1. Bioconversion of raw glycerol into single cell oil rich in γ- linolenic acid S. Bellou, A. Makri, S. Fakas & G. Aggelis Unit of Microbiology, Division of Genetics; Cell & Development Biology, Dpt of Biology; University of Patras; Greece Figure 1. Model fitted on the experimental data from cultures of Thamnidium elegans growing on raw glycerol. (■) Lipid-free biomass (Xf, g l-1), (▲) raw glycerol (Glol, g l-1), and (●) lipids (g l-1). Two lots of independent cultures were conducted by using different inocula. Acknowledgements Financial support was provided by the project ‘‘Kinetics of growth of oleaginous microorganisms and dynamics of biosynthesis of polyunsaturated fatty acids” funded by the University of Patras (Project K. Karatheodori) and the project “Biodiesel production from agro-industrial by-products’’ funded by the Greek Fuel Company DRACOIL SA. Figure 3. Fatty acid composition of lipid fractions in late exponential phase. NL: Neutral lipids; G+S: Glycolipids plus sphingolipids; P: Phospholipids. ME: Mid exponential growth phase; LE: Late exponential growth phase; S: Stationary phase. Figure 2. Lipid composition during growth of Thamnidium elegans on raw glycerol. NL: Neutral lipids; G+S: Glycolipids plus sphingolipids; P: Phospholipids. ME: Mid exponential growth phase; LE: Late exponential growth phase; S: Stationary phase. Samples were taken for lipid analysis at time points indicated by arrows in Figure 1. Results and Discussion Th. elegans grew very well on raw glycerol as sole carbon source and showed remarkable biomass synthesis, while significant lipid quantities were accumulated into the fungal mycelia (i.e. up to 47.9%, wt/wt oil in dry biomass) (Figure 1). Glycerol was gradually consumed during growth and exhausted after 240 h of incubation. Lipid accumulation lagged far behind nitrogen exhaustion, starting 120h after inoculation. A fluctuation in the lipid composition during the various growth phases was noticed (Figure 2). At the beginning of growth, the produced lipids were rich in polar lipids (glycolipids plus sphingolipids (G+S) and phospholipids (P)). Neutral lipids (NL), however, were the major constituent of Th. elegans lipids. During growth, NL accumulated in the mycelia, which resulted at the production of lipids containing more than 80% (w/w) NL. Lipid analysis by TLC showed that NL comprised mainly from triacylglycerol, while diacylglycerol and monoacylglycerol were minor components. G+S fraction comprised mostly of monogalactosyl- and digalactosyl-diacylglycerol. P fraction contained phosphatidylcholine, phosphatidylethanolamine, and lower quantities of phosphatidylinositol and phosphatedylserine. Thamnidium elegans microbial lipids contained mostly oleic acid (C18:1Δ9), followed by palmitic (C16:0) and linoleic (C18:2 Δ9,12) acids, while stearic (C16:0) and γ-linolenic (GLA, C18:3 Δ6,9,12) acids were found in lower amounts. The GLA content of the oil was higher during active growth (9.9% w/w), while in the lipogenic phase GLA content started to decrease, reaching 7.3% (w/w) in the produced oil. Fatty acid analysis of the lipid fractions in late exponential phase showed that the P fraction was particularly enriched in PUFAs, while NL and G+S contained lower and almost equal amounts of PUFAs (Figure 3). NL contained very high amounts of C18:1 (almost 50% w/w), while G+S had the highest C16:0 content among lipid fractions. Raw glycerol seems like a promising substrate for SCO and GLA production by Thamnidium elegans. Taking into account the zero cost of raw glycerol, a bioprocess for SCO production using raw glycerol as feedstock may be visualized. References [1] Certik and Shimizu (1999). J Biosci. Bioeng. 87,1-142. [2] Kenny et al., (2000). Int. J. Cancer. 85, 643-648. [3] Papanikolaou, S. et al., (2007). European Journal of Lipid Science and Technology, 109, 1060-1070. [4] Fakas, S., et al., (2008a). Bioresource Technology, 99, 5986–5990. [5] Fakas, S., et al., (2008b). Journal of Applied Microbiology, 105, 1062 - 1070. [6] Fakas et al. (2006). Appl Microbiol Biotechnol., 73, 676–683. Introduction Oleaginous microorganisms, having the ability to accumulate more than 20% w/w lipid in their biomass, have long been used for the production of lipids designated as single cell oils (SCOs). SCOs are sources of polyunsaturated fatty acids (PUFAs) having great pharmaceutical and nutraceutical interest [1]. Microbial PUFAs production has been established because of the lack of alternative sources of these fatty acids. I.e. gamma linolenic acid (GLA) is among PUFAs that have potential commercial interest, because there are no abundant sources of this fatty acid in nature. Interest in GLA production has emerged lately, because of its selective anticancer properties [2]. However, the GLA to be used in cancer treatment should be of the highest purity which then makes SCOs the best GLA sources, since plant oils contain high amounts of other PUFAs along with GLA, making its purification very difficult. As a result, several researchers focused their interest to develop processes for GLA production by waste media because these media have zero or even negative cost [3, 4, 5]. Several by-products have been used as substrates for SCOs production originating mainly from agro-industries. Raw glycerol is produced during biodiesel industry. The administration of raw glycerol is a major problem for bio-diesel industry so using it as a carbon substrate is an alternative way to convert glycerol into value-added products such as PUFAs. The aim of this work was to study the production of GLA-rich SCOs by the oleaginous Zygomycete Thamnidium elegans grown on raw glycerol and the lipid composition during growth. Materials and Methods Microorganisms: Thamnidium elegans CCF-1465 Culture conditions: 250 ml Erlenmeyer flasks containing 50 ml of a liquid medium were incubated in a rotary shaker at T=28°C and 180 rpm. Medium: Glycerol at 100 g/l, supplemented with minerals and yeast extract. Nitrogen was the limiting growth factor. HPLC analysis: Glycerol was determined in filtered (through 0.2 μ m pore size bacteriological filter, Whatman) aliquots of the culture by an HPLC apparatus (Ultimate 3000, Dionex, Germering, Germany) equipped with a Shodex SH1011 and a R.I. Detector, under the following conditions: sample volume 20 μl, mobile phase 0.001 N H2SO4, flow rate 1.0 ml/min and column temperature 500 C.. Lipid extraction: According to Folch protocol. Lipid fractionation: by using a column of silicic acid activated by heating overnight at 80 °C. Successive applications of dichloromethane, acetone and methanol produced fractions containing neutral lipids (NL), glycolipids plus sphingolipids (G+S) and phospholipids (P), respectively. GC analysis: Fatty acid analysis was performed after trans-methylation according to the AFNOR method [6], in an Agilent Technologies 7890 A device equipped with a HP-88 (J&W scientific) column (60 m x 0.25 mm). Conditions: carrier gas helium, flow rate 2 ml/min, oven T=200 °C, injector T=250 °C, detector (FID) T=280 °C.