Glucose is the typical carbon source for producing microbial polyunsaturated fatty acids (PUFA) with single cell microorganisms such as thraustochytrids. We assessed the use of a fish oil derived glycerol by-product (raw glycerol), produced by a fish oil processing plant, as a carbon source to produce single cell oil rich in polyunsaturated fatty acids (PUFA), notably docosahexaenoic acid (DHA). These results were compared to those obtained when using analytical grade glycerol, and glucose. The thraustochytrid strain tested produced similar amounts of oil and PUFA when grown with both types of glycerol, and results were also similar to those obtained using glucose. After 6 days of fermentation, approximately 320 mg/g of oil, and 145 mg/g of PUFA were produced with all carbon sources tested. All oils produced by our strain were 99.95% in the triacylglycerol form. To date, this is the first report of using raw glycerol derived from fish oil for producing microbial triglyceride oil rich in PUFA. (c) 2010 Elsevier Inc. All rights reserved.