Corecovery of Lipids and Fermentable Sugars from Rhodosporidium toruloides Using Ionic Liquid Cosolvents: Application of Recycle to Batch Fermentation Godwin Severa University of Hawaii at Manoa, Hawaii Natural Energy Inst. 1680 East West Road, POST 109, Honolulu, Hawaii 96822 Guneet Kumar Suganit Systems Inc., 10903 Hunt Club Road Reston, VA 20190-3912 Michael J. Cooney University of Hawaii at Manoa, Hawaii Natural Energy Inst. 1680 East West Road, POST 109, Honolulu, Hawaii 96822 DOI 10.1002/btpr.1952 Published online August 12, 2014 in Wiley Online Library (wileyonlinelibrary.com) This work evaluates the ability of an ionic liquid-methanol cosolvent system to extract lip- ids and recycle fermentable sugars recovered from oil-bearing Rhodosporidium toruloides grown in batch culture on defined media using glucose and xylose as carbon sources. Growth on the recycled mixed carbon substrate was successful with glucose consumed before xylose and overall cell mass to lipid yields (Y P/X ) between 57% and 61% (w/w rela- tive to whole dried cell mass) achieved. Enzymatic hydrolysis of the delipified carbohydrate fraction recovered approximately 9%–11% (w/w) of the whole dried cell mass as fermenta- ble sugars, which were successfully recycled as carbon sources without further purification. In total, up to 70% (w/w) of the whole dried cell mass was recovered as lipids and fermenta- ble sugars and the substrate to lipid yields (Y P/S ) was increased from 0.12 to 0.16 g lipid/g carbohydrate consumed, highlighting the promise of this approach to process lipid bearing cell biomass. V C 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1239– 1242, 2014 Keywords: ionic liquids, Rhodosporidium toruloides, lipids, fermentable sugars, enzyme sac- charization, cosolvent extraction Introduction The growing demand for renewable energy has spurred extensive research on oleaginous species of microalgae and yeast as potential sources of lipids for biofuel production. 1–4 Various strategies have been applied to optimize both cell mass density and cell mass to lipid yield (Y P/X ) using batch and fed-batch operation. 1,2,4–6 While batch fermentations are simpler to execute, fed-batch fermentations achieve higher cell mass concentrations and cell mass to lipid yields (Y P/X ) through implementation of two-phase growth processes wherein cell mass growth is first pursued on non-nitrogen limited (low C/N) media and lipid accumulation is subse- quently pursued on a nitrogen limited (high C/N) media. 2 Less researched are methodologies to improve the sub- strate to lipid yield (Y P/S ). The cost of producing biofuel via fermentation of lipid bearing cell mass is dominated by the cost of carbon feedstock, fermentation (including recovery and purification of lipid), and processing the recovered lipids to biofuel (e.g., biodiesel). Fermentation and lipid processing are mature technologies that offer limited opportunity for additional cost savings; for example, the cost of producing biodiesel is dominated by the cost of the lipid feedstock (88%). 7 Reducing the cost of carbon feedstock, therefore, remains the most promising opportunity to reduce the full cost of commercialization. 3 Methods proposed to achieve this include the development of successful low cost processes to hydrolyze lignocellulosic biomass to simple sugars—mainly fermentable glucose and xylose, 8 the reuse of waste sugars (e.g., recovered food processing waste streams), 9 and adding enzyme extracts (e.g., Eschericia coli (E. coli)) to improve xylose update in ethanol producing Saccharomyces. 10 This last interesting approach uses extracts of E. coli culture as a source of xylose isomerase enzymes that promote ethanol productivities on feed- stock that contained xylose. Another pathway is through increasing the substrate to lipid yield (Y P/S ). Recently, Severa et al. 11 demonstrated the application of ionic liquid based cosolvents to simultane- ously extract lipids and pretreat the carbohydrate fraction of oil seed biomass (jatropha and safflower seeds) for success- ful enzymatic hydrolysis of the carbohydrate fraction to fer- mentable sugars. As spent yeast cell mass contains glucose, mannose, and other sugars that have been shown to be effi- ciently used by oleaginous yeast, 12 the extraction system of Severa et al. was applied to the lipid bearing red yeast R. toruloides to investigate whether fermentable sugars could be recovered and recycled. If successful the process could be used to reduce the total amount of fermentable sugars Correspondence concerning this article should be addressed to M. J. Cooney at mcooney@hawaii.edu. V C 2014 American Institute of Chemical Engineers 1239