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Cultivation of previously uncultured microorganisms with a continuous-flow down-flow hanging sponge (DHS) bioreactor, using a syntrophic archaeon culture obtained from deep marine sediment as a case study

  • Flemming, H.-C. & Wuertz, S. Bacteria and archaea on Earth and their abundance in biofilms. Nat. Rev. Microbiol. 17, 247–260 (2019).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Inagaki, F. et al. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor. Science 349, 420–424 (2015).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Bar-On, Y. M., Phillips, R. & Milo, R. The biomass distribution on Earth. Proc. Natl Acad. Sci. USA 115, 6506–6511 (2018).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • D’Hondt, K. et al. Microbiome innovations for a sustainable future. Nat. Microbiol. 6, 138–142 (2021).

    PubMed 
    Article 
    CAS 

    Google Scholar 

  • Lloyd, K. G., Steen, A. D., Ladau, J., Yin, J. & Crosby, L. Phylogenetically novel uncultured microbial cells dominate earth microbiomes. mSystems 3, e00055-18 (2018).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Lewis, W. H., Tahon, G., Geesink, P., Sousa, D. Z. & Ettema, T. J. G. Innovations to culturing the uncultured microbial majority. Nat. Rev. Microbiol. 19, 225–240 (2021).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Nayfach, S. et al. A genomic catalog of Earth’s microbiomes. Nat. Biotechnol. 39, 499–509 (2021).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Rinke, C. et al. Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437 (2013).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Hugenholtz, P. Exploring prokaryotic diversity in the genomic era. Genome Biol. 3, REVIEWS0003 (2002).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Engelen, B. & Imachi, H. Cultivation of subseafloor prokaryotic life in developments in marine geology. In Earth and Life Processes Discovered from Subseafloor Environment. Vol. 7 (eds. Stein, R., Blackman, D., Inagaki, F. & Larsen, H.-L.) 197–209 (Elsevier, 2014).

  • Baker, B. J., Appler, K. E. & Gong, X. New microbial biodiversity in marine sediments. Annu. Rev. Mar. Sci. 13, 161–175 (2021).

    Article 

    Google Scholar 

  • Hoshino, T. et al. Global diversity of microbial communities in marine sediment. Proc. Natl Acad. Sci. USA 117, 27587–27597 (2020).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Tahon, G., Geesink, P. & Ettema, T. J. G. Expanding archaeal diversity and phylogeny: past, present, and future. Annu. Rev. Microbiol. 75, 359–381 (2021).

    PubMed 
    Article 
    CAS 

    Google Scholar 

  • Bhattarai, S., Cassarini, C. & Lens, P. N. L. Physiology and distribution of archaeal methanotrophs that couple anaerobic oxidation of methane with sulfate reduction. Microbiol. Mol. Biol. Rev. 83, e00074-18 (2019).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Krukenberg, V. et al. Gene expression and ultrastructure of meso- and thermophilic methanotrophic consortia. Environ. Microbiol. 20, 1651–1666 (2018).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Wegener, G., Krukenberg, V., Ruff, S. E., Kellermann, M. Y. & Knittel, K. Metabolic capabilities of microorganisms involved in and associated with the anaerobic oxidation of methane. Front. Microbiol. 7, 46 (2016).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Agrawal, L. K. et al. Treatment of raw sewage in a temperate climate using a UASB reactor and the hanging sponge cubes process. Water Sci. Technol. 36, 433–440 (1997).

    CAS 
    Article 

    Google Scholar 

  • Tyagi, V. K. et al. Future perspectives of energy saving down-flow hanging sponge (DHS) technology for wastewater valorization—a review. Rev. Environ. Sci. Biotechnol. 20, 389–418 (2021).

    Article 

    Google Scholar 

  • Namita Maharjan, N. et al. Downflow hanging sponge system: a self-sustaining option for wastewater treatment. In Wastewater Treatment (IntechOpen, London, UK, 2020) Available at https://www.intechopen.com/online-first/74120

  • Nurmiyanto, A. & Ohashi, A. Downflow hanging sponge (DHS) reactor for wastewater treatment—a short review. MATEC Web Conf. 280, 05004 (2019).

    CAS 
    Article 

    Google Scholar 

  • Hatamoto, M., Okubo, T., Kubota, K. & Yamaguchi, T. Characterization of downflow hanging sponge reactors with regard to structure, process function, and microbial community compositions. Appl. Microbiol. Biotechnol. 102, 10345–10352 (2018).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Tandukar, M., Uemura, S., Ohashi, A. & Harada, H. Combining UASB and the ‘fourth generation’ down-flow hanging sponge reactor for municipal wastewater treatment. Water Sci. Technol. 53, 209–218 (2006).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Chuang, H.-P. et al. Microbial community that catalyzes partial nitrification at low oxygen atmosphere as revealed by 16S rRNA and amoA genes. J. Biosci. Bioeng. 104, 525–528 (2007).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Imachi, H. et al. Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor. ISME J. 5, 1913–1925 (2011).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Aoki, M. et al. A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor. PLoS ONE 9, e105356 (2014).

    PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 

  • Kato, S. et al. Biotic manganese oxidation coupled with methane oxidation using a continuous-flow bioreactor system under marine conditions. Water Sci. Technol. 76, 1781–1795 (2017).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Imachi, H. et al. Cultivable microbial community in 2-km-deep, 20-million-year-old subseafloor coalbeds through ~1000 days anaerobic bioreactor cultivation. Sci. Rep. 9, 2305 (2019).

    PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 

  • Imachi, H. et al. Pelolinea submarina gen. nov., sp. nov., an anaerobic, filamentous bacterium of the phylum Chloroflexi isolated from subseafloor sediment. Int. J. Syst. Evol. Microbiol. 64, 812–818 (2014).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Imachi, H. et al. Sedimentibacter acidaminivorans sp. nov., an anaerobic, amino-acid-utilizing bacterium isolated from marine subsurface sediment. Int. J. Syst. Evol. Microbiol. 66, 1293–1300 (2016).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Imachi, H. et al. Isolation of an archaeon at the prokaryote-eukaryote interface. Nature 577, 519–525 (2020).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Miyazaki, M. et al. Sphaerochaeta multiformis sp. nov., an anaerobic, psychrophilic bacterium isolated from subseafloor sediment, and emended description of the genus Sphaerochaeta. Int. J. Syst. Evol. Microbiol. 64, 4147–4154 (2014).

    PubMed 
    Article 
    CAS 

    Google Scholar 

  • Miyazaki, M. et al. Spirochaeta psychrophila sp. nov., a psychrophilic spirochaete isolated from subseafloor sediment, and emended description of the genus Spirochaeta. Int. J. Syst. Evol. Microbiol. 64, 2798–2804 (2014).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Nakahara, N. et al. Aggregatilinea lenta gen. nov., sp. nov., a slow-growing, facultatively anaerobic bacterium isolated from subseafloor sediment, and proposal of the new order Aggregatilineales ord. nov. within the class Anaerolineae of the phylum Chloroflexi. Int. J. Syst. Evol. Microbiol. 69, 1185–1194 (2019).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Spang, A. et al. Complex archaea that bridge the gap between prokaryotes and eukaryotes. Nature 521, 173–179 (2015).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Zaremba-Niedzwiedzka, K. et al. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature 541, 353–358 (2017).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Liu, Y. et al. Expanded diversity of Asgard archaea and their relationships with eukaryotes. Nature 593, 553–557 (2021).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Ruff, S. E. et al. Global dispersion and local diversification of the methane seep microbiome. Proc. Natl Acad. Sci. USA 112, 4015–4020 (2015).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Chadwick, G. et al. Comparative genomics reveals electron transfer and syntrophic mechanisms differentiating methanotrophic and methanogenic archaea. PLoS Biol. 20, e3001508 (2022).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Cario, A., Oliver, G. C. & Rogers, K. L. Exploring the deep marine biosphere: challenges, innovations, and opportunities. Front. Earth Sci. 7, 225 (2019).

    Article 

    Google Scholar 

  • Jørgensen, B. B. & Boetius, A. Feast and famine—microbial life in the deep-sea bed. Nat. Rev. Microbiol. 5, 770–781 (2007).

    PubMed 
    Article 
    CAS 

    Google Scholar 

  • Hoehler, T. M. & Jørgensen, B. B. Microbial life under extreme energy limitation. Nat. Rev. Microbiol. 11, 83–94 (2013).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Schink, B. & Stams, A. J. M. Syntrophism among prokaryotes. In The Prokaryotes: Prokaryotic Communities and Ecophysiology (eds. Rosenberg, E. et al.) 471–493 (Springer, 2013).

  • de Bok, F. A. M. et al. The first true obligately syntrophic propionate-oxidizing bacterium, Pelotomaculum schinkii sp. nov., co-cultured with Methanospirillum hungatei, and emended description of the genus Pelotomaculum. Int. J. Syst. Evol. Microbiol. 55, 1697–1703 (2005).

    PubMed 
    Article 
    CAS 

    Google Scholar 

  • Imachi, H. et al. Pelotomaculum propionicicum sp. nov., an anaerobic, mesophilic, obligately syntrophic, propionate-oxidizing bacterium. Int. J. Syst. Evol. Microbiol. 57, 1487–1492 (2007).

    PubMed 
    Article 

    Google Scholar 

  • Qiu, Y.-L. et al. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the first cultured anaerobe capable of degrading phenol to acetate in obligate syntrophic associations with a hydrogenotrophic methanogen. Appl. Environ. Microbiol. 74, 2051–2058 (2008).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Matsushita, S. et al. Anti-bacterial effects of MnO2 on the enrichment of manganese-oxidizing bacteria in downflow hanging sponge reactors. Microbes Environ. 35, ME20052 (2020).

    PubMed Central 

    Google Scholar 

  • Momper, L. et al. Rectinema subterraneum sp. nov, a chemotrophic spirochaete isolated from the deep terrestrial subsurface. Int. J. Syst. Evol. Microbiol. 70, 4739–4747 (2020).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Chuang, H.-P., Ohashi, A., Imachi, H., Tandukar, M. & Harada, H. Effective partial nitrification to nitrite by down-flow hanging sponge reactor under limited oxygen condition. Water Res. 41, 295–302 (2007).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Hatamoto, M., Koshiyama, Y., Kindaichi, T., Ozaki, N. & Ohashi, A. Enrichment and identification of methane-oxidizing bacteria by using down-flow hanging sponge bioreactors under low methane concentration. Ann. Microbiol. 61, 683–687 (2010).

    Article 
    CAS 

    Google Scholar 

  • Hatamoto, M., Yamamoto, H., Kindaichi, T., Ozaki, N. & Ohashi, A. Biological oxidation of dissolved methane in effluents from anaerobic reactors using a down-flow hanging sponge reactor. Water Res. 44, 1409–1418 (2010).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Hatamoto, M., Miyauchi, T., Kindaichi, T., Ozaki, N. & Ohashi, A. Dissolved methane oxidation and competition for oxygen in down-flow hanging sponge reactor for post-treatment of anaerobic wastewater treatment. Bioresour. Technol. 102, 10299–10304 (2011).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Hatamoto, M. et al. Potential of nitrous oxide conversion in batch and down-flow hanging sponge bioreactor systems. Sustain. Environ. Res. 24, 117–128 (2014).

    Google Scholar 

  • Cao, L. T. T. et al. Biological oxidation of Mn(II) coupled with nitrification for removal and recovery of minor metals by downflow hanging sponge reactor. Water Res. 68, 545–553 (2015).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Yamaguchi, T. et al. A novel approach for toluene gas treatment using a downflow hanging sponge reactor. Appl. Microbiol. Biotechnol. 102, 5625–5634 (2018).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Matsushita, S. et al. Production of biogenic manganese oxides coupled with methane oxidation in a bioreactor for removing metals from wastewater. Water Res. 130, 224–233 (2018).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Onodera, T. et al. Characterization of the retained sludge in a down-flow hanging sponge (DHS) reactor with emphasis on its low excess sludge production. Bioresour. Technol. 136, 169–175 (2013).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Miyaoka, Y., Hatamoto, M., Yamaguchi, T. & Syutsubo, K. Eukaryotic community shift in response to organic loading rate of an aerobic trickling filter (down-flow hanging sponge reactor) treating domestic sewage. Microb. Ecol. 73, 801–814 (2016).

    PubMed 
    Article 
    CAS 

    Google Scholar 

  • Park, M.-O., Ikenaga, H. & Watanabe, K. Phage diversity in a methanogenic digester. Microb. Ecol. 53, 98–103 (2006).

    PubMed 
    Article 
    CAS 

    Google Scholar 

  • Wu, Q. & Liu, W.-T. Determination of virus abundance, diversity and distribution in a municipal wastewater treatment plant. Water Res. 43, 1101–1109 (2009).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Chien, I.-C., Meschke, J. S., Gough, H. L. & Ferguson, J. F. Characterization of persistent virus-like particles in two acetate-fed methanogenic reactors. PLoS One 8, e81040 (2013).

    PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 

  • Girguis, P. R., Orphan, V. J., Hallam, S. J. & DeLong, E. F. Growth and methane oxidation rates of anaerobic methanotrophic archaea in a continuous-flow bioreactor. Appl. Environ. Microbiol. 69, 5472–5482 (2003).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Zhang, Y., Maignien, L., Zhao, X., Wang, F. & Boon, N. Enrichment of a microbial community performing anaerobic oxidation of methane in a continuous high-pressure bioreactor. BMC Microbiol. 11, 137 (2011).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Sauer, P., Glombitza, C. & Kallmeyer, J. A system for incubations at high gas partial pressure. Front. Microbiol. 3, 25 (2012).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Bhattarai, S. et al. Enrichment of sulfate reducing anaerobic methane oxidizing community dominated by ANME-1 from Ginsburg Mud Volcano (Gulf of Cadiz) sediment in a biotrickling filter. Bioresour. Technol. 259, 433–441 (2018).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Cassarini, C. et al. Enrichment of anaerobic methanotrophs in biotrickling filters using different sulfur compounds as electron acceptor. Environ. Eng. Sci. 36, 431–443 (2018).

    Article 
    CAS 

    Google Scholar 

  • Cassarini, C. et al. Anaerobic methane oxidation coupled to sulfate reduction in a biotrickling filter: reactor performance and microbial community analysis. Chemosphere 236, 124290 (2019).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Adams, M. M., Hoarfrost, A. L., Bose, A., Joye, S. B. & Girguis, P. R. Anaerobic oxidation of short-chain alkanes in hydrothermal sediments: potential influences on sulfur cycling and microbial diversity. Front. Microbiol. 4, 110 (2013).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Machdar, I., Sekiguchi, Y., Sumino, H., Ohashi, A. & Harada, H. Combination of a UASB reactor and a curtain type DHS (downflow hanging sponge) reactor as a cost-effective sewage treatment system for developing countries. Water Sci. Technol. 42, 83–88 (2000).

    CAS 
    Article 

    Google Scholar 

  • Judd, S. The status of membrane bioreactor technology. Trends Biotechnol. 26, 109–116 (2008).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Smith, A. L. et al. Perspectives on anaerobic membrane bioreactor treatment of domestic wastewater: a critical review. Bioresour. Technol. 122, 149–159 (2012).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Meulepas, R. J. W. et al. Enrichment of anaerobic methanotrophs in sulfate-reducing membrane bioreactors. Biotechnol. Bioeng. 104, 458–470 (2009).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Jagersma, G. C. et al. Microbial diversity and community structure of a highly active anaerobic methane-oxidizing sulfate-reducing enrichment. Environ. Microbiol. 11, 3223–3232 (2009).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Lettinga, G., van Velsen, A. F. M., Hobma, S. W., de Zeeuw, W. & Klapwijk, A. Use of the upflow sludge blanket (USB) reactor concept for biological wastewater treatment, especially for anaerobic treatment. Biotechnol. Bioeng. 22, 699–734 (1980).

    CAS 
    Article 

    Google Scholar 

  • Sekiguchi, Y., Kamagata, Y., Nakamura, K., Ohashi, A. & Harada, H. Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules. Appl. Environ. Microbiol. 65, 1280–1288 (1999).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Tawfik, A., Ohashi, A. & Harada, H. Sewage treatment in a combined up-flow anaerobic sludge blanket (UASB)-down-flow hanging sponge (DHS) system. Biochem. Eng. J. 29, 210–219 (2006).

    CAS 
    Article 

    Google Scholar 

  • Onodera, T. et al. Development of a sixth-generation down-flow hanging sponge (DHS) reactor using rigid sponge media for post-treatment of UASB treating municipal sewage. Bioresour. Technol. 152, 93–100 (2014).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Tandukar, M., Ohashi, A. & Harada, H. Performance comparison of a pilot-scale UASB and DHS system and activated sludge process for the treatment of municipal wastewater. Water Res. 41, 2697–2705 (2007).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Hallam, S. J. et al. Reverse methanogenesis: testing the hypothesis with environmental genomics. Science 305, 1457–1462 (2004).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Nauhaus, K., Albrecht, M., Elvert, M., Boetius, A. & Widdel, F. In vitro cell growth of marine archaeal-bacterial consortia during anaerobic oxidation of methane with sulfate. Environ. Microbiol. 9, 187–196 (2007).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Wegener, G., Niemann, H., Elvert, M., Hinrichs, K. & Boetius, A. Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane. Environ. Microbiol. 10, 2287–2298 (2008).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Hu, H., Natarajan, V. P. & Wang, F. Towards enriching and isolation of uncultivated archaea from marine sediments using a refined combination of conventional microbial cultivation methods. Mar. Life Sci. Technol. 3, 231–242 (2021).

    Article 
    CAS 

    Google Scholar 

  • Balch, W. E., Fox, G. E., Magrum, L. J., Woese, C. R. & Wolfe, R. S. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43, 260–296 (1979).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Yokooji, Y. et al. Pantoate kinase and phosphopantothenate synthetase, two novel enzymes necessary for CoA biosynthesis in the Archaea. J. Biol. Chem. 284, 28137–28145 (2009).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Moench, T. & Zeikus, J. G. An improved preparation method for a titanium (III) media reductant. J. Microbiol. Methods 1, 199–202 (1983).

    CAS 
    Article 

    Google Scholar 

  • Miyashita, A. et al. Development of 16S rRNA gene-targeted primers for detection of archaeal anaerobic methanotrophs (ANMEs). FEMS Microbiol. Lett. 297, 31–37 (2009).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Sekiguchi, Y. et al. Sequence-specific cleavage of 16S rRNA for rapid and quantitative detection of particular groups of anaerobes in bioreactors. Water Sci. Technol. 52, 107–113 (2005).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • Meechan, P. J. & Wilson, C. Use of ultraviolet lights in biological safety cabinets: a contrarian view. Appl. Biosaf. 11, 222–227 (2006).

    Article 

    Google Scholar 

  • Imachi, H., Sekiguchi, Y., Kamagata, Y., Ohashi, A. & Harada, H. Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge. Appl. Environ. Microbiol. 66, 3608–3615 (2000).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Stams, A. J., Grolle, K. C., Frijters, C. T. & van Lier, J. B. Enrichment of thermophilic propionate-oxidizing bacteria in syntrophy with Methanobacterium thermoautotrophicum or Methanobacterium thermoformicicum. Appl. Environ. Microbiol. 58, 346–352 (1992).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • Uchino, Y. & Suzuki, K. A simple preparation of liquid media for the cultivation of strict anaerobes. J. Pet. Environ. Biotechnol. S3, 001 (2011).

    Google Scholar 

  • Akinyemi, T. S., Shao, N. & Whitman, W. B. Genus Methanothrix. In Bergey’s Manual of Systematics of Archaea and Bacteria (John Wiley & Sons, 2020).


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