, Volume 15, Issue 1, pp 77–87

Probing the redox metabolism in the strictly anaerobic, extremely thermophilic, hydrogen-producing Caldicellulosiruptorsaccharolyticus using amperometry

  • Natalie Kostesha
  • Karin Willquist
  • Jenny Emneus
  • Ed W. J. van Niel
Original Paper


Changes in the redox metabolism in the anaerobic, extremely thermophilic, hydrogen-forming bacterium Caldicellulosiruptor saccharolyticus were probed for the first time in vivo using mediated amperometry with ferricyanide as a thermotolerant external mediator. Clear differences in the intracellular electron flow were observed when cells were supplied with different carbon sources. A higher electrochemical response was detected when cells were supplied with xylose than with sucrose or glucose. Moreover, using the mediated electrochemical method, it was possible to detect differences in the electron flow between cells harvested in the exponential and stationary growth phases. The electron flow of C. saccharolyticus was dependent on the NADH- and reduced ferredoxin generation flux and the competitive behavior of cytosolic and membrane-associated oxidoreductases. Sodium oxamate was used to inhibit the NADH-dependent lactate dehydrogenase, upon which more NADH was directed to membrane-associated enzymes for ferricyanide reduction, leading to a higher electrochemical signal. The method is noninvasive and the results presented here demonstrate that this method can be used to accurately detect changes in the intracellular electron flow and to probe redox enzyme properties of a strictly anaerobic thermophile in vivo.


Caldicellulosiruptor saccharolyticus Lactate regulation Anaerobiosis Electrochemistry Mediated amperometry Ferricyanide 


  1. Almeida JRM, Roder A, Modig T, Laadan B, Liden G, Gorwa-Grauslund MF (2008) NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 78(6):939–945CrossRefPubMedGoogle Scholar
  2. Angenent LT, Karim K, Al-Dahhan MH, Domiguez-Espinosa R (2004) Production of bioenergy and biochemicals from industrial and agricultural wastewater. Trends Biotechnol 22(9):477–485CrossRefPubMedGoogle Scholar
  3. Arrigo AP (1999) Gene expression and the thiol redox state. Free Radic Biol Med 27(9–10):936–944CrossRefPubMedGoogle Scholar
  4. Bielen AAM, Willquist K, Engman J, van der Oost J, van Niel EWJ, Kengen SWM (2010) Pyrophosphate as a central energy carrier in the hydrogen-producing extremely thermophilic Caldicellulosiruptor saccharolyticus. FEMS Microbiol Lett 307(1):48–54CrossRefPubMedGoogle Scholar
  5. Bloomfield V, Alberty RA (1963) Abortive complexes in dehydrogenase reactions. J Biol Chem 238(8):2817–2822Google Scholar
  6. Brown DM, Upcroft JA, Edwards MR, Upcroft P (1998) Anaerobic bacterial metabolism in the ancient eukaryote Giardia duodenalis. Int J Parasitol 28(1):149–164CrossRefPubMedGoogle Scholar
  7. Buchanan BB, Schurmann P, Wolosiuk RA, Jacquot JP (2002) The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond. Photosynth Res 73(1–3):215–222CrossRefPubMedGoogle Scholar
  8. Catterall K, Morris K, Gladman C, Zhao HJ, Pasco N, John R (2001) The use of microorganisms with broad range substrate utilisation for the ferricyanide-mediated rapid determination of biochemical oxygen demand. Talanta 55(6):1187–1194CrossRefPubMedGoogle Scholar
  9. Chaubey A, Malhotra BD (2002) Mediated biosensors. Biosens Bioelectron 17(6–7):441–456CrossRefPubMedGoogle Scholar
  10. de Vrije T, Mars AE, Budde MA, Lai MH, Dijkema C, de Waard P, Claassen PAM (2007) Glycolytic pathway and hydrogen yield studies of the extreme thermophile Caldicellulosiruptor saccharolyticus. Appl Microbiol Biotechnol 74(6):1358–1367CrossRefPubMedGoogle Scholar
  11. de Vrije T, Bakker RR, Budde MAW, Lai MH, Mars AE, Claassen PAM (2009) Efficient hydrogen production from the lignocellulosic energy crop Miscanthus by the extreme thermophilic bacteria Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana. Biotechnol Biofuels 2(12):12CrossRefPubMedGoogle Scholar
  12. Gao H, Zhao MH, Zhang XL, Jin WR (2006) Measurement of enzyme activity in single cells by voltammetry using a microcell with a positionable dual electrode. Anal Chem 78(1):231–238CrossRefPubMedGoogle Scholar
  13. Harwood GW, Pouton CW (1996) Amperometric enzyme biosensors for the analysis of drugs and metabolites. Adv Drug Deliv Rev 18(2):163–191CrossRefGoogle Scholar
  14. Heiskanen A, Spégel C, Kostesha N, Lindahl S, Ruzgas T, Emneus J (2009) Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells. Anal Biochem 384(1):11–19CrossRefPubMedGoogle Scholar
  15. Ikeda T (2004) A novel electrochemical approach to the characterization of oxidoreductase reactions. Chem Record 4(3):192–203CrossRefGoogle Scholar
  16. Ikeda T, Kano K (2001) An electrochemical approach to the studies of biological redox reactions and their applications to biosensors, bioreactors, and biofuel cells. J Biosci Bioeng 92(1):9–18CrossRefPubMedGoogle Scholar
  17. Kim I, Yun H, Iwahashi H, Jin I (2006) Genome-wide expression analyses of adaptive response against medadione-induced oxidative stress in Saccharomyces cerevisiae KNU5377. Process Biochem 41(11):2305–2313CrossRefGoogle Scholar
  18. Kolthoff IM, Lingane JJ (1952) Polarography. Interscience Publishers, New YorkGoogle Scholar
  19. Kondo T, Ikeda T (1999) An electrochemical method for the measurements of substrate-oxidizing activity of acetic acid bacteria using a carbon-paste electrode modified with immobilized bacteria. Appl Microbiol Biotechnol 51(5):664–668CrossRefGoogle Scholar
  20. Kostesha N, Heiskanen A, Spegel C, Hahn-Hagerdal B, Gorwa-Grauslund MF, Emneus J (2009a) Real-time detection of cofactor availability in genetically modified living Saccharomyces cerevisiae cells—simultaneous probing of different geno- and phenotypes. Bioelectrochemistry 76(1–2):180–188CrossRefPubMedGoogle Scholar
  21. Kostesha NV, Almeida JRM, Heiskanen AR, Gorwa-Grauslund MF, Hahn-Hagerdal B, Emneus J (2009b) Electrochemical probing of in vivo 5-hydroxymethyl furfural reduction in Saccharomyces cerevisiae. Anal Chem 81(24):9896–9901CrossRefPubMedGoogle Scholar
  22. Kraemer JT, Bagley DM (2007) Improving the yield from fermentative hydrogen production. Biotechnol Lett 29(5):685–695CrossRefPubMedGoogle Scholar
  23. Krylov SN, Zhang ZR, Chan NWC, Arriaga E, Palcic MM, Dovichi NJ (1999) Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry. Cytometry 37(1):14–20CrossRefPubMedGoogle Scholar
  24. Mashego MR, Rumbold K, De Mey M, Vandamme E, Soetaert W, Heijnen JJ (2007) Microbial metabolomics: past, present and future methodologies. Biotechnol Lett 29(1):1–16CrossRefPubMedGoogle Scholar
  25. Nakamura H, Nakamura K, Yodoi J (1997) Redox regulation of cellular activation. Annu Rev Immunol 15:351–369CrossRefPubMedGoogle Scholar
  26. Powis G, Briehl M, Oblong J (1995) Redox signaling and the control of cell-growth and death. Pharmacol Ther 68(1):149–173CrossRefPubMedGoogle Scholar
  27. Rainey FA, Donnison AM, Janssen PH, Saul D, Rodrigo A, Bergquist PL, Danie RM, Stackebrandt E, Morgan HW (1994) Description of Caldicellulosiruptor saccharolyticus gen-nov, sp, nov. an obligately anaerobic, extremely thermophilic, cellulolytic bacterium. FEMS Microbiol Lett 120(3):263–266CrossRefPubMedGoogle Scholar
  28. Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30(11):1191–1212CrossRefPubMedGoogle Scholar
  29. Shaw AJ, Jenney E, Adams MWW, Lynd LR (2008) End-product pathways in the xylose fermenting bacterium Thermoanaerobacterium saccharolyticum. Enzyme Microb Technol 42(6):453–458CrossRefGoogle Scholar
  30. Spegel CF, Heiskanen AR, Kostesha N, Johanson TH, Gorwa-Grauslund MF, Koudelka-Hep M, Emneus J, Ruzgas T (2007) Amperometric response from the glycolytic versus the pentose phosphate pathway in Saccharomyces cerevisiae cells. Anal Chem 79(23):8919–8926CrossRefPubMedGoogle Scholar
  31. Takayama K, Kurosaki T, Ikeda T (1993) Mediated electrocatalysis at a biocatalyst electrode based on a bacterium, Gluconobacter industrius. J Electroanal Chem 356(1–2):295–301CrossRefGoogle Scholar
  32. van de Werken HJG, Verhaart MRA, van Fossen AL, Willquist K, Lewis DL, Nichols JD, Goorissen HP, Mongodin EF, Nelson KE, van Niel EWJ, Stams AJM, Ward DE, de Vos WM, van der Oost J, Kelly RM, Kengen SWM (2008) Hydrogenomics of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus. Appl Environ Microbiol 74(21):6720–6729CrossRefPubMedGoogle Scholar
  33. van Fossen AL, Lewis DL, Nichols JD, Kelly RM (2008) Polysaccharide degradation and synthesis by extremely nermophilic anaerobes. Incredible anaerobes: from physiology to genomics to fuels. Ann Acad Sci, New York 1125:322–337CrossRefGoogle Scholar
  34. van Fossen AL, Verhaart MR, Kengen SM, Kelly RM (2009) Carbohydrate utilization patterns for the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus reveal broad growth substrate preferences. Appl Environ Microbiol 75:7718–7724CrossRefGoogle Scholar
  35. van Niel EWJ, Claassen PAM, Stams AMJ (2003) Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus. Biotechnol Bioeng 81(3):255–262CrossRefPubMedGoogle Scholar
  36. Walsh KAJ, Daniel RM, Morgan HW (1983) A soluble NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from Thermus aquaticus strain T351. Biochem J 209(2):427–433PubMedGoogle Scholar
  37. Williamson D, Lund P, Krebs HA (1967) Redox state of free nicotinamide adenine dinucleotide in cytoplasm and mitochondria of rat liver. Biochem J 103(2):514–527PubMedGoogle Scholar
  38. Willquist K (2010) Physiology of Caldicellulosiruptor saccharolyticus: a hydrogen cell factory. Doctoral thesis, Department of Applied Microbiology, Lund University, LundGoogle Scholar
  39. Willquist K, van Niel EWJ (2010) Lactate formation in Caldicellulosiruptor saccharolyticus is regulated by the energy carriers pyrophosphate and ATP. Metab Eng 12(3):282–290CrossRefPubMedGoogle Scholar
  40. Willquist K, Claassen PAM, van Niel EWJ (2009) Evaluation of the influence of CO2 on hydrogen production by Caldicellulosiruptor saccharolyticus. Int J Hydrogen Energy 34(11):4718–4726CrossRefGoogle Scholar
  41. Zhang ZQ, Yu J, Stanton RC (2000) A method for determination of pyridine nucleotides using a single extract. Anal Biochem 285(1):163–167CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2010

Authors and Affiliations

  • Natalie Kostesha
    • 1
  • Karin Willquist
    • 2
  • Jenny Emneus
    • 1
  • Ed W. J. van Niel
    • 2
  1. 1.Department of Micro- and NanotechnologyTechnical University of DenmarkKgs. LyngbyDenmark
  2. 2.Department of Applied MicrobiologyLund UniversityLundSweden

Personalised recommendations