Applied Microbiology and Biotechnology

, Volume 100, Issue 18, pp 7989–8002 | Cite as

Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state

  • Jutta Speda
  • Mikaela A. Johansson
  • Bengt-Harald Jonsson
  • Martin KarlssonEmail author
Biotechnologically relevant enzymes and proteins


Novel enzymes that are stable in diverse conditions are intensively sought because they offer major potential advantages in industrial biotechnology, and microorganisms in extreme environments are key sources of such enzymes. However, most potentially valuable enzymes are currently inaccessible due to the pure culturing problem of microorganisms. Novel metagenomic and metaproteomic techniques that circumvent the need for pure cultures have theoretically provided possibilities to identify all genes and all proteins in microbial communities, but these techniques have not been widely used to directly identify specific enzymes because they generate vast amounts of extraneous data.

In a first step towards developing a metaproteomic approach to pinpoint targeted extracellular hydrolytic enzymes of choice in microbial communities, we have generated and analyzed the necessary conditions for such an approach by the use of a methanogenic microbial community maintained on a chemically defined medium. The results show that a metabolic steady state of the microbial community could be reached, at which the expression of the targeted hydrolytic enzymes were suppressed, and that upon enzyme induction a distinct increase in the targeted enzyme expression was obtained. Furthermore, no cross talk in expression was detected between the two focal types of enzyme activities under their respective inductive conditions. Thus, the described approach should be useful to generate ideal samples, collected before and after selective induction, in controlled microbial communities to clearly discriminate between constituently expressed proteins and extracellular hydrolytic enzymes that are specifically induced, thereby reducing the analysis to only those proteins that are distinctively up-regulated.


Microbial community Enzyme discovery Metaproteomics Biogas Cellulase Protease 



The technical support from Tekniska Verken i Linköping for running and maintaining the bioreactors is greatly appreciated.

Compliance with ethical standards


This work was financially supported by the Swedish Research Council (grant to Martin Karlsson, number 621-2009-4150) and InZymes Biotech AB.

Conflict of interest

Martin Karlsson is affiliated to both InZymes Biotech AB and Linköping University.

Ethical approval

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


  1. Allison SD, Vitousek PM (2005) Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biol Biochem 37:937–944CrossRefGoogle Scholar
  2. Allison SD, Weintraub MN, Gartner TB, Waldrop MP (2011) Evolutionary-economic principles as regulators of soil enzyme production and ecosystem. In: Shukla G, Varma A (eds) Soil enzymology, soil biology 22. Springer-Verlag, Berlin, pp. 229–243Google Scholar
  3. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169PubMedPubMedCentralGoogle Scholar
  4. Barber RD (2007) Methanogenesis: ecology. In: Encyclopedia of life sciences. John Wiley and Sons, Ltd. Hoboken, USA. doi: 10.1002/9780470015902.a0000475.pub2
  5. Batstone DJ, Keller J, Angelidaki I, Kalyuzhnyi SV, Pavlostathis SG, Rozzi A, Sanders WT, Siegrist H, Vavilin VA (2002) The IWA anaerobic digestion model No 1 (ADM1). Water Sci Technol 45:65–73Google Scholar
  6. Boubaker F, Ridha BC (2008) Implementation of IWA anaerobic digestion model No. 1 (ADM1) for simulating the thermophilic anaerobic co-digestion of olive mill wastewater with olive mill solid waste in a semi-continuous tubular digester. Chem Eng J 141:75–88CrossRefGoogle Scholar
  7. Buswell AM and Hatfield DW (1936) Bulletin No. 32, anaerobic fermentations. State of Illinois department of registration and education, division of the state water survey, Urbana, USAGoogle Scholar
  8. Carle-Urioste JC, Escobar-Vera J, El-Gogary S, Henrique-Silva F, Torigoi E, Crivellaro O, Herrera-Estrella A, El-Dorry H (1997) Cellulase induction in Trichoderma reesei by cellulose requires its own basal expression. J Biol Chem 272:10169–10174CrossRefPubMedGoogle Scholar
  9. Coleman DJ, Studler MJ, Naleway JJ (2007) A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-β-D-cellobioside. Anal Biochem 371:146–153CrossRefPubMedGoogle Scholar
  10. Dar SA, Kleerebezem R, Stams AJM, Kuenen JG, Muyzer G (2008) Competition and coexistence of sulfate-reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio. Appl Microbiol Biotechnol 78:1045–1055CrossRefPubMedPubMedCentralGoogle Scholar
  11. Demirjian DC, Moris-Varas F, Cassidy CS (2001) Enzymes from extremophiles. Curr Opin Chem Biol 5:144–151CrossRefPubMedGoogle Scholar
  12. Drosg B, Braun R, Bochmann G, Al Saedi T (2013) Analysis and characterization of biogas feedstocks. In: Wellinger A, Murphy J, Baxter D (eds) The biogas handbook: science, production and applications. Woodhead Publishing Ltd, Sawston, p. 76Google Scholar
  13. Egli T, Zinn M (2003) The concept of multiple-nutrient-limited growth of microorganisms and its application in biotechnological processes. Biotechnol Adv 22:35–43CrossRefGoogle Scholar
  14. Elbeshbishy E, Nakhla G (2012) Batch anaerobic co-digestion of proteins and carbohydrates. Bioresource Technol 116:170–178CrossRefGoogle Scholar
  15. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana press, New York, pp. 571–607CrossRefGoogle Scholar
  16. Geisseler D, Horwath WR (2008) Regulation of extracellular protease activity in soil in response to different sources and concentrations of nitrogen and carbon. Soil Biol Biochem 40:3040–3048CrossRefGoogle Scholar
  17. Gurung N, Ray S, Bose S, Rai V (2013) A broader view: microbial enzymes and their relevance in industries, medicine and beyond. Biomed Res Int, vol 2013, article ID 329191Google Scholar
  18. Han SO, Cho HY, Yukawa H, Inui M, Doi RH (2004) Regulation of expression of cellulosomes and noncellulosomal (hemi) cellulolytic enzymes in Clostridium cellulovorans during growth on different carbon sources. J Bacteriol 186:4218–4227CrossRefPubMedPubMedCentralGoogle Scholar
  19. Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5:245–249CrossRefGoogle Scholar
  20. Johnson EA, Madia A, Demain AL (1981) Chemically defined minimal medium for growth of the anaerobic cellulolytic thermophile Clostridium thermocellum. Appl Environ Microbiol 41:1060–1062PubMedPubMedCentralGoogle Scholar
  21. Kim IJ, Lee HJ, Choi I-G, Kim KH (2014) Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulase. Appl Microbiol Biotechnol 98:8469–8480CrossRefPubMedGoogle Scholar
  22. Kirk O, Borchert TV, Fuglsang CC (2002) Industrial enzyme applications. Curr Opin Biotech 13:345–351CrossRefPubMedGoogle Scholar
  23. Kopečný J, Hodrová B (1997) The effect of yellow affinity substance on cellulases of Ruminococcus flavefaciens. Lett Appl Microbiol 25:191–196CrossRefPubMedGoogle Scholar
  24. Kuhad RC, Gupta R, Singh A (2011) Microbial cellulases and their industrial applications. Enz Res, vol 2011, article ID 280696Google Scholar
  25. Langer M, Gabor EM, Liebeton K, Meurer G, Niehaus F, Schulze R, Eck J, Lorenz P (2006) Metagenomics: an inexhaustible access to nature’s diversity. Biotechnol J 1:815–821CrossRefPubMedGoogle Scholar
  26. Li S, Yang X, Yang S, Zhu M, Wang X (2012) Technology prospecting on enzymes: application, marketing and engineering. Comput Struct Biotechnol J 2:1–11Google Scholar
  27. Ljungdahl LG, Pettersson B, Eriksson KE, Wiegel J (1983) A yellow affinity substance involved in the cellulolytic system of Clostridium thermocellum. Curr Microbiol 9:195–199CrossRefGoogle Scholar
  28. Lorenz P, Eck J (2005) Metagenomics and industrial applications. Nat Rev Micro 3:510–516CrossRefGoogle Scholar
  29. Mienda BS, Yahya A, Galadima IA, Shamsir MS (2014) An overview of microbial proteases for industrial applications. Res J Pharm Biol Chem Sci 5:388–396Google Scholar
  30. Mould FL, Morgan R, Kliem KE, Krystallidou E (2005) A review and simplification of the in vitro incubation medium. Anim Feed Sci Tech 123:155–172CrossRefGoogle Scholar
  31. Niehaus F, Bertoldo C, Kähler M, Antranikian G (1999) Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol Biotechnol 5:711–729CrossRefGoogle Scholar
  32. Nordell E, Moestedt J, Karlsson M (2011) Biogas producing laboratory reactor. SE Patent 1150954–4Google Scholar
  33. Parawira W (2012) Enzyme research and applications in biotechnological intensification of biogas production. Crit Rev Biotechnol 32:172–186CrossRefPubMedGoogle Scholar
  34. Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF (2007) Stability of biocatalysts. Curr Opin Chem Biol 11:220–225CrossRefGoogle Scholar
  35. Rappe MS, Giovannoni SJ (2003) The uncultured microbial majority. Annu Rev Microbiol 57:369–394CrossRefPubMedGoogle Scholar
  36. Rinke C, Schwientek P, Sczyrba A, Ivanova NN, Anderson IJ, Cheng J-F, Darling A, Malfatti S, Swan BK, Gies EA, Dodsworth JA, Hedlund BP, Tsiamis G, Sievert SM, Liu W-T, Eisen JA, Hallam SJ, Kyrpides NC, Stepanauskas R, Rubin EM, Hugenholtz P, Woyke T (2013) Insights into the phylogeny and coding potential of microbial dark matter. Nature 499:431–437CrossRefPubMedGoogle Scholar
  37. Rogowska-Wrzesinska A, Le Bihan M-C, Thaysen-Andersen M, Roepstorff P (2013) 2D gels still have a niche in proteomics. J Proteome 88:4–13CrossRefGoogle Scholar
  38. Rothschild LJ, Mancinelli RL (2001) Life in extreme environments. Nature 409:1092–1101CrossRefPubMedGoogle Scholar
  39. Schloss PD, Handelsman J (2003) Biotechnological prospects from metagenomics. Curr Opin Biotechnol 14:303–310CrossRefPubMedGoogle Scholar
  40. Sharpton TJ (2014) An introduction to the analysis of shotgun metagenomic data. Front Plant Sci 5:209CrossRefPubMedPubMedCentralGoogle Scholar
  41. Shink B (2002) Synergistic interactions in the microbial world. A Van Leeuw J Microb 81:257–261CrossRefGoogle Scholar
  42. Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A (2013) Role and significance of β- glucosidases in the hydrolysis of cellulose for bioethanol production. Biores Technol 127:500–507CrossRefGoogle Scholar
  43. SS-EN 12176 (1998) Characterization of sludges—determination of pH. STD-23050. Swedish Standards Institute, Stockholm, SwedenGoogle Scholar
  44. SS-EN ISO 9963–2 (1994) Water quality—determination of alkalinity—part 2: determination of carbonate alkalinity. STD-18780. Swedish Standards Institute, Stockholm, SwedenGoogle Scholar
  45. Staley JT, Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:369–394CrossRefGoogle Scholar
  46. Sternberg D, Mandels GR (1979) Induction of cellulolytic enzymes in Trichoderma reesei by sophorose. J Bacteriol 139:761–769PubMedPubMedCentralGoogle Scholar
  47. Stewart EJ (2012) Growing unculturable bacteria. J Bacteriol 194:4151–4160CrossRefPubMedPubMedCentralGoogle Scholar
  48. Streit WR, Schmitz RA (2004) Metagenomics—the key to the uncultured microbes. Curr Opin Microbiol 7(5):492–498CrossRefPubMedGoogle Scholar
  49. Sweeney MD, Xu F (2012) Biomass converting enzymes as industrial biocatalysts for fuels and chemicals: recent developments. Catalysts 2:244–263CrossRefGoogle Scholar
  50. Torsvik V, Øvreås L, Thingstad TF (2002) Prokaryotic diversity—magnitude, dynamics, and controlling factors. Science 296:1064–1066CrossRefPubMedGoogle Scholar
  51. Wilmes P, Bond PL (2004) The application of two-dimensional polyacrylamide gel electrophoresis and downstream analyses to a mixed community of prokaryotic microorganisms. Environ Microbiol 6:911–920CrossRefPubMedGoogle Scholar
  52. Zinder SH (1984) Microbiology of anaerobic conversion of organic wastes to methane: recent developments. ASM News 50:294–298Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Jutta Speda
    • 1
  • Mikaela A. Johansson
    • 1
  • Bengt-Harald Jonsson
    • 1
  • Martin Karlsson
    • 1
    • 2
    Email author
  1. 1.Molecular Biotechnology, Department of Physics, Chemistry and BiologyLinköping UniversityLinköpingSweden
  2. 2.InZymes Biotech ABLinköpingSweden

Personalised recommendations