Abstract
Methanotrophs have recently gained interest as biocatalysts for mitigation of greenhouse gas emission and conversion of methane to value-added products; however, their slow growth has, at least partially, hindered their industrial application. A rapid isolation technique that specifically screens for the fastest-growing methanotrophs was developed using continuous cultivation with gradually increased dilution rates. Environmental samples collected from methane-rich environments were enriched in continuously stirred tank reactors with unrestricted supply of methane and air. The reactor was started at the dilution rate of 0.1 h−1, and the dilution rates were increased with an increment of 0.05 h−1 until the reactor was completely washed out. The shifts in the overall microbial population and methanotrophic community at each step of the isolation procedure were monitored with 16S rRNA amplicon sequencing. The predominant methanotrophic groups recovered after reactor operations were affiliated to the gammaproteobacterial genera Methylomonas and Methylosarcina. The methanotrophic strains isolated from the reactor samples collected at their respective highest dilution rates exhibited specific growth rates up to 0.40 h−1; the highest value reported for methanotrophs. The novel isolation method developed in this study significantly shortened the time and efforts needed for isolation of methanotrophs from environmental samples and was capable of screening for the methanotrophs with the fastest growth rates.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Auman AJ, Stolyar S, Costello AM, Lidstrom ME (2000) Molecular characterization of methanotrophic isolates from freshwater lake sediment. Appl Environ Microbiol 66:5259–5266. https://doi.org/10.1128/aem.66.12.5259-5266.2000
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 7:335–336. https://doi.org/10.1038/nmeth.f.303
Chin KJ, Liesack W, Janssen PH (2001) Opitutus terrae gen. nov., sp. nov., to accommodate novel strains of the division ‘Verrucomicrobia’ isolated from rice paddy soil. Int J Syst Evol Microbiol 51:1965–1968. https://doi.org/10.1099/00207713-51-6-1965
Conrado R, Gonzalez R (2014) Envisioning the bioconversion of methane to liquid fuels. Science 343:621–623. https://doi.org/10.1126/science.1246929
Crombie AT, Murrell JC (2014) Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris. Nature 510:148–151. https://doi.org/10.1038/nature13192
Demidenko A, Akberdin IR, Allemann M, Allen EE, Kalyuzhnaya MG (2017) Fatty acid biosynthesis pathways in Methylomicrobium buryatense 5G(B1). Front Microbiol 7:2167. https://doi.org/10.3389/fmicb.2016.02167
Dong T, Fei Q, Genelot M, Smith H, Laurens LML, Watson MJ, Pienkos PT (2017) A novel integrated biorefinery process for diesel fuel blendstock production using lipids from the methanotroph, Methylomicrobium buryatense. Energ Convers Manage 140:62–70. https://doi.org/10.1016/j.enconman.2017.02.075
Feeley JC, Gibson RJ, Gorman GW, Langford NC, Rasheed JK, Mackel DC, Baine WB (1979) Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol 10:437–441
Fei Q, Guarnieri MT, Tao L, Laurens LML, Dowe N, Pienkos PT (2014) Bioconversion of natural gas to liquid fuel: opportunities and challenges. Biotechnol Adv 32:596–614. https://doi.org/10.1016/j.biotechadv.2014.03.011
Hanke A, Hamann E, Sharma R, Geelhoed JS, Hargesheimer T, Kraft B, Meyer V, Lenk S, Osmers H, Wu R, Makinwa K, Hettich RL, Banfield JF, Tegetmeyer HE, Strous M (2014) Recoding of the stop codon UGA to glycine by a BD1-5/SN-2 bacterium and niche partitioning between Alpha- and Gammaproteobacteria in a tidal sediment microbial community naturally selected in a laboratory chemostat. Front Microbiol 5:231. https://doi.org/10.3389/fmicb.2014.00231
Haynes CA, Gonzalez R (2014) Rethinking biological activation of methane and conversion to liquid fuels. Nat Chem Biol 10:331–339. https://doi.org/10.1038/nchembio.1509
Henard CA, Smith H, Dowe N, Kalyuzhnaya MG, Pienkos PT, Guarnieri MT (2016) Bioconversion of methane to lactate by an obligate methanotrophic bacterium. Sci Rep 6:21585. https://doi.org/10.1038/srep21585
Hilger HA, Cranford DF, Barlaz MA (2000) Methane oxidation and microbial exopolymer production in landfill cover soil. Soil Biol Biochem 32:457–467. https://doi.org/10.1016/S0038-0717(99)00101-7
Hirayama H, Abe M, Miyazaki M, Nunoura T, Furushima Y, Yamamoto H, Takai K (2014) Methylomarinovum caldicuralii gen. nov., sp. nov., a moderately thermophilic methanotroph isolated from a shallow submarine hydrothermal system, and proposal of the family Methylothermaceae fam. nov. Int J Syst Evol Microbiol 64:989–999. https://doi.org/10.1099/ijs.0.058172-0
Hirayama H, Fuse H, Abe M, Miyazaki M, Nakamura T, Nunoura T, Furushima Y, Yamamoto H, Takai K (2013) Methylomarinum vadi gen. nov., sp. nov., a methanotroph isolated from two distinct marine environments. Int J Syst Evol Microbiol 63:1073–1082. https://doi.org/10.1099/ijs.0.040568-0
Ho A, Angel R, Veraart AJ, Daebeler A, Jia Z, Kim SY, Kerckhof F-M, Boon N, Bodelier PLE (2016) Biotic interactions in microbial communities as modulators of biogeochemical processes: methanotrophy as a model system. Front Microbiol 7:1285. https://doi.org/10.3389/fmicb.2016.01285
Hoefman S, van der Ha D, De Vos P, Boon N, Heylen K (2012) Miniaturized extinction culturing is the preferred strategy for rapid isolation of fast-growing methane-oxidizing bacteria. Microbial Biotechnol 5:368–378. https://doi.org/10.1111/j.1751-7915.2011.00314.x
Hugenholtz P, Goebel BM, Pace NR (1998) Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180:4765–4774
Joseph SJ, Hugenholtz P, Sangwan P, Osborne CA, Janssen PH (2003) Laboratory cultivation of widespread and previously uncultured soil bacteria. Appl Environ Microbiol 69:7210–7215. https://doi.org/10.1128/aem.69.12.7210-7215.2003
Kalyuzhnaya MG, Yang S, Rozova ON, Bringel F, Smalley NE, Clubb J, Konopka M, Orphan VJ, Beck D, Trotsenko YA, Vuilleumier S, Khmelenina VN, Lidstrom ME (2013) Highly efficient methane biocatalysis revealed in methanotrophic bacterium. Nat Commun 4:2785. https://doi.org/10.1038/ncomms3785
Kopylova E, Noé L, Touzet H (2012) SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28:3211–3217. https://doi.org/10.1093/bioinformatics/bts611
Matsuki T, Watanabe K, Fujimoto J, Miyamoto Y, Takada T, Matsumoto K, Oyaizu H, Tanaka R (2002) Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces. Appl Environ Microbiol 68:5445–5451. https://doi.org/10.1128/aem.68.11.5445-5451.2002
Olah GA, Goeppert A, Prakash GKS (2009) Production of methanol: from fossil fuels and bio-sources to chemical carbon dioxide recycling. In: Olah GA, Goeppert A, Prakash S (eds) Beyond oil and gas: the methanol economy, 2nd edn. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, pp 233–278
Oshkin IY, Beck DAC, Lamb AE, Tchesnokova V, Benuska G, McTaggart TL, Kalyuzhnaya MG, Dedysh SN, Lidstrom ME, Chistoserdova L (2014) Methane-fed microbial microcosms show differential community dynamics and pinpoint taxa involved in communal response. ISME J 9:1119–1129. https://doi.org/10.1038/ismej.2014.203
Pachauri RK, Meyer L, Pattner G-K, Stocker T (2015) IPCC, 2014: climate change 2014: synthesis report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change
Pieja AJ, Sundstrom ER, Criddle CS (2012) Cyclic, alternating methane and nitrogen limitation increases PHB production in a methanotrophic community. Bioresour Technol 107:385–392. https://doi.org/10.1016/j.biortech.2011.12.044
Pine L, George JR, Reeves MW, Harrell WK (1979) Development of a chemically defined liquid medium for growth of Legionella pneumophila. J Clin Microbiol 9:615–626
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541. https://doi.org/10.1128/aem.01541-09
Semrau JD, DiSpirito A, Yoon S (2010) Methanotrophs and copper. FEMS Microbiol Rev 34:496–531. https://doi.org/10.1111/j.1574-6976.2010.00212.x
Semrau JD, DiSpirito AA, Vuilleumier S (2011) Facultative methanotrophy: false leads, true results, and suggestions for future research. FEMS Microbiol Lett 323:1–12. https://doi.org/10.1111/j.1574-6968.2011.02315.x
Sheets JP, Ge X, Li Y-F, Yu Z, Li Y (2016) Biological conversion of biogas to methanol using methanotrophs isolated from solid-state anaerobic digestate. Bioresour Technol 201:50–57. https://doi.org/10.1016/j.biortech.2015.11.035
Spain JC, Nishino SF (1987) Degradation of 1,4-dichlorobenzene by a Pseudomonas sp. Appl Environ Microbiol 53:1010–1019
Strong PG, Xie S, Clarke WP (2015) Methane as a resource: can the methanotrophs add value? Environ Sci Technol 49:4001–4018. https://doi.org/10.1021/es504242n
Strong PJ, Kalyuzhnaya M, Silverman J, Clarke WP (2016) A methanotroph-based biorefinery: potential scenarios for generating multiple products from a single fermentation. Bioresour Technol 215:314–323. https://doi.org/10.1016/j.biortech.2016.04.099
van den Berg EM, van Dongen U, Abbas B, van Loosdrecht MCM (2015) Enrichment of DNRA bacteria in a continuous culture. ISME J 9:2153–2161. https://doi.org/10.1038/ismej.2015.26
Whittenbury R, Phillips KC, Wilkinson JF (1970) Enrichment, isolation and some properties of methane-utilizing bacteria. Microbiology 61:205–218. https://doi.org/10.1099/00221287-61-2-205
Wise MG, McArthur JV, Shimkets LJ (1999) Methanotroph diversity in landfill soil: isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis. Appl Environ Microbiol 65:4887–4897
Yan J, Im J, Yang Y, Löffler FE (2013) Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity. Phil Trans Royal Soc Lond B 368:20120320. https://doi.org/10.1098/rstb.2012.0320
Yan X, Chu F, Puri AW, Fu Y, Lidstrom ME (2016) Electroporation-based genetic manipulation in type I methanotrophs. Appl Environ Microbiol 82:2062–2069. https://doi.org/10.1128/aem.03724-1
Funding
This research was financially supported by the National Research Foundation of Korea (NRF) (grant no. 2015M3D3A1A01064881) and the “R&D Center for Reduction of Non-CO2 Greenhouse Gases” (grant no. 2017002420002) funded by Korea Ministry of Environment (MOE) as “Global Top Environment R&D Program”. The authors were also financially supported by Korea Ministry of Land, Infrastructure and Transport (MOLIT) as U-City Master and Doctor Course Grant Program and the Brain Korea 21 Plus Project (grant no. 21A20132000003).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
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.
Electronic supplementary material
ESM 1
(XLSX 134 kb)
Rights and permissions
About this article
Cite this article
Kim, J., Kim, D.D. & Yoon, S. Rapid isolation of fast-growing methanotrophs from environmental samples using continuous cultivation with gradually increased dilution rates. Appl Microbiol Biotechnol 102, 5707–5715 (2018). https://doi.org/10.1007/s00253-018-8978-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-018-8978-5