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Analytical Characterization of Fermentation Inhibitors in Biomass Pretreatment Samples Using Liquid Chromatography, UV-Visible Spectroscopy, and Tandem Mass Spectrometry

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Biofuels

Part of the book series: Methods in Molecular Biology ((MIMB,volume 581))

Summary

A variety of chemicals are produced upon pretreatment of lignocellulosic biomass. Aliphatic acids, aromatic acids, aldehydes, and phenolic compounds are of particular interest due to their presumed inhibitory influence on downstream enzymatic or microbial steps in biomass-to-ethanol conversion. Herein, we describe a series of analytical protocols that collectively enable quantitative monitoring of 40 potential fermentation inhibitors in biomass pretreatment samples. Solid samples are accommodated by first employing pressurized fluid extraction to generate an aqueous “wash stream.” Sample preparation for liquids involves an initial precipitation-filtration step, followed by liquid–liquid extraction and reconstitution of extracts in water. Samples are analyzed using high–performance liquid chromatography (HPLC) in combination with ultraviolet (UV) absorbance and tandem mass spectrometry (MS/MS) detection. A standard addition approach is utilized for quantitation to alleviate complications arising from co-extracted sample matrix.

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References

  1. Mielenz, J. R. (2001) Ethanol production from biomass: technology and commercialization status. Curr Opin Microbiol. 4, 324–329

    Article  CAS  Google Scholar 

  2. Zaldivar, J., Nielsen, J., and Olsson, L. (2001) Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl. Microbiol. Biotechnol. 56, 17–34

    Article  CAS  Google Scholar 

  3. Lynd, L. R., Wyman, C. E., and Gerngross, T. U. (1999). Biocommodity engineering. Biotechnol. Prog. 15, 777–793

    Article  CAS  Google Scholar 

  4. Berlin, A., Balakshin, M., Gilkes, N., Kadla, J., Maximenko, V., Kubo, S., Saddler, J.(2006) Inhibition of cellulase, xylanase and β-glucosidase activities by softwood lignin preparations. J. Biotechnol. 125, 198–209

    Article  CAS  Google Scholar 

  5. Klinke, H. B., Thomesen, A. B., and Ahring, B. K. (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl. Microbiol. Biotechnol. 66, 10–26

    Article  CAS  Google Scholar 

  6. Saha, B. C. (2003) Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30, 279–291

    Article  CAS  Google Scholar 

  7. Taherzadeh, M. J., Eklund, R., Gustafsson, L., Niklasson, C., and Liden, G. (1997) Characterization and fermentation of dilute-acid hydrolyzates from wood. Ind. Eng. Chem. Res. 36, 4659–4665

    Article  CAS  Google Scholar 

  8. Ranatunga, T. D., Jervis, J., Helm, R. F., McMillan, J. D., and Woodey, R. J. (2000) The effect of overliming on the toxicity of dilute acid pretreated ligncellulosics: the role of inorganics, uronic acids and ether-soluble organics. Enzyme Microb. Technol. 27, 240–247

    Article  CAS  Google Scholar 

  9. Larsson, S., Reimann, A., Nilvebrant, N.-O., and Jonsson, L. J. (1999) Comparison of different methods for the detoxification of lignocellulose hydrolysates of spruce. Appl. Biochem. Biotechnol. 77/79, 91–103

    Article  Google Scholar 

  10. Alves, L. A., Felipe, M. G. A., Silva, J. B. A. E., Silva, S. S., and Prata, A. M. R. (1998). Pretreatment of sugarcane bagasse hemicellulose hydrolysate for xylitol production by Candida guilliermondi. Appl. Biochem. Biotechnol. 70/72, 89–98

    Article  Google Scholar 

  11. Leonard, R. H. and Hajny, G. J. (1945) Fermentation of wood sugars to ethyl alcohol Ind. Eng. Chem. 37, 390–395

    Article  CAS  Google Scholar 

  12. Martinez, A., Rodriguez, M. E., Wells, M. L., York, S. W., Preston, J. F., and Ingram, L. (2001). Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnol. Prog. 17, 287–293

    Article  CAS  Google Scholar 

  13. Martin, C., Galbe, M., Wahlbom, C., HahnHagerdal, B., and Jonsson, L. (2002) Ethanol production from enzymatic hydrolysates of sugarcane bagasse using recombinantxylose-utilising Saccharomyces cerevisiae. Enzyme Microb. Technol. 31, 274–282

    Article  CAS  Google Scholar 

  14. Mohagheghi, A., Ruth, M., and Schell, D. J. (2006) Conditioning hemicellulose hydrolysates for fermentation: effects of overliming pH on sugar and ethanol yields. Process Biochem. 41, 1806–1811

    Article  CAS  Google Scholar 

  15. Millati, R., Niklasson, C., and Taherzadeh, M. J. (2002) Effect of pH, time and temperature of overliming on detoxification of dilute-acid hydrolyzates for fermentation by Saccharomyces cerevisiae. Process Biochem. 38, 515–522

    Article  CAS  Google Scholar 

  16. Luo, C., Brink, D. L., and Blanch, H. W. (2002) Identification of potential fermentation inhibitors in conversion of hybrid poplar hydrolyzate to ethanol. Biomass Bioenergy 22, 125–138

    Article  CAS  Google Scholar 

  17. Tran, A. V. and Chambers, R. P. (1986) Ethanol Fermentation of Red Oak Acid Prehydrolysate by the Yeast Pichia stipitis CBS 5776. Enzyme Microb. Technol. 18, 312–331

    Google Scholar 

  18. Weil, J. R., Dien, B.; Bothast, R.; Hend-rickson, R.; Mosier, N. S.; Ladisch, M. R. (2002) Removal of fermentation inhibitors formed during pretreatment of biomass by polymeric adsorbents. Ind. Eng. Chem. Res. 41, 6132–6138

    Article  CAS  Google Scholar 

  19. Nichols, N. N., Dien, B. S., Guisado, G. M., Lopez, M. J. (2005) Bioabatement to remove inhibitors from biomass-derived sugar hydrolysates. Appl. Biochem. Biotechnol. 121–124, 379–390

    Article  Google Scholar 

  20. Chen, S.-F., Mowery, R. A., Chambliss, C.K., and van Walsum, G. P. (2007) Pseudo reaction kinetics of organic degradation products in dilute-acid-catalyzed corn stover pretreatment hydrolysates. Biotechnol. Bioeng. 98, 1135–1145

    Article  CAS  Google Scholar 

  21. Kim, S. and Holtzapple, M. T. (2005) Lime pretreatment and enzymatic hydrolysis of corn stover. Biores. Technol. 96, 1994–2006

    Article  CAS  Google Scholar 

  22. Cantarella, M., Cantarella, L., Gallifuoco, A., Spera, A., and Alfani, F. (2004) Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF. Biotechnol. Prog. 20, 200–206

    Article  CAS  Google Scholar 

  23. Klinke, H. B., Thomsen, A. B., and Ahring, B. K. (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl. Microbiol. Biotechnol. 66, 10–26

    Article  CAS  Google Scholar 

  24. Chen, S.-F., Mowery, R. A., Castleberry, V. A., van Walsum, G. P., and Chambliss, C. K. (2006) High-performance liquid chromatography method for simultaneous determination of aliphatic acid, aromatic acid and neutral degradation products in biomass pretreatment hydrolysates. J. Chromatogr. A 1104, 54–61

    Article  CAS  Google Scholar 

  25. Snyder, L. R., Kirkland, J. J., and Glajch, J. L. (1997) Practical HPLC Method Development. Wiley, New York, NY

    Google Scholar 

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Acknowledgments

This work was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2005–35504–16355. The authors would also like to acknowledge the following collaborators for providing pretreatment samples: Dr. G. Peter van Walsum (University of Maine); Dr. Bruce E. Dale, Dr. Balan Venkatesh, and Mr. Shishir Chundawat (Michigan State University); Dr. Nancy N. Nichols (USDA-ARS, Peoria, IL); Dr. Joy D. Peterson (University of Georgia).

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Authors and Affiliations

  1. Department of Chemistry & Biochemistry, Baylor University, Waco, TX, USA

    Lekh N. Sharma, Christopher Becker & C. Kevin Chambliss

Authors
  1. Lekh N. Sharma
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  2. Christopher Becker
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  3. C. Kevin Chambliss
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Editors and Affiliations

  1. Bioconversion Science & Technology, Oak Ridge National Laboratory, Bethel Valley Road 1, Oak Ridge, 37831-6341, U.S.A.

    Jonathan R. Mielenz

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Sharma, L.N., Becker, C., Chambliss, C.K. (2009). Analytical Characterization of Fermentation Inhibitors in Biomass Pretreatment Samples Using Liquid Chromatography, UV-Visible Spectroscopy, and Tandem Mass Spectrometry. In: Mielenz, J. (eds) Biofuels. Methods in Molecular Biology, vol 581. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-214-8_10

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  • DOI: https://doi.org/10.1007/978-1-60761-214-8_10

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-213-1

  • Online ISBN: 978-1-60761-214-8

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