Conductive carbon nanoparticles inhibit methanogens and stabilize hydrogen production in microbial electrolysis cells

  • Kazuki Fujinawa
  • Misa Nagoya
  • Atsushi Kouzuma
  • Kazuya WatanabeEmail author
Bioenergy and biofuels


Nanosized conductive carbon materials have been reported to stimulate methanogenesis by anaerobic microbiomes, while other studies have shown their antimicrobial activities. The present study examined effects of conductive carbon nanoparticles (carbon black Vulcan, CB) on methanogenesis from glucose by anaerobic sludge. We found that a relatively high concentration (e.g., 2% w/v) of CB entirely inhibited the methanogenesis, where a substantial amount of acetate was accumulated after degradation of glucose. Quantitative real-time PCR assays and metabarcoding of 16S rRNA amplicons revealed that, while bacteria were stably present irrespective of the presence and absence of CB, archaea, in particular methanogens, were largely decreased in the presence of CB. Pure-culture experiments showed that methanogenic archaea were more seriously damaged by CB than fermentative bacteria. These results demonstrate that CB specifically inhibits methanogens in anaerobic sludge. We attempted to supplement cathode chambers of microbial electrolysis cells with CB for inhibiting methanogenesis from hydrogen, demonstrating that hydrogen is stably produced in the presence of CB.


Methanogenesis Methanogen Metabarcoding Conductive nanoparticle Microbial electrolysis cells 



This study was funded by JSPS KAKENHI (Grant Number 15H01753).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2019_9946_MOESM1_ESM.pdf (557 kb)
ESM 1 (PDF 556 kb)


  1. Bhattacharyya A, Majumder NS, Basak P, Mukherji S, Roy D, Nag S, Haldar A, Chattopadhyay D, Mitra S, Bhattacharyya M, Ghosh A (2015) Diversity and distribution of archaea in the mangrove sediment of Sundarbans. Archaea 2015:968582CrossRefGoogle Scholar
  2. Catal T, Lesnik KL, Liu H (2015) Suppression of methanogenesis for hydrogen production in single-chamber microbial electrolysis cells using various antibiotics. Bioresour Technol 187:77–83CrossRefGoogle Scholar
  3. Chae KJ, Choi MJ, Kim KY, Ajayi FF, Chang IS, Kim IS (2010) Selective inhibition of methanogens for the improvement of biohydrogen production in microbial electrolysis cells. Int J Hydrog Energy 35:13379–13386CrossRefGoogle Scholar
  4. Chen S, Rotaru A-E, Shrestha PM, Malvankar NS, Liu F, Fan W (2014) Promoting interspecies electron transfer with biochar. Sci Rep 4:5019CrossRefGoogle Scholar
  5. Daffonchio D, Thaveesri J, Verstraete W (1995) Contact angle measurement and cell hydrophobicity of granular sludge from upflow anaerobic sludge bed reactors. Appl Environ Microbiol 61:3676–3680Google Scholar
  6. Ferry JG (2012) Methanogenesis: ecology, physiology, biochemistry & genetics. Springer Science & Business Media, BerlinGoogle Scholar
  7. Freguia S, Rabaey K, Yuan Z, Keller J (2007) Electron and carbon balances in microbial fuel cells reveal temporary bacterial storage behavior during electricity generation. Environ Sci Technol 41:2915–2921CrossRefGoogle Scholar
  8. Fu L, Zhou T, Wang J, You L, Lu Y, Yu L, Zhou S (2019) NanoFe3O4 as solid electron shuttles to accelerate acetotrophic methanogenesis by Methanosarcina barkeri. Front Microbiol 10:388CrossRefGoogle Scholar
  9. Hang J, Desai V, Zavaljevski N, Yang Y, Lin X, Satya RV, Martinez LJ, Blaylock JM, Jarman RG, Thomas SJ, Kuschner RA (2014) 16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles. Microbiome 2:31CrossRefGoogle Scholar
  10. Hou Y, Luo H, Liu G, Zhang R, Li J, Fu S (2014) Improved hydrogen production in the microbial electrolysis cell by inhibiting methanogenesis using ultraviolet irradiation. Environ Sci Technol 48:10482–10488CrossRefGoogle Scholar
  11. Ishii S, Kosaka T, Hori K, Hotta Y, Watanabe K (2005) Coaggregation facilitates interspecies hydrogen transfer between Pelotomaculum thermopropionicum and Methanothermobacter thermautotrophicus. Appl Environ Microbiol 71:7838–7845CrossRefGoogle Scholar
  12. Kang S, Pinault M, Pfefferle LD, Elimelech M (2007) Single-walled carbon nanotubes exhibit strong antimicrobial activity. Langmuir 23:8670–8673CrossRefGoogle Scholar
  13. Kato S, Hashimoto K, Watanabe K (2012a) Microbial interspecies electron transfer mediated by electric currents through conductive minerals. Proc Natl Acad Sci U S A 109:10042–10049CrossRefGoogle Scholar
  14. Kato S, Hashimoto K, Watanabe K (2012b) Methanogenesis facilitated by electric syntrophy via (semi) conductive iron-oxide minerals. Environ Microbiol 14:1646–1654CrossRefGoogle Scholar
  15. Kouzuma A, Kato S, Watanabe K (2015) Microbial interspecies interactions: recent findings in syntrophic consortia. Front Microbiol 6:477Google Scholar
  16. Li LL, Tong ZH, Fang CY, Chu J, Yu HQ (2015) Response of anaerobic granular sludge to single-wall carbon nanotube exposure. Water Res 70:1–8CrossRefGoogle Scholar
  17. Liu F, Rotaru AE, Shrestha PM, Malvankar NS, Nevin KP, Lovley DR (2012) Promoting direct interspecies electron transfer with activated carbon. Energy Environ Sci 5:8982–8989CrossRefGoogle Scholar
  18. Logan BE, Call D, Cheng S, Hamelers HV, Sleutels TH, Jeremiasse AW, Rozendal RA (2008) Microbial electrolysis cells for high yield hydrogen gas production from organic matter. Environ Sci Technol 42:8630–8640CrossRefGoogle Scholar
  19. Martins G, Salvador AF, Pereira L, Alves MM (2018) Methane production and conductive materials: a critical review. Environ Sci Technol 52:10241–10253CrossRefGoogle Scholar
  20. Mata-Alvarez J, Mace S, Llabres P (2000) Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresour Technol 74:3–16CrossRefGoogle Scholar
  21. Mayer F, Lurz R, Schoberth S (1977) Electron microscopic investigation of the hydrogen-oxidizing acetate-forming anaerobic bacterium Acetobacterium woodii. Arch Microbiol 115:207–213CrossRefGoogle Scholar
  22. Moore AD, Holmes SM, Roberts EP (2012) Evaluation of porous carbon substrates as catalyst supports for the cathode of direct methanol fuel cells. RSC Adv 2:1669–1674CrossRefGoogle Scholar
  23. Nepal D, Balasubramanian S, Simonian AL, Davis VA (2008) Strong antimicrobial coatings: single-walled carbon nanotubes armored with biopolymers. Nano Lett 8:1896–1901CrossRefGoogle Scholar
  24. Newton GJ, Mori S, Nakamura R, Hashimoto K, Watanabe K (2009) Analyses of current-generating mechanisms of Shewanella loihica PV-4 and Shewanella oneidensis MR-1 in microbial fuel cells. Appl Environ Microbiol 75:7674–7681CrossRefGoogle Scholar
  25. Park HS, Kim BH, Kim HS, Kim HJ, Kim GT, Kim M, Chang IS, Park YK, Chang HI (2001) A novel electrochemically active and Fe (III)-reducing bacterium phylogenetically related to Clostridium butyricum isolated from a microbial fuel cell. Anaerobe 7:297–306CrossRefGoogle Scholar
  26. Pérez-Rodríguez S, Pastor E, Lázaro MJ (2018) Electrochemical behavior of the carbon black Vulcan XC-72R: influence of the surface chemistry. Int J Hydrog Energy 43:7911–7922CrossRefGoogle Scholar
  27. Rotaru AE, Shrestha PM, Liu F, Markovaite B, Chen S, Nevin KP, Lovley DR (2014) Direct interspecies electron transfer between Geobacter metallireducens and Methanosarcina barkeri. Appl Environ Microbiol 80:4599–4605CrossRefGoogle Scholar
  28. Rozendal RA, Hamelers HVM, Euverink GJW, Metz SJ, Buisman CJN (2006) Principle and perspectives of hydrogen production through biocatalyzed electrolysis. Int J Hydrog Energy 31:1632e40CrossRefGoogle Scholar
  29. Salvador AF, Martins G, Melle-Franco M, Serpa R, Stams AJ, Cavaleiro AJ, Pereira A, Alves MM (2017) Carbon nanotubes accelerate methane production in pure cultures of methanogens and in a syntrophic coculture. Environ Microbiol 19:2727–2739CrossRefGoogle Scholar
  30. Shah FA, Mahmood Q, Rashid N, Pervez A, Raja IA, Shah MM (2015) Co-digestion, pretreatment and digester design for enhanced methanogenesis. Renew Sust Energ Rev 42:627–642CrossRefGoogle Scholar
  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. Stams AJ, Plugge CM (2009) Electron transfer in syntrophic communities of anaerobic bacteria and archaea. Nat Rev Microbiol 7:568–577CrossRefGoogle Scholar
  33. Steinberg LM, Regan JM (2009) mcrA-targeted real-time quantitative PCR method to examine methanogen communities. Appl Environ Microbiol 75:4435–4442CrossRefGoogle Scholar
  34. Summers ZM, Fogarty HE, Leang C, Franks AE, Malvankar NS, Lovley DR (2010) Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria. Science 330:1413–1415CrossRefGoogle Scholar
  35. Takahashi S, Miyahara M, Kouzuma A, Watanabe K (2016) Electricity generation from rice bran in microbial fuel cells. Bioresour Bioprocess 3:50CrossRefGoogle Scholar
  36. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefGoogle Scholar
  37. Watanabe K, Kodama Y, Harayama S (2001) Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting. J Microbiol Methods 44:253–262CrossRefGoogle Scholar
  38. Wen YH, Shao L, Zhao PC, Wang BY, Cao GP, Yang YS (2017) Carbon coated stainless steel mesh as a low-cost and corrosion-resistant current collector for aqueous rechargeable batteries. J Mater Chem 5:15752–15758CrossRefGoogle Scholar
  39. Xu S, He C, Luo L, Liu F, He P, Cui L (2015) Comparing activated carbon of different particle sizes on enhancing methane generation in upflow anaerobic digester. Bioresour Technol 196:606–612CrossRefGoogle Scholar
  40. Yadav T, Mungray AA, Mungray AK (2016) Effect of multiwalled carbon nanotubes on UASB microbial consortium. Environ Sci Pollut Res 23:4063–4072CrossRefGoogle Scholar
  41. Yeates C, Gillings MR, Davison AD, Altavilla N, Veal DA (1998) Methods for microbial DNA extraction from soil for PCR amplification. Biol Proced Online 1:40–47CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Kazuki Fujinawa
    • 1
    • 2
  • Misa Nagoya
    • 1
  • Atsushi Kouzuma
    • 1
  • Kazuya Watanabe
    • 1
    Email author
  1. 1.School of Life SciencesTokyo University of Pharmacy and Life SciencesHachiojiJapan
  2. 2.Ichikawa Co. Ltd.TokyoJapan

Personalised recommendations