Biocatalysis and Agricultural Biotechnology 2 (2013) 339 343

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Biocatalysis and Agricultural Biotechnology

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Original Research Paper

Inuence of culture conditions on lipid production by Candida sp. LEB-M3 using glycerol from biodiesel synthesis
Susan Hartwig Duarte a,n, Gislaine Ghiselli b, Francisco Maugeri a
a b

Laboratory of Bioprocess Engineering, Faculty of Food EngineeringUNICAMP, Campinas, Brazil Instrumental and Chemical Analysis CentreEMBRAPA AGROENERGY, Braslia, Brazil

art ic l e i nf o
Article history: Received 27 May 2013 Received in revised form 4 July 2013 Accepted 16 July 2013 Available online 24 July 2013 Keywords: Fatty acids Biodiesel Raw glycerol Lipid Linolenic acid

a b s t r a c t
The goal of this work was to use glycerol produced from biodiesel synthesis to grow Candida sp. LEB-M3 and select signicant variables of culture conditions on lipid production. This yeast was isolated from a Brazilian biome which showed capacity to accumulate up to 55% lipids (w/w) and convert about 43% of glycerol into lipids. Production and lipid prole at different growth temperatures were studied. The experimental design showed that glycerol, FeCl3 6H2O, yeast hydrolyzate and temperature were signicant on the lipid production. Cultivation at 23 1C promoted the highest concentration of lipids about 9.9 g/L. However, the lipid proles for the different growth temperatures were similar, with high concentrations of linoleic acid (C18:2) (4555%) and smaller amount of gamma linolenic acid (C18:3) (25%), both essential fatty acids, leading to the conclusion that lipid produced by Candida sp. LEB-M3 has potential to be used both as a feedstock for biodiesel production or as a source for essential fatty acids. & 2013 Elsevier Ltd. All rights reserved.

1. Introduction Biodiesel has received considerable attention in recent years because it is a biodegradable, renewable and non-toxic fuel, contributing to the environment by emitting less polluting gases in the atmosphere than regular diesel (Antolin et al., 2002). Brazil is a large producer and consumer of biodiesel besides the conditions for cultivation of oil plants, raw material for biodiesel production, are favorable in several areas (Silva et al., 2009). The traditional production of biodiesel using vegetable oils has economic impacts due to their high costs and the fact that they are also used for food, thus it is a raw material of low viability (Marchetti et al., 2008). Moreover, biodiesel produced from vegetable oils generates about 10% glycerol as the main by-product, whose generation in excess may represent an environmental problem, since currently the market does not absorb the entire production (Silva et al., 2009). According to ANP (Agncia Nacional do Petrleo, Gs Natural e Biocombustveis) in 2011 Brazilian market produced 2.6 million m of biodiesel, which resulted over than 273 million m of glycerin. Therefore, there is an increased

n Correspondence to: Department of Food Engineering, Faculty of Food Engineering, University of CampinasUNICAMP, Rua Monteiro Lobato 80, Baro Geraldo, CEP: 13083-862 Campinas, SP, Brazil. Tel.: +55 19 35214052. E-mail addresses: susanduarte@hotmail.com, susan@fea.unicamp.br (S.H. Duarte).

interest in exploring alternatives for the production of lipids to produce biofuel and also to use glycerol as a carbon source (Chi et al., 2007; Easterling et al., 2009; Kaur et al., 2012; Papanikolaou et al., 2008). Oleaginous microorganisms are able to accumulate 20% or more of their biomass in lipids, mainly in the form of triacylglycerol (TAG) (Ratledge, 2005), which can be used to produce biodiesel by transesterication process, where ester bonds in TAG are broken leading to two products: fatty acid methyl esters and glycerol. These microorganisms present great industrial potential because of their ability to store lipids with properties and composition often similar to those of animal and vegetable products (Papanikolaou et al., 2008). When it comes to use of the residual glycerol from biodiesel in culture media, without prior purication, advantages over the traditional use of pure glycerol as a substrate are observed, mainly with respect to lower cost and higher lipid production. However relatively few studies have reported the use of this substrate as the sole carbon source (Papanikolaou et al., 2008). In order to obtain an alternative for the use of glycerol generated in the synthesis of biodiesel, as well as to study the production of microbial lipids as an alternative for biodiesel production, this study sought to select signicant variables in the production of lipids and in the growth of Candida sp. LEB-M3 on glycerol from biodiesel, including medium components and cultivation conditions, and also to study the inuence of temperature on fatty acid composition.

1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bcab.2013.07.001

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2. Materials and methods 2.1. Microorganism The culture was isolated from owers found in the Pantanal byMaugeri and Hernalsteens (2007) and screened as a oleaginous yeast strain identied of Candida sp. LEB-M3 (Laboratory of Bioprocess Engineering, UNICAMP, Brazil) in previous work (Duarte et al., 2013). The yeast was maintained on GYMP (yeast, malt, glucose, peptone) agar slant at 5 1C, as stock cultures. Before each culture, colonies were reactivated on GYMP slants composed of 20.0 g/L glucose, 5.0 g/L yeast extract, 10.0 g/L malt extract, 2.0 g/L KH2PO4 and 20.0 g/L agar, pH 5.5 and incubated at 30 1C for 48 h. 2.2. Preparation of inoculum Two tubes of the microbial culture on slant GYMP were scraped with 10 mL of 0.1% peptone water for removal of the microorganism cells and transferred into Erlenmeyer asks containing 180 mL of culture medium composed of: 30.0 g/L glycerol, 7.0 g/L KH2PO4, 2.5 g/L Na2HPO4, 1.5 g/L MgSO4 7H2O, 0.15 g/L CaCl2, 0.15 g/L FeCl3 6H2O, 0.02 g/L ZnSO4 7H2O, 0.06 g/L MnSO4 H2O, 0.5 g/L (NH4)2SO4 and 0.5 g/L yeast extract, pH 6.0 (Papanikolaou and Aggelis, 2002). The inoculum was cultivated at 28 1C in shaken asks (New Brunswick Scientic model Innova 4430) at 185 rpm. The cell concentration was monitored by counting in a Neubauer chamber until reaching approximately 1 108 cells/mL (Zhang et al., 2005). 2.3. Flask cultures For selection of the signicant variables in the production of lipids, a PlackettBurman design was performed (Rodrigues and Iemma 2012). Eight independent variables were studied: concentrations of crude glycerol (carbon source) (2040 g/L), FeCl3 6H2O (00.2 g/L), MnSO4 H2O (00.06 g/L), MgSO4 7H2O (01.0 g/L), (NH4)2SO4 (inorganic salts) (0.20.6 g/L), yeast hydrolyzate Prodex Lacs (Prodesa S.A., Campinas, Brazil) (1.03.0 g/L), initial pH (5.5 6.5) and cultivation temperature (2535 1C). The asks were inoculated with 10% (v/v) of the inoculum and maintained in the incubator at the determined temperature for each test, and samples were taken at pre-set intervals. Two types of glycerol were used in the experiments: commercial glycerol, for inoculum, and crude glycerol (from the synthesis of biodiesel), containing 42.4% (w/v) glycerol, free fatty acids, methanol, salts and other impurities, obtained by transesterication of soybean oil with methanol without any previous treatment, which was kindly supplied by SP-Bio, Sumar, Brazil. The amount of crude glycerol added to the culture medium was determined by considering the desired concentration of the carbon source substrate. Different experimental levels of the PlackettBurman design were carried out, totaling 12 trials and three replications at the central point. The dependent variables studied were the lipid content (g lipid/100 g of biomass), lipid concentrations (g/L of fermented medium) and lipid yield (g lipid/g of glycerol 100). 2.4. Inuence of temperature on lipid production According of the results from the PlacketBurman design, values of each variable were chosen to study the inuence of temperature. Cultures were grown in Erlenmeyer asks containing 180 mL of culture medium composed of: 30.0 g/L crude glycerol, 7.0 g/L KH2PO4, 2.5 g/L Na2HPO4, 0.15 g/L CaCl2, 0.02 g/L ZnSO4 7H2O, 0.4 g/L (NH4)2SO4 and 3.0 g/L yeast hydrolyzate, at pH 6.0. The inoculum used was the same as that in the tests

carried out in the PlackettBurman design. Flasks were inoculated with 10% (v/v) of the inoculum at the desired temperature, and 185 rpm. 2.5. Analysis 2.5.1. Cell growth Samples were collected and centrifuged at 785g (Dupont Sorvall centrifuge model RC 26 Plus). After removing the supernatant, the cells were washed once with distilled water, centrifuged again and re-suspended in a known volume of water. Absorbance was then measured at 600 nm. The absorbance values were converted to cell concentration (g/L) using a biomass standard curve (Easterling et al., 2009; Duarte et al., 2013). 2.5.2. Glycerol concentration Samples of culture medium were rst diluted and ltered through 0.22 mm lters. The analysis of glycerol was performed in HPLC (Varian 9095), using an HPX-87H column, a mobile phase composed of 0.005 N H2SO4, pH 2.6 at a ow of 0.6 mL/min, and a RI detector. Concentrations of glycerol (retention time about 15 min) were calculated based on calibration curves constructed for this compound using external standards. 2.5.3. Lipids extraction The dried biomass was treated with a 2 M HCl solution to rupture the cell wall then lipid concentration was determined using the Bligh and Dyer method (Bligh and Dyer, 1959), followed by re-extraction (Manirakiza et al., 2001). The chloroform phase, containing the lipids, was evaporated and lipids were measured by dry weight. 2.5.4. Determination of fatty acids Determination of fatty acids was performed after the lipid fraction was esteried to obtain the fatty acid methyl esters (Metcalfe et al., 1966). The identication and quantication of fatty acids was performed on a Varian 3800 GC, gas chromatograph with 1 mL sample manual injection, a Carbowax (30 m 0.25 mm 0.25 mm) chromatographic column and a ame ionization detector (FID). Analysis conditions were: injector temperature 230 1C, detector 250 1C, initial column temperature 140 1C for 20 min, 2.5 1C/min to 220 1C and 10 min at 220 1C; 1.6 mL/min gas ow (N2), split ratio 1:100, gas ow in the detector: 30/30/ 300N2/H2/synthetic ar. Fatty acids were identied by direct comparison of retention times with standards obtained from SigmaAldrich and quantied by normalization of areas. 2.6. Statistical methods For the PlackettBurman design the level of signicance was determined by the Student's-test and to evaluate the results a 90% condence interval (p0.1) was used. Tests of the inuence of temperature on lipid production were performed in triplicate; data was treated by the ANOVA and Tukey test to determine signicant differences between the results at the different temperatures studied, at 95% condence (p0.05). The software Statistica 7.0 was used to analyze the results.

3. Results and discussion 3.1. Selection of variables for the production of lipids The coded values of the independent variables and the results of the PlackettBurman design are presented in Table 1. The highest lipid accumulation was observed in assay 10, where the

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lipid content was 55.02%, lipid concentration of 13.14 g/L and yield of 38.69%, in which the variables glycerol, (NH4)2SO4, yeast hydrolyzate and initial pH were at the highest studied levels and MgSO4 7H2O, FeCl3 6H2O, MnSO4 H2O and temperature at the lowest levels. However, the best conversion occurred in assay 7, where the variables glycerol, (NH4)2SO4 concentrations and temperature were encountered at the lowest level ( 1) and the remaining variables at the highest level (+1). Considering the effect of estimates for the three responses in Table 2, it can be seen that only the variable glycerol had a signicant effect (p0.1) on the lipid content response at 90% condence. For the concentration of lipids, the variables FeCl3 6H2O and temperature showed a signicant negative effect, while glycerol and yeast hydrolyzate concentrations and initial pH showed a signicant positive effect. For the yield of lipids, temperature and yeast hydrolyzate had signicant effects at 90% condence, indicating that organic nitrogen sources are more benecial to lipid production of Candida sp. LEB-M3 than inorganic nitrogen sources, which was also observed with Trichosporon fermentans (Zhu et al., 2008). Curvature was a signicant effect decreasing the standard error and preventing that effects of the variables were masked (Papanikolaou and Aggelis, 2002). The compounds MgSO4 7H2O, MnSO4 H2O and FeCl3 6H2O presented insignicant effects or signicant and negative effects (Table 2), which means that be removed from the medium. Concerning glycerol, whose effect is signicant and positive for
Table 1 PlackettBurman design (coded values) and responses in lipid content (%) lipid concentration (g/L) and yield (%) at the end of cultivation. Assays Independent variablesa X1 X2 X3 X4 X5 X6 X7 X8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 + + + + + + + + + + + + 0 0 0 0 0 0 + + + + + + 0 0 0 + + + + + + 0 0 0 + + + + + + 0 0 0 + + + + + + 0 0 0 + + + + + + 0 0 0 + + + + + + 0 0 0 Lipids Content (%) 47.68 53.18 28.10 41.16 48.85 51.37 37.84 31.18 40.37 55.02 33.61 47.70 21.74 22.81 19.59 Concentration (g/L) 5.53 6.28 3.83 7.70 10.09 8.43 7.64 4.34 6.05 13.14 5.62 6.35 3.48 3.86 3.14 Yield (%) 16.90 17.27 20.38 23.13 28.75 22.01 43.24 21.02 32.19 38.69 25.92 32.34 10.91 12.16 9.62

lipid content and lipid concentration, it should be kept at the central level, because at higher concentrations there are considerable glycerol residues at the end of fermentations which is undesirable, as shown by Fig. 1. Furthermore it is possible to reduce the cultivation time when the goal is total consumption of glycerol. For yeast hydrolyzate concentrations, whose effect is signicant and positive for lipid concentration and lipid yield, it is maintained at the highest level (+1). Regarding pH, since it is signicant only for the lipid concentration, it will be kept at the central level for the next step, as well as (NH4)2SO4, which is necessary for cell growth as an inorganic nitrogen source. Temperature is an important variable, showing a negative effect for all responses and should be studied in specic experiments at lower temperatures. Moreover there are studies reporting in the literature that temperature can be cause inuence on fatty acid prole formation and saturation, and then it is an important variable to study separately on prole lipids and accumulation of this (Ruangudom and Punpeng, 2011; Leathers and Scragg, 1989).

3.2. Inuence of temperature on the production of lipids As shown by Fig. 2, the growth of Candida sp. LEB-M3 on medium containing glycerol from biodiesel synthesis is slightly different according to the fermentation temperature. It can be seen that at 23 1C growth reached the stationary phase after 192 h of culture, where biomass reached the highest concentration (19.7 7 1.07 g/L), while at 25 1C and 27 1C the stationary phase is not reached even after 240 h of cultivation. Lipid content showed no signicant difference between the temperatures, as shown in Table 3. However, the concentration of lipids at 23 1C is signicantly different from the others, reaching 9.90 7 0.87 g/L. This result is due to the fact that for the determination of lipid concentration, the amount of biomass produced is taken into account for the concentration of lipids, which was higher at 23 1C as shown in Fig. 2. Lipid yield was highest at 23 and 25 1C according to the above results. Ruangudom and Punpeng (2011) studied the inuence of temperature on lipid production by the yeast Rhodosporidium toruloides TISTR 5123 from sugar cane juice; they found that temperature was important variable and with the cultivation at 20 1C reached a maximum value of lipid content about 55%. Also, the microbial lipid prole, quantity, productivity and conversion efciency are inuenced by various process conditions (Beopoulos et al., 2009). However, in this present work temperature was signicant only for lipid content, as previously stated (Table 3).

a X1: glycerol, X2: MgSO4 7H2O, X3: FeCl3 6H2O, X4: MgSO4 7H2O, X5: (NH4)2SO4, X6: yeast hydrolyzate, X7: initial pH, X8: temperature.

Table 2 Effect estimates for the dependents variables (signicant at 90% condence). Factor Lipid content (%) Effect (%) Mean Curvature Glycerol MgSO4 7H2O FeCl3 6H2O MnSO4 H2O (NH4)2SO4 Yeast hydrolyzate Initial pH Temperature 43.00 43.25 13.07 1.69 6.90 1.81 1.04 0.44 1.28 0.21 St error 1.84 8.27 3.69 3.69 3.69 3.69 3.69 3.69 3.69 3.69 t (5) 23.24 5.22 3.53 0.45 1.86 0.49 0.28 0.12 0.34 0.05 p-value o 0.001 0.003 0.016 0.666 0.121 0.644 0.788 0.908 0.742 0.956 Lipid concentration (g/L) Effect (g/L) 7.08 7.17 2.89 0.20 1.67 0.13 1.12 2.02 1.28 2.08 St error 0.30 1.36 0.61 0.61 0.61 0.61 0.61 0.61 0.61 0.61 t (5) 23.13 5.23 4.71 0.33 2.73 0.21 1.84 3.30 2.10 3.40 p-value o 0.001 0.003 0.005 0.753 0.040 0.836 0.125 0.021 0.089 0.019 Lipid yield (%) Effect (%) 26.82 31.84 4.72 1.11 4.74 1.55 0.70 8.08 4.53 8.53 St error 1.61 7.20 3.22 3.22 3.22 3.22 3.22 3.22 3.22 3.22 t (5) 16.65 4.42 1.46 0.34 1.47 0.48 0.21 2.51 1.40 2.65 p-value o 0.001 0.006 0.202 0.743 0.200 0.648 0.835 0.053 0.218 0.045

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25

25

Biomass (g/L)

Biomass (g/L)

20 15 10 5 0 24 48 72 96 120 144 168 192 216 240

20 15 10 5 0 0 24 48 72 96 120 144 168 192 216 240

Time (h)
40 35 40 35

Time (h)

Glycerol (g/L)

Glycerol (g/L)

30 25 20 15 10 5 0

30 25 20 15 10 5 0

24

48

72

96 120 144 168 192 216 240

24

48

72

96 120 144 168 192 216 240

Time (h)

Time (h)

Fig. 1. Time courses for biomass growth and glycerol consumption at three different initial glycerol concentrations, (a) and (c) for the assays: 1 (), 2 (), 3 (), 4 (), 5 (), 6 (), 7 (), 8 (), and 9 (+); (b) and (d) for the assays 10 (), 11 (), 12 (), 13 (), 14 (), and 15 ().
25 20 35 30

Glycerol (g/L)
0 24 48 72 120 144 168 192 216 240

Biomass (g/L)

25 20 15 10 5 0 0 24 48 72 120 144 168 192 216 240

15 10 5 0

Time (h)

Time (h)

Fig. 2. Time course of Candida sp. LEB-M3 growth and glycerol consumption at different temperatures: (o) 23 1C, () 25 1C e and () 27 1C.

Table 3 Mean and standard deviations for lipid content (%), lipid concentration (g/L) and lipid yield (%) according to Tukey's test of statistical analysisa. Temperature (1C) Lipid content (%) Lipid concentration (g/L) Lipid yield (%)

Table 4 Fatty acid composition (% w/w of total lipid) in the biomass of Candida sp. LEB-M3 grown in culture media containing biodiesel glycerol at different temperatures and comparison with the prole of other vegetable oils [25]. Fatty acid 23 1C 25 1C 27 1C Soybean Sunower Palm

23 25 27

50.18 7 2.75a 47.65 7 3.7a 44.14 7 1.63a

9.90 7 0.87a 7.65 7 0.44b 7.70 7 0.84b

32.07 7 3.39a 28.53 7 2.81a,b 22.67 7 0.36b

Saturated Fatty acid C16:0 12.9 C18:0 3.1

9.8 3.5

14.2 1.4 0.51 27.4 52.3 3.6

11.4 4.4 20.8 53.8 9.3

7.1 4.7 25.5 62.4

42.6 4.4 0.3 40.5 10.1 0.2

a Same letters in a column means no signicant differences between values (p o 0.05).

3.3. Fatty acid prole In the cells cultivated at 23 1C (Table 4), the fatty acid prole was 16.06% saturated fatty acids, especially palmitic acid (C16:0), and 35.32% monounsaturated fatty acids, with emphasis on oleic acid (C18:1). However the amount of polyunsaturated fatty acids was higher, being 46.73% of the total, with prominence of the essential linoleic acid (C18:2), making up 44.6% and gamma linolenic acid (C18:3) (GLA) composing 2.13%. In the cells cultivated at 25 1C, the percentage of saturated fatty acids was 13.26%, composed primarily of C16:0, while of the 26.78% monounsaturated fatty acids, C18:1 stood out. Polyunsaturated fatty acids accounted for 60.0% in this case, of which C18:2 made up 54.9% and C18:3 5.10%. In cells cultivated at 27 1C, the percentage of saturated fatty acids was 15.66%, with prominence

Monounsaturated fatty acids C16:1 0.6 0.4 C18:1 34.0 25.5 Polyunsaturated fatty acids C18:2 44.6 54.9 C18:3 2.1 5.1

of the C16:0, while oleic acid C18:1 was prominent among monounsaturated fatty acids. The polyunsaturated fatty acids reach a total of 55.94%, with 52.32% of C18:2 and 3.62% of C18:3. The current results are in agreement with other studies, where it was observed that the oleaginous yeast strain Cryptococcus curvatus NRRLY-1511 produced lipids consisting mainly of 30.68% (C18:2), 22.66% (C18:1) and 16.74% (C16:0) (El Fadaly et al., 2009). Papanikolaou et al. (2004) also reported the microbial production of lipids containing 3.5% of GLA, corresponding to 1619 mg of GLA per gram of dry biomass. In the present study the amount of GLA

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produced by Candida sp. LEB-M3 ranged between 10.65 and 24.3 mg/g of biomass (dry weight basis), depending on the fermentation temperature, which represents signicant production of this essential fatty acid when using residual glycerol from biodiesel as a substrate. Evans and Ratledge (1983) reported the lipid production by Candida curvata in medium containing xylose as the carbon source, where the fatty acid prole consisted of 15% stearic acid (18:0) and 4% of linoleic acid (18:2). Li et al. (2010), when studying the production of lipids by the yeast Rhodotorula mucilaginosa TJY15a using cassava starch as the substrate, obtained a fatty acid prole composed of 63.5% C18:1 and 22.3% C16:0, and 5.7% C18:2, the only polyunsaturated fatty acid. When comparing the fatty acid proles produced by Candida sp. LEB-M3 for the different temperatures, it can be found that at 25 1C there was a higher polyunsaturated fatty acid production, followed by the temperature of 27 1C, which showed a similar prole, and nally at 23 1C was the lowest production of essential fatty acids. It seems that temperatures above 23 1C are less suitable for increased production of these acids. Single-cell oils, those obtained from microorganisms, are now accepted as biotechnological products playing key roles in the supply of major polyunsaturated fatty acids (PUFA), such as the gamma linolenic acid produced in this work, which are known to be essential for human nutrition and development (Ratledge, 2005). The lipids produced by Candida sp. LEB-M3 at all temperatures can be used as feedstock for biodiesel production because the composition on fatty acids is similar to the composition of vegetable oils commonly used for biodiesel production (Akoh et al., 2007) mainly soybean oil, and still can be used as a source of essential fatty acids for use in foods.

References
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4. Conclusion The PlackettBurman screening experimental design enabled the establishment of important variables for lipid production by the yeast Candida sp. LEB-M3 from biodiesel based glycerol, which included the glycerol concentration, yeast hydrolyzate concentration, pH and temperature. The yeast was able to accumulate up to 55% (dry weigh basis) lipids and convert about 43% of glycerol into lipids. The fatty acid prole showed similarity at the three temperatures, with a predominance of C18:2, but during cultivation at 23 1C there was increased production of C18:1 and lower production of linoleic acid when compared to the other temperatures. Lipids produced by Candida sp. LEB-M3 represent a signicant source of this essential fatty acid when using residual glycerol from biodiesel as a substrate. Acknowledgments The authors would like to thank the Laboratory of Ecology and Biotechnology of Yeasts (Federal University of Minas Gerais) and CAPES for their nancial support.