Applied Microbiology and Biotechnology

, Volume 98, Issue 17, pp 7645–7657 | Cite as

Identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover

  • Irnayuli R. Sitepu
  • Mingjie Jin
  • J. Enrique Fernandez
  • Leonardo da Costa Sousa
  • Venkatesh Balan
  • Kyria L. Boundy-Mills
Bioenergy and biofuels


Microbial oil is a potential alternative to food/plant-derived biodiesel fuel. Our previous screening studies identified a wide range of oleaginous yeast species, using a defined laboratory medium known to stimulate lipid accumulation. In this study, the ability of these yeasts to grow and accumulate lipids was further investigated in synthetic hydrolysate (SynH) and authentic ammonia fiber expansion (AFEX™)-pretreated corn stover hydrolysate (ACSH). Most yeast strains tested were able to accumulate lipids in SynH, but only a few were able to grow and accumulate lipids in ACSH medium. Cryptococcus humicola UCDFST 10-1004 was able to accumulate as high as 15.5 g/L lipids, out of a total of 36 g/L cellular biomass when grown in ACSH, with a cellular lipid content of 40 % of cell dry weight. This lipid production is among the highest reported values for oleaginous yeasts grown in authentic hydrolysate. Preculturing in SynH media with xylose as sole carbon source enabled yeasts to assimilate both glucose and xylose more efficiently in the subsequent hydrolysate medium. This study demonstrates that ACSH is a suitable medium for certain oleaginous yeasts to convert lignocellullosic sugars to triacylglycerols for production of biodiesel and other valuable oleochemicals.


Lignocellulosic Cryptococcus humicola Biodiesel Energy Oleochemics 



Some yeasts used in this study isolated and identified as part of a collaborative project with the Government of the Republic of Indonesia, funded by Grant Number U01TW008160 from the NIH Fogarty International Center, the NIH Office of Dietary Supplements, the National Science Foundation and the Department of Energy. This project was supported by the USDA Agricultural Food Research Initiative of the National Food and Agriculture, USDA, Grant #35621-04750. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health, the Office of Dietary Supplements, the National Science Foundation, the Department of Energy, or the Department of Agriculture. Atit Kanti and Agustinus Joko Nugroho of LIPI Biology, Indonesia, and Sarah Asih Faulina of FORDA, Ministry of Forestry, Indonesia, were thanked for the help with isolation of Indonesia microbes. Erin Cathcart and Silviana Tjahyono of UC Davis are thanked for their technical assistance. The authors thanked the anonymous reviewers for providing valuable feedback that significantly improved the quality of the manuscript.


  1. Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG (2011) Oily yeasts as oleaginous cell factories. Appl Microbiol Biot 90(4):1219–1227CrossRefGoogle Scholar
  2. Alvira P, Tomás-Pejó E, Ballesteros M, Negro M (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 101(13):4851–4861PubMedCrossRefGoogle Scholar
  3. Atabani A, Silitonga A, Badruddin I, Mahlia T, Masjuki H, Mekhilef S (2012) A comprehensive review on biodiesel as an alternative energy resource and its characteristics. Renew Sust Energ Rev 16:2070–2093CrossRefGoogle Scholar
  4. Balan V, Bals B, Chundawat SS, Marshall D, Dale B (2009) Lignocellulosic biomass pretreatment using AFEX. In: Mielenz JR (ed) Biofuels. Methods in Molecular Biology, vol 581. Humana Press, pp 61-77Google Scholar
  5. Beopoulos A, Mrozova Z, Thevenieau F, Le Dall M-T, Hapala I, Papanikolaou S, Chardot T, Nicaud J-M (2008a) Control of lipid accumulation in the yeast Yarrowia lipolytica. Appl Environ Microbiol 74(24):7779–7789PubMedCentralPubMedCrossRefGoogle Scholar
  6. Beopoulos A, Mrozova Z, Thevenieau F, Le Dall MT, Hapala I, Papanikolaou S, Chardot T, Nicaud JM (2008b) Control of lipid accumulation in the yeast Yarrowia lipolytica. Appl Environ Microbiol 74(24):7779–7789. doi: 10.1128/AEM.01412-08 PubMedCentralPubMedCrossRefGoogle Scholar
  7. Beopoulos A, Chardot T, Nicaud JM (2009) Yarrowia lipolytica: a model and a tool to understand the mechanisms implicated in lipid accumulation. Biochimie 91(6):692–696. doi: 10.1016/j.biochi.2009.02.004 PubMedCrossRefGoogle Scholar
  8. Chen X, Li Z, Zhang X, Hu F, Ryu DD, Bao J (2009) Screening of oleaginous yeast strains tolerant to lignocellulose degradation compounds. Appl Biochem Biotechnol 159(3):591–604PubMedCrossRefGoogle Scholar
  9. Chen X-f, Huang C, Xiong L, L-l M (2012) Microbial oil production from corncob acid hydrolysate by Trichosporon cutaneum. Biotechnol Lett 34(6):1025–1028PubMedCrossRefGoogle Scholar
  10. Chundawat SPS, Vismeh R, Sharma LN, Humpula JF, da Costa Sousa L, Chambliss CK, Jones AD, Balan V, Dale BE (2010) Multifaceted characterization of cell wall decomposition products formed during ammonia fiber expansion (AFEX) and dilute acid based pretreatments. Bioresour Technol 101(21):8429–8438. doi: 10.1016/j.biortech.2010.06.027 PubMedCrossRefGoogle Scholar
  11. Cohen Z, Ratledge C (2005) Single Cell Oils. AOCS Press, ChampaignGoogle Scholar
  12. Dekker RF (1989) Biodegradation of the hetero-1, 4-linked xylans. Plant cell wall polymers. American Chemical Society, Washington, pp 619–629CrossRefGoogle Scholar
  13. Galafassi S, Cucchetti D, Pizza F, Franzosi G, Bianchi D, Compagno C (2012) Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis. Bioresour Technol 111:398–403PubMedCrossRefGoogle Scholar
  14. Gao Q, Cui Z, Zhang J, Bao J (2014) Lipid fermentation of corncob residues hydrolysate by oleaginous yeast Trichosporon cutaneum. Bioresour Technol 152:552–556Google Scholar
  15. Gong Z, Wang Q, Shen H, Hu C, Jin G, Zongbao K (2012) Co-fermentation of cellobiose and xylose by Lipomyces starkeyi for lipid production. Bioresour Technol 117:20–24PubMedCrossRefGoogle Scholar
  16. Huang C, Zong M-H, Wu H, Liu Q-P (2009) Microbial oil production from rice straw hydrolysate by Trichosporon fermentans. Bioresour Technol 100(19):4535–4538PubMedCrossRefGoogle Scholar
  17. Huang C, Chen X-F, Xiong L, Chen X-D, Ma L-L (2012a) Oil production by the yeast Trichosporon dermatis cultured in enzymatic hydrolysates of corncobs. Bioresource Technology 110(0):711–714 doi: 10.1016/j.biortech.2012.01.077
  18. Huang C, Wu H, Li R-F, Zong M-H (2012b) Improving lipid production from bagasse hydrolysate with Trichosporon fermentans by response surface methodology. New Biotechnol 29(3):372–378CrossRefGoogle Scholar
  19. Huang C, Wu H, Liu Z, Cai J, Lou W-y, Zong M-h (2012c) Effect of organic acids on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans. Biotechnol Biofuels 5(4)Google Scholar
  20. Huang C, Chen X-F, Xiong L, Chen X-D, Ma L-L, Chen Y (2013a) Single cell oil production from low-cost substrates: the possibility and potential of its industrialization. Biotechnol Adv 31:129–139PubMedCrossRefGoogle Scholar
  21. Huang C, Chen X-F, Xiong L, Yang X-Y, Chen X-D, Ma L-L, Chen Y (2013b) Microbial oil production from corncob acid hydrolysate by oleaginous yeast Trichosporon coremiiforme. Biomass Bioenergy 49:273–278CrossRefGoogle Scholar
  22. Jin M, Gunawan C, Balan V, Dale BE (2012) Consolidated bioprocessing (CBP) of AFEX™-pretreated corn stover for ethanol production using Clostridium phytofermentans at a high solids loading. Biotechnol Bioeng 109(8):1929–1936. doi: 10.1002/bit.24458 PubMedCrossRefGoogle Scholar
  23. Lau MW, Dale BE (2009) Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST). Proc Natl Acad Sci 106(5):1368–1373. doi: 10.1073/pnas.0812364106 PubMedCentralPubMedCrossRefGoogle Scholar
  24. Li Y, Zhao Z, Bai F (2007) High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzym Microb Technol 41(3):312–317. doi: 10.1016/j.enzmictec.2007.02.008 CrossRefGoogle Scholar
  25. Li Q, Du W, Dehua L (2008a) Perspectives of microbial oils for biodiesel production. Appl Microbiol Biotechnol 80:749–756PubMedCrossRefGoogle Scholar
  26. Li Q, Du W, Liu D (2008b) Perspectives of microbial oils for biodiesel production. Appl Microbiol Biotechnol 80(5):749–756PubMedCrossRefGoogle Scholar
  27. Li B-Z, Balan V, Yuan Y-J, Dale BE (2010a) Process optimization to convert forage and sweet sorghum bagasse to ethanol based on ammonia fiber expansion (AFEX) pretreatment. Bioresour Technol 101(4):1285–1292PubMedCrossRefGoogle Scholar
  28. Li M, Liu G-L, Chi Z, Chi Z-M (2010b) Single cell oil production from hydrolysate of cassava starch by marine-derived yeast Rhodotorula mucilaginosa TJY15a. Biomass Bioenergy 34(1):101–107CrossRefGoogle Scholar
  29. Liu W, Wang Y, Yu Z, Bao J (2012) Simultaneous saccharification and microbial lipid fermentation of corn stover by oleaginous yeast Trichosporon cutaneum. Bioresour Technol 118:13–18PubMedCrossRefGoogle Scholar
  30. Nicaud J-M, Chardot T, Beopoulos A (2010) New mutant yeast strains capable of accumulating a large of lipids. WO Patent 2,010,004,141Google Scholar
  31. Rodrigues F, Ludovico P, Leão C (2006) Sugar metabolism in yeasts: an overview of aerobic and anaerobic glucose catabolism Biodiversity and ecophysiology of yeasts. Springer, pp 101-121Google Scholar
  32. Rossi M, Amaretti A, Raimondi S, Leonardi A (2011) Getting lipids for biodiesel production from oleaginous fungi. Biodiesel–feedstocks and processing technologies. InTech, Rijeka, pp 71–92Google Scholar
  33. Shi S, Valle-Rodriguez J, Siewers V, Nielsen J (2011) Prospects for microbial biodiesel production. Biotechol J 6:277–285CrossRefGoogle Scholar
  34. 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(2):321–328PubMedCentralPubMedCrossRefGoogle Scholar
  35. Sitepu IR, Sestric R, Ignatia L, Levin D, Bruce German J, 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 144:360–369PubMedCrossRefGoogle Scholar
  36. Sitepu I, Selby T, Lin T, Zhu S, Boundy-Mills K (2014) Carbon source utilization and inhibitor tolerance of 45 oleaginous yeast species. J Ind Microbiol Biotechnol:1-10 doi: 10.1007/s10295-014-1447-y
  37. Stanier R (1946) Some aspects of microbiological research in Germany BIOS Final Report No 691, Item No 24. British Intelligence Objectives Sub-Committee, LondonGoogle Scholar
  38. Thiru M, Sankh S, Rangaswamy V (2011) Process for biodiesel production from Cryptococcus curvatus. Bioresour Technol 102:10436–10440PubMedCrossRefGoogle Scholar
  39. Thomas KC, Hynes SH, Ingledew WM (2002) Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids. Appl Environ Microbiol 68(4):1616–1623. doi: 10.1128/aem.68.4.1616-1623.2002 PubMedCentralPubMedCrossRefGoogle Scholar
  40. Tsigie YA, Wang C-Y, Truong C-T, Ju Y-H (2011) Lipid production from Yarrowia lipolytica Po1g grown in sugarcane bagasse hydrolysate. Bioresour Technol 102(19):9216–9222. doi: 10.1016/j.biortech.2011.06.047 PubMedCrossRefGoogle Scholar
  41. Wang Q, Guo F-J, Rong Y-J, Chi Z-M (2012) Lipid production from hydrolysate of cassava starch by Rhodosporidium toruloides 21167 for biodiesel making. Renewable EnergyGoogle Scholar
  42. Wiebe MG, Koivuranta K, Penttilä M, Ruohonen L (2012) Lipid production in batch and fed-batch cultures of Rhodosporidium toruloides from 5 and 6 carbon carbohydrates. BMC Biotechnol 12(1):26PubMedCentralPubMedCrossRefGoogle Scholar
  43. Woodbine M (1959) Microbial fat: micro-organisms as potential fat producers progress in industrial microbiology. pp 179-245Google Scholar
  44. Yu X, Zheng Y, Dorgan K, Chen S (2011) Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid. Bioresour Technol 102:6134–6140PubMedCrossRefGoogle Scholar
  45. Zhao X, Kong X, Hua Y, Feng N, Zhao Z (2008) Medium optimization for lipid production through co-fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi. Eur J Lipid Sci Technol 110:405–412CrossRefGoogle Scholar
  46. Zhu L, Zong M, Wu H (2008) Efficient lipid production with Trichosporon fermentans and its use for biodiesel preparation. Bioresour Technol 99:7881–7885PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Irnayuli R. Sitepu
    • 1
    • 2
  • Mingjie Jin
    • 3
  • J. Enrique Fernandez
    • 1
  • Leonardo da Costa Sousa
    • 3
  • Venkatesh Balan
    • 3
  • Kyria L. Boundy-Mills
    • 1
  1. 1.Phaff Yeast Culture Collection, Department of Food Science and TechnologyUniversity of CaliforniaDavisUSA
  2. 2.Forestry Research and Development Agency (FORDA)The Ministry of ForestryBogorIndonesia
  3. 3.Biomass Conversion Research Laboratory, Department of Chemical Engineering and Material ScienceMichigan State UniversityEast LansingUSA

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