., a thermophilic anaerobic bacterium pertaining to the phylum “Chloroflexi”, isolated from a deep hot aquifer in the Aquitaine Basin. Syst Appl Microbiol 34:494–497CrossRefGoogle Scholar
  34. 34.
    Regueiro L, Spirito CM, Usack JG, Hospodsky D, Werner JJ, Angenent LT (2015) Comparing the inhibitory thresholds of dairy manure co-digesters after prolonged acclimation periods: part 2—correlations between microbiomes and environment. Water Res 87:458–466CrossRefGoogle Scholar
  35. 35.
    Al-Shorgani NKN, Isa MHM, Yusoff WMW, Kalil MS, Hamid AA (2016) Isolation of a Clostridium acetobutylicum strain and characterization of its fermentation performance on agricultural wastes. Renew Energy 86:459–465CrossRefGoogle Scholar
  36. 36.
    Steppe M, Nieuwerburgh FV, Vercauteren G, Boyena F, Eeckhaut V, Deforce D, Haesebrouck F, Ducatelle R, Immerseel FV (2014) Safety assessment of the butyrate-producing Butyricicoccus pullicaecorum strain 25-3T, a potential probiotic for patients with inflammatory bowel disease, based on oral toxicity tests and whole genome sequencing. Food Chem Toxicol 72:129–137CrossRefGoogle Scholar
  37. 37.
    Kampfer P (1995) Physiological and chemotaxonomic characterization of filamentous bacteria belonging to the genus Haliscomenobacter. Syst Appl Microbiol 18:363–367CrossRefGoogle Scholar
  38. 38.
    Cavaleiro AJ, Sousa DZ, Alves MM (2010) Methane production from oleate: assessing the bioaugmentation potential of Syntrophomonas zehnderi. Water Res 44:4940–4947CrossRefGoogle Scholar
  39. 39.
    Wu C, Liu X, Dong X (2006) Syntrophomonas erecta subsp. sporosyntropha subsp. nov., a spore-forming bacterium that degrades short chain fatty acids in co-culture with methanogens. Syst Appl Microbiol 29:457–462CrossRefGoogle Scholar
  40. 40.
    Scholten JC, Culley DE, Brockman FJ, Wu G, Zhang W (2007) Evolution of the syntrophic interaction between Desulfovibrio vulgaris and Methanosarcina barkeri: involvement of an ancient horizontal gene transfer. Biochem Biophys Res Commun 352:48–54CrossRefGoogle Scholar
  41. 41.
    Fernandes L, Lucas MS, Maldonado MI, Oller I, Sampaio A (2014) Treatment of pulp mill wastewater by Cryptococcus podzolicus and solar photo-Fenton: a case study. Chem Eng J 245:158–165CrossRefGoogle Scholar
  42. 42.
    Ntougias S, Baldrian P, Ehaliotis C, Nerud F, Antoniou T, Merhautová V, Zervakis GI (2012) Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus. Chemosphere 88:620–626CrossRefGoogle Scholar
  43. 43.
    Argun H, Kargi F, Kapdan IK (2008) Light fermentation of dark fermentation effluent for bio-hydrogen production by different Rhodobacter species at different initial volatile fatty acid (VFA) concentrations. Int J Hydrogen Energy 33:7405–7412CrossRefGoogle Scholar
  44. 44.
    Hiraishi Akira, Muramatsu K, Urata K (1995) Characterization of new denitrifying Rhodobacter strains isolated from photosynthetic sludge for wastewater treatment. J Ferment Bioeng 79:39–44CrossRefGoogle Scholar
  45. 45.
    Zhou Z, Qiao W, Xing C, Shen X, Hu D, Wang L (2014) A micro-aerobic hydrolysis process for sludge in situ reduction: performance and microbial community structure. Bioresour Technol 173:452–456CrossRefGoogle Scholar
  46. 46.
    de Gannes V, Eudoxie G, Hickey WJ (2013) Prokaryotic successions and diversity in composts as revealed by 454-pyrosequencing. Bioresour Technol 133:573–580CrossRefGoogle Scholar
  47. 47.
    Tian M, Zhao Fangqing, Shen X, Chu K, Wang J, Chen S, Guo Y, Liu H (2015) The first metagenome of activated sludge from full-scale anaerobic/anoxic/oxic (A2O) nitrogen and phosphorus removal reactor using Illumina sequencing. J Environ Sci-China 35:181–190CrossRefGoogle Scholar
  48. 48.
    Razaviarani V, Buchanan ID (2014) Reactor performance and microbial community dynamics during anaerobic co-digestion of municipal wastewater sludge with restaurant grease waste at steady state and overloading stages. Bioresour Technol 172:232–240CrossRefGoogle Scholar
  49. 49.
    Levican A, Collado L, Aguilar C, Yustes C, Diéguez AL, Romalde JL, Figueras MJ (2012) Arcobacter bivalviorum sp. nov. and Arcobacter venerupis sp. nov., new species isolated from shellfish. Syst Appl Microbiol 35:133–138CrossRefGoogle Scholar
  50. 50.
    Li R, X-a Ning, Sun J, Wang Y, Liang J, Lin M, Zhang Y (2015) Decolorization and biodegradation of the Congo red by Acinetobacter baumannii YNWH 226 and its polymer production’s flocculation and dewatering potential. Bioresour Technol 194:233–239CrossRefGoogle Scholar
  51. 51.
    Yang L, Ren Y-X, Liang X, Zhao S-Q, J-p Wang, Xia Z-H (2015) Nitrogen removal characteristics of a heterotrophic nitrifier Acinetobacter junii YB and its potential application for the treatment of high-strength nitrogenous wastewater. Bioresour Technol 193:227–233CrossRefGoogle Scholar
  52. 52.
    Srinivasan S, Aslan A, Xagoraraki I, Alocilja E, Rose JB (2011) Escherichia coli, enterococci, and Bacteroides thetaiotaomicron qPCR signals through wastewater and septage treatment. Water Res 45:2561–2572CrossRefGoogle Scholar
  53. 53.
    Eribe ER, Olsen I (2008) Leptotrichia species in human infections. Anaerobe 14:131–137CrossRefGoogle Scholar
  54. 54.
    Bibby K, Viau E, Peccia J (2010) Pyrosequencing of the 16S rRNA gene to reveal bacterial pathogen diversity in biosolids. Water Res 44:4252–4260CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.School of Chemical Engineering and EnergyZhengzhou UniversityZhengzhouChina
  2. 2.Zhongyuan Environmental Protection Co. Ltd.ZhengzhouChina
  3. 3.Zhengzhou Sewage Purification Co. Ltd.ZhengzhouChina

Personalised recommendations