Applied Microbiology and Biotechnology

, Volume 100, Issue 2, pp 927–937 | Cite as

Bioaugmentation of anaerobic sludge digestion with iron-reducing bacteria: process and microbial responses to variations in hydraulic retention time

  • Gahyun Baek
  • Jaai Kim
  • Seung Gu Shin
  • Changsoo LeeEmail author
Environmental biotechnology


Although anaerobic digestion (AD) is a widely used option to manage waste activated sludge (WAS), there are some drawbacks related to its slow reaction rate and low energy productivity. This study examined an anaerobic WAS digester, augmented with an iron-reducing microbial consortium, relative to changes in microbial community structure and process performance at decreasing hydraulic retention times (HRTs) of 20 to 10 days. The enhanced methanation performance (approximately 40 % increase in methane yield) by the bioaugmentation was sustained until the HRT was decreased to 12.5 days, under Fe3+-rich conditions (ferric oxyhydroxide, 20 mM Fe). Enhanced iron-reducing activity was evidenced by the increased Fe2+ to total Fe ratio maintained above 50 % during the stable operational phases. A further decrease in HRT to 10 days resulted in a significant performance deterioration, along with a drop in the Fe2+ to total Fe ratio to <35 %, after four turnovers of operation. Prevailing existence of putative iron-reducing bacteria (IRBs) was identified by denaturing gradient gel electrophoresis (DGGE), with Spirochaetaceae- and Thauera-related organisms being dominant members, and clear dominance shifts among them with respect to decrease in HRT were observed. Lowering HRT led to evident shifts in bacterial community structure likely associated with washout of IRBs, leading to decreases in iron respiration activity and AD performance at a lower HRT. The bacterial community structure shifted dynamically over phases, and the community transitions correlated well with the changes in process performance. Overall, the combined biostimulation and bioaugmentation investigated in this study proved effective for enhanced methane recovery from anaerobic WAS digestion, which suggests an interesting potential for high-rate AD.


Anaerobic digestion Bioaugmentation Ferric oxyhydroxide Iron-reducing bacteria Microbial community structure 



This research was supported by the National Research Foundation of Korea (NRF) through Basic Science Research Program (2014R1A1A1002329) granted by the Ministry of Science, ICT, and Future Planning and also through International Cooperation Program managed by NRF (2013K2A1A2054369). The authors are also grateful for the support of Korea Ministry of Environment (MOE) through a Waste-to-Energy Human Resource Development Project.

Conflict of interest

The authors declare that they have no competing interest.

Ethical statement

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


  1. Ahring BK (2003) Biomethanation I, vol 81. Springer, New YorkCrossRefGoogle Scholar
  2. APHA-AWWA-WEF (2005) Standard methods for the examination of water and wastewater, 21st edn. American Public Health Association, WashingtonGoogle Scholar
  3. Baek G, Kim J, Lee C (2014) Influence of ferric oxyhydroxide addition on biomethanation of waste activated sludge in a continuous reactor. Bioresour Technol 166:596–601PubMedCrossRefGoogle Scholar
  4. Boone DR, Castenholz RW (2001) Bergey’s manual of systematic bacteriology, Vol 1: The Archaea and the Deeply Branching and Phototrophic Bacteria, 2nd edn. Springer.Google Scholar
  5. Brenner DJ, Krieg NR, Staley JT (2005) Bergey’s manual of systematic bacteriology, Vol 2: The Proteobacteria, 2nd edn. Springer.Google Scholar
  6. Brown TL, LeMay, Jr. HE, Bursten BE, Murphy CJ, Woodward PM, Stoltzfus MW (2014) Chemistry: the central science, 13th edn. Prentice Hall.Google Scholar
  7. Chakraborty A, Picardal F (2013) Neutrophilic, nitrate-dependent, Fe (II) oxidation by a Dechloromonas species. World J Microbiol Biotechnol 29:617–623PubMedCrossRefGoogle Scholar
  8. Chaudhuri SK, Lovley DR (2003) Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nat Biotech 21:1229–1232CrossRefGoogle Scholar
  9. Cheng S, Xing D, Call DF, Logan BE (2009) Direct biological conversion of electrical current into methane by electromethanogenesis. Environ Sci Technol 43:3953–3958PubMedCrossRefGoogle Scholar
  10. Cruz Viggi C, Rossetti S, Fazi S, Paiano P, Majone M, Aulenta F (2014) Magnetite particles triggering a faster and more robust syntrophic pathway of methanogenic propionate degradation. Environ Sci Technol 48:7536–7543PubMedCrossRefGoogle Scholar
  11. Dinh HT, Kuever J, Muszmann M, Hassel AW, Stratmann M, Widdel F (2004) Iron corrosion by novel anaerobic microorganisms. Nature 427:829–832PubMedCrossRefGoogle Scholar
  12. Duldhardt I, Gaebel J, Chrzanowski L, Nijenhuis I, Härtig C, Schauer F, Heipieper HJ (2010) Adaptation of anaerobically grown Thauera aromatica, Geobacter sulfurreducens and Desulfococcus multivorans to organic solvents on the level of membrane fatty acid composition. Microb Biotechnol 3:201–209PubMedPubMedCentralCrossRefGoogle Scholar
  13. Garcia J-L, Patel BKC, Ollivier B (2000) Taxonomic, phylogenetic, and ecological diversity of methanogenic archaea. Anaerobe 6:205–226PubMedCrossRefGoogle Scholar
  14. Graber JR, Breznak JA (2004) Physiology and nutrition of Treponema primitia, an H2/CO2-acetogenic Spirochete from Termite hindguts. Appl Environ Microbiol 70:1307–1314PubMedPubMedCentralCrossRefGoogle Scholar
  15. Hori T, Müller A, Igarashi Y, Conrad R, Friedrich MW (2010) Identification of iron-reducing microorganisms in anoxic rice paddy soil by 13C-acetate probing. Isme J 4:267–278PubMedCrossRefGoogle Scholar
  16. Kato S, Hashimoto K, Watanabe K (2012) Methanogenesis facilitated by electric syntrophy via (semi) conductive iron-oxide minerals. Environ Microbiol 14:1646–1654PubMedCrossRefGoogle Scholar
  17. Kato S, Ikehata K, Shibuya T, Urabe T, Ohkuma M, Yamagishi A (2015) Potential for biogeochemical cycling of sulfur, iron and carbon within massive sulfide deposits below the seafloor. Environ Microbiol 17:1817–1835PubMedCrossRefGoogle Scholar
  18. Kiely PD, Regan JM, Logan BE (2011) The electric picnic: synergistic requirements for exoelectrogenic microbial communities. Curr Opin Biotechnol 22:378–385PubMedCrossRefGoogle Scholar
  19. Kim J, Lee S, Lee C (2013) Comparative study of changes in reaction profile and microbial community structure in two anaerobic repeated-batch reactors started up with different seed sludges. Bioresour Technol 129:495–505PubMedCrossRefGoogle Scholar
  20. Kim S-J, Park S-J, Cha I-T, Min D, Kim J-S, Chung W-H, Chae J-C, Jeon CO, Rhee S-K (2014) Metabolic versatility of toluene-degrading, iron-reducing bacteria in tidal flat sediment, characterized by stable isotope probing-based metagenomic analysis. Environ Microbiol 16:189–204PubMedCrossRefGoogle Scholar
  21. Kong Y, Nielsen JL, Nielsen PH (2005) Identity and ecophysiology of uncultured actinobacterial polyphosphate-accumulating organisms in full-scale enhanced biological phosphorus removal plants. Appl Environ Microbiol 71:4076–4085PubMedPubMedCentralCrossRefGoogle Scholar
  22. Kostka JE, Dalton DD, Skelton H, Dollhopf S, Stucki JW (2002) Growth of iron (III)-reducing bacteria on clay minerals as the sole electron acceptor and comparison of growth yields on a variety of oxidized iron forms. Appl Environ Microbiol 68:6256–6262PubMedPubMedCentralCrossRefGoogle Scholar
  23. Lentini CJ, Wankel SD, Hansel CM (2012) Enriched iron (III)-reducing bacterial communities are shaped by carbon substrate and iron oxide mineralogy. Front Microbiol 3:1–19CrossRefGoogle Scholar
  24. Li H, Chang J, Liu P, Fu L, Ding D, Lu Y (2014) Direct interspecies electron transfer accelerates syntrophic oxidation of butyrate in paddy soil enrichments. Environ Microbiol 5:1533–1547Google Scholar
  25. Liu F, Rotaru A-E, Shrestha PM, Malvankar NS, Nevin KP, Lovley DR (2015) Magnetite compensates for the lack of a pilin-associated c-type cytochrome in extracellular electron exchange. Environ Microbiol 17:648–655PubMedCrossRefGoogle Scholar
  26. Lovley DR (2006) Bug juice: harvesting electricity with microorganisms. Nat Rev Micro 4:497–508CrossRefGoogle Scholar
  27. Lovley DR, Phillips EJ (1986) Organic matter mineralization with reduction of ferric iron in anaerobic sediments. Appl Environ Microbiol 51:683–689PubMedPubMedCentralGoogle Scholar
  28. McCune B, Grace JB, Urban DL (2002) Analysis of ecological communities. MjM Software Design, Glenden BeachGoogle Scholar
  29. Nguyen HTT, Le VQ, Hansen AA, Nielsen JL, Nielsen PH (2011) High diversity and abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated sludge systems. FEMS Microbiol Ecol 76:256–267PubMedCrossRefGoogle Scholar
  30. Park B, Ahn J, Kim J, Hwang S (2004) Use of microwave pretreatment for enhanced anaerobiosis of secondary sludge. Wat Sci Technol 50:17–23Google Scholar
  31. Park T-J, Ding W, Cheng S, Brar MS, Ma APY, Tun HM, Leung FC (2014) Microbial community in microbial fuel cell (MFC) medium and effluent enriched with purple photosynthetic bacterium (Rhodopseudomonas sp.). AMB Express 4:22PubMedPubMedCentralCrossRefGoogle Scholar
  32. Pelletier E, Kreimeyer A, Bocs S, Rouy Z, Gyapay G, Chouari R, Rivière D, Ganesan A, Daegelen P, Sghir A (2008) “Candidatus Cloacamonas acidaminovorans”: genome sequence reconstruction provides a first glimpse of a new bacterial division. J Bacteriol 190:2572–2579PubMedPubMedCentralCrossRefGoogle Scholar
  33. Risso C, Sun J, Zhuang K, Mahadevan R, DeBoy R, Ismail W, Shrivastava S, Huot H, Kothari S, Daugherty S, Bui O, Schilling C, Lovley D, Methe B (2009) Genome-scale comparison and constraint-based metabolic reconstruction of the facultative anaerobic Fe(III)-reducer Rhodoferax ferrireducens. BMC Genomics 10:447PubMedPubMedCentralCrossRefGoogle Scholar
  34. Rotaru A-E, Shrestha PM, Liu F, Shrestha M, Shrestha D, Embree M, Zengler K, Wardman C, Nevin KP, Lovley DR (2014) A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane. Energ Environ Sci 7:408–415CrossRefGoogle Scholar
  35. Vu AT, Nguyen NC, Leadbetter JR (2004) Iron reduction in the metal-rich guts of wood-feeding termites. Geobiology 2:239–247CrossRefGoogle Scholar
  36. Weon H-Y, Kim B-Y, Schumann P, Kroppenstedt RM, Noh H-J, Park C-W, Kwon S-W (2007) Knoellia aerolata sp. nov., isolated from an air sample in Korea. Int J Syst Evol Microbiol 57:2861–2864PubMedCrossRefGoogle Scholar
  37. Yamada C, Kato S, Ueno Y, Ishii M, Igarashi Y (2015) Conductive iron oxides accelerate thermophilic methanogenesis from acetate and propionate. J Biosci Bioeng 119:678–682PubMedCrossRefGoogle Scholar
  38. Yashiro Y, Sakai S, Ehara M, Miyazaki M, Yamaguchi T, Imachi H (2011) Methanoregula formicica sp. nov., a methane-producing archaeon isolated from methanogenic sludge. Int J Syst Evol Microbiol 61:53–59PubMedCrossRefGoogle Scholar
  39. Zhang D, Chen Y, Zhao Y, Zhu X (2010) New sludge pretreatment method to improve methane production in waste activated sludge digestion. Environ Sci Technol 44:4802–4808PubMedCrossRefGoogle Scholar
  40. Zhang Y, Feng Y, Yu Q, Xu Z, Quan X (2014) Enhanced high-solids anaerobic digestion of waste activated sludge by the addition of scrap iron. Bioresour Technol 159:297–304PubMedCrossRefGoogle Scholar
  41. Zhang J, Zhang Y, Quan X, Chen S (2015) Enhancement of anaerobic acidogenesis by integrating an electrochemical system into an acidogenic reactor: effect of hydraulic retention times (HRT) and role of bacteria and acidophilic methanogenic Archaea. Bioresour Technol 179:43–49PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Gahyun Baek
    • 1
  • Jaai Kim
    • 1
  • Seung Gu Shin
    • 2
  • Changsoo Lee
    • 1
    Email author
  1. 1.School of Urban and Environmental EngineeringUlsan National Institute of Science and Technology (UNIST)UlsanRepublic of Korea
  2. 2.School of Environmental Science and EngineeringPOSTECHPohangRepublic of Korea

Personalised recommendations