Raw materials
Soybean oil was purchased from Qinhuangdao Jinhai Grain & Oil Industrial Co., Ltd. (Qinhaungdao, China). Its fatty acid composition was palmitic acid 20.03%, 4.85% stearic acid, 24.17% oleic acid, 47.03% linoleic acid and 3.92% α-linolenic acid. Olive oil was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Heptadecanoic acid methyl ester (chromatographically pure) was from Sigma, USA. Yarrowia lipolytica strain was from China Agricultural University. (The strain was deposited at the China General Microbiological Culture Collection as CGMCC 2707.) Its lipase was produced by Qinhuangdao Leading Science & Technology Co., Ltd. Qinhuangdao, China. Soybean powder was obtained from a local market. All other reagents were obtained commercially and were of analytical grade.
Lipase preparation
The Y. lipolytica strain CGMCC 2707 was stored at -80°C in tubes containing 25% (vol/vol) glycerol solution. For the preparation of inoculum, cells were transferred to YPD medium (20 g of tryptone, 10 g of yeast extract and 20 g dextrose per liter autoclaved for 15 minutes at 121°C) two times for activation, and they were then incubated at 28°C. Activated cells were inoculated into fermentation medium, which contained 60 g of soybean powder, 90 g of soybean oil, 2.5 g of K2HPO4, 0.5 g of MgSO4·7H2O and 2 g of (NH4)2SO4 per liter of distilled water. Thirty-liter cultures were grown in a 50-l fermentor with agitation at 500 rpm and 1:1 vvm air flow at 28°C, and pH was adjusted to 6.5 by using 10 N KOH. The lipase produced reached 8,000 Uw/ml after 90 to 110 hours of fermentation (Uw refers to the hydrolysis activity of lipase). Lipase solution was obtained by the removal of cells by centrifugation (4,000 × g for 20 minutes).
Lipase in the supernatant was precipitated by the addition of three volumes of acetone. The precipitate was washed with acetone and dried at room temperature. The activity of the enzyme powder was 140,000 Uw/g.
Immobilization of lipase on fabric membrane
Lipase was immobilized by using an established immobilization procedure [16]. Briefly, 0.1 g of fabric (approximately 9 cm2) was presoaked for 1 hour in 10 ml of coimmobilization solution consisting of 0.5 g of gluten, 0.2 g of lecithin, 0.2 g of polyethylene glycol 6000 and 0.1 g of magnesium chloride. Fabric membranes were dried at room temperature and used as supports for the immobilization of lipase. Membranes were added into 10 ml of enzyme solution (5,000 to 10,000 Uw/ml), stirred for 2 to 3 hours, taken out and dried at room temperature under a vacuum. The activity of immobilized lipase, determined by using an olive oil emulsion method after grinding at 0°C, was 10,000 Uw/g membrane.
Lipase activity determination
Hydrolysis activity of lipase (Uw) was determined by using the olive oil emulsion method. One hydrolysis activity unit was defined as the amount of enzyme required to release 1 μM fatty acid per minute under assay conditions [6].
The esterification activity of lipase (Ue) was determined by using a lauric acid and lauryl alcohol reaction system. One esterification activity unit was defined as the amount of enzyme required to release 1 μM lauric lauryl ester per minute under assay conditions. The substrate was an equimolar mixture of lauric acid with lauryl alcohol at a final concentration of 0.1 mM in n-hexane solvent. The reaction was initiated by adding 0.01 g of lipase (pure or diluted depending on the activity of lipase), continued by incubation for 20 minutes at 40°C and stopped by the addition of 15 ml of ethanol. Enzyme activity was determined by titration of the remaining lauric acid with 100 mM sodium hydroxide. Esterification activity was calculated on the basis of the release of lauric lauryl alcohol using the following formula:
where V0 and VNaOH are, respectively, the volumes of NaOH consumed by titration of the mixture at the beginning (0 minutes) and end (20 minutes) of the reaction.
Hydrolysis reaction
Small-scale hydrolysis was conducted at 40°C in a 50 ml screw-cap tube containing 2 g of soybean oil, 200 Uw lipase broth and 1.2 ml of water, with agitation on an orbital shaker (180 rpm) for 28 to 48 hours. At defined intervals, 0.8 ml of the reaction mixture was removed and separated into oil and water phases by centrifugation (10,000 × g for 5 minutes). The oil phase was analyzed as described in the Analytical methods section.
Large-scale hydrolysis was conducted by filling a 1-l reaction vessel with 200 g of soybean oil, 20,000 Uw lipase broth, 120 ml of water and 10 g of sodium stearate and then agitating the mixture at 180 rpm at 40°C. When the degree of hydrolysis reached 90%, the reaction mixture was acidified with 3 N sulfate until the pH of the water layer was 4.5. The water layer was then removed, and the remaining oil layer was washed twice with hot water (70°C to 80°C). The oil layer was vacuum-distilled at 93 to 98 kPa, and distillates were collected at 220°C to 260°C. The resulting fatty acid fraction was used for esterification as described below.
Esterification reaction
Fatty acid was esterified using immobilized lipase membrane in 50-ml stoppered flasks without organic solvent. The reaction was performed with 2.82 g of oleic acid or FFA and 0.33 g of lipase membrane, and 192 μl of ethanol were added every 1 hour (oleic acid:ethanol molar ratio, 1:0.3) until theoretical molar ratio was reached. The mixture was incubated with agitation at 130 rpm at 30°C. Molecular sieves (Figure 5A) were added for 1 hour to eliminate water. Immobilized lipase and fatty acid were preheated in a 30°C incubator for 30 minutes, and the reaction was started by the addition of ethanol to the system. Experiments were replicated three or more times, and the results are presented as mean values.
Adsorbed water and lipase membranes were recovered from the reaction solution by filtration, and 15% (wt/vol) NaOH solution was added according to the amount of remaining fatty acid. The solution was stirred slowly for 30 minutes and then left undisturbed so that the aqueous and organic phases could separate. The organic phase was washed twice with two volumes of water to remove unreacted ethanol and dehydrated by decompression distillation. The final product, ethyl ester (biodiesel), was obtained with 95% recovery.
Analytical methods
TLC
Silica gel plates (Whatman Inc. Shaihai, China) were heated at 110°C for 1 hour prior to use. Oil phase samples obtained as described in the small-scale hydrolysis reaction section were dissolved in acetone to form a 10 mg/ml solution, and 10-μl capillary spots were subjected to TLC analysis. The spots were sprayed with a 20 volume percent solution of sulfuric acid in ethanol developed with petroleum ether/ethyl ether/acetic acid (80:30:1 ratio, volume fraction) and visualized by heating at 100°C for 30 to 50 seconds.
Gas chromatography was conducted to quantify the composition of fatty acids and FAEEs. At a predefined time, 20-μl samples were taken and centrifuged. A quantity of 5 μl of the upper phase thus obtained was dissolved in n-hexane and analyzed using a GC-2010 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a capillary column (HP-INNOWax columns, 30 m-0.25 mm-0.25 μm; J & W Scientific Columns, Agilent Technologies, Palo Alto, USA) and a flame ionizing detector. Injection was performed in split mode (1:30), with injection and detection temperatures of 260°C and 280°C, respectively. Samples (1 μl) were injected at an oven temperature of 240°C and held for 10 minutes. The carrier gas was nitrogen at a flow rate of 30 ml/min.
Hydrolysis degree was calculated as the acid value of the hydrolyzed oil sample as a percentage of the saponification value of soybean oil.
The degree of esterification was calculated as the reduction of acid value (obtained by titration of aliquots of mixture taken at the beginning and end of the reaction) as a percentage of fatty acid value.
Water content was measured by using a Karl-Fisher WS-3 trace moisture analyzer (Shandong Zibo Corson Instruments, Zibo, China) [17].
The viscosity of the oil or reaction mixture was measured using a viscometer (RVDV-II+PRO; Brookfield Engineering Laboratories, Middleboro, MA, USA).