Potential of Hydrogen Fermentative Pathways in Marine Thermophilic Bacteria: Dark Fermentation and Capnophilic Lactic Fermentation in Thermotoga and Pseudothermotoga Species

  • Laura Dipasquale
  • Nirakar Pradhan
  • Giuliana d’Ippolito
  • Angelo FontanaEmail author
Part of the Grand Challenges in Biology and Biotechnology book series (GCBB)


Hydrogen is a clean energy vector that could help to face the current environmental issues of greenhouse gas emissions and, over a longer time scale, to replace the depleting nonrenewable fuels. Biological production by fermentation of waste and residues has the potential to surrogate the current technologies of production of this gas. In this chapter we report a summary of the fermentative pathways related to hydrogen production in the thermophilic microorganisms of the genera Thermotoga and Pseudothermotoga that embrace several marine species with the highest hydrogen yields among eubacteria. The contribution includes a brief review of dark fermentation (DF) and capnophilic lactic fermentation (CLF), the two processes related to hydrogen synthesis in these organisms, together with a discussion of new data concerning the distribution of CLF in these bacteria. The data show a varied scenario with different metabolic capabilities spread across the two genera. Under standard conditions, CLF is active only in few species of Thermotoga genus. The study underlines the great potential of these microbes in the valorization of agro-food waste and production of fuel and chemicals. In particular, the metabolic and biochemical diversity of Thermotoga and Pseudothermotoga species, together with their resilience to different environmental conditions, suggests the possibility to overtake many of the bottlenecks related to operational factors such as substrates, temperature, pH, hydraulic retention time, and hydrogen partial pressure.



This work is based upon research supported by the PON01_02740 project “Sfruttamento Integrato di Biomasse Algali in Filiera Energetica di Qualità” (SIBAFEQ), Programma Operativo Nazionale—Ricerca e Competitività 2007–2013 and the European Horizon-2020 project “Biological routes for CO2 conversion into chemical building blocks BioRECO2VER)” (Project ID: 760431). The authors are especially grateful to FERRERO SPA and SEPE SRL for the ideative contribution and technical support.


  1. 1.
    Dipasquale L, d’Ippolito G, Fontana A (2014) Capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana: an unexpected deviation from the dark fermentation model. Int J Hydrog Energy 39:4857–4862CrossRefGoogle Scholar
  2. 2.
    d’Ippolito G, Dipasquale L, Fontana A (2014) Recycling of carbon dioxide and acetate as lactic acid by the hydrogen-producing bacterium Thermotoga neapolitana. ChemSusChem 7:2678–2683CrossRefPubMedGoogle Scholar
  3. 3.
    Thauer RK, Jungermann K, Decker K (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180PubMedPubMedCentralGoogle Scholar
  4. 4.
    Pradhan N, Dipasquale L, d’Ippolito G, Fontana A, Panico A, Pirozzi F, Lens PNL, Esposito G (2016) Model development and experimental validation of capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana. Water Res 99:225–234CrossRefPubMedGoogle Scholar
  5. 5.
    Pradhan N, Dipasquale L, d’Ippolito G, Panico A, Lens PNL, Esposito G, Fontana A (2017) Hydrogen and lactic acid synthesis by the wild-type and a laboratory strain of the hyperthermophilic bacterium Thermotoga neapolitana DSMZ 4359T under capnophilic lactic fermentation conditions. Int J Hydrogen Energy.
  6. 6.
    Frock AD, Notey JS, Kelly RM (2010) The genus Thermotoga: recent developments. Environ Technol 31:1169–1181CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bhandari V, Gupta RS (2014) Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations. Anton van Leeuwenhoek 105:143–168CrossRefGoogle Scholar
  8. 8.
    Mori K, Yamazoe A, Hosoyama A, Ohji S, Fujita N, Ishibashi J, Kimura H, Suzuki K (2014) Thermotoga profunda sp. nov. and Thermotoga caldifontis sp. nov., anaerobic thermophilic bacteria isolated from terrestrial hot springs. Int J Syst Evol Microbiol 64:2128–2136CrossRefPubMedGoogle Scholar
  9. 9.
    Huber R, Hannig M (2006) Chapter 12.1. Thermotogales. Prokaryotes 7:899–922Google Scholar
  10. 10.
    Huber R, Langworthy TA, Konig H, Thomm M, Woese CR, Sleytr UB, Stetter KO (1986) Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90 °C. Arch Microbiol 144:324–333CrossRefGoogle Scholar
  11. 11.
    Belkin S, Wirsen CO, Jannasch HW (1986) A new sulfur-reducing, extremely thermophilic eubacterium from a submarine thermal vent. Appl Environ Microbiol 51:1180–1185PubMedPubMedCentralGoogle Scholar
  12. 12.
    Jannasch HW, Huber R, Belkin S, Stetter KO (1988) Thermotoga neapolitana sp. nov. of the extremely thermophilic, eubacterial genus Thermotoga. Arch Microbiol 150:103–104CrossRefGoogle Scholar
  13. 13.
    Takahata Y, Nishijima M, Hoaki T, Maruyama T (2001) Thermotoga petrophila sp. nov. and Thermotoga naphthophila sp. nov., two hyperthermophilic bacteria from the Kubiki oil reservoir in Niigata, Japan. Int J Syst Evol Microbiol 51:1901–1909CrossRefPubMedGoogle Scholar
  14. 14.
    Ravot G, Magot M, Fardeau ML, Patel BKC, Prensier G, Egan A, Garcia JL, Ollivier B (1995) Thermotoga elfii sp. nov, a novel thermophilic bacterium from an African oil-producing well. Int J Syst Bacteriol 45:308–314CrossRefPubMedGoogle Scholar
  15. 15.
    Jeanthon C, Reysenbach AL, L’Haridon S, Gambacorta A, Pace NR, Glenat P, Prieur D (1995) Thermotoga subterranea sp. nov., a new thermophilic bacterium isolated from a continental oil reservoir. Arch Microbiol 164:91–97CrossRefPubMedGoogle Scholar
  16. 16.
    Fardeau ML, Ollivier B, Patel BKC, Magot M, Thomas P, Rimbault A, Rocchiccioli F, Garcia JL (1997) Thermotoga hypogea sp. nov., a xylanolytic, thermophilic bacterium from an oil-producing well. Int J Syst Bacteriol 47:1013–1019CrossRefPubMedGoogle Scholar
  17. 17.
    Windberger E, Huber R, Trincone A, Fricke H, Stetter KO (1989) Thermotoga thermarum sp. nov. and Thermotoga neapolitana occurring in African continental solfataric springs. Arch Microbiol 151:506–512CrossRefGoogle Scholar
  18. 18.
    Balk M, Weijma J, Stams AJM (2002) Thermotoga lettingae sp. nov., a novel thermophilic, methanol-degrading bacterium isolated from a thermophilic anaerobic reactor. Int J Syst Evol Microbiol 52:1361–1368PubMedGoogle Scholar
  19. 19.
    d’Ippolito G, Dipasquale L, Vella FM, Romano I, Gambacorta A, Cutignano A, Fontana A (2010) Hydrogen metabolism in the extreme thermophile Thermotoga neapolitana. Int J Hydrog Energy 35:2290–2295CrossRefGoogle Scholar
  20. 20.
    Pradhan N, Dipasquale L, d’Ippolito G, Panico A, Lens PN, Esposito G, Fontana A (2015) Hydrogen production by the thermophilic bacterium Thermotoga neapolitana. Int J Mol Sci 16:12578–12600CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Johnston J, Mayo MC, Khare A (2005) Hydrogen: the energy source for the 21st century. Technovation 25:569–585CrossRefGoogle Scholar
  22. 22.
    Khanna N, Das D (2013) Biohydrogen production by dark fermentation. WIREs Energy Environ 2:401–421CrossRefGoogle Scholar
  23. 23.
    Schut GJ, Adams MWW (2009) The iron-hydrogenase of Thermotoga maritima utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen production. J Bacteriol 191:4451–4457CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Wu SY, Hung CH, Lin CN, Chen HW, Lee AS, Chang JS (2006) Fermentative hydrogen production and bacterial community structure in high-rate anaerobic bioreactors containing silicone-immobilized and self-flocculated sludge. Biotechnol Bioeng 93:934–946CrossRefPubMedGoogle Scholar
  25. 25.
    Eriksen NT, Nielsen TM, Iversen N (2008) Hydrogen production in anaerobic and microaerobic Thermotoga neapolitana. Biotechnol Lett 30:103–109CrossRefPubMedGoogle Scholar
  26. 26.
    Kyazze G, Martinez-Perez N, Dinsdale R, Premier GC, Hawkes FR, Guwy AJ, Hawkes DL (2006) Influence of substrate concentration on the stability and yield of continuous biohydrogen production. Biotechnol Bioeng 93:971–979CrossRefPubMedGoogle Scholar
  27. 27.
    Munro SA, Zinder SH, Walker LP (2009) The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature, oxygen, and pH on hydrogen production. Biotechnol Prog 25:1035–1034CrossRefPubMedGoogle Scholar
  28. 28.
    Greening C, Biswas A, Carere CR, Jackson CJ, Taylor MC, Stott MB, Cook GM, Morales SE (2016) Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival. ISME J 10:761–777CrossRefPubMedGoogle Scholar
  29. 29.
    Frey M (2002) Hydrogenases: hydrogen-activating enzymes. Chembiochem 3:153–160CrossRefPubMedGoogle Scholar
  30. 30.
    Vignais PM, Billoud B, Meyer J (2001) Classification and phylogeny of hydrogenases. FEMS Microbiol Rev 25:455–501CrossRefPubMedGoogle Scholar
  31. 31.
    Albertini M, Vallese F, Di Valentin M, Berto P, Giacometti GM, Costantini P, Carbonera D (2014) The proton iron-sulfur cluster environment of the [FeFe]-hydrogenase maturation protein HydF from Thermotoga neapolitana. Int J Hydrog Energy 39:18574–18582CrossRefGoogle Scholar
  32. 32.
    Peters JW, Schut GJ, Boyd ES, Mulder DW, Shepard EM, Broderick JB, King PW, Adams MWW (2015) [FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation. Biochim Biophys Acta 1853:1350–1369CrossRefPubMedGoogle Scholar
  33. 33.
    Herrmann G, Jayamani E, Mai G, Buckel W (2008) Energy conservation via electron-transferring flavoprotein in anaerobic bacteria. J Bacteriol 190:784–791CrossRefPubMedGoogle Scholar
  34. 34.
    Buckel W, Thauer RK (2013) Energy conservation via electron bifurcating ferredoxin reduction and proton/Na+ translocating ferredoxin oxidation. Biochim Biophys Acta Bioenerg 1827:94–113CrossRefGoogle Scholar
  35. 35.
    Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike T (2000) Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresour Technol 73:59–65CrossRefGoogle Scholar
  36. 36.
    Valdez-Vazquez I, Ríos-Leal E, Carmona-Martínez A, Munos-Paez KM, Poggi-Varaldo HM (2006) Improvement of biohydrogen production from solid wastes by intermittent venting and gas flushing of batch reactors headspace. Environ Sci Technol 40:3409–3415CrossRefPubMedGoogle Scholar
  37. 37.
    Wolfe AJ (2005) The acetate switch. Microbiol Mol Biol Rev 69:12–50CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Butler CS, Lovley DR (2016) How to sustainably feed a microbe: strategies for biological production of carbon-based commodities with renewable electricity. Front Microbiol 7:1–6CrossRefGoogle Scholar
  39. 39.
    Martin WF (2012) Hydrogen, metals, bifurcating electrons, and proton gradients: the early evolution of biological energy conservation. FEBS Lett 586:485–493CrossRefPubMedGoogle Scholar
  40. 40.
    Furdui C, Ragsdale SW (2000) The role of pyruvate ferredoxin oxidoreductase in pyruvate synthesis during autotrophic growth by the Wood-Ljungdahl pathway. J Biol Chem 275:28494–28499CrossRefPubMedGoogle Scholar
  41. 41.
    Bock AK, Schonheit P, Teixeira M (1997) The iron-sulfur centers of the pyruvate:ferredoxin oxidoreductase from Methanosarcina barkeri (Fusaro). FEBS Lett 414:209–212CrossRefPubMedGoogle Scholar
  42. 42.
    Berg IA, Ramos-Vera WH, Petri A, Huber H, Fuchs G (2010) Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota. Microbiology 156:256–269CrossRefPubMedGoogle Scholar
  43. 43.
    Braakman R, Smith E (2012) The emergence and early evolution of biological carbon-fixation. PLoS Comput Biol 8:e1002455CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Werkman CH, Wood HG (2000) Heterotrophic assimilation of carbon dioxide. Adv Enzymol Relat Areas Mol Biol 74:135–182Google Scholar
  45. 45.
    Reilly S (1980) The carbon dioxide requirements of anaerobic bacteria. J Med Microbiol 13:573–579CrossRefPubMedGoogle Scholar
  46. 46.
    Kim S-H, Han S-K, Shin H-S (2004) Feasibility of biohydrogen production by anaerobic co-digestion of food waste and sewage sludge. Int J Hydrog Energy 29:1607–1616CrossRefGoogle Scholar
  47. 47.
    Willquist K, Claassen PAM, van Niel EWJ (2009) Evaluation of the influence of CO2 on hydrogen production by Caldicellulosiruptor saccharolyticus. Int J Hydrog Energy 34:4718–4726CrossRefGoogle Scholar
  48. 48.
    Willquist K, Zeidan AA, van Niel EWJ (2010) Physiological characteristics of the extreme thermophile Caldicellulosiruptor saccharolyticus: an efficient hydrogen cell factory. Microb Cell Factor 9:89–105CrossRefGoogle Scholar
  49. 49.
    Cappelletti M, Bucchi G, Mendes JDS, Alberini A, Fedi S, Bertin L, Frascari D (2012) Biohydrogen production from glucose, molasses and cheese whey by suspended and attached cells of four hyperthermophilic Thermotoga strains. J Chem Technol Biotechnol 87:1291–1301CrossRefGoogle Scholar
  50. 50.
    Dipasquale L, Adessi A, d’Ippolito G, Rossi F, Fontana A, De Philippis R (2015) Introducing capnophilic lactic fermentation in a combined dark-photo fermentation process: a route to unparalleled H2 yields. Appl Microbiol Biotechnol 99:1001–1010CrossRefPubMedGoogle Scholar
  51. 51.
    Li C, Fang HHP (2007) Fermentative hydrogen production from wastewater and solid wastes by mixed cultures. Crit Rev Environ Sci Technol 37:1–39CrossRefGoogle Scholar
  52. 52.
    Atsumi S, Higashide W, Liao JC (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat Biotechnol 27:1177–1180CrossRefPubMedGoogle Scholar
  53. 53.
    Hania WB, Postec A, Aüllo T, Ranchou-Peyruse A, Erauso G, Brochier-Armanet C, Hamdi M, Ollivier B, Saint-Laurent S, Magot M, Fardeau ML (2013) Mesotoga infera sp. nov., a mesophilic member of the order Thermotogales, isolated from an underground gas storage aquifer. Int J Syst Evol Microbiol 63:3003–3008CrossRefPubMedGoogle Scholar
  54. 54.
    Nesbø CL, Bradnan DM, Adebusuyi A, Dlutek M, Petrus AK, Foght J, Doolittle WF, Noll KM (2012) Mesotoga prima gen. nov., sp. nov., the first described mesophilic species of the Thermotogales. Extremophiles 16:387–393CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Laura Dipasquale
    • 1
  • Nirakar Pradhan
    • 2
    • 1
  • Giuliana d’Ippolito
    • 1
  • Angelo Fontana
    • 1
    Email author
  1. 1.Bio-Organic Chemistry Unit, Consiglio Nazionale delle Ricerche—Institute of Biomolecular ChemistryNational Research Council of ItalyPozzuoli (Naples)Italy
  2. 2.Department of Civil and Mechanical EngineeringUniversity of Cassino and Southern LazioCassinoItaly

Personalised recommendations