Skip to main content
SpringerLink
Log in
Book cover

Microbial Lipid Production pp 249–283Cite as

Screening for Oily Yeasts Able to Convert Hydrolysates from Biomass to Biofuels While Maintaining Industrial Process Relevance

  • Protocol
  • First Online:

  • 1229 Accesses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1995))

Abstract

Research has recently intensified to discover new oleaginous yeast strains able to function quickly and efficiently in low-cost lignocellulosic hydrolysates to produce high-quality lipids for use in biodiesel and chemicals. Detailed techniques are given here for ranking candidate yeast strains based on conversion of hydrolysate sugars to lipids and then optimizing cultivation conditions for best performers in a 96-well aerobic microcultivation format. A full battery of assays applicable to high throughput of small-volume samples are described for efficiently evaluating cell biomass production, lipid accumulation, fatty acid composition, and sugar utilization. Original data is additionally presented on the validation of the microtechnique for GC analysis of lipid composition in yeast since this application involved modification of a previously published assay for microalgae.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG (2011) Oily yeasts as oleaginous cell factories. Appl Microbiol Biotechnol 90(4):1219–1227

    Article  CAS  Google Scholar 

  2. Leiva-Candia D, Pinzi S, Redel-Macías M, Koutinas A, Webb C, Dorado M (2014) The potential for agro-industrial waste utilization using oleaginous yeast for the production of biodiesel. Fuel 123:33–42

    Article  CAS  Google Scholar 

  3. Sitepu IR, Garay LA, Sestric R, Levin D, Block DE, German JB, Boundy-Mills KL (2014) Oleaginous yeasts for biodiesel: current and future trends in biology and production. Biotechnol Adv 32(7):1336–1360

    Article  CAS  Google Scholar 

  4. Jin M, Slininger PJ, Dien BS, Waghmode SB, Moser BR, Sousa LC, Orjuela A, Balan V (2015) Microbial lipid based lignocellulosic biorefinery: feasibility and challenges. Trends Biotechnol 33(1):43–54

    Article  CAS  Google Scholar 

  5. Thorpe R, Ratledge C (1972) Fatty acid distribution in triglycerides of yeasts grown on glucose or n-alkanes. J Gen Microbiol 72:151–163

    Article  CAS  Google Scholar 

  6. Slininger PJ, Dien BS, Kurtzman CP, Moser BR, Bakota EL, Thompson SR, O’Bryan PJ, Cotta MA, Balan V, Jin M (2016) Comparative lipid production by oleaginous yeasts in hydrolyzates of lignocellulosic biomass and process strategy for high titers. Biotechnol Bioeng 113:1676–1690

    Article  CAS  Google Scholar 

  7. US Department of Energy (2011) U.S. billion-ton update: biomass supply for a bioenergy and bioproducts industry (R.D. Perlack and B.J. Stokes (Leads)), Oak Ridge, TN: ORNL/TM-2011/224. Oak Ridge National laboratory, p 227

    Google Scholar 

  8. Dugar D, Stephanopoulos G (2011) Relative potential of biosynthetic pathways for biofuels and bio-based products. Nat Biotechnol 29(12):1074–1078

    Article  CAS  Google Scholar 

  9. Ratledge C, Cohen Z (2008) Microbial and algal oils: do they have a future for biodiesel or as commodity oils? Lipid Technol 20(7):155–160

    Article  Google Scholar 

  10. Bates G, Keyser P, Harper C, Waller J (2008) Using switchgrass for forage (https://utextension.tennessee.edu/publications/documents/SP701-B.pdf). UT Biofuels Initiative, University of Tennessee Institute of Agriculture

  11. Dien BS, Jung H-J, Vogel K, Casler M, Lamb J, Iten L, Mitchell R, Sarath G (2006) Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass. Biomass Bioenergy 30(10):880–891

    Article  CAS  Google Scholar 

  12. Dien BS, O’Bryan P, Hector R, Iten L, Mitchell R, Qureshi N, Sarath G, Vogel K, Cotta M (2013) Conversion of switchgrass to ethanol using dilute ammonium hydroxide pretreatment: influence of ecotype and harvest maturity. Environ Technol 34(13–14):1837–1848

    Article  CAS  Google Scholar 

  13. Dien BS, Zhu J, Slininger PJ, Kurtzman CP, Moser BR, O’Bryan PJ, Gleisner R, Cotta MA (2016) Conversion of SPORL pretreated Douglas fir forest residues into microbial lipids with oleaginous yeasts. RSC Adv 6(25):20695–20705

    Article  CAS  Google Scholar 

  14. Koutinas A, Chatzifragkou A, Kopsahelis N, Papanikolaou S, Kookos I (2014) Design and techno-economic evaluation of microbial oil production as a renewable resource for biodiesel and oleochemical production. Fuel 116:566–577

    Article  CAS  Google Scholar 

  15. Dien BS, Slininger PJ, Kurtzman CP, Moser BR, O’Bryan PJ (2016) Identification of superior lipid producing Lipomyces and Myxozyma yeasts. AIMS Environ Sci 3(1):1–20

    Article  CAS  Google Scholar 

  16. Quarterman J, Slininger PJ, Kurtzman CP, Thompson SR, Dien BS (2017) A survey of yeast from the Yarrowia clade for lipid production in dilute acid pretreated lignocellulosic biomass hydrolysate. Appl Microbiol Biotechnol 101(8):3319–3334

    Article  CAS  Google Scholar 

  17. Liu ZL, Slininger PJ, Dien BS, Berhow MA, Kurtzman CP, Gorsich SW (2004) Adaptive response of yeast to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. J Ind Microbiol Biotechnol 31:345–352

    Article  CAS  Google Scholar 

  18. Weete JD (1980) Chapter 2: Fungal lipids. In: Weete JD (ed) Lipid biochemistry of fungi and other organisms. Plenum Press, New York, pp 9–48

    Chapter  Google Scholar 

  19. Sitepu IR, Sestric R, Ignatia L, Levin D, Bruce GJ, Gillies LA, Almada LA, Boundy-Mills KL (2013) Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeasts species. Bioresour Technol 144:360–369

    Article  CAS  Google Scholar 

  20. Hounslow E, Noirel J, Gilmour DJ, Wright PC (2017) Lipid quantification techniques for screening oleaginous species of microalgae for biofuel production. Eur J Lipid Sci Technol 119(2):1500469

    Article  Google Scholar 

  21. Bligh EG (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8):911–917

    Article  CAS  Google Scholar 

  22. Sitepu I, Ignatia L, Franz A, Wong D, Faulina S, Tsui M, Kanti A, Boundy-Mills K (2012) An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species. J Microbiol Methods 91:321–328

    Article  CAS  Google Scholar 

  23. Govender T, Ramanna L, Rawat I, Bux F (2012) BODIPY staining, an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae. Bioresour Technol 114:507–511

    Article  CAS  Google Scholar 

  24. Krygsman P, Barrett A (2004) Simple methods for measuring total oil content by bench-top NMR. In: D.L. Luthira (ed), Oil extraction and analysis, AOCS Press, Champagin, Illinois pp 152–165

    Google Scholar 

  25. Cheng Y-S, Zheng Y, VanderGheynst JS (2011) Rapid quantitative analysis of lipids using a colorimetric method in a microplate format. Lipids 46(1):95–103

    Article  CAS  Google Scholar 

  26. Johnson K, Ellis G, Toothill C (1977) The sulfophosphovanillin reaction for serum lipids: a reappraisal. Clin Chem 23(9):1669–1678

    CAS  PubMed  Google Scholar 

  27. Knight JA, Anderson S, Rawle JM (1972) Chemical basis of the sulfo-phospho-vanillin reaction for estimating total serum lipids. Clin Chem 18(3):199–202

    CAS  PubMed  Google Scholar 

  28. Laurens LM, Quinn M, Van Wychen S, Templeton DW, Wolfrum EJ (2012) Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by in situ transesterification. Anal Bioanal Chem 403(1):167–178

    Article  CAS  Google Scholar 

  29. Izard J, Limberger RJ (2003) Rapid screening method for quantitation of bacterial cell lipids from whole cells. J Microbiol Methods 55(2):411–418

    Article  CAS  Google Scholar 

  30. Wang J, Li R, Lu D, Ma S, Yan Y, Li W (2009) A quick isolation method for mutants with high lipid yield in oleaginous yeast. World J Microbiol Biotechnol 25(5):921–925

    Article  CAS  Google Scholar 

  31. Slininger PJ, Shea-Andersh MA, Thompson SR, Dien BS, Kurtzman CP, Balan V, da Costa Sousa L, Uppugundla N, Dale BE, Cotta MA (2015) Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid-and base-pretreated biomass hydrolyzate at high solids loading. Biotechnol Biofuels 8(1):60

    Article  Google Scholar 

  32. Gao C, Xiong W, Zhang Y, Yuan W, Wu Q (2008) Rapid quantitation of lipid in microalgae by time-domain nuclear magnetic resonance. J Microbiol Methods 75(3):437–440

    Article  CAS  Google Scholar 

  33. Moreton R (1989) Yeast lipid estimation by enzymatic and nuclear magnetic resonance methods. Appl Environ Microbiol 55(11):3009–3011

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Ravimannan SP (2016) Soy flour as alternative culture media for yeasts. Global J Sci Front Res 16(3):75–78

    Google Scholar 

  35. Zabriski DW, Armiger WB, Phillips DH, Albano PA (1980) Fermentation media formulation. In: Trader’s guide to fermentation media formulation. Trader’s Protein Memphis, TN, pp 1–39

    Google Scholar 

  36. Kennedy M, Krouse D (1999) Strategies for improving fermentation medium performance: a review. J Ind Microbiol Biotechnol 23(6):456–475

    Article  CAS  Google Scholar 

  37. Webb SR (1971) Small incomplete factorial experiment designs for two- and three-level factors. Technometrics 13(2):243–256

    Article  Google Scholar 

  38. Donaldson M (2012) Development of a rapid, simple assay of plasma total carotenoids. BMC Res Notes 5(1):521

    Article  CAS  Google Scholar 

  39. Tinoi J, Rakariyatham N, Deming R (2005) Simplex optimization of carotenoid production by Rhodotorula glutinis using hydrolyzed mung bean waste flour as substrate. Process Biochem 40(7):2551–2557

    Article  CAS  Google Scholar 

  40. Hart DJ, Scott KJ (1995) Development and evaluation of an HPLC method for the analysis of carotenoids in foods, and the measurement of the carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chem 54(1):101–111

    Article  CAS  Google Scholar 

  41. Rodriguez-Amaya DB (2001) A guide to carotenoid analysis in foods. ILSI Press, International Life Sciences Institute, Washington, DC

    Google Scholar 

Download references

Acknowledgments

The authors would like to thank Maureen Shea-Andersh for her valuable technical assistance in support of the oleaginous yeast project.

Disclaimer: The mention of trade names or commercial products in this chapter is solely for the purpose of providing specific information and does not imply any recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.

Author information

Authors and Affiliations

  1. Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL, USA

    Patricia J. Slininger, Bruce S. Dien, Joshua C. Quarterman & Stephanie R. Thompson

  2. Mycotoxin Prevention and Applied Microbiology Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL, USA

    Cletus P. Kurtzman

Authors
  1. Patricia J. Slininger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bruce S. Dien
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joshua C. Quarterman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephanie R. Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cletus P. Kurtzman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Patricia J. Slininger .

Editor information

Editors and Affiliations

  1. Department of Engineering Technology, Biotechnology Program College of Technology, University of Houston, Houston, TX, USA

    Venkatesh Balan

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Slininger, P.J., Dien, B.S., Quarterman, J.C., Thompson, S.R., Kurtzman, C.P. (2019). Screening for Oily Yeasts Able to Convert Hydrolysates from Biomass to Biofuels While Maintaining Industrial Process Relevance. In: Balan, V. (eds) Microbial Lipid Production. Methods in Molecular Biology, vol 1995. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9484-7_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9484-7_16

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9483-0

  • Online ISBN: 978-1-4939-9484-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation