Biodiesel production from microalgae oil catalyzed by a recombinant lipase

Biodiesel production from microalgae oil catalyzed by a recombinant lipase

Bioresource Technology 180 (2015) 47–53 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/b...

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Bioresource Technology 180 (2015) 47–53

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Biodiesel production from microalgae oil catalyzed by a recombinant lipase Jinjin Huang, Ji Xia, Wei Jiang, Ying Li ⇑, Jilun Li State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, Beijing 100193, PR China

h i g h l i g h t s  Chlorella vulgaris oil is an excellent substrate for biodiesel production.  Lipase GH2 can be efficiently catalytic microalgae oil into FAME and FAEE.  A simple process flow – ‘‘one step method’’.

a r t i c l e

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Article history: Received 26 September 2014 Received in revised form 20 December 2014 Accepted 22 December 2014 Available online 30 December 2014 Keywords: Recombinant lipase Microalgae oil Esterification reaction Biodiesel Conversion efficiency

a b s t r a c t A recombinant Rhizomucor miehei lipase was constructed and expressed in Pichia pastoris. The target enzyme was termed Lipase GH2 and it can be used as a free enzyme for catalytic conversion of microalgae oil mixed with methanol or ethanol for biodiesel production in an n-hexane solvent system. Conversion rates of two major types of biodiesel, fatty acid methyl ester (FAME) and fatty acid ethyl ester (FAEE), reached maximal values (>90%) after 24 h. The process of FAME production is generally more simple and economical than that of FAEE production, even though the two processes show similar conversion rates. In spite of the damaging effect of ethanol on enzyme activity, we successfully obtained ethyl ester by the enzymatic method. Our findings indicate that Lipase GH2 is a useful catalyst for conversion of microalgae oil to FAME or FAEE, and this system provides efficiency and reduced costs in biodiesel production. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction There is increasing interest in alternative or new energy sources because of the environmental impacts and declining supplies of fossil fuels. Biodiesel, a sustainable, nontoxic, biodegradable diesel fuel substitute, can be used in current diesel car infrastructure without major engine modifications (Luque et al., 2010). Biodiesel is usually obtained by transesterification of fat and vegetable oils with alcohol (usually methanol or ethanol) in the presence of a catalyst, with resulting production of a fatty acid methyl ester (FAME) or fatty acid ethyl ester (FAEE) that can be used as a biofuel (Almeida et al., 2012). The major factor limiting development and use of biodiesel is its high production cost, of which feedstock expense accounts for Abbreviations: FAME, fatty acid methyl ester; FAEE, fatty acid ethyl ester; MAG, monoacylglycerol; 1,3-DAG and 1,2-DAG, diacylglycerols; TAG, triacylglycerol; FFAs, free fatty acids; C/M, chloroform:methanol (v/v). ⇑ Corresponding author. Tel.: +86 10 62733751. E-mail address: [email protected] (Y. Li). http://dx.doi.org/10.1016/j.biortech.2014.12.072 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

>75% (Lim and Teong, 2010). Feedstock prices are a crucial factor in strategies to make biodiesel competitive with fossil fuels. Three ‘‘generations’’ are considered in the development of biodiesel feedstocks. First generation feedstocks were based on edible vegetable oils, e.g., soybean and sunflower oils (Ahmad et al., 2011). Second generation feedstocks, intended to reduce dependence on edible vegetable oils, were based on nonfood sources such as jatropha, jojoba oil, waste oil, recycled oil, and animal fats (Ahmad et al., 2011; Pinzi et al., 2014). Second generation feedstocks can be produced from non-agricultural land, thereby eliminating competition with food production. Nevertheless, development of first and second generation feedstocks is limited by high cost, inefficiency, and unsustainability (Ahmad et al., 2011; Balat and Balat, 2010; Leung et al., 2010). Research attention is now focused on the third generation biodiesel feedstock: microalgae. In comparison with first and second generation feedstocks, microalgae is considered a more promising alternative source for biodiesel production because of its high oil productivity, rapid reproduction, no requirement of arable

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land or fresh water, and enrichment in useful or valuable coproducts (Pinzi et al., 2014; Minowa et al., 1995). Production of biodiesel from microalgae involves several steps, including cell cultivation, cell harvesting, oil extraction, and biodiesel synthesis (Goncalves et al., 2013). Many research studies have focused on cultivation of microalgae for increased lipid productivity, but relatively few have addressed the chemical or enzymatic conversion of microalgae oil to biodiesel (Lai et al., 2012a). Enzymatic conversation methods typically have lower operating costs and higher product purity than do chemical methods. Some lipases have been for enzymatic conversion of microalgae oil to biodiesel, e.g., NovozymÒ 435 (Da Ros et al., 2012), extracellular lipase from Bacillus sp. (Sivaramakrishnan and Muthukumar, 2012), Penicillium expansum lipase (Lai et al., 2012b), immobilized Burkholderia lipase (Tran et al., 2013), and immobilized Candida sp. 99–125 lipase (Li et al., 2007). Methanol has traditionally been the alcohol most commonly used in biodiesel production, but there is now increasing emphasis on use of ethanol for production of FAEE. Both FAME and FAEE preparation methods have unique advantages and disadvantages (Mohamad Firdaus et al., 2014). Methanol is less expensive than ethanol, but oil is less soluble in methanol, and methanolysis involves limited mass transfer during the transesterification process. Ethanol is safer to use, renewable, has higher oil solubility, and the mass transfer limitation is not as great; however, ethanol is more expensive than methanol (Zhang et al., 2014). In a recent study, we used various strategies (optimization of signal peptide codons, addition of target gene propeptide, optimized gene dosage) to enhance expression level of a recombinant lipase (termed Lipase GH2) in the methylotrophic yeast Pichia pastoris. The unique properties of Lipase GH2 made it more suitable than other lipases for conversion of microalgae oil to FAME (Huang et al., 2014). Depend on Lipase GH2 was obtained, the aims of the present study were to (i) evaluate the ability of Lipase GH2 in combination with ethanol to catalyze production of FAEE from microalgae oil; (ii) further optimize the reaction conditions and improve the conversion rate of FAME and FAEE by Lipase GH2 in a n-hexane system; (iii) comparison of the differences in FAME and FAEE preparation process by using Lipase GH2. 2. Methods 2.1. Enzyme preparation Construction of a recombinant P. pastoris strain (ma-2pRMLX33) was described in our previous report (Huang et al., 2014). The strain highly expressed Rhizomucor miehei lipase (termed Lipase GH2), and this enzyme displayed high lipase activity. The strain was flask-cultured in BMGY/BMMY medium as described by Hu et al. (2013), and target protein expression was induced by daily addition of 1% methanol. Optical density (OD600) and enzyme activity were measured throughout the culture period as described by Guan et al. (2010), except that our reaction buffer was 0.1 M sodium dihydrogen phosphate/citric acid, pH 6.0, 35 °C. After fermentation, the supernatant were collected by 5 min centrifugation at 6000 rpm and stored at 4 °C. 2.2. Microalgae oil extraction and detection Chlorella vulgaris (CV) powder was kindly provided by Dr. Peng Pu (State Key Laboratory of Catalytic Material and Reaction Engineering, Research Institute of Petroleum Processing, SINOPEC, Beijing, China). Dried CV powder (0.5 g) was mixed with 20 mL deionized water. CV cell walls were disrupted by sonication

(200 W, ultrasonic 5 s, intermittent 5 s, 60 times), and the disrupted cells were mixed with biphasic solvent (chloroform/methanol [C/M], 1:2) or hexane in a shaker (200 rpm, 30 min). The mixture was centrifuged (6000 rpm, 10 min) to form two layers. The upper hexane layer that contained microalgae oil was collected, and crude oil was obtained by evaporation in a hood at room temperature. Free fatty acids (FFAs) in the extracted crude oil were analyzed by gas chromatography (GC) and thin-layer chromatography (TLC). In brief, 10 lL of the upper layer was mixed with 40 lL hexane, and 2 lL of the mixture was subjected to TLC (Guan et al., 2010). FFAs in the microalgae oil were analyzed using a GC system (model 6890A, Agilent Technologies; Santa Clara, CA, USA) equipped with a J&W DB-23 capillary column (60 m  250 lm  0.25 lm). Preparation of standards and samples, and testing conditions, were as described by Li et al. (2011). These experiments were performed at the Feed Detection Center of the Chinese Ministry of Agriculture. 2.3. Biodiesel preparation Extracted crude oil as above was used as a substrate for biodiesel production using the organic solvent n-hexane. Short-chain alcohols (methanol or ethanol) were used separately as another substrate. Free Lipase GH2 was used as a biocatalyst. We performed a series of experiments to determine optimal reaction conditions (temperature, water content, alcohol/oil molar ratio, procedure for adding alcohol, Lipase GH2 amount). The following optimal reaction conditions were used, except when stated otherwise: 0.3 g microalgae oil, 90 lL methanol or 217 lL ethanol, 600 lL n-hexane, 150 lL Lipase GH2, incubation temperature 30 °C, shaking at 150 rpm for 24 h. Enzyme solution in reaction was recovered by centrifugal, then the enzyme solution was added in the next new reaction system to detected the reuse of Lipase GH2. 2.4. Detection of enzyme-catalyzed reaction products FAME or FAEE in the reaction mixture were analyzed by a GC (model GC522, Wufeng, China) equipped with an HP-INNOWax capillary column (30 m  0.25 mm  0.25 lm; Agilent). Heptadecanoic acid methyl ester was dissolved in hexane (10 mg/mL) as internal standard. The reaction mixture was centrifuged (12,000 rpm, 5 min) and solvent was eliminated by drying. The dried sample (40 mg) was mixed thoroughly with 400 lL internal standard for GC analysis as described by Guan et al. (2010). 3. Results and discussion 3.1. Collection of efficient liquid enzyme P. pastoris is frequently used as a host for expression of heterologous proteins because it can be tightly regulated by a eukaryotic promoter (alcohol oxidase I; AOX1) and can tolerate a broad pH range (3.0–7.0) (Soyaslan and Calik, 2011). To date, 500 heterologous proteins have been expressed in P. pastoris systems (Yu et al., 2010). R. miehei lipase is a strong 1,3-specific lipase customarily used for production of biodiesel. We recently generated a recombinant R. miehei lipase (Lipase GH2) in P. pastoris by addition of the target gene propeptide, optimization of signal peptide codons, and optimization of gene dosage (Huang et al., 2014). The recombinant P. pastoris strain contained two copies of the R. miehei lipase precursor gene, and displayed maximal lipase production in flask culture. We cultured the recombinant strain in shake flasks as described in our previous report, and measured cell growth and enzyme

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activity every day. Cell density reached OD600 = 35 at day 5, and the maximal enzyme activity was 1200 U/mL (Fig. 1). The supernatant was collected by centrifugation and showed target protein concentration 0.8 mg/mL. The supernatant was used directly as catalyst in conversion experiments, and could be stored at 4 °C for >6 months because enzyme stability and tolerance were enhanced by glycosylation (Huang et al., 2014).

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C20:5n-3; five double bonds) and docosahexaenoic acid (DHA, C22:6n-3; six double bonds), are easily oxidized during storage and therefore undesirable as components of biodiesel (Chisti, 2007). The oil extracted from CV contained almost no polyunsaturated fatty acids with P4 double bonds and was therefore suitable for preparation of high-quality biodiesel. 3.3. Optimized conditions for Lipase GH2-catalyzed preparation of biodiesel

3.2. Extraction and quality testing of microalgae oil Techniques used for laboratory-scale microalgal lipid extraction include organic solvent extraction and supercritical fluid extraction. For organic solvent extraction, the solvent should be volatile for low-energy distillation from crude microalgae oil. Extraction using C/M 1:2 is faster than hexane extraction; however, C/M is more toxic than hexane (Halim et al., 2012). In a comparison of five organic solvent mixtures for extracting lipids from Botryococcus braunii cells, Lee et al. (1998) found that C/M gave the highest total lipid yield. Two extraction solvents (C/M 1:2 and hexane) were used in the present study, and the results are compared in Fig. 2A. Starting from 1 g CV powder, the amount of crude microalgae oil obtained was 160 mg using C/M but only 24 mg using hexane. The crude oil extraction rate was 7-fold higher for C/M than for hexane. In view of its shorter extraction time and greater extraction efficiency, C/M was used as extraction solvent in subsequent experiment. Microalgae oil extracted from CV powder by C/M was analyzed by TLC and GC. For TLC analysis (Fig. S1), the standard samples (lanes1–4) were monoacylglycerol (MAG), diacylglycerols (1,3and 1,2-DAG), FFAs, and triacylglycerol (TAG). The major components of microalgae oil were found to be FFAs and TAG (lane 5). GC was performed for qualitative and quantitative analysis of fatty acids (Table S1). The proportions of various fatty acids in microalgae oil are summarized in Fig. 2B. The microalgae oil (1 g) extracted from CV powder contained 508.26 mg fatty acids. By comparison with FAME standards, seven peaks were identified as myristic acid (C14:0, 0.56%), palmitic acid (C16:0, 11.8%), palmitoleic acid (C16:1, 0.97%), stearic acid (C18:0, 1.28%), oleic acid (C18:1, 17.02%), linoleic acid (C18:2, 8.50%), and linolenic acid (C18:3, 9.06%). The 508 mg fatty acids contained 487 mg C16– C18 fatty acids 96% of the total (Table S1). The common types of fatty acid found in all microalgae are C14:0, C16:0, C18:0, C18:1, C18:2, and C18:3; however, their proportions vary depending on the species (Amaro et al., 2011). The contents of other fatty acid residues are species-specific (Mata et al., 2010). Some microalgae are enriched in polyunsaturated fatty acids. Polyunsaturated fatty acids having >4 double bonds, e.g., eicosapentaenoic acid (EPA,

R. miehei lipase is a common catalyst used for biodiesel in the form of free, immobilization or whole-cell biocatalysts (Guan et al., 2010; Jin et al., 2013; Ognjanovic et al., 2008). This lipase is 1,3-specific, and was used in combination with nonspecific mono- and diacylglycerol lipases for methanolysis of soybean oil (Guan et al., 2010). R. miehei lipase has been used for catalysis of FAME or FAEE production from soybean, rapeseed, and tallow oil (Mohamad Firdaus et al., 2014). Most recent studies of enzymatic production of biodiesel from microalgae are focused on FAME production. Very few studies have addressed FAEE production by conversion of microalgae oil. In a study of biodiesel production by transesterification reaction of microalgae oil using various acyl acceptors (methanol, ethanol, 2-propanol, n-butanol), Pushpendra et al. (2013) found that only methanol gave a substantial yield, with a conversion rate of 44%. The substrates used for FAEE production with R. miehei lipase as catalyst are limited to second generation biodiesel feedstocks. Nelson et al. (1996) used soybean oil, rapeseed, and tallow as raw materials, R. miehei lipase as catalyst, and methanol and ethanol as short-chain alcohol donors in a study of optimal conversion of TAG to alkyl esters, and obtained yields ranging from 70% to 98%. Bergamasco et al. (2013) used R. miehei lipase immobilized on highly crystalline PVA microspheres as a biocatalyst for transesterification of soybean oil to generate FAEE, and obtained 66.3% yield. In order to first try and prove that by using the free lipase transesterification reaction strategy is feasible or not? And whether the crude microalgae extract catalyzed by free lipase GH2 can be obtained ideal result? To ensure the experimental repeatability, small amount catalytic reaction was performed in the present study. We used microalgae oil from dried CV powder as raw material, a glycosylated precursor of R. miehei lipase (Lipase GH2) highly expressed in P. pastoris as catalyst, and methanol and ethanol as short-chain alcohol donors for biodiesel production. Enzymatic reaction is affected by many factors, including reaction temperature, water content, enzyme amount, and substrate ratio. We performed a series of experiments to determine optimal conditions for FAME and FAEE production, and obtained the results described in the following sections.

Fig. 1. Cell density (measured as OD600) and enzyme activity during flask fermentation of the recombinant P. pastoris strain. At day 5, cell growth of the strain reached OD600 35, and enzyme activity was maximal (1200 U/mL).

3.3.1. Reaction temperature The trend of temperature effect on the enzymatic reaction was the same regardless of whether methanol or ethanol was used as short-chain alcohol donor. At a reaction temperature of 30 °C, the maximal Lipase GH2-catalyzed conversion rate was 87% for FAME and 86% for FAEE (Fig. 3A). Lower conversion rates were obtained for reaction temperatures above or below 30 °C. The effect of temperature on enzymatic reactions has three major aspects. (i) Temperature affects collisions between substrate molecules. As temperature rises, the number of collisions between substrate molecules increases, resulting in an accelerated reaction rate. (ii) Enzyme proteins undergo progressive denaturation as temperature rises, resulting in decreased reaction rate. (iii) Esterification reactions are endothermic (Gumel et al., 2011); an increase in reaction temperature tends to push the reaction process in the synthesis direction. As these three aspects approach their equilibrium, the reaction rate becomes maximal.

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Fig. 2. Extraction and quality detection of microalgae oil from CV powder. (A) Extraction rate using C/M vs. hexane as solvent. 1 g CV powder yielded 160 mg crude microalgae oil when C/M 1:2 used as solvent, but only 24 mg crude oil when hexane was used. The extraction rate was 7-fold higher for C/M than for hexane. (B) Qualitative and quantitative analysis of fatty acids in microalgae oil by GC. The major fatty acid components were myristic acid (C14:0, 0.56%), palmitic acid (C16:0, 11.8%), palmitoleic acid (C16:1, 0.97%), stearic acid (C18:0, 1.28%), oleic acid (C18:1, 17.02%), linoleic acid (C18:2, 8.50%), and linolenic acid (C18:3, 9.06%). The oil extracted from CV is a suitable substrate for biodiesel production.

Fig. 3. Effects of reaction temperature, water content, and alcohol/oil molar ratio on Lipase GH2-catalyzed biodiesel production from microalgae oil. (A) Reaction temperature. 30 °C was the optimal temperature for FAME and FAEE production. (B) Water content (volume ratio of water to oil). The optimal water contents were 0.2 for FAME production and 0.5 for FAEE production. (C) Alcohol/oil molar ratio. For FAME production, maximal conversion rate (90%) was obtained with methanol/oil molar ratio 3:1 to 5:1. For FAEE production, maximal conversion rate (95%) was obtained with ethanol/oil ratio 5:1.

3.3.2. Water content The effect of water content on enzymatic reactions has two major aspects. (i) Water provides an oil–water interface necessary for lipase catalysis. The reaction rate is reduced by a water content that is either too low (resulting in decreased contact area between the lipase and substrate) or too high (resulting in dilution of lipase concentration). (ii) Water is a reaction product in the biodiesel

preparation process. An excessive water content pushes the reaction in the reverse direction and reduces conversion rate. We determined optimal water content [quality ratio of water to oil] for FAME or FAEE production (Fig. 3B). The optimal water content was 0.2 for FAME production (conversion rate 85.5%) and 0.5 for FAEE production (conversion rate 86.4%). In both cases, conversion rate was lower for water content below the optimal value.

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Why did optimal water content differ for FAME vs. FAEE production using the same catalyst? One possible explanation is the differential tolerance of Lipase GH2 to methanol (40%) vs. ethanol (30%) (Huang et al., 2014). Methanol and ethanol were each mixed in an alcohol/oil molar ratio 3:1 and were added in equal amounts to the reaction system at times 0, 3, and 6 h. A higher concentration of either methanol or ethanol in the mixture resulted in reduced enzyme stability and tolerance, and lower catalytic activity. Methanol concentration was 33% for water content 0.2 but 50% for water content 0.1, resulting in damage to the enzyme. Ethanol concentration was 22% for water content 0.5 but 42% for water content 0.2, which exceeded the ethanol tolerance of Lipase GH2 (30%). Thus, even though the esterification reactions were catalyzed by the same enzyme, the differing natures of the substrates resulted in different reaction conditions. 3.3.3. Alcohol/oil molar ratio Methanol and ethanol serve as reaction substrates for biodiesel production, but are harmful to proteins in excessive proportions. Addition of appropriate amounts of alcohol to the reaction mixture can enhance reaction velocity and degree of esterification. We determined the optimal amount of alcohol added to the reaction (Fig. 3C). Maximal conversion rates were obtained with methanol: oil ratio 3:1 to 5:1 (86.6%) and ethanol: oil ratio 5:1 (87.5%). Higher methanol or ethanol proportions reduced conversion efficiency. The proportion of ethanol needed for FAEE production was greater than the proportion of methanol needed for FAME production. Either alcohol in excess is reduced lipase activity.

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3.3.4. Alcohol additional strategy We observed that addition of an appropriate amount of alcohol to the reaction mixture could enhance reaction velocity and esterification degree. On the other hand, high alcohol concentrations may denature proteins (Meng et al., 2011). We therefore designed a series of experimental steps for addition of methanol or ethanol to the reaction mixture with minimal protein (enzyme) denaturing effect. A 3:1 methanol/oil molar ratio was established by 1-step, 2step, and 3-step addition methods. Addition of methanol by the 1step method resulted in inhibited reaction and a 50% conversion rate. Addition of methanol by either the 2- or 3-step method resulted in an increase of conversion rate to 90% (Fig. 4A). Damage to the enzyme was significantly reduced by 2-step addition of methanol. A 5:1 ethanol/oil molar ratio was established by 1-, 2-, 3-, 4-, and 5-step addition methods. The conversion rate was 50% for the 1- and 2-step methods, and increased to 75% for the 3-step and 95% for the 4-step method (Fig. 4B). These findings indicate that Lipase GH2 activity was inhibited by ethanol added by 1-, 2, and 3-step methods, whereas the 4-step method had significantly less damaging effect on the enzyme. High concentrations of methanol or ethanol cause enzyme denaturation and inactivation. These two alcohols are usually added by a step-wise strategy to reduce enzyme damage. Because the tolerance of Lipase GH2 to methanol and ethanol differed, the amount of each alcohol added per step was necessarily different. Lipase GH2 has various advantages over other lipases in regard to FAEE production. The ethanol tolerance of Lipase GH2 is greater

Fig. 4. Effects of alcohol additional strategy and enzyme content on Lipase GH2-catalyzed biodiesel production from microalgae oil. (A) Methanol addition strategy. A 3:1 methanol/oil molar ratio was established by 1-, 2-, and 3-step addition methods. (B) Ethanol addition strategy. A 5:1 ethanol/oil molar ratio was established by 1-, 2-, 3-, 4-, and 5-step addition methods. (C) Lipase GH2 content and FAME production. (D) Lipase GH2 content and FAEE production.

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Table 1 Optimal conditions used for Lipase GH2-catalyzed production of FAME and FAEE from microalgae oil. Reaction conditions

FAME

FAEE

Reaction temperature (°C) Water content (quality ratio of water to oil) Alcohol/oil (molar ratio) Alcohol additional strategy (steps) Enzyme content (U/g) Conversion rate (%) Reuse of Lipase GH2 (times)

30 0.2 3:1 2 160 95 >5

30 0.5 5:1 4 560 95 1

Fig. 5. TLC analysis of reaction mixture components for FAME and FAEE production under optimal conditions. Lane 1: monoacylglycerol (MAG). Lane 2: diacylglycerols (1,3- and 1,2-DAG). Lane 3: oleic acid (FFA). Lane 4: triacylglycerol (TAG). Lane 5: FAME. Lane 6: FAEE. Lane 7 and 8: samples for FAME production at 0 and 24 h. Lane 9 and 10: samples for FAEE production at 0 and 24 h.

than that of Yarrowia lipolytica lipase. In the present study, an ethanol/oil molar ratio of 5:1 was used and ethanol was added to the reaction mixture 4 times with a conversion rate >95%. In a Y. lipolytica lipase-catalyzed system, the ethanol/oil molar ratio used was only 2:1, and ethanol was added 5 times with a final conversion rate 90% (Meng et al., 2011). 3.3.5. Lipase GH2 content To reduce the production cost of FAME and FAEE, we optimized the amount of Lipase GH2 added to the reaction mixture in each case. As Lipase GH2 content increased from 40 to 160 U/g, the FAME conversion rate increased gradually to 95% (Fig. 4C). There was no further increase in conversion rate at higher lipase content. Thus, 160 U/g Lipase GH2 is appropriate for catalysis of FAME production. In the case of FAEE production, the conversion rate increased gradually to 95% as Lipase GH2 content increased from 40 to 560 U/g (Fig. 4D). Thus, 560 U/g Lipase GH2 is appropriate for catalysis of FAEE production. 3.3.6. Reuse of Lipase GH2 Lipase GH2 was repeatedly reused to evaluate the stability of the catalysis system (Fig. S2). When Lipase GH2 was used for FAME production, the conversion rate was still 90% after 5 batches (Fig. S2A). In contrast, for FAEE production, the conversion rate declined from 95% in the first batch to 42% in the second batch (Fig. S2B), presumably because of the damaging effect of ethanol on the enzyme. Optimal conditions for Lipase GH2-catalyzed production of FAME vs. FAEE from microalgae oil are compared in Table 1. The strong catalytic ability of Lipase GH2 for FAME and FAEE synthesis (conversion rate P 90%) is also demonstrated by TLC results (Fig. 5). Significant differences in effects on FAME and FAEE production were observed for each of the experimental conditions tested: reaction temperature, water content, alcohol/oil molar

ratio, alcohol additional strategy, and enzyme content. There are two major explanations for these differences: (1) Lipase GH2 has different tolerance of methanol vs. ethanol. Ethanol had a substantially greater inhibitory effect on the enzyme activity than did methanol. In contrast, Nelson et al. (1996) reported that methanol had a greater inhibitory effect on R. miehei lipase (lipozyme IM60, a commercial immobilized lipase produced by Novozymes, Denmark), possibly catalyzed by immobilized enzyme and free enzyme is different. In fact, different sources of enzyme can also influence the catalytic properties of the enzyme. For example, Meng et al. (2011) found the more tolerance in methanol of Y. lipolytica lipase than ethanol which is differs from that of Watanabe et al. (2007) who reported that the inhibitory effect of methanol on immobilized Candida antarctica lipase was much higher than that of ethanol, which might be due to the difference in lipase source (Meng et al., 2011; Watanabe et al., 2007). Therefore, the catalytic reaction system of enzyme and substrate needs reasonable matching. (2) The one-carbon difference between methanol (CH3OH) and ethanol (CH3CH2OH), in terms of enzyme substrate preference, may lead to differences in catalytic ability of the two short-chain alcohol donors. When using Lipase GH2 as catalyst, the process of FAME production is generally more simple and economical than that of FAEE production, even though the two processes show similar conversion rates. Lipase GH2 appears to have a great advantage in production of biodiesel (FAME and FAEE) from microalgae oil. Microalgae oil consists mainly of FFAs, TAG, DAGs, and MAG, with FFAs as the major component. Lipase GH2 functions as a direct catalyst in the esterification and transesterification reaction for 1-step synthesis of FAME or FAEE. In spite of the damaging effect of ethanol on enzyme activity, we successfully obtained ethyl ester by the enzymatic method. The reaction conditions for FAEE and recovery of target product from the reaction system clearly need to be investigated further. 4. Conclusion This is the first investigation of differences in Lipase GH2-catalyzed production of FAME vs. FAEE, two types of biodiesel. Microalgae oil was extracted from Chlorella vulgaris, an excellent feedstock for biodiesel production. Optimal conditions were determined for production of FAME and FAEE, both of which showed high conversion rates in an n-hexane system. Lipase GH2 shows great potential as an efficient catalyst for production of biodiesel (FAME or FAEE) from microalgae oil. Acknowledgements This study was supported by the Chinese High Technology Research and Development Program (Grant No. 2013AA065802), Chinese Scientific and Technical Supporting Programs (Grant No. 2013BAD10B01), and the Undergraduate Innovation Program of China Agricultural University (Grant No. J1103520). The authors are grateful to Dr. S. Anderson for English editing of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 12.072. References Ahmad, A., Yasin, N., Derek, C., Lim, J., 2011. Microalgae as a sustainable energy source for biodiesel production: a review. Renew. Sustain. Energy Rev. 15, 584– 593.

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