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Enzymatic production of microalgal biodiesel in ionic liquid [BMIm][PF6]
Fuel 95 (2012) 329–333
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Enzymatic production of microalgal biodiesel in ionic liquid [BMIm][PF6] Jing-Qi Lai, Zhang-Li Hu, Peng-Wei Wang, Zhen Yang ⇑ College of Life Sciences, Shenzhen University, Shenzhen, Guangdong 518060, China
a r t i c l e
i n f o
Article history: Received 17 July 2011 Received in revised form 1 November 2011 Accepted 1 November 2011 Available online 15 November 2011 Keywords: Lipase Ionic liquid Biodiesel Microalgae
a b s t r a c t Microalgae have been taken as a sustainable energy source for biodiesel production. In this work, oils were extracted from microalgae Botryococcus braunii (two strains, BB763 and BB764), Chlorella vulgaris (CV), and Chlorella pyrenoidosa (CP). The highest lipid content was produced from CV (40.7%, w/w), whereas the lowest from CP (2.2%, w/w). The major fatty acid components of the microalgal oil from CP include myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3), and their mass proportion is 1:3.9:1.9:4.2:4.3:13.8. Enzymatic production of biodiesel from the microalgal oil was investigated, catalyzed by two immobilized lipases, Penicillium expansum lipase (PEL) and Candida antarctica lipase B (Novozym 435), in two solvent systems: an ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate, [BMIm][PF6]) and an organic solvent (tert-butanol). The effect of the following factors on the production yield was studied for all the four reaction systems: methanol/oil molar ratio, reaction temperature, solvent volume, and water content. Under optimal conditions, both enzymes induced significantly higher yields in the IL (90.7% and 86.2%) relative to that obtained in tert-butanol (48.6% and 44.4%), while the PEL-catalyzed conversions were comparable to or slightly higher than those catalyzed by Novozym 435. These results demonstrate that ionic liquids offer a promising new type of solvent for enzymatic production of microalgal biodiesel and that PEL can be employed as an efficient catalyst for such application. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel, a common term for long chain fatty acid methyl esters (FAMEs), has been generally regarded as a renewable, biodegradable, and non-toxic biofuel that shows great promise of substituting fossil diesel. As compared to the previous two generations of biodiesel feedstocks (i.e., food and non-food crops), the third generation, microalgae, has appeared to be a sustainable energy source for biodiesel production [1–5]. Advantages of utilizing microalgae as the biodiesel feedstock include [1,2,6]: easy cultivation and fast reproduction, high oil content, no competition for water and land resources, and being eco-friendly by reducing eutrophication in the aquatic environment while using less fertilizers. However, much of the published work related to the development of microalgal biodiesel has been focused on the research regarding the algae cultivation in order to increase the lipid productivity, whereas there have been very few studies concerning the conversion of microalgal oils to biodiesel, either chemically or enzymatically. Although lipase-catalyzed transesterification with methanol (MeOH) for biodiesel production has been proven to be more environmentally friendly as compared to the chemical ⇑ Corresponding author. Tel.: +86 755 2653 4152; fax: +86 755 2653 4277. E-mail address: [email protected] (Z. Yang). 0016-2361/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fuel.2011.11.001
approaches requiring either acid or alkali catalysts [7], so far there has been only one group that has reported research work about enzymatic production of biodiesel from microalgal oils: Wu and colleagues have produced biodiesel by heterotrophic fermentation of the green microalga Chlorella protothecoides and subsequent transesterification catalyzed by immobilized lipase from Candida sp. 99–125 [8,9]. The advantageous enzymatic process has been hampered by the lack of suitable solvents and efficient enzymes. The lipase most commonly used in biodiesel production is Novozym 435, an immobilized lipase B from Candida antarctica (CALB). It is too expensive for wide application at industrial scales. On the other hand, enzymatic production of biodiesel has been mainly conducted in solvent-free or organic solvent systems, which have suffered from drawbacks such as the use of contaminating organic solvents, low productivity, difficult product separation, and loss of enzyme activity due to the methanolinduced inactivation and accumulation of glycerol (a by-product) around the enzyme. Alternative for conventional volatile organic solvents, ionic liquids (ILs) have been experiencing a rapid growth as a promising new type of reaction medium for biocatalytic processes due to their attractive features such as low volatility and tunable solvent properties and their ability of presenting excellent enzymatic performance [10,11]. Unfortunately, however, their use in enzymatic biodiesel production is very limited [12–22]. Indeed, all these examples have shown the potential of using ILs as the
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reaction medium for biodiesel production. But it has to be stressed that enzymatic conversion of microalgal oils to biodiesel has never been carried out in ILs and that Novozym 435 was always used as the catalyst for all these applications. In order to provide solutions to the issues addressed above, we have recently focused on investigating the catalytic performance of Penicillium expansum lipase (PEL) in ionic liquids, in hope of introducing a less expensive yet efficient lipase and a new solvent type for producing microalgal biodiesel. Unlike the commonly used and widely investigated lipases (such as CALB), PEL has received only limited attention from researchers in terms of its potential applications in biocatalytic processes. Previously, this enzyme has been utilized in catalyzing biodiesel production from corn oil [23] and waste oil [24] in organic media. Our recent study [21] has demonstrated that this enzyme offers a significantly enhanced activity (in both hydrolysis and transesterification) in the ionic liquid [BMIm][PF6] than in organic solvents such as hexane and tert-butanol. More importantly, the IL [BMIm][PF6] provides a promising reaction medium for lipase-catalyzed biodiesel production from corn oil, with a high conversion obtained in this IL (86%) superior to that obtained in tert-butanol (52%), an organic solvent most commonly used for biodiesel production, and in the solvent-free system (14%) [21,22]. PEL has also shown to be highly tolerant to methanol in these reaction media [22]. To extend our interest in enzymatic biodiesel production, here we report for the first time the enzymatic conversion of microalgal oils to biodiesel in ionic liquids. The biodiesel productivity was investigated, with both PEL and Novozym 435 as the catalyst in two different solvents: the ionic liquid [BMIm][PF6] vs. the organic solvent tert-butanol. For all these four reaction systems, the effect of the following variables was examined in order to achieve a maximal conversion yield: MeOH/oil molar ratio, reaction temperature, water content, and solvent volume. The oil contents of four microalgae (Chlorella pyrenoidosa (CP), Chlorella vulgaris (CV), and two strains of Botryococcus braunii (BB763 and BB764)) have been compared, but the algal oil extracted from CP was employed as the substrate for this study simply because its powders can be purchased to provide sufficient algal oil for our investigation on biodiesel production. 2. Materials and methods 2.1. Materials The algae powder of C. pyrenoidosa (CP) was purchased from Luyuan Biotechnology Inc., Shenzhen, China. C. vulgaris (CV) and two strains of B. Braunii (BB763 and BB764) were provided by the Institute of Eco-Environmental Sciences at Shenzhen University. Lipase from P. expansum (PEL, 10,000 U/g, determined by hydrolysis of olive oil in 50 mM, pH 9.4 glycine/NaOH buffer at 36 °C) was kindly donated by Shenzhen Leveking Bioengineering Co. Ltd., China. This enzyme was produced by spraying the concentrated supernatant from fermentation with addition of a certain amount of starch as a thickening agent. Novozym 435 (C. antarctica lipase B immobilized on acrylic resins) was purchased from Novozymes (China) Investment Co. Ltd. Fatty acid methyl esters (FAMEs) used for GC characterization were of chromatographic purity from Sigma–Aldrich China Inc. The ionic liquid [BMIm][PF6] (99%) was obtained from ShangHai Cheng Jie Chemical Co. Ltd. All other reagents used were of analytical grade from local manufacturers in China. 2.2. Lipid extraction from microalgae Lipids from the algae powders were extracted by Soxhlet extraction, using hexane as the extraction solvent and lasting for
8 h. After extraction, the solvent was removed by means of rotary evaporation, and the water present in the algal oil sample was further removed by heating in an oven at 105 °C until the sample reached a constant weight. The crude oil content for each microalga was then estimated as the percentage ratio of this oil weight to the weight of the dry algal powder used for extraction. The resulting oils were transparent and looked dark green. 2.3. Characterization of the microalgal lipids The saponification value of the lipid sample was determined based on its hydrolysis reaction with KOH, in the unit of mg of KOH required to saponify 1 g of the lipid sample. The average molecular weight was estimated from the saponification value using the formula: MW = 168,300/SV, where MW is the molecular weight and SV is the saponification value. For determination of the acid value (defined as the mg of KOH required to neutralize the free fatty acid released from 1 g of the lipid sample), the lipid sample was prepared by dissolving 1.0 g of the microalgal oil in 95% ethanol/ethyl ether solution (1:2, v/v, 30 mL), followed by addition of saturated NaCl aqueous solution (30 mL). The solution was then titrated with 0.1 N KOH. The fatty acid composition of the oil extracted from C. pyrenoidosa (CP) was estimated by thorough methanolysis of the oil with 0.5 M NaOH in methanol and subsequent analysis of the products with a Trace2000 gas chromatography (Thermal Co., Germany) equipped with a SUPELCOWAX 10 column (30 m 0.32 mm 0.25 lm, Agilent Technologies, USA) [21]. 2.4. Biodiesel production The oil extracted from C. pyrenoidosa (CP) was employed as the substrate. The general procedures of the enzymatic reactions, both in the ionic liquid [BMIm][PF6] and in the organic solvent tert-butanol, and GC analysis of the biodiesel products have been described in [21]. In both reaction systems, the following variables were studied in order to determine the operation conditions that could maximize the yield of biodiesel: MeOH/oil molar ratio, reaction temperature, solvent volume, and water content. Unless otherwise stated, the following reaction conditions were used for converting 0.5 g of microalgal oil: 72 lL of MeOH, 1 mL of [BMIm][PF6] or 0.5 mL of tert-butanol, 100 mg of the PEL or 50 mg of Novozym 435, conducted at 40 °C for 48 h with agitation of 220 rpm. Both solvents were dried over molecular sieves prior to use. Errors associated with the FAME yields were all within the range of ±10%. 3. Results and discussion 3.1. Characterization of the microalgal oil The powders of C. pyrenoidosa (CP) contained a crude lipid content of 2.2% (w/w), estimated after being extracted with hexane (Section 2.2); and the lipid sample possessed a saponification value of 200.3, an acid value of 2.52, and a molecular weight of 840.3, as have been determined by following the methods described in Section 2.3. Microalgae are known to exhibit a great variability in lipid content, ranging between 15% and 80% [3,5]. The variations depend on different species, growing conditions, and extraction methods used [4], which has also been verified in our study. Extraction with hexane caused a higher lipid content than that with petroleum ether (data not shown). In our study, B. Braunii (2 strains, BB763 and BB764), C. vulgaris (CV), and C. pyrenoidosa (CP) have been selected for comparison of their oil contents, with the highest level produced from CV (40.7%, w/w). Chlorella and Botryococcus have been
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usually employed as candidate feedstocks for biodiesel production simply because they can contribute a high lipid content [3,5]. These microalgae are phototrophic, and their cultivations take place by using sunlight as energy source and atmospheric CO2 as carbon source, following photosynthesis. To serve as the biodiesel feedstock, these autotrophic microalgae should therefore be more advantageous than the heterotrophic ones which have to consume a significant amount of glucose during cultivation [8,9]. Although with a disappointingly low lipid level (2.2%), C. pyrenoidosa (CP) was employed as the oil contributor in this study simply because of its sufficient supply. Fig. 1 shows the GC diagram of the methanolysis products of the oil extracted from the CP biomass. By comparing to the standard fatty acid methyl esters, six peaks can be identified as derived from myristic acid (C14:0, peak A), palmitic acid (C16:0, peak B), stearic acid (C18:0, peak D), oleic acid (C18:1, peak E), linoleic acid (C18:2, peak F), and linolenic acid (C18:3, peak G). These are also the fatty acids that fresh water microalga species can commonly synthesize [5]. The mass proportion of these fatty acids is 1:3.9:1.9:4.2:4.3:13.8, as can be determined by GC analysis. This clearly confirms that microalgal oils differ from most vegetable oils in being rich in polyunsaturated fatty acids [6,8]. In spite of this, Li et al. [8] have already demonstrated that the properties of biodiesel from Chlorella were comparable to conventional diesel fuel and complied with the US Standard for biodiesel. Therefore, it is feasible to add microalgal oils into the list of choices for biodiesel feedstocks. 3.2. Lipase-catalyzed biodiesel production from microalgal oil In view of our previous finding that the ionic liquid [BMIm][PF6], as compared to the commonly used organic solvent tert-butanol, offers a superior solvent for PEL-catalyzed biodiesel production from corn oil [21,22], it is necessary to evaluate the performance of these two solvent systems for converting microalgal oils to biodiesel, catalyzed by PEL in comparison with Novozym 435. Fatty acid methyl esters can indeed be produced from the microalgal oils in these two solvent systems. Reactions were carried out within 48 h in order to assure of the thorough conversions. The following factors were examined in both solvent systems, catalyzed by the two lipases (Fig. 2): MeOH/oil molar ratio, reaction
Fig. 1. GC diagram of the methanolysis products of the oil extracted from Chlorella pyrenoidosa (CP). Seven peaks are identified as: (A) myristic acid methyl ester, C14:0; (B) palmitic acid methyl ester, C16:0; (C) heptadecanoic acid methyl ester, C17:0, as the internal standard for GC analysis; (D) stearic acid methyl ester, C18:0; (E) oleic acid methyl ester, C18:1; (F) linoleic acid methyl ester, C18:2; and (G) linolenic acid methyl ester, C18:3.
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temperature, water content, and solvent volume. Obviously, the conversion yield depends on these four variables. In particular, methanol is a co-substrate to the enzymatic process but too much of it would cause enzyme inactivation, because methanol is wellknown as a strong denaturant to proteins. The highest conversion was obtained by PEL and Novozym 435 in both solvent systems when the molar ratio of methanol to oil was set at a stoichiometric quantity of 3:1 and 4:1, respectively. The optimal reaction temperatures for both enzymes were also determined to be 50 °C in the IL and 40 °C in tert-butanol. Based on the results obtained in Fig. 2, the optimal reaction conditions were set up and summarized in Table 1. Under these conditions, the FAME yields obtained in the IL can reach 90.7% and 86.2% when catalyzed by PEL and Novozym 435, respectively, while the corresponding conversions obtained in tert-butanol were respectively 86.2% and 44.4% (Fig. 3). Obviously, PEL elicited a remarkable yield for biodiesel production in the IL, comparable to or slightly higher than the result obtained by Novozym 435, and thus offers an excellent enzyme alternative for efficient transformation of microalgal oils to biodiesel. For both enzymes tested, the conversion obtained in [BMIm][PF6] was always much higher than the one obtained in tert-butanol, substantiating that the ionic liquid is a superior solvent for lipase-catalyzed biodiesel production from microalgal oils. Our previous work has already verified that the use of [BMIm][PF6] rather than tert-butanol as the reaction medium yielded a significantly enhanced conversion of corn oil to biodiesel [21,22]. However, from other researchers there have been very limited reports so far on carrying out biodiesel production in ILs [12–20]. Among the 23 different ILs for screening in [14], only [EMIm][TfO] (1ethyl-3-methylimidazolium trifluoromethylsulfonate) exhibited a slightly increased conversion as compared to the one obtained in tert-butanol. All other ILs tested in these references either showed a much lower production yield than the conventional organic solvents such as tert-butanol [14] or isopropanol [20], or have not been involved in such comparison. It has to be noted that although with limited published work on using ILs as the advanced reaction medium for biodiesel production, a trend of switching from random screening and selection of ILs to their rational design seems to be obvious recently. For instance, Zhao’s group has been active in designing ILs which are compatible with biodiesel production: ether-functionalized ILs [14] and eutectic ILs [15]. The Spanish group [16–19] has done excellent work in developing a highly efficient, homogeneous and recyclable enzymatic process for biodiesel production by designing hydrophobic immidazolium ILs with large alkyl side chains (e.g., 1-methyl-3-octadecylimidazolium bis(trifluoromethylsulfonyl)imide), [C18MIm][NTf2]), so that both the enzyme, the solvent, and the products were able to be recycled. In support for this, de los Ríos et al. [20] have tested biocatalytic transesterification of sunflower and waste cooking oils in 10 different ILs based on imidazolium and pyridinium cations, and have concluded that the enzyme activity was enhanced by increasing cation hydrophobicity and decreasing anion nucleophilicity. Indeed, designing biocompatible ILs for biocatalytic processes such as biodiesel production is challenging, and more work has to be done in order for us to have a better understanding about the various impacts of ILs on the enzyme performance, so that general rules can be worked out to guide our job of designing ILs for each specific biocatalytic transformation [25]. Although we have to admit that at the moment the high cost of ILs is still a big obstacle hindering their industrial applications, the use of these nonconventional solvents as the reaction medium has shown a great advantage in improving the enzymatic processes, and the advanced research on rational design of less expensive yet more efficient ILs will bring a bright future for the development of enzymatic production of biodiesel.
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80
FAME yield (%)
100
Novozym 435, [BMIm][PF6] Novozym 435, tert-butanol PEL, [BMIm][PF6] PEL, tert-butanol
A
Novozym 435, [BMIm][PF6] Novozym 435, tert-butanol PEL, [BMIm][PF6] PEL, tert-butanol
B 80
FAME yield (%)
100
60 40 20
60
40
20
0
0
1:2
1:1
2:1
3:1
4:1
5:1
30
35
MeOH/oil molar ratio
100
C
Novozym 435, [BMIm][PF6] PEL, [BMIm][PF6]
Novozym 435, tert-butanol PEL, tert-butanol
45
50
o
55
60
100
D
Novozym 435, [BMIm][PF6] PEL, [BMIm][PF6]
Novozym 435, tert-butanol PEL, tert-butanol
80
FAME yield (%)
80
FAME yield (%)
40
Reaction temperature ( C)
60 40
60 40 20
20 0
0 0
1
2
3
4
5
1
H2O% (w/w, based on oil weight)
2
3
4
5
6
Solvent (mL/g, based on oil weight)
Fig. 2. Effect of the following factors on production of microalgal biodiesel: (A) MeOH/oil molar ratio; (B) reaction temperature; (C) water content; (D) solvent volume. Please refer to Section 2.4 for experimental details. Table 1 Optimal conditions used for biodiesel production from microalgal oil (extracted from the CP biomass) in both tert-butanol and [BMIm][PF6], catalyzed by PEL and Novozym 435, respectively. Lipase
PEL
Novozym 435
Solvent
tertButanol
[BMIm][PF6]
tertButanol
[BMIm][PF6]
Microalgal oil Reaction time Enzyme dosage Solvent volume MeOH/oil molar ratio H2O addition Reaction temperature
0.5 g 48 h 100 mg 0.5 mL 3:1
0.5 g 48 h 100 mg 1 mL 3:1
0.5 g 48 h 50 mg 0.5 mL 4:1
0.5 g 48 h 50 mg 1 mL 4:1
15 lL 40 °C
15 lL 50 °C
0 lL 40 °C
5 lL 50 °C
4. Conclusions Herein we have reported the first investigation on enzymatic production of microalgal biodiesel. Oils extracted from microalgae (e.g., from Chlorella) can be taken as an excellent feedstock for biodiesel production. The ionic liquid [BMIm][PF6] offers a promising solvent for this enzymatic transformation, with a greater conversion yield obtained relative to the one obtained in the commonly used organic solvent tert-butanol. On the other hand, the lipase from P. expansum has also shown its potential as an efficient catalyst for the production of microalgal biodiesel in place of Novozym 435, the most commonly used commercialized enzyme for this application. Acknowledgment This work was financially supported by the Foundation of the Science and Technology Program of Shenzhen.
100
FAME yield (%)
80
References 60
40 PEL, [BMIm][PF6] PEL, tert-butanol
20
Novozym 435, [BMIm][PF6] Novozym 435, tert-butanol
0 0
10
20
30
40
50
60
Reaction time (hr) Fig. 3. The time course of the biodiesel production from microalgal oil in both [BMIm][PF6] and tert-butanol, catalyzed by PEL and Novozym 435, respectively, under optimal conditions (see Table 1).
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Fuel journal homepage: www.elsevier.com/locate/fuel
Enzymatic production of microalgal biodiesel in ionic liquid [BMIm][PF6] Jing-Qi Lai, Zhang-Li Hu, Peng-Wei Wang, Zhen Yang ⇑ College of Life Sciences, Shenzhen University, Shenzhen, Guangdong 518060, China
a r t i c l e
i n f o
Article history: Received 17 July 2011 Received in revised form 1 November 2011 Accepted 1 November 2011 Available online 15 November 2011 Keywords: Lipase Ionic liquid Biodiesel Microalgae
a b s t r a c t Microalgae have been taken as a sustainable energy source for biodiesel production. In this work, oils were extracted from microalgae Botryococcus braunii (two strains, BB763 and BB764), Chlorella vulgaris (CV), and Chlorella pyrenoidosa (CP). The highest lipid content was produced from CV (40.7%, w/w), whereas the lowest from CP (2.2%, w/w). The major fatty acid components of the microalgal oil from CP include myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3), and their mass proportion is 1:3.9:1.9:4.2:4.3:13.8. Enzymatic production of biodiesel from the microalgal oil was investigated, catalyzed by two immobilized lipases, Penicillium expansum lipase (PEL) and Candida antarctica lipase B (Novozym 435), in two solvent systems: an ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate, [BMIm][PF6]) and an organic solvent (tert-butanol). The effect of the following factors on the production yield was studied for all the four reaction systems: methanol/oil molar ratio, reaction temperature, solvent volume, and water content. Under optimal conditions, both enzymes induced significantly higher yields in the IL (90.7% and 86.2%) relative to that obtained in tert-butanol (48.6% and 44.4%), while the PEL-catalyzed conversions were comparable to or slightly higher than those catalyzed by Novozym 435. These results demonstrate that ionic liquids offer a promising new type of solvent for enzymatic production of microalgal biodiesel and that PEL can be employed as an efficient catalyst for such application. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel, a common term for long chain fatty acid methyl esters (FAMEs), has been generally regarded as a renewable, biodegradable, and non-toxic biofuel that shows great promise of substituting fossil diesel. As compared to the previous two generations of biodiesel feedstocks (i.e., food and non-food crops), the third generation, microalgae, has appeared to be a sustainable energy source for biodiesel production [1–5]. Advantages of utilizing microalgae as the biodiesel feedstock include [1,2,6]: easy cultivation and fast reproduction, high oil content, no competition for water and land resources, and being eco-friendly by reducing eutrophication in the aquatic environment while using less fertilizers. However, much of the published work related to the development of microalgal biodiesel has been focused on the research regarding the algae cultivation in order to increase the lipid productivity, whereas there have been very few studies concerning the conversion of microalgal oils to biodiesel, either chemically or enzymatically. Although lipase-catalyzed transesterification with methanol (MeOH) for biodiesel production has been proven to be more environmentally friendly as compared to the chemical ⇑ Corresponding author. Tel.: +86 755 2653 4152; fax: +86 755 2653 4277. E-mail address: [email protected] (Z. Yang). 0016-2361/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fuel.2011.11.001
approaches requiring either acid or alkali catalysts [7], so far there has been only one group that has reported research work about enzymatic production of biodiesel from microalgal oils: Wu and colleagues have produced biodiesel by heterotrophic fermentation of the green microalga Chlorella protothecoides and subsequent transesterification catalyzed by immobilized lipase from Candida sp. 99–125 [8,9]. The advantageous enzymatic process has been hampered by the lack of suitable solvents and efficient enzymes. The lipase most commonly used in biodiesel production is Novozym 435, an immobilized lipase B from Candida antarctica (CALB). It is too expensive for wide application at industrial scales. On the other hand, enzymatic production of biodiesel has been mainly conducted in solvent-free or organic solvent systems, which have suffered from drawbacks such as the use of contaminating organic solvents, low productivity, difficult product separation, and loss of enzyme activity due to the methanolinduced inactivation and accumulation of glycerol (a by-product) around the enzyme. Alternative for conventional volatile organic solvents, ionic liquids (ILs) have been experiencing a rapid growth as a promising new type of reaction medium for biocatalytic processes due to their attractive features such as low volatility and tunable solvent properties and their ability of presenting excellent enzymatic performance [10,11]. Unfortunately, however, their use in enzymatic biodiesel production is very limited [12–22]. Indeed, all these examples have shown the potential of using ILs as the
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reaction medium for biodiesel production. But it has to be stressed that enzymatic conversion of microalgal oils to biodiesel has never been carried out in ILs and that Novozym 435 was always used as the catalyst for all these applications. In order to provide solutions to the issues addressed above, we have recently focused on investigating the catalytic performance of Penicillium expansum lipase (PEL) in ionic liquids, in hope of introducing a less expensive yet efficient lipase and a new solvent type for producing microalgal biodiesel. Unlike the commonly used and widely investigated lipases (such as CALB), PEL has received only limited attention from researchers in terms of its potential applications in biocatalytic processes. Previously, this enzyme has been utilized in catalyzing biodiesel production from corn oil [23] and waste oil [24] in organic media. Our recent study [21] has demonstrated that this enzyme offers a significantly enhanced activity (in both hydrolysis and transesterification) in the ionic liquid [BMIm][PF6] than in organic solvents such as hexane and tert-butanol. More importantly, the IL [BMIm][PF6] provides a promising reaction medium for lipase-catalyzed biodiesel production from corn oil, with a high conversion obtained in this IL (86%) superior to that obtained in tert-butanol (52%), an organic solvent most commonly used for biodiesel production, and in the solvent-free system (14%) [21,22]. PEL has also shown to be highly tolerant to methanol in these reaction media [22]. To extend our interest in enzymatic biodiesel production, here we report for the first time the enzymatic conversion of microalgal oils to biodiesel in ionic liquids. The biodiesel productivity was investigated, with both PEL and Novozym 435 as the catalyst in two different solvents: the ionic liquid [BMIm][PF6] vs. the organic solvent tert-butanol. For all these four reaction systems, the effect of the following variables was examined in order to achieve a maximal conversion yield: MeOH/oil molar ratio, reaction temperature, water content, and solvent volume. The oil contents of four microalgae (Chlorella pyrenoidosa (CP), Chlorella vulgaris (CV), and two strains of Botryococcus braunii (BB763 and BB764)) have been compared, but the algal oil extracted from CP was employed as the substrate for this study simply because its powders can be purchased to provide sufficient algal oil for our investigation on biodiesel production. 2. Materials and methods 2.1. Materials The algae powder of C. pyrenoidosa (CP) was purchased from Luyuan Biotechnology Inc., Shenzhen, China. C. vulgaris (CV) and two strains of B. Braunii (BB763 and BB764) were provided by the Institute of Eco-Environmental Sciences at Shenzhen University. Lipase from P. expansum (PEL, 10,000 U/g, determined by hydrolysis of olive oil in 50 mM, pH 9.4 glycine/NaOH buffer at 36 °C) was kindly donated by Shenzhen Leveking Bioengineering Co. Ltd., China. This enzyme was produced by spraying the concentrated supernatant from fermentation with addition of a certain amount of starch as a thickening agent. Novozym 435 (C. antarctica lipase B immobilized on acrylic resins) was purchased from Novozymes (China) Investment Co. Ltd. Fatty acid methyl esters (FAMEs) used for GC characterization were of chromatographic purity from Sigma–Aldrich China Inc. The ionic liquid [BMIm][PF6] (99%) was obtained from ShangHai Cheng Jie Chemical Co. Ltd. All other reagents used were of analytical grade from local manufacturers in China. 2.2. Lipid extraction from microalgae Lipids from the algae powders were extracted by Soxhlet extraction, using hexane as the extraction solvent and lasting for
8 h. After extraction, the solvent was removed by means of rotary evaporation, and the water present in the algal oil sample was further removed by heating in an oven at 105 °C until the sample reached a constant weight. The crude oil content for each microalga was then estimated as the percentage ratio of this oil weight to the weight of the dry algal powder used for extraction. The resulting oils were transparent and looked dark green. 2.3. Characterization of the microalgal lipids The saponification value of the lipid sample was determined based on its hydrolysis reaction with KOH, in the unit of mg of KOH required to saponify 1 g of the lipid sample. The average molecular weight was estimated from the saponification value using the formula: MW = 168,300/SV, where MW is the molecular weight and SV is the saponification value. For determination of the acid value (defined as the mg of KOH required to neutralize the free fatty acid released from 1 g of the lipid sample), the lipid sample was prepared by dissolving 1.0 g of the microalgal oil in 95% ethanol/ethyl ether solution (1:2, v/v, 30 mL), followed by addition of saturated NaCl aqueous solution (30 mL). The solution was then titrated with 0.1 N KOH. The fatty acid composition of the oil extracted from C. pyrenoidosa (CP) was estimated by thorough methanolysis of the oil with 0.5 M NaOH in methanol and subsequent analysis of the products with a Trace2000 gas chromatography (Thermal Co., Germany) equipped with a SUPELCOWAX 10 column (30 m 0.32 mm 0.25 lm, Agilent Technologies, USA) [21]. 2.4. Biodiesel production The oil extracted from C. pyrenoidosa (CP) was employed as the substrate. The general procedures of the enzymatic reactions, both in the ionic liquid [BMIm][PF6] and in the organic solvent tert-butanol, and GC analysis of the biodiesel products have been described in [21]. In both reaction systems, the following variables were studied in order to determine the operation conditions that could maximize the yield of biodiesel: MeOH/oil molar ratio, reaction temperature, solvent volume, and water content. Unless otherwise stated, the following reaction conditions were used for converting 0.5 g of microalgal oil: 72 lL of MeOH, 1 mL of [BMIm][PF6] or 0.5 mL of tert-butanol, 100 mg of the PEL or 50 mg of Novozym 435, conducted at 40 °C for 48 h with agitation of 220 rpm. Both solvents were dried over molecular sieves prior to use. Errors associated with the FAME yields were all within the range of ±10%. 3. Results and discussion 3.1. Characterization of the microalgal oil The powders of C. pyrenoidosa (CP) contained a crude lipid content of 2.2% (w/w), estimated after being extracted with hexane (Section 2.2); and the lipid sample possessed a saponification value of 200.3, an acid value of 2.52, and a molecular weight of 840.3, as have been determined by following the methods described in Section 2.3. Microalgae are known to exhibit a great variability in lipid content, ranging between 15% and 80% [3,5]. The variations depend on different species, growing conditions, and extraction methods used [4], which has also been verified in our study. Extraction with hexane caused a higher lipid content than that with petroleum ether (data not shown). In our study, B. Braunii (2 strains, BB763 and BB764), C. vulgaris (CV), and C. pyrenoidosa (CP) have been selected for comparison of their oil contents, with the highest level produced from CV (40.7%, w/w). Chlorella and Botryococcus have been
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usually employed as candidate feedstocks for biodiesel production simply because they can contribute a high lipid content [3,5]. These microalgae are phototrophic, and their cultivations take place by using sunlight as energy source and atmospheric CO2 as carbon source, following photosynthesis. To serve as the biodiesel feedstock, these autotrophic microalgae should therefore be more advantageous than the heterotrophic ones which have to consume a significant amount of glucose during cultivation [8,9]. Although with a disappointingly low lipid level (2.2%), C. pyrenoidosa (CP) was employed as the oil contributor in this study simply because of its sufficient supply. Fig. 1 shows the GC diagram of the methanolysis products of the oil extracted from the CP biomass. By comparing to the standard fatty acid methyl esters, six peaks can be identified as derived from myristic acid (C14:0, peak A), palmitic acid (C16:0, peak B), stearic acid (C18:0, peak D), oleic acid (C18:1, peak E), linoleic acid (C18:2, peak F), and linolenic acid (C18:3, peak G). These are also the fatty acids that fresh water microalga species can commonly synthesize [5]. The mass proportion of these fatty acids is 1:3.9:1.9:4.2:4.3:13.8, as can be determined by GC analysis. This clearly confirms that microalgal oils differ from most vegetable oils in being rich in polyunsaturated fatty acids [6,8]. In spite of this, Li et al. [8] have already demonstrated that the properties of biodiesel from Chlorella were comparable to conventional diesel fuel and complied with the US Standard for biodiesel. Therefore, it is feasible to add microalgal oils into the list of choices for biodiesel feedstocks. 3.2. Lipase-catalyzed biodiesel production from microalgal oil In view of our previous finding that the ionic liquid [BMIm][PF6], as compared to the commonly used organic solvent tert-butanol, offers a superior solvent for PEL-catalyzed biodiesel production from corn oil [21,22], it is necessary to evaluate the performance of these two solvent systems for converting microalgal oils to biodiesel, catalyzed by PEL in comparison with Novozym 435. Fatty acid methyl esters can indeed be produced from the microalgal oils in these two solvent systems. Reactions were carried out within 48 h in order to assure of the thorough conversions. The following factors were examined in both solvent systems, catalyzed by the two lipases (Fig. 2): MeOH/oil molar ratio, reaction
Fig. 1. GC diagram of the methanolysis products of the oil extracted from Chlorella pyrenoidosa (CP). Seven peaks are identified as: (A) myristic acid methyl ester, C14:0; (B) palmitic acid methyl ester, C16:0; (C) heptadecanoic acid methyl ester, C17:0, as the internal standard for GC analysis; (D) stearic acid methyl ester, C18:0; (E) oleic acid methyl ester, C18:1; (F) linoleic acid methyl ester, C18:2; and (G) linolenic acid methyl ester, C18:3.
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temperature, water content, and solvent volume. Obviously, the conversion yield depends on these four variables. In particular, methanol is a co-substrate to the enzymatic process but too much of it would cause enzyme inactivation, because methanol is wellknown as a strong denaturant to proteins. The highest conversion was obtained by PEL and Novozym 435 in both solvent systems when the molar ratio of methanol to oil was set at a stoichiometric quantity of 3:1 and 4:1, respectively. The optimal reaction temperatures for both enzymes were also determined to be 50 °C in the IL and 40 °C in tert-butanol. Based on the results obtained in Fig. 2, the optimal reaction conditions were set up and summarized in Table 1. Under these conditions, the FAME yields obtained in the IL can reach 90.7% and 86.2% when catalyzed by PEL and Novozym 435, respectively, while the corresponding conversions obtained in tert-butanol were respectively 86.2% and 44.4% (Fig. 3). Obviously, PEL elicited a remarkable yield for biodiesel production in the IL, comparable to or slightly higher than the result obtained by Novozym 435, and thus offers an excellent enzyme alternative for efficient transformation of microalgal oils to biodiesel. For both enzymes tested, the conversion obtained in [BMIm][PF6] was always much higher than the one obtained in tert-butanol, substantiating that the ionic liquid is a superior solvent for lipase-catalyzed biodiesel production from microalgal oils. Our previous work has already verified that the use of [BMIm][PF6] rather than tert-butanol as the reaction medium yielded a significantly enhanced conversion of corn oil to biodiesel [21,22]. However, from other researchers there have been very limited reports so far on carrying out biodiesel production in ILs [12–20]. Among the 23 different ILs for screening in [14], only [EMIm][TfO] (1ethyl-3-methylimidazolium trifluoromethylsulfonate) exhibited a slightly increased conversion as compared to the one obtained in tert-butanol. All other ILs tested in these references either showed a much lower production yield than the conventional organic solvents such as tert-butanol [14] or isopropanol [20], or have not been involved in such comparison. It has to be noted that although with limited published work on using ILs as the advanced reaction medium for biodiesel production, a trend of switching from random screening and selection of ILs to their rational design seems to be obvious recently. For instance, Zhao’s group has been active in designing ILs which are compatible with biodiesel production: ether-functionalized ILs [14] and eutectic ILs [15]. The Spanish group [16–19] has done excellent work in developing a highly efficient, homogeneous and recyclable enzymatic process for biodiesel production by designing hydrophobic immidazolium ILs with large alkyl side chains (e.g., 1-methyl-3-octadecylimidazolium bis(trifluoromethylsulfonyl)imide), [C18MIm][NTf2]), so that both the enzyme, the solvent, and the products were able to be recycled. In support for this, de los Ríos et al. [20] have tested biocatalytic transesterification of sunflower and waste cooking oils in 10 different ILs based on imidazolium and pyridinium cations, and have concluded that the enzyme activity was enhanced by increasing cation hydrophobicity and decreasing anion nucleophilicity. Indeed, designing biocompatible ILs for biocatalytic processes such as biodiesel production is challenging, and more work has to be done in order for us to have a better understanding about the various impacts of ILs on the enzyme performance, so that general rules can be worked out to guide our job of designing ILs for each specific biocatalytic transformation [25]. Although we have to admit that at the moment the high cost of ILs is still a big obstacle hindering their industrial applications, the use of these nonconventional solvents as the reaction medium has shown a great advantage in improving the enzymatic processes, and the advanced research on rational design of less expensive yet more efficient ILs will bring a bright future for the development of enzymatic production of biodiesel.
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80
FAME yield (%)
100
Novozym 435, [BMIm][PF6] Novozym 435, tert-butanol PEL, [BMIm][PF6] PEL, tert-butanol
A
Novozym 435, [BMIm][PF6] Novozym 435, tert-butanol PEL, [BMIm][PF6] PEL, tert-butanol
B 80
FAME yield (%)
100
60 40 20
60
40
20
0
0
1:2
1:1
2:1
3:1
4:1
5:1
30
35
MeOH/oil molar ratio
100
C
Novozym 435, [BMIm][PF6] PEL, [BMIm][PF6]
Novozym 435, tert-butanol PEL, tert-butanol
45
50
o
55
60
100
D
Novozym 435, [BMIm][PF6] PEL, [BMIm][PF6]
Novozym 435, tert-butanol PEL, tert-butanol
80
FAME yield (%)
80
FAME yield (%)
40
Reaction temperature ( C)
60 40
60 40 20
20 0
0 0
1
2
3
4
5
1
H2O% (w/w, based on oil weight)
2
3
4
5
6
Solvent (mL/g, based on oil weight)
Fig. 2. Effect of the following factors on production of microalgal biodiesel: (A) MeOH/oil molar ratio; (B) reaction temperature; (C) water content; (D) solvent volume. Please refer to Section 2.4 for experimental details. Table 1 Optimal conditions used for biodiesel production from microalgal oil (extracted from the CP biomass) in both tert-butanol and [BMIm][PF6], catalyzed by PEL and Novozym 435, respectively. Lipase
PEL
Novozym 435
Solvent
tertButanol
[BMIm][PF6]
tertButanol
[BMIm][PF6]
Microalgal oil Reaction time Enzyme dosage Solvent volume MeOH/oil molar ratio H2O addition Reaction temperature
0.5 g 48 h 100 mg 0.5 mL 3:1
0.5 g 48 h 100 mg 1 mL 3:1
0.5 g 48 h 50 mg 0.5 mL 4:1
0.5 g 48 h 50 mg 1 mL 4:1
15 lL 40 °C
15 lL 50 °C
0 lL 40 °C
5 lL 50 °C
4. Conclusions Herein we have reported the first investigation on enzymatic production of microalgal biodiesel. Oils extracted from microalgae (e.g., from Chlorella) can be taken as an excellent feedstock for biodiesel production. The ionic liquid [BMIm][PF6] offers a promising solvent for this enzymatic transformation, with a greater conversion yield obtained relative to the one obtained in the commonly used organic solvent tert-butanol. On the other hand, the lipase from P. expansum has also shown its potential as an efficient catalyst for the production of microalgal biodiesel in place of Novozym 435, the most commonly used commercialized enzyme for this application. Acknowledgment This work was financially supported by the Foundation of the Science and Technology Program of Shenzhen.
100
FAME yield (%)
80
References 60
40 PEL, [BMIm][PF6] PEL, tert-butanol
20
Novozym 435, [BMIm][PF6] Novozym 435, tert-butanol
0 0
10
20
30
40
50
60
Reaction time (hr) Fig. 3. The time course of the biodiesel production from microalgal oil in both [BMIm][PF6] and tert-butanol, catalyzed by PEL and Novozym 435, respectively, under optimal conditions (see Table 1).
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