Maximizing biodiesel production from Yarrowia

Bioresource Technology 111 (2012) 201–207

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Maximizing biodiesel production from Yarrowia lipolytica Po1g biomass using subcritical water pretreatment Yeshitila Asteraye Tsigie a, Lien Huong Huynh b, Ibrahim Nasser Ahmed a, Yi-Hsu Ju a,⇑ a b

Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Rd., Sec.4, Taipei 106-07, Taiwan Department of Chemical Engineering, Can Tho University, 3-2 Street, Can Tho City, Viet Nam

a r t i c l e

i n f o

Article history: Received 12 November 2011 Received in revised form 4 February 2012 Accepted 6 February 2012 Available online 22 February 2012 Keywords: Biodiesel Fatty acid methyl ester Microbial oil Sub-critical water Neutral lipid

a b s t r a c t The yeast Yarrowia lipolytica Po1g is one of the oleaginous microorganisms with a potential for biodiesel production. Sub-critical water (SCW) treatment has been known as an effective method for increasing the amount of extractable lipids in microorganisms. In this work, the amount of neutral lipids and fatty acid profiles in neutral lipids extracted from Y. lipolytica Po1g with and without SCW pre-treatment were investigated. The effects of temperature (125, 150 or 175 °C), amount of water (20, 30 or 40 mL/g biomass) and time (10, 20 or 30 min) showed that maximum neutral lipid (42.69%, w/w) could be achieved at 175 °C using 20 mL water for 20 min. The maximum neutral lipid from unpretreated samples was 23.21%. No difference in fatty acid profiles was observed, but long chain fatty acids were observed in higher amount in SCW pretreated samples. SCW pretreatment increased biodiesel yield twofold. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Microbial lipid has already been developed as sources of highvalued oils including cocoa butter equivalent (CBE) and polyunsaturated fatty acids (Papanikolaou and Aggelis, 2010). Recently, it has also been suggested as a potential feedstock for biodiesel industry (Adamczak et al., 2009; Easterling et al., 2009; Tsigie et al., 2011), because microbial lipid can be produced using various cheap feedstocks, especially renewable biomaterials such as lignocelluloses. Biodiesel is an attractive replacement for petroleum diesel because it is domestically available, biodegradable, compatible with existing diesel engines, and reduces tailpipe emissions of most criteria air pollutants (Janaun and Ellis, 2010). Biodiesel, produced from vegetable oils and animal fats, is rather an attractive alternative for its biodegradable, non-toxic and clean renewable characteristics as well as the similar properties to the conventional diesel fuels. Although biodiesel has presently been used in many countries, the high cost of biodiesel has become one of the major obstacles for its further development and wide application. Besides, the use of vegetable oils as raw material for biodiesel pro-

Abbreviations: SCW, sub-critical water; FAME, fatty acid methyl ester; FFA, free fatty acids; MAG, monoacylglycerides; DAG, diacylglycerides; TAG, triacylglyce rides; TLC, thin layer chromatography; GC, gas chromatography. ⇑ Corresponding author. Tel.: +886 2 27376612; fax: +886 2 27376644. E-mail address: [email protected] (Y.-H. Ju). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.052

duction would compete with edible oils, thus leading to the soar of food price. Using recovered animal fats and used frying oils as feedstock can efficiently reduce the price of biodiesel, however, the amount of waste oils is limited and cannot meet the increasing needs for clean renewable fuels (Zhu et al., 2008). Chemically, biodiesel is a fatty acid alkyl ester, commonly known as fatty acid methyl ester (FAME) that is produced via esterification and/or transesterification of various lipid sources in the presence of a base, acid, enzyme or solid catalyst (Knothe, 2005). Microbial oils, otherwise referred to as single cell oils (SCO) produced by various microorganisms, are now believed as a potential feedstock for biodiesel production due to their specific characteristics such as: they are not affected either by seasons or by climates, they own high lipid content, can be produced from a wide variety of sources with short period of production, especially from the residues with abundant nutrition, and so on (Papanikolaou et al., 2004; Xue et al., 2006). The oleaginous yeast Yarrowia lipolytica can accumulate large amount of lipid and through its efficient mechanism, it can break down hydrophobic substrates. The complete sequencing of its genome and application of methods of genetic manipulation have enabled researchers to use this yeast for biotechnological applications as reviewed by Beopoulos et al. (2009). This oleaginous yeast can also be developed into a versatile and high-throughput microbial factory that, by using specific enzymatic pathways from hydrocarbonclastic bacteria, efficiently mobilizes lipids by directing its versatile lipid metabolism towards the production of industrially

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valuable lipid-derived compounds such as wax esters (WE), isoprenoid-derived compounds (carotenoids, polyenic carotenoid ester), polyhydroxyalkanoates (PHAs) and free hydroxylated fatty acids (HFAs) (Sabirova et al., 2011). Subcritical water (SCW) treatment is an environmentally friendly technique with a wide range of applications, such as extraction, hydrolysis, and wet oxidation of organic compounds (Holliday et al., 1998; Kruse and Dinjus, 2007). SCW is defined as hot water at temperatures ranging between 100 and 374 °C under high pressure to maintain water in the liquid state. Dielectric constant, which can be changed by temperature, is the most important factor when using water as an extraction solvent; it decreases from 80 (at room temperature) to 27 (at 250 °C) which is almost equal to that of ethanol at ambient temperature (Herrero et al., 2006). Thus, SCW can be used for extraction of organic compounds instead of using organic solvents which are environmentally unacceptable. On the other hand, SCW has been widely used for hydrolysis of organic compounds. Recently growing attention has led to extensive research activities using SCW for hydrolysis and conversion of biomass and carbohydrates to useful compounds (Bicker et al., 2005; Kruse and Gawlik, 2002; Salak Asghari and Yoshida, 2006; Sasaki et al., 1998; Yoshida et al., 1999). SCW has also been known as a uniquely special medium or solvent for various chemical reactions and extraction of compounds. SCW extraction is able to selectively extract different classes of compounds at various temperatures since the polarity of water changes with temperature as long as the water is maintained at its sub-critical condition. Moreover, SCW can also act as an effective catalyst for a hydrolysis or biodegradation reaction. It has been demonstrated by Galkin and Lunin (2005) that SCW can completely remove a wide variety of organic pollutant from industrial waste within only a few minutes. SCW, therefore, has been becoming a promising extractant and catalyst (Rogalinski et al., 2005). An experimental design is a fast, economic, and effective way to systematically investigate the effect of several variables simultaneously (multivariate data analysis). In the experimental study of M variables and N experiments, an M N matrix constitutes a variable space (X). A response variable (Y) for each experiment is necessary for the analysis of the experimental data (Jalbani et al., 2006). In the present work, the effect of SCW pretreatment of Y. lipolytica Po1g biomass on lipid and biodiesel production was investigated. A 23 two-level factorial design was applied to study three factors (temperature, amount of water added to the biomass and pretreatment time) which are believed to play important roles in the pretreatment of Y. lipolytica Po1g biomass for the extraction of lipid using organic solvents. The effect of temperature, pretreatment time and amount of water on the amount of extractable lipids and the fatty acid profile of subcritical water pretreated biomass was also studied. Finally, comparison of the results from untreated and SCW treated biomass samples was made.

2. Methods 2.1. Materials All solvents and reagents were either gas chromatography (GC) or analytical reagent grade, obtained from commercial sources. For GC analysis, all the standards were purchased from Acros Organics (New Jersey, USA) and Sigma Aldrich (St. Louis, MO 63103, USA). Bacto peptone was supplied by Becton Drive (Sparks, MD 21152, USA). Thin layer chromatography (TLC) aluminum plates (20 cm 20 cm 250 cm) were purchased from Merck KGaA (Darmstadt, Germany). Qualitative filter paper (grade No. 2,

0.26 mm thickness, 80% collection efficiency and grade No. 5C) was obtained from Advantec (Tokyo, Japan). 2.2. Microorganism, media preparation, precultivation and cultivation Y. lipolytica Po1g cells were obtained from YEASTERN Biotech Co. Ltd. (Taipei, Taiwan). The cells were maintained on YPDA medium at 4 °C. The preculture was performed on precultivation medium (g/L, D-glucose 20, peptone 10, yeast extract 10) at 26 °C and 160 rpm shaking for 24 h. Then, the preculture was inoculated to the culture medium with a ratio of 1:10 (v/v). The culture medium in the flask consisted of detoxified sugar cane bagasse hydrolysate (SCBH, with a total sugar concentration of 20 g/L) and 5 g/L of peptone. Yeast extract (5 g/L), containing leucine (6.2%), was added to the medium. Cultivation was performed in 500 mL conical flasks each containing 250 mL culture media in an orbital shaker incubator model LM-570 (Chemist Scientific Co., Taiwan) at 26 °C and 160 rpm. The required amount of biomass was then collected for subsequent culturing experiments. The procedures in our previous work were followed (Tsigie et al., 2011). 2.3. Biomass pretreatment and sample preparation To compare the amount of extractable lipid, neutral lipid and determine fatty acid profiles, biomass was prepared in two ways. The first type consisted of unpretreated freeze-dried biomass of Y. lipolytica Po1g. The second type of biomass was prepared by subcritical water pretreatment of freeze-dried biomass samples with subcritical water extractor. The effect of subcritical water pretreatment was studied by considering three variables: temperature (125–175 °C), amount of water added to 1 g biomass (10–30 mL), pretreatment time (10–30 min). The equipment for subcritical water extraction used in this research was constructed by Ju-Shan Industrial Co. Ltd. in Taiwan. There are three main parts in this equipment, subcritical reactor, heater, and control devices. The reactor was made from stainless steel, and the total inner volume was about 90 mL. It is 25 mm thick and can withstand an estimated maximum operation pressure of 100 MPa. Ten M8 screws which can afford 12.8 tons of tensile force were used for tightening the reactor with its cap. Two layers of spacers were put between the cap and the reactor (Kasim et al., 2009). A thermocouple and a pressure gauge were connected to the reactor. The process was run under batch mode. For subcritical water extraction, nitrogen gas (99.9% purity) purchased from Dong-Xing Company (Taiwan) was used to maintain constant pressure (13 bar) inside the reactor. Freeze-dried biomass (1 g) was dissolved in deionized water (10–30 mL) in the high pressure reactor. Temperature inside the reactor was measured by a thermocouple and controlled at either 125, 150 or 175 °C. The pressure inside the reactor was maintained at 13 bar by using nitrogen to assure that water remained as liquid. This condition was maintained for either 10, 20 or 30 min and then the system was allowed to cool to 30 °C. After that the pretreated biomass was collected, separated from the aqueous phase by vacuum filtration and then subjected to freeze drying (Freeze Zone – 2.5 l freeze dry system-Model 7670520, Labconco Corporation, Kansas city, USA). The freeze-dried, SCW pretreated biomass was then collected and stored 4 °C before use. 2.4. Experimental design A 23 factorial design was used in this study. The optimization study was conducted with Design-Expert 8 software, by means of customized design of experiments. The experiments were done in random sequence. Upon completion of all the experimental

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runs, the obtained responses were fitted in a linear model using regression analysis. The three pretreatment variables under investigation and their high and low experimental levels and center points are given in Table 1. Two replicates were done for all treatment combinations. ANOVA was used to show the validity of the model used for the experiment and the following equations were used to determine the contents of the corresponding responses. Crude Lipid ¼ 71:33 þ 6:71A þ 1:82B 1:23C 3:54AB 1:71AC þ 0:95BC Neutral Lipid ¼ 32:03 þ 7:87A þ 0:25B þ 0:17C 0:087AB 0:31AC þ 0:94BC Wax and Gum ¼ 27:58 þ 0:77A 0:016B þ 3:40C þ 0:85AB 3:42AC þ 0:59BC

ð1Þ ð2Þ ð3Þ

2.5. Lipid content and fatty acids determination Lipid extraction from dried biomass (with or without subcritical water pretreatment) was performed according to the modified procedure of Bligh and Dyer (1959). Lipid was extracted with a mixture of hexane: methanol (2:1, v/v) for 4 h. The extracted lipid was centrifuged (3500g) to obtain a clear supernatant and the solvent was removed by evaporation under vacuum. TLC was used to qualitatively identify the components of the crude single cell oil (SCO) extracted from the biomass. The crude SCO was dewaxed and degummed as described by Rajam et al., (2005) and Vandana et al., (2001). Crude SCO (0.5 g) was dissolved in 15 mL water (60 °C) and the water soluble fraction was separated from the insoluble fraction by vacuum filtration. Then the insoluble fraction was dissolved in acetone and kept at 60 °C for 1 h to obtain clear solution. After allowing the contents to cool to room temperature (25 °C), the solution was then kept at 4 °C for 24 h to crystallize the remaining waxes and phospholipids. The insoluble fraction was separated by vacuum filtration. The filtrate was collected and subjected to another solvent crystallization at 5 °C for 24 h. The solid phase was separated by vacuum filtration, the filtrate was collected and then acetone was evaporated by using a rotary evaporator (650 mm Hg, 60 °C). The wax content was determined after the water soluble fraction was subjected to freeze drying. The dewaxed and degummed SCO obtained was used for determining the lipid content and fatty acid profile as described elsewhere (Tsigie et al., 2011). The lipid content and the fatty acid profile were determined by gas chromatography (GC-17A, Shimadzu, Japan) with a flame ionization detector. Separations were carried out on a DB5-HT capillary column (30 m x 0.32 mm; Agilent Technologies, USA). Temperatures of the injector and the detector were both set at 370 °C. The temperature of the column was started at 80 °C, and was increased to 365 °C at a rate of 15 °C/min and maintained at 365 °C for 10 min. The total run time was 29 min. The split ratio was 1:50 using nitrogen as the carrier gas with a linear velocity of 30 cm/s at 80 °C. Twenty milligrams sample was dissolved in 1 mL ethyl acetate, and 0.5 lL sample was taken and injected into the GC. 2.6. Determination of FAMEs contents Crude microbial oil (1 g) was added to a 100 mL tube with a Teflon-lined screw cap. Fifteen milliliters ethanol was added and the mixture was titrated with 1 M KOH/ in ethanol standard

solution. The mixture was then heated at 60 °C in water bath for 2 h until the reaction was completed, as verified by analytical TLC (silica gel; eluted with a mixture solvent, hexane/ethyl acetate/acetic acid = 90:10:1, v/v/v) and GC. The mixture containing the saponified matter was acidified to pH 2 using sulfuric acid and the reaction was completed in about 1 day. Water was then added to the mixture to stop the reaction, and unsaponifiable matter was separated by extraction using n-hexane and water. The hexane extract, which contained the fatty acids and acylglycerols, was collected and removed. The fatty acids were then converted into their corresponding FAMEs by heating with boron trifluoride (BF3) in methanol and analyzed by GC and TLC. External standard calibration curves were obtained by using 0.2–20 mg pure standards. A 37 component FAME mix (Sigma–Aldrich, Bellefonte, USA) standard was used to identify individual FAME in the product. Chromatographic analysis was performed using a GC-2010 gas chromatograph (Shimadzu, Japan) equipped with a flame ionization detector. The column used was a Rtx2330 10% cyanopropylphenyl – 90% biscyanopropyl polysiloxane column (30 m 0.25 mm i.d., (Restek, Bellefonte, PA). The operating conditions were set as follows. The injector and detector temperatures were set at 250 °C. The column temperature was held at 150 °C for 2 min, and then raised to 250 °C at 5 °C/min and held for 8 min. Hydrogen flow, air flow and make up flow were set at 50.0 mL/min, 500.0 mL/min and 30 mL/min, respectively while the linear velocity and purge flow were 8.0 cm/s and 3.0 mL/min, respectively. The biodiesel yield from Y. lipolytica Po1g biomass (either untreated or treated with SCW hydrolysis) was calculated by:

Biodiesel yield ð%Þ ¼

Biodiesel massðgÞ 100% Dry biomassðgÞ

ð4Þ

3. Results and discussion 3.1. Lipid, wax and gum contents of Y. lipolytica Po1g After extraction with hexane and methanol (2:1, v/v) for 4 h, the crude lipid, neutral lipid, wax and gum contents of the samples were determined gravimetrically and the results are summarized in Table 2. The maximum crude lipid, neutral lipid and wax and gum contents obtained when freeze-dried, unpretreated Y. lipolytica Po1g biomass was extracted using hexane: methanol (2:1) for 4 h are 51.54%, 23.21% and 29.33%, respectively. After the biomass was pretreated with SCW (20 mL water per gram dry biomass) at 175 °C for 10 min and extracted with the same solvent for 4 h, the maximum crude lipid, neutral lipid and wax and gum contents obtained are 84.79%, 42.69% and 34.39%, respectively. The maximum crude lipid content of the SCW pretreated Y. lipolytica Po1g samples in this study (84.79%) is almost equivalent to that of cells of Rhodococcus opacus PD630 which accounted for up to 87% of the cellular dry weight (Alvarez and Steinbüchel, 2002). However, only 51.54% lipid content can be obtained from the untreated Y. lipolytica Po1g biomass. It has been reported that SCW pretreatment of activated sludge increases the neutral lipid content almost four times compared to that of the untreated one (Huynh et al., 2010). Under sub-critical

Table 1 The experimental parameters and their high and low levels and center points for the lipid extraction from dried subcritical water pretreated biomass. Variables

Low level (1)

Center point (0)

High level (+1)

A: Temperature (°C) B: Amount of H2O (mL/g biomass) C: Time (min)

125 20 10

150 30 20

175 40 30

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Table 2 Crude lipid, neutral lipid, wax and gum contents of Y. lipolytica Po1g with SCW pretreatment. Run

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Factors

Responses (%,w/w)

A

B

C

Crude lipid

Neutral lipid

Wax and gum

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0

61.97 84.68 72.03 73.13 57.44 84.79 73.13 79.67 67.22 75.85 69.6 60.44 77.69 76.89 75.82 65.04 59.28 69.29

26.61 42.67 23.82 26.78 23.92 37.65 37.88 42.46 21.04 41.04 26.99 24.41 42.53 38.03 38.26 32.73 21.01 28.71

19.31 29.71 18.82 30.94 21.62 25.71 29.85 32.04 17.4 29.17 33.81 32.92 28.82 23.22 22.68 32.01 34.03 34.39

conditions, water which is rich in H+ and OH can act either as a good and non-toxic medium for chemical reaction or as an efficient acid/base catalyst (Galkin and Lunin, 2005). SCW can also be employed especially for milder hydrolysis reaction (Quitain et al., 2002). Therefore, as a catalyst for hydrolysis of biological compounds from biomass, SCW can easily maximize the amount of extractable lipids from Y. lipolytica Po1g biomass, which is significantly higher than that from biomass without SCW pretreatment.

3.2. Effects of pretreatment parameters on composition of extract The experimental results shown in Table 2 were analyzed using analysis of variance (ANOVA) to develop a regression model for the determination of crude lipid, neutral lipid and wax and gum contents. Among the single parameters investigated, temperature played a significant role (p < 0.0001) on the amount of extractable crude lipid. Amount of water (p = 0.0316) was the second factor that significantly affecting the crude lipid amount. The parameter (time temperature) also showed a p value less than 0.0001, indicating there is a considerable interaction effect between reaction time and reaction temperature on the amount of crude lipid extracted. On the other hand, the p value of the parameter (amount of water time) is 0.2239 indicating that this combination of factors is less significant in determining the amount of extractable crude lipid. Theoretically, an increase in temperature at a constant pressure increases oil extraction (Brunner, 1994). The effect of temperature on palm oil yield from palm mesocarp using sub-critical 1,1,1,2tetrafluoroethane was studied and it was found that oil yield increased from 48.57% to 66.06% as temperature was increased from 40 to 80 °C at constant pressure (Mustapa et al., 2009). The highest extractable crude lipid content from SCW pretreated sample (84.79%) in this study occurred at high pretreatment temperature (175 °C). The crude lipid yield obtained at the highest temperature can be attributed to the improved oil dissolution due to the increase in mass transfer rate at higher temperature. Brunner (1994) explained that a higher amount of extract during lipid extraction under sub-critical conditions can be attributed to the increasing solute volatility and/or mass transfer rate at higher temperature. In this study, SCW acted as an effective catalyst that facilitated hydrolysis or biodegradation reaction when Y. lipolytica Po1g

biomass was treated with SCW at higher temperature, as stated elsewhere (Rogalinski et al., 2005). The neutral lipid content of SCW pretreated samples was significantly affected by temperature while extractable wax and gum content was much significantly affected by hydrolysis time and the combined effect of temperature and time. Statistical results show that temperature (p < 0.0001) was the most significant factor which played a significant role in determining the amount of extractable neutral lipids from biomass. The maximum amount of neutral lipids (42.67%) was obtained at the highest temperature (175 °C). The maximum wax and gum content of the untreated biomass sample in this study is 29.33% while that of the SCW pretreated sample was 34.39% using 30 mL water and heating the mixture in sub-critical conditions for 20 min at 150 °C. The increase in the amount of extractable wax and gum content is, most probably, attributed to the increase in dissolution of wax and gum in water under the sub-critical conditions.

3.3. Neutral lipid profiles of lipid extracted from Y. lipolytica Po1g The effect of temperature (125, 150 or 175 °C) on neutral lipid profile was investigated with treatment time and amount of water kept constant at 10 min and 20 mL/g dry biomass, respectively. Gas chromatography results of the neutral lipid profiles of untreated and SCW pretreated microbial biomass samples show the existence of different amounts of free fatty acid (FFA), monoacylglycerides (MAG), diacylglycerides (DAG) and triacylglycerides (TAG). A comparison of the results is summarized in Table 3. The data are averages of at least two independent experiments. Neutral lipid extracted from the untreated Y. lipolytica Po1g biomass that was cultivated in sugarcane bagasse hydrolysate contains mostly FFA (up to 78.98%). The TAG content of the neutral lipids from the untreated sample is very low (3.41%), as shown in Table 3. In our previous study, it was found that 81.35% (w/w) of the neutral lipid from Y. lipolytica Po1g is FFA (Tsigie et al., 2011), which is consistent with the results of this study on the untreated biomass sample (78.89%). In general, when glucose is used as the substrate for lipid synthesis in various oleaginous yeasts or molds, neutral lipids accumulate principally as TAG (80–90%, w/w, of the total lipids). FFA represents only a marginal storage compound under this condition (Meesters and Eggink, 1996). The amount of FFA in lipids differs depending on media type, cultivation time and the micro-organism used. A study by Beopoulos et al. (2008) showed that growth on medium containing hydrophobic compounds as (co)substrates resulted in the accumulation of storage lipid containing considerable amount of FFA (30–40 wt.% of total lipids). They found that the maximum total fatty acid level was observed at 11 h cultivation time which reached 50.0% of the dry weight for the wild type strain and 70.1% of the dry weight for the Dgut2 strain, which indicates that type of strain and medium have significant effects on the amount of FFA in the lipid. The reason for such unusually high FFA content in this study (78.89%) is still unknown. Mobilization of accumulated lipids in oleaginous microorganisms occurs as a consequence of three different metabolic states,

Table 3 FFA, MAG, DAG and TAG contents (%) of neutral lipid from Y. lipolytica Po1g biomass. Untreated

FFA MAG DAG TAG

78.98 ± 1.23 8.89 ± 0.98 4.56 ± 0.76 3.41 ± 0.45

SCW pretreated 125 °C

150 °C

175 °C

48.07 ± 0.45 11.22 ± 2.85 13.93 ± 1.4 26.79 ± 1.9

54.33 ± 1.26 13.15 ± 2.36 13.03 ± 1.87 19.51 ± 1.76

61.35 ± 1.7 10.17 ± 0.4 11.94 ± 0.06 16.57 ± 1.36

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(i) during the exponential phase of growth, when storage lipids are used for membrane lipid synthesis to support cellular growth and division, (ii) during stationary phase, when, upon nutrient depletion, FFAs are liberated rather slowly from the TAG and subjected to peroxisomal b-oxidation, and (iii) when cells exit starvation conditions, e.g., from stationary phase, and enter a vegetative growth cycle, and upon carbon supplementation, lipid depots are very rapidly degraded to FFA (Kurat et al., 2006). The high FFA value in this work might be due to nutrient depletion which is accompanied by the rapid degradation of lipids to FFA. When biomass of Y. lipolytica Po1g was pretreated with SCW, the amount of FFA that could be extracted was suppressed by the increasing amount of acylglycerides extracted, as can be seen from the results in Table 3. Generally, the amount of TAGs that can be extracted from Y. lipolytica Po1g by SCW is very high (26.79% pretreated at 125 °C) compared to that of the untreated samples (3.41%). The DAG and MAG amounts from the pretreated samples are almost constant at different temperatures, but higher than that of the untreated samples. Therefore, the highest amount of TAGs from Y. lipolytica Po1g that can be extracted is using SCW at 125 °C. As clearly indicated in Fig. 1, temperature has a significant effect on the amount of FFA and acylglycerides can be extracted from Y. lipolytica Po1g. Increasing temperature from 125 to 175 °C reduces the amount of TAG from 26.79% to 16.57% while the FFA content increases from 48.07% to 61.35% which implies that more acylglycerols were hydrolyzed at higher temperature. The amount of MAG, DAG and TAG in general decreases as temperature is raised from 125 to 175 °C. The same trend was observed during the continuous hydrolysis of Cuphea seed oil in SCW (Eller et al., 2011). The conversion of Cuphea seed oil TAG to FFA increased with temperature and leveled off at about 330 °C. At 300 and 310 °C, the percentage of FFA was only 60% and 70%, respectively. At temperatures higher than 330 °C there was no further increase in FFA. The neutral lipid content of activated sludge increased four times from 2.1% to 7.87% when the activated sludge was pre-treated by SCW. However, there are little differences in the compositions of neutral lipids obtained (Huynh et al., 2010), which agrees with the results of this work. Therefore, the neutral lipid content that can be extracted from Y. lipolytica Po1g increases almost four times when the biomass is pretreated with SCW.

3.4. Fatty acid composition of neutral lipids of Y. lipolytica Po1g The effect of temperature of SCW pretreatment, with the amount of water and pretreatment time kept constant at 20 mL/g

biomass and 10 min, respectively, on fatty acid profile in the extracted neutral lipids was investigated. The fatty acid compositions of neutral lipids from untreated and SCW pretreated biomass were analyzed by GC and the results are presented in Table 4. Basically, fatty acid profiles in untreated and SCW pretreated biomass samples are similar. Oleic acid (C18:1) is the most abundant fatty acid, with a content of 46.78% and 56.09% (125 °C) in the neutral extracted from the untreated and pretreated sample, respectively, which is consistent with the results reported by Tsigie et al. (2011). According to a study on the biosynthesis of lipids and organic acids by Y. lipolytica strain that was cultivated on glucose, it was found that oleic acid, palmitic acid, palmitoleic acid and stearic acid were the major fatty acids in the cellular fatty acid profile of the lipid produced (Papanikolaou et al., 2009). The same trend was observed when this yeast was cultivated in glycerol (Makri et al., 2010), which is quite similar to the result of this work. In general, the amount of long chain fatty acids such as lingoceric acid (C24:0) and those greater than 24 carbons increases with increasing temperature. Such long chain fatty acids were either not detected or they were found in trace amount in the lipids of untreated samples. On the other hand, the amount of short chain fatty acids such as capric acid (C10:0) decreases with increasing temperature, but its amount is higher in lipids of the untreated samples. The increase in temperature might have led to the hydrolysis of acylglycerides into FFAs facilitating the production of fatty acids with longer carbon chain. 3.5. FAMEs of lipids from untreated and SCW treated samples After transesterification reactions and purification of the products, the FAME profiles of the samples are determined by GC2010 and the results are summarized in Table 5. The SCW treatment at 175 °C gave the highest lipid content. Lipid obtained under this condition was employed for producing FAMEs. The FAMEs profiles of the two oil samples (treated or untreated) are very similar. Oleic acid methyl ester (C18:1n9c) was the most abundant FAME in both types of oil samples followed by palmitic acid methyl ester (C16:0) and linoleic acid methyl ester (C18:2n6c). This is in agreement with the fatty acids profiles except linoleic acid (C18:2n6C), which was not detected using GC 17A. This may be due to the separation inefficiency of the column used which could not separate the unsaturated C18 fatty acids very well. There is little difference in the FAMEs profiles except a slight increase in the contents of FAMEs with longer carbon chains in the pretreated

Table 4 Fatty acid composition of oil extracted from untreated and SCW pretreated biomass samples at different temperature. Data are averages of three independent experiments.

Fig. 1. Composition of the neutral lipids obtained from untreated Y. lipolytica Po1g samples and SCW pretreated samples at different temperatures.

a b

Type of fatty acid

Amount of fatty acid (% peak area) Unpretreated

SCW pretreated 125 °C

150 °C

175 °C

C10:0 C13:0 C14:0 C16:0 C16:1 C18:0 C18:1 C20:0 C22:0 C24:0 C > 24

5.92 ± 0.01 1.65 ± 0.02 0.94 ± 0.01 28.6 ± 1.28 3.1 ± 0.20 6.25 ± 0.05 46.78 ± 2.98 2.67 ± 0.23 2.38 ± 0.01 1.71 ± 0.45 NDb

0.01 ± 0.00 0.19 ± 0.00 0.75 ± 0.01 24.63 ± 1.01 4.36 ± 0.51 5.37 ± 0.02 56.09 ± 2.77 1.64 ± 0.12 1.5 ± 0.01 3.7 ± 0.67 1.76 ± 0.18

Tracea 0.31 ± 0.00 0.35 ± 0.01 25 ± 2.01 6.82 ± 0.01 4.23 ± 0.09 54.8 ± 2.56 0.54 ± 0.00 1.21 ± 0.02 4.35 ± 0.23 2.39 ± 0.19

0.36 ± 0.00 Trace 0.56 ± 0.02 24.27 ± 0.90 10.48 ± 1.26 8.16 ± 1.08 42.52 ± 1.23 1.08 ± 0.00 2.93 ± 0.03 6.48 ± 1.26 3.16 ± 1.08

Trace = less than 0.01%. ND = Not detected.

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Table 5 FAME compositions of neutral lipids from untreated and SCW treated Y. lipolytica Po1g biomass. Data are averages of two independent experiments.

*

Type of FAME

SCW treated (175 °C)

Untreated

Capric acid methyl ester (C10:0) Lauric acid methyl ester (C12:0) Tridecanoic acid methyl ester (C13:0) Myristic acid methyl ester (C14:0) Myristoleic acid methyl ester (C14:1) Pentadecanoic acid methyl ester (C15:0) Palmitic acid methyl ester (C16:0) Palmitoleic acid methyl ester (C16:1) Stearic acid methyl ester (C18:0) Oleic acid methyl ester (C18:1n9c) Linoleic acid methyl ester (C18:2n6c) Arachidic acid methyl ester (C20:0) Behenic acid methyl ester (C22:0) cis-11,14,17-Eicosatrienoic acid methyl ester (C20:3n3) Lignoceric acid methyl ester (C24:0) Others

0.11 ± 0.00 0.18 ± 0.01 0.19 ± 0.00 0.37 ± 0.00 0.42 ± 0.01 0.41 ± 0.01 22.87 ± 1.05 6.32 ± 0.30 6.83 ± 0.00 37.11 ± 0.42 13.97 ± 0.10 2.56 ± 0.00 1.58 ± 0.10 0.31 ± 0.03

1.29 ± 0.01 0.73 ± 0.02 0.54 ± 0.02 1.46 ± 0.20 0.38 ± 0.01 0.23 ± 0.00 27.79 ± 0.95 8.59 ± 0.32 9.09 ± 0.41 33.75 ± 1.02 11.62 ± 0.23 1.06 ± 0.05 0.57 ± 0.10 ND*

2.65 ± 0.20 4.12 ± 0.01

0.09 ± 0.00 3.11 ± 0.20

ND = Not detected.

samples. This may probably be due to the acid behavior of the hydrogen ions of water molecules under subcritical condition that facilitated the evolution of long chain fatty acids. Under sub-critical conditions, water which is rich in H+ and OH can act either as a good and non-toxic medium for chemical reaction or as an efficient acid/base catalyst (Galkin and Lunin, 2005). The maximum experimental biodiesel yield achieved from untreated biomass of Y. lipolytica Po1g was 21.86 (%, w/w) while that of SCW treated samples was 40.21 (%, w/w), which shows the potential of SCW pretreatment in significantly enhancing biodiesel yield. It increases the biodiesel yield twofold by increasing the amount of extractable neutral lipids from the yeast biomass. 4. Conclusion The results of this study show that it is possible to increase the amount of extractable crude lipid from 51.53% to 84.75% and the amount of neutral lipid from 23.21% to 42.67% if Y. lipolytica Po1g biomass is pretreated with SCW. The results also suggest that solvent extraction alone is not effective enough for the complete recovery of neutral lipids from Y. lipolytica Po1g and SCW plays a significant role in increasing the amount of extractable neutral lipid from this yeast and hence raises the biodiesel yield. Therefore, this yeast can be a good source of biodiesel. Acknowledgements The authors would like to acknowledge the National Science Council of Taiwan (NSC100-2623-E-011-001-ET) and National Taiwan University of Science and Technology (100H451403) for financing this work. References Adamczak, M., Bornscheuer, U.T., Bednarski, W., 2009. The application of biotechnological methods for the synthesis of biodiesel. Eur. J. Lipid Sci. Technol. 111 (8), 800–813. Alvarez, H.A., Steinbüchel, A.S., 2002. Triacylglycerols in prokaryotic microorganisms. Appl. Microbiol. Biotechnol. 60 (4), 367–376. Beopoulos, A., Cescut, J., Haddouche, R., Uribelarrea, J.-L., Molina-Jouve, C., Nicaud, J.-M., 2009. Yarrowia lipolytica as a model for bio-oil production. Prog. Lipid Res. 48 (6), 375–387. Beopoulos, A., Mrozova, Z., Thevenieau, F., Le Dall, M.-T., Hapala, I., Papanikolaou, S., Chardot, T., Nicaud, J.-M., 2008. Control of Lipid Accumulation in the Yeast Yarrowia lipolytica. Appl. Environ. Microbiol. 74 (24), 7779–7789.

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