Characterization of Hydrogen Production by Escherichia coli Wild-type and Mutants of Hydrogenases Utilizing Xylose as Fermentation Substrate

  • Anna Poladyan
  • Armen TrchounianEmail author


Xylose, one of the most common sugars derived from lignocellulosic material, can be fermented by bacteria. Escherichia coli uses natural sugars to produce biomass and H2. The latter can be formed from the formate via formate hydrolysis (FHL) during the fermentation of xylose or glucose; [Ni-Fe]-hydrogenase (Hyd) is involved in H2 formation. The growth, H2 production characteristics of E. coli BW25113 (wild-type), and ΔhyaB, ΔhybC, ΔhycE, or ΔhyfG mutant strains with deletions of catalytic subunits of Hyd-1 to Hyd-4, respectively, were monitored with addition of 0, 0.5, to 1% xylose on peptone and minimal salt-based mediums, pH 5.5 and pH 7.5. At pH 5.5, with the growth of bacteria on both media, H2 produced in the early logarithmic phase (1.40–0.02 mmol H2 L−1), whereas at pH 7.5 it is noticeably delayed. Results showed that during the fermentation of xylose, Hyd-3 and Hyd-4 are important for both bacterial growth and production of H2, mainly at pH 5.5. In addition, high xylose concentrations can stimulate the activity of the Hyd-1 enzyme during bacterial growth at pH 7.5. These results are new and important for developing advanced H2 production technologies using xylose as a feedstock.


Bioenergy and biomass Xylose fermentation and pH H2 production and hydrogenase enzymes Escherichia coli 


Funding Information

This work was supported by research grants of the Committee of Science, Ministry of Education and Science of Armenia, to AT (#AB16-37 and 18T-F045) and Armenian National Science and Education Fund (USA) research grant to AP (NS-Biotech-5285).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Chu S, Majumdar A (2012) Opportunities and challenges for a sustainable energy future. Nature 488:294–303CrossRefGoogle Scholar
  2. 2.
    Sánchez OJ, Cardona CA (2008) Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresour Technol 99:5270–5295CrossRefGoogle Scholar
  3. 3.
    Kumar G, Bakonyi P, Periyasamy S, Kim SH, Nemestóthy N, Bélafi-Bakó K (2015) Lignocellulose biohydrogen: practical challenges and recent progress. Renew Sustain Energy Rev 44:728–773CrossRefGoogle Scholar
  4. 4.
    Kapdan IK, Kargi F (2006) Bio-hydrogen production from waste materials. J Enzyme Microb Technol 38:569–582CrossRefGoogle Scholar
  5. 5.
    Cheng CL, Lo YC, Lee KS, Lee DJ, Lin CY, Chang JS (2011) Biohydrogen production from lignocellulosic feedstock. Bioresour Technol 102:8514–5818CrossRefGoogle Scholar
  6. 6.
    Sargsyan H, Gabrielyan L, Trchoinian A (2016) Novel approach of ethanol waste utilization: biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains. Int J Hydrogen Energy 41:2377–2382CrossRefGoogle Scholar
  7. 7.
    Kwak S, Jin YS (2017) Production of fuels and chemicals from xylose by engineered Saccharomyces cerevisiae: a review and perspective. Microb Cell Fact 16:1. CrossRefGoogle Scholar
  8. 8.
    Trchounian K, Sawers G, Trchounian A (2017) Improving biohydrogen productivity by microbial dark- and photo-fermentations: novel data and future approaches. Renew Sustain Energy Rev. 80:1201–1216CrossRefGoogle Scholar
  9. 9.
    Kumar AK, Sharma S (2017) Recent updates on different methods of pretreatment of lignocellulosic feedstocks: a review. Bioresour Bioprocess 4:7. CrossRefGoogle Scholar
  10. 10.
    Gonzalez EJ, Long PC, Antoniewicz MR (2017) Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis. Metab Eng 39:9–18CrossRefGoogle Scholar
  11. 11.
    Trchounian K, Poladyan A, Vassilian A, Trchounian A (2012) Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and the F0F1-ATPase. Crit Rev Biochem Mol Biol 47:236–249CrossRefGoogle Scholar
  12. 12.
    Luo Y, Zhang T, Wu H (2014) The transport and mediation mechanisms of the common sugars. Biotechnol Adv 32:905–919CrossRefGoogle Scholar
  13. 13.
    Xia T, Sriram N, Lee SA, Altman R, Urbauer LJ, Altman E, Eiteman MA (2017) Glucose consumption in carbohydrate mixtures by phosphotransferase-system mutants of Escherichia coli. Microbiology 163:866–877CrossRefGoogle Scholar
  14. 14.
    Khankal R, Chin JW, Cirino PC (2008) Role of xylose transporters in xylitol production from engineered Escherichia coli. J Biotechnol 134:246–252CrossRefGoogle Scholar
  15. 15.
    Desai TA, Rao CV (2010) Regulation of arabinose and xylose metabolism in Escherichia coli. Appl Environ Microbiol 76:1524–1532CrossRefGoogle Scholar
  16. 16.
    Trchounian A (2015) Mechanisms for hydrogen production by different bacteria during mixed-acid and photo-fermentation and perspectives of hydrogen production biotechnology. Crit Rev Biotechnol 35:103–113CrossRefGoogle Scholar
  17. 17.
    Sargent F (2016) The model [NiFe]-hydrogenases of Escherichia coli. Adv Microb Physiol 68:433–507CrossRefGoogle Scholar
  18. 18.
    Trchounian K, Pinske C, Sawers G, Trchounian A (2012) Characterization of Escherichia coli [NiFe]-hydrogenase distribution during fermentative growth at different pHs. Cell Biochem Biophys. 62:433–440CrossRefGoogle Scholar
  19. 19.
    Bagramyan K, Mnatsakanyan N, Poladian A, Vassilian A, Trchounian A (2002) The roles of hydrogenases 3 and 4, and the F0F1-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH. FEBS Lett 516:172–178CrossRefGoogle Scholar
  20. 20.
    Maeda T, Sanchez-Torres V, Wood TK (2007) Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities. Appl Microbiol Biotechnol 76:1035–1042CrossRefGoogle Scholar
  21. 21.
    Trchounian K, Trchounian A (2015) Hydrogen producing activity by Escherichia coli hydrogenase 4 (hyf) depends on glucose concentration. Int J Hydrogen Energy 39:16914–16918CrossRefGoogle Scholar
  22. 22.
    Gonzalez R, Tao H, Shanmugam KT, York SW, Ingram LO (2002) Global gene expression differences associated with changes in glycolytic flux and growth rate in Escherichia coli during the fermentation of glucose and xylose. Biotechnol Prog 18:6–20CrossRefGoogle Scholar
  23. 23.
    Koirala S, Wang X, Rao CV (2016) Reciprocal regulation of L-arabinose and D-xylose metabolism in Escherichia coli. J Bacteriol 198:386–393CrossRefGoogle Scholar
  24. 24.
    Hasona A, Kim Y, Healy FG, Ingram LO, Shanmugam KT (2004) Pyruvate formate lyase and acetate kinase are essential for anaerobic growth of Escherichia coli on xylose. J Bacteriol 186:7593–7600CrossRefGoogle Scholar
  25. 25.
    Poladyan A, Trchounian K, Vasilian A, Trchouian A (2018) Hydrogen production by Escherichia coli using brewery waste: optimal pretreatment of waste and role of different hydrogenases. Renew Energy 115:931–936CrossRefGoogle Scholar
  26. 26.
    Poladyan A, Baghdasaryan L, Trchounian A (2018) Escherichia coli wild type and hydrogenase mutant cells growth and hydrogen production upon xylose and glycerol co-fermentation in media with different buffer capacities. Int J Hydrogen Energy 43(33):15870–15879CrossRefGoogle Scholar
  27. 27.
    Paliy O, Gunasekera TS (2007) Growth of E. coli BL21 in minimal media with different gluconeogenic carbon sources and salt contents. Appl Microbiol Biotechnol 73:1169–1172CrossRefGoogle Scholar
  28. 28.
    Poladyan A, Avagyan A, Vassilian A, Trchounian A (2013) Oxidative and reductive routes of glycerol and glucose fermentation by Escherichia coli batch cultures and their regulation by oxidizing and reducing reagents at different pHs. Curr Microbiol 66:49–55CrossRefGoogle Scholar
  29. 29.
    Vassilian A, Trchounian A (2009) Environment oxidation-reduction potential and redox sensing by bacteria. In Bacterial membranes (ed. A. Trchounian). Research Signpost, Kerala (India) pp. 163-95.Google Scholar
  30. 30.
    Eiteman MA, Lee SA, Altman E (2008) A co-fermentation strategy to consume sugar mixtures effectively. J Biol Eng 2(3):1–8. Google Scholar
  31. 31.
    Ammar EM, Wang X, Rao CV (2018) Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose. Sci Rep 8:609. CrossRefGoogle Scholar
  32. 32.
    Kim JH, David E, Block, David A, Mills (2010) Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass. Appl Microbiol Biotechnol 88:1077–1085CrossRefGoogle Scholar
  33. 33.
    Mnatsakanyan N, Bagramyan K, Trchounian A (2004) Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogen lyase at acidic pH and in the presence of external formate. Cell Biochem Biophys 41:357–366CrossRefGoogle Scholar
  34. 34.
    Rossmann R, Sawers G, Bock A (1991) Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon. Mol Microbiol 5:2807–2814CrossRefGoogle Scholar
  35. 35.
    Ghosh D, Hallenbeck PC (2009) Fermentative hydrogen yields from different sugars by batch cultures of metabolically engineered Escherichia coli DJT135. Int J Hydrogen Energy 34:7979–7982CrossRefGoogle Scholar
  36. 36.
    Zhao L, Wang Z-H, Wu J-T, Ren H-R, Yang S-S, Nan J, Cao G-L, Sheng C, Nan JW, Ren N-Q (2019) Co-fermentation of a mixture of glucose and xylose to hydrogen by Thermoanaerobacter thermosaccharolyticum W16: characteristics and kinetics. Int J Hydrogen Energy Epub Mar 12.
  37. 37.
    Pinske C, Sawers RG (2016) Anaerobic formate and hydrogen metabolism. Eco Sal Plus 7(1).
  38. 38.
    Trchounian K, Blbulyan S, Trchounian A (2013) Hydrogenase activity and proton-motive force generation by Escherichia coli during glycerol fermentation. J Bioenerg Biomembr 45:253–260CrossRefGoogle Scholar
  39. 39.
    Noguchi K, Riggins DP, Eldahan KC, Kitko RD, Slonczewski JL (2010) Hydrogenase-3 contributes to anaerobic acid resistance of Escherichia coli. PloS one 5(4):e10132CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biochemistry, Microbiology and Biotechnology, Biology FacultyYerevan State UniversityYerevanArmenia
  2. 2.Research Institute of Biology, Biology FacultyYerevan State UniversityYerevanArmenia

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