Skip to main content
SpringerLink
Log in

Isolation and Cultivation of Anaerobes

  • Chapter
  • First Online:
Anaerobes in Biotechnology

Part of the book series: Advances in Biochemical Engineering/Biotechnology ((ABE,volume 156))

Abstract

Anaerobic microorganisms play important roles in different biotechnological processes. Their complex metabolism and special cultivation requirements have led to less isolated representatives in comparison to their aerobic counterparts. In view of that, the isolation and cultivation of anaerobic microorganisms is still a promising venture, and conventional methodologies as well as considerations and modifications are presented here. An insight into new methodologies and devices as well as a discussion on future perspectives for the cultivation of anaerobes may open the prospects of the exploitation of these microorganisms as a source for biotechnology.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Abbreviations

FCM:

Flow cytometry

NGS:

Next generation sequencing

PVC:

Poly(vinyl chloride)

References

  1. Goldstein EJ (1995) Anaerobes under assault: from cottage industry to industrialization of medicine and microbiology. Clin Infect Dis 20:S112–S116

    Article  Google Scholar 

  2. Antoni D, Zverlov VV, Schwarz WH (2007) Biofuels from microbes. Appl Microbiol Biotechnol 77:23–35

    Article  CAS  Google Scholar 

  3. Kumar M, Gayen K (2011) Developments in biobutanol production: new insights. Appl Energy 88:1999–2012

    Article  CAS  Google Scholar 

  4. Tracy BP, Jones SW, Fast AG, Indurthi DC, Papoutsakis ET (2012) Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications. Curr Opin Biotechnol 23:364–381

    Article  CAS  Google Scholar 

  5. Khanal SK (2008) Anaerobic biotechnology for bioenergy production. Principles and applications. Wiley-Blackwell, USA

    Book  Google Scholar 

  6. Brulc JM, Antonopoulos DA, Miller MEB, Wilson MK, Yannarell AC, Dinsdale EA, Edwards RE, Frank ED, Emerson JB, Wacklin P, Coutinho PM, Henrissat B, Nelson KE, White BA (2009) Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases. Proc Nat Acad Sci 106:1948–1953

    Article  CAS  Google Scholar 

  7. Buerger S, Spoering A, Gavrish E, Leslin C, Ling L, Epstein SS (2012) Microbial scout hypothesis and microbial discovery. Appl Environ Microbiol 78:3229–3233

    Article  CAS  Google Scholar 

  8. Epstein SS (2013) The phenomenon of microbial uncultivability. Curr Opin Microbiol 16:636–642

    Article  CAS  Google Scholar 

  9. Zehnder AJB, Stumm W (1988) Geochemistry and biogeochemistry of anaerobic habitats. In: Zehnder AJB (ed) Biology of anaerobic microorganisms. John Wiley, New York, pp 1–38

    Google Scholar 

  10. Tamaru Y, Miyake H, Kuroda K, Ueda M, Doi RH (2010) Comparative genomics of the mesophilic cellulosome‐producing Clostridium cellulovorans and its application to biofuel production via consolidated bioprocessing. Environ Technol 31:889–903

    Article  CAS  Google Scholar 

  11. Gosalbes MJ, Durbán A, Pignatelli M, Abellan JJ, Jiménez-Hernández N, Pérez-Cobas AE, Latorre A, Moya A (2011) Metatranscriptomic approach to analyze the functional human gut microbiota. PLoS One 6, e17447. doi:10.1371/journal.pone.0017447

    Article  CAS  Google Scholar 

  12. Vanwonterghem I, Jensen PD, Ho DP, Batstone DJ, Tyson GW (2014) Linking microbial community structure, interactions and function in anaerobic digesters using new molecular techniques. Curr Opin Biotechnol 27:55–64

    Article  CAS  Google Scholar 

  13. Qi M, Wang P, O'Toole N, Barboza PS, Ungerfeld E, Leigh MB, Selinger LB, Butler G, Tsang A, McAllister TA, Forster RJ (2011) Snapshot of the eukaryotic gene expression in muskoxen rumen—a metatranscriptomic approach. PLoS One 6, e20521. doi:10.1371/journal.pone.0020521

    Article  CAS  Google Scholar 

  14. Lü F, Bize A, Guillot A, Monnet V, Madigou C, Chapleur O, Mazéus L, He P, Bouchez T (2014) Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity. ISME J 8:88–102

    Article  Google Scholar 

  15. Busk PK, Lange L (2013) Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs. Appl Environ Microbiol 79:3380–3391

    Article  CAS  Google Scholar 

  16. Nielsen HB, Almeida M, Juncker AS, Rasmussen S, Li J, Sunagawa S, Ehrlich SD (2014) Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes. Nat Biotechnol 32:822–828

    Article  CAS  Google Scholar 

  17. Aßhauer KP, Wemheuer B, Daniel R, Meinicke P (2015) Tax4Fun: predicting functional profiles from metagenomic 16S rRNA data. Bioinformatics 31:2882–2884

    Google Scholar 

  18. Lagier JC, Hugon P, Khelaifia S, Fournier PE, La Scola B, Raoult D (2015) The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota. Clin Microbiol Rev 28:237–264

    Article  Google Scholar 

  19. Ishoey T, Woyke T, Stepanauskas R, Novotny M, Lasken RS (2008) Genomic sequencing of single microbial cells from environmental samples. Curr Opin Microbiol 11:198–204

    Article  CAS  Google Scholar 

  20. Stepanauskas R (2012) Single cell genomics: an individual look at microbes. Curr Opin Microbiol 15:613–620

    Article  CAS  Google Scholar 

  21. Vasdekis AE, Stephanopoulos G (2015) Review of methods to probe single cell metabolism and bioenergetics. Metab Eng 27:115–135

    Article  CAS  Google Scholar 

  22. Li K, Bihan M, Methé BA (2013) Analyses of the stability and core taxonomic memberships of the human microbiome. PLoS One 8, e63139

    Article  CAS  Google Scholar 

  23. Faith JJ, Guruge JL, Charbonneau M, Subramanian S, Seedorf H, Goodman AL, Clemente JC, Knight R, Heath AC, Leibel RL, Rosenbaum M, Gordon JI (2013) The long-term stability of the human gut microbiota. Science 341:1237439

    Article  Google Scholar 

  24. Caron DA, Worden AZ, Countway PD, Demir E, Heidelberg KB (2009) Protists are microbes too: a perspective. ISME J 3:4–12

    Article  CAS  Google Scholar 

  25. Lange L (2014) The importance of fungi and mycology for addressing major global challenges. IMA Fungus 5:463–471

    Article  Google Scholar 

  26. Hugerth LW, Muller EE, Hu YO, Lebrun LA, Roume H, Lundin D, Wilmes P, Andersson AF (2014) Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia. PloS One 9, e95567

    Article  Google Scholar 

  27. Rettedal EA, Gumpert H, Sommer MO (2014) Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria. Nat Commun 5:4714

    Article  CAS  Google Scholar 

  28. Ma L, Kim J, Hatzenpichler R, Karymov MA, Hubert N, Hanan IM, Chang EB, Ismagilov RF (2014) Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project’s most wanted taxa. Proc Nat Acad Sci 111:9768–9773

    Article  CAS  Google Scholar 

  29. Hentges DJ (1996) Anaerobes: general characteristics. In: Baron S (ed) Medical microbiology, 4th edn. University of Texas Press, Galvestone

    Google Scholar 

  30. Speers AM, Cologgi DL, Reguera G (2009) Anaerobic cell culture. Curr Protoc Microbiol 12:4F:A.4F.1–A.4F.16

    Google Scholar 

  31. Plugge CM (2005) Anoxic media design, preparation, and considerations. Methods Enzymol 397:3–16

    Article  CAS  Google Scholar 

  32. Fröhlich J, König H (2000) New techniques for isolation of single prokaryotic cells. FEMS Microbiol Rev 24:567–572

    Article  Google Scholar 

  33. Börner RA, Aliaga MTA, Mattiasson B (2013) Microcultivation of anaerobic bacteria single cells entrapped in alginate microbeads. Biotechnol Lett 35:397–405

    Article  Google Scholar 

  34. Eini A, Sol A, Coppenhagen-Glazer S, Skvirsky Y, Zini A, Bachrach G (2013) Oxygen deprivation affects the antimicrobial action of LL-37 as determined by microplate real-time kinetic measurements under anaerobic conditions. Anaerobe 22:20–24

    Article  CAS  Google Scholar 

  35. Zeidan AA, Van Niel EW (2010) A quantitative analysis of hydrogen production efficiency of the extreme thermophile Caldicellulosiruptor owensensis OL T. Int J Hydrogen Energ 35:1128–1137

    Article  CAS  Google Scholar 

  36. Lehmann-Richter S, Grosskopf R, Liesack W, Frenzel P, Conrad R (1999) Methanogenic archaea and CO2-dependent methanogenesis on washed rice roots. Environ Microbiol 1:159–166

    Article  CAS  Google Scholar 

  37. Edwards AN, Suárez JM, McBride SM (2013) Culturing and maintaining Clostridium difficile in an anaerobic environment. J Vis Exp 79, e50787. doi:10.3791/50787

    Google Scholar 

  38. Hungate RE (1969) A roll tube method for cultivation of strict anaerobes. Methods Microbiol 3:117–132

    Article  Google Scholar 

  39. Bryant MP (1972) Commentary on the Hungate technique for culture of anaerobic bacteria. Am J Clin Nutr 25:1324–1328

    CAS  Google Scholar 

  40. Balch WE, Wolfe RS (1976) New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressureized atmosphere. Appl Environ Microbiol 32:781–791

    CAS  Google Scholar 

  41. Zengler K (2013) To grow or not to grow: isolation and cultivation procedures in the genomic age. In: Fredricks DN (ed) The human microbiota: how microbial communities affect health and disease. Wiley, Hoboken. doi:10.1002/9781118409855.ch12

    Google Scholar 

  42. Lagier JC, Edouard S, Pagnier I, Mediannikov O, Drancourt M, Raoult D (2015) Current and past strategies for bacterial culture in clinical microbiology. Clin Microbiol Rev 28:208–236

    Article  Google Scholar 

  43. McSweeney CS, Denman SE, Mackie RI (2005) Rumen bacteria. Methods in gut microbial ecology for ruminants. Springer, The Netherlands, pp 23–37

    Book  Google Scholar 

  44. Khelaifia S, Raoult D, Drancourt M (2013) A versatile medium for cultivating methanogenic archaea. PLos One. doi:10.1371/journal.pone.0061563

    Google Scholar 

  45. Brioukhanov AL, Netrusov AI (2012) The positive effect of exogenous hemin on a resistance of strict anaerobic archaeon Methanobrevibacter arboriphilus to oxidative stresses. Curr Microbiol 65:375–383

    Article  CAS  Google Scholar 

  46. Bräuer SL, Yashiro E, Ueno NG, Yavitt JB, Zinder SH (2006) Characterization of acid-tolerant H2/CO2-utilizing methanogenic enrichment cultures from an acidic peat bog in New York State. FEMS Microbiol Ecol 57:206–216

    Article  Google Scholar 

  47. Carbonero F, Oakley BB, Purdy KJ (2010) Improving the isolation of anaerobes on solid media: the example of the fastidious Methanosaeta. J Microbiol Methods 80:203–220

    Article  CAS  Google Scholar 

  48. La Scola B, Khelaifia S, Lagier JC, Raoult D (2014) Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report. Eur J Clin Microbiol Infect Dis 33:1781–1783

    Article  Google Scholar 

  49. Millis KK, Weaver KH, Rabenstein DL (1993) Oxidation/reduction potential of glutathione. J Org Chem 58:4144–4146

    Article  CAS  Google Scholar 

  50. Haitjema CH, Solomon KV, Henske JK, Theodorou MK, O'Malley MA (2014) Anaerobic gut fungi: advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production. Biotechnol Bioeng 111:1471–1482

    Article  CAS  Google Scholar 

  51. Vlková E, Salmonová H, Bunešová V, Geigerová M, Rada V, Musilová Š (2015) A new medium containing mupirocin, acetic acid, and norfloxacin for the selective cultivation of bifidobacteria. Anaerobe 34:27–33

    Article  Google Scholar 

  52. Khalid A, Kausar F, Arshad M, Mahmood T, Ahmed I (2012) Accelerated decolorization of reactive azo dyes under saline conditions by bacteria isolated from Arabian seawater sediment. Appl Microbiol Biotechnol 96:1599–1606

    Article  CAS  Google Scholar 

  53. Razo-Flores E, Luijten M, Donlon B, Lettinga G, Field J (1997) Biodegradation of selected azo dyes under methanogenic conditions. Water Sci Technol 36:65–72

    Article  CAS  Google Scholar 

  54. Hara S, Hashidoko Y, Desyatkin RV, Hatano R, Tahara S (2009) High rate of N2 fixation by East Siberian cryophilic soil bacteria as determined by measuring acetylene reduction in nitrogen-poor medium solidified with gellan gum. Appl Environ Microbiol 75:2811–2819

    Article  CAS  Google Scholar 

  55. Tamaki H, Hanada S, Sekiguchi Y, Tanaka Y, Kamagata Y (2009) Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment. Environ Microbiol 11:1827–1834

    Google Scholar 

  56. Nakamura K, Tamaki H, Kang MS, Mochimaru H, Lee ST, Nakamura K, Kamagata Y (2011) A six-well plate method: less laborious and effective method for cultivation of obligate anaerobic microorganisms. Microbes Environ 26:301–306

    Google Scholar 

  57. Nyonyo T, Shinkai T, Tajima A, Mitsumori M (2013) Effect of media composition, including gelling agents, on isolation of previously uncultured rumen bacteria. Lett Appl Microbiol 56:63–70

    Article  CAS  Google Scholar 

  58. Scherer PA, Müller E, Lippert H, Wolff G (1988) Multielement analysis of agar and gelrite impurities investigated by inductively coupled plasma emission spectrometry as well as physical properties of tissue culture media prepared with agar on the gellan gum gelrite. Acta Horticult 226:655–658

    Article  Google Scholar 

  59. Zhang F, Zhang Y, Chen Y, Dai K, van Loosdrecht MC, Zeng RJ (2015) Simultaneous production of acetate and methane from glycerol by selective enrichment of hydrogenotrophic methanogens in extreme-thermophilic (70°C) mixed culture fermentation. Appl Energy 148:326–333

    Article  CAS  Google Scholar 

  60. Schiel-Bengelsdorf B, Dürre P (2012) Pathway engineering and synthetic biology using acetogens. FEBS Lett 586:2191–2198

    Article  CAS  Google Scholar 

  61. Olson DG, Sparling R, Lynd LR (2015) Ethanol production by engineered thermophiles. Curr Opin Biotechnol 33:130–141

    Article  CAS  Google Scholar 

  62. Panagiotopoulos IA (2015) Dark fermentative hydrogen production from lignocellulosic biomass. Production of hydrogen from renewable resources. Springer, The Netherlands, pp 3–40

    Google Scholar 

  63. Zeldes BM, Keller MW, Loder AJ, Straub CT, Adams MW, Kelly RM (2015) Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals. Front Microbiol 6:1209

    Article  Google Scholar 

  64. Gunnigle E, Nielsen JL, Fuszard M, Botting CH, Sheahan J, O'Flaherty V, Abram F (2015) Functional responses and adaptation of mesophilic microbial communities to psychrophilic anaerobic digestion. FEMS Microbiol Ecol 91:132

    Google Scholar 

  65. Nolla-Ardèvol V, Strous M, Tegetmeyer HE (2015) Anaerobic digestion of the microalga Spirulina at extreme alkaline conditions: biogas production, metagenome, and metatranscriptome. Front Microbiol 6:597

    Article  Google Scholar 

  66. Johnson DB (2014) Biomining—biotechnologies for extracting and recovering metals from ores and waste materials. Curr Opin Biotechnol 30:24–31

    Article  CAS  Google Scholar 

  67. Joblin KN (2005) Methanogenic archaea. In: Methods in gut microbial ecology for ruminants. Springer, The Netherlands, pp 47–53

    Google Scholar 

  68. Latif H, Zeidan AA, Nielsen AT, Zengler K (2014) Trash to treasure: production of biofuels and commodity chemicals via syngas fermenting microorganisms. Curr Opin Biotechnol 27:79–87

    Article  CAS  Google Scholar 

  69. Dianou D, Ueno C, Ogiso T, Kimura M, Asakawa S (2012) Diversity of cultivable methane-oxidizing bacteria in microsites of a rice paddy field: investigation by cultivation method and fluorescence in situ hybridization (FISH). Microbes Environ 27:278–287

    Article  Google Scholar 

  70. Löffler FE, Sanford RA, Ritalahti KM (2005) Enrichment, cultivation, and detection of reductively dechlorinating bacteria. Methods Enzymol 397:77–111

    Article  Google Scholar 

  71. Kaeberlein T, Lewis K, Epstein SS (2002) Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296:1127–1129

    Article  CAS  Google Scholar 

  72. Thompson H, Rybalka A, Moazzez R, Dewhirst FE, Wade WG (2015) In vitro culture of previously uncultured oral bacterial phylotypes. Appl Environ Microbiol 81:8307–8314

    Article  CAS  Google Scholar 

  73. McBain AJ, Bartolo RG, Catrenich CE, Charbonneau D, Ledder RG, Gilbert P (2003) Growth and molecular characterization of dental plaque microcosms. J Appl Microbiol 94:655–664

    Article  CAS  Google Scholar 

  74. Bollmann A, Lewis K, Epstein SS (2007) Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates. Appl Environ Microbiol 73:6386–6390

    Article  CAS  Google Scholar 

  75. Aoi Y, Kinoshita T, Hata T, Ohta H, Obokata H, Tsuneda S (2009) Hollow-fiber membrane chamber as a device for in situ environmental cultivation. Appl Environ Microbiol 75:3826–3833

    Article  CAS  Google Scholar 

  76. Gross A, Schoendube J, Zimmermann S, Steeb M, Zengerle R, Koltay P (2015) Technologies for single-cell isolation. Int J Mol Sci 16:16897–16919

    Article  CAS  Google Scholar 

  77. Müller S, Hübschmann T, Kleinsteuber S, Vogt C (2012) High resolution single cell analytics to follow microbial community dynamics in anaerobic ecosystems. Methods 57:338–349

    Article  Google Scholar 

  78. Hamilton-Brehm SD, Vishnivetskaya TA, Allman SL, Mielenz JR, Elkins JG (2012) Anaerobic high-throughput cultivation method for isolation of thermophiles using biomass-derived substrates. Methods Mol Biol 908:153–168

    CAS  Google Scholar 

  79. Weaver JC, Williams GB, Klibanov A, Demain AL (1988) Gel microdroplets: rapid detection and enumeration of individual microorganisms by their metabolic activity. Nat Biotech 6:1084–1089

    Article  CAS  Google Scholar 

  80. Manome A, Zhang H, Tani Y, Katsuragi T, Kurane R, Tsuchida T (2001) Application of gel microdroplet and flow cytometry techniques to selective enrichment of non-growing bacterial cells. FEMS Microbiol Lett 197:29–33

    Article  CAS  Google Scholar 

  81. Akselband Y, Cabral C, Castor TP, Chikarmane HM, McGrath P (2006) Enrichment of slow-growing marine microorganisms from mixed cultures using gel microdrop (GMD) growth assay and fluorescence-activated cell sorting. J Exp Mar Biol Ecol 329:196–205

    Article  CAS  Google Scholar 

  82. Zengler K, Toledo G, Rappé M, Elkins J, Mathur EJ, Short JM, Keller M (2002) Cultivating the uncultured. Proc Natl Acad Sci 99:15681–15686

    Article  CAS  Google Scholar 

  83. Fitzsimons MS, Novotny M, Lo CC, Dichosa AE, Yee-Greenbaum JL, Snook JP, Gu W, Chertkov O, Davenport KW, McMurry K, Reitenga KG, Daughton AR, He J, Johnson SL, Gleasner CD, Wills PL, Parson-Quintana B, Chain PC, Detter JC, Lasken RG, Han CS (2013) Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome. Genome Res 23:878–888

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden

    Rosa Aragão Börner

  2. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark

    Rosa Aragão Börner

Authors
  1. Rosa Aragão Börner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rosa Aragão Börner .

Editor information

Editors and Affiliations

  1. Biotechnology, Department of Chemistry Center for Chemistry & Chemical Engineering, Lund University, Lund, Sweden

    Rajni Hatti-Kaul

  2. Biotechnology, Department of Chemistry Center for Chemistry & Chemical Engineering, Lund University, Lund, Sweden

    Gashaw Mamo

  3. Biotechnology, Department of Chemistry Center for Chemistry & Chemical Engineering, Lund University, Lund, Sweden

    Bo Mattiasson

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Börner, R.A. (2016). Isolation and Cultivation of Anaerobes. In: Hatti-Kaul, R., Mamo, G., Mattiasson, B. (eds) Anaerobes in Biotechnology. Advances in Biochemical Engineering/Biotechnology, vol 156. Springer, Cham. https://doi.org/10.1007/10_2016_1

Download citation

Publish with us

Policies and ethics

Search

Navigation