Bioresource Technology 187 (2015) 346–353

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Enzymatic production of biodiesel from Nannochloropsis gaditana lipids: Influence of operational variables and polar lipid content Elvira Navarro López, Alfonso Robles Medina ⇑, Pedro A. González Moreno, María J. Jiménez Callejón, Luis Esteban Cerdán, Lorena Martín Valverde, Beatriz Castillo López, Emilio Molina Grima Area of Chemical Engineering, University of Almería, 04120 Almería, Spain

h i g h l i g h t s  Saponifiable lipids (SLs) were extracted from wet microalgal biomass.  SLs were transformed to biodiesel by lipase catalyzed transesterification.  The importance of polar lipids and SL purity for biodiesel conversion is showed.  Influence of solvent and methanol/SL ratio on lipase stability is discussed.  94.7% of microalgal SLs were transformed to biodiesel in the optimized conditions.

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Article history: Received 13 January 2015 Received in revised form 25 March 2015 Accepted 26 March 2015 Available online 2 April 2015 Keywords: Biodiesel Microalga Lipase Transesterification Polar lipid

a b s t r a c t Fatty acid methyl esters (FAMEs, biodiesel) were produced from Nannochloropsis gaditana wet biomass (12% saponifiable lipids, SLs) by extraction of SLs and lipase catalyzed transesterification. Lipids were extracted by ethanol (96%)–hexane, and 31% pure SLs were obtained with 85% yield. When the lipids were degummed, SL purity increased to 95%. Novozym 435 was selected from four lipases tested. Both the lipidic composition and the use of t-butanol instead of hexane increased the reaction velocity and the conversion, since both decreased due to the adsorption of polar lipids on the lipase immobilization support. The best FAME yield (94.7%) was attained at a reaction time of 48 h and using 10 mL of t-butanol/g SL, 0.225 g N435/g SL, 11:1 methanol/SL molar ratio and adding the methanol in three steps. In these conditions the FAME conversion decreased by 9.8% after three reaction cycles catalyzed by the same lipase batch. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Biodiesel is defined as the mono alkyl esters of long chain fatty acids derived from vegetable oils or animal fats for use in compression-ignition (diesel) engines. Compared to the two previous generations of biodiesel feedstocks (i.e., food and non-food crops), the third generation, microalgae, has appeared to be a suitable energy source for biodiesel production. Algae present higher photosynthetic efficiency, biomass productivity and growth velocity than the majority of oleaginous cultures (Chisti, 2007). However, the production cost of high grade algae oils constitutes an obstacle in the short term. The main reason is that the ⇑ Corresponding author at: Area of Chemical Engineering, Department of Engineering, University of Almería, 04120 Almería, Spain. Tel.: +34 950 015065; fax: +34 950 015484. E-mail address: [email protected] (A. Robles Medina). http://dx.doi.org/10.1016/j.biortech.2015.03.126 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

operational conditions leading to high grade oil in microalgae are usually those providing low growth rates: low temperature, low light intensity and nitrogen deficiency (Molina, 1999). The major cost factor in biodiesel production is the oil, and while the possibility of reducing the production costs of vegetable oils is limited, this possibility is unlimited for microalgae oil, improving some critical steps in the microalgae cultivation (Li et al., 2007). Nannochloropsis is one of the most promising algae for biodiesel production due to their high biomass accumulation rate, high lipid content, successful cultivation at large scale using natural sunlight, optimal growing on brackish water (thus their large-scale cultivation would not threaten the limited freshwater resources) and their natural resistance to predation and to be overrun by competitive algal species (Ma et al., 2014; Hallenbeck et al., 2015). But the genus consists of six species and hundreds of strains. Between the six genuses, Nannochloropsis gaditana is one of the most studied for the production of biodiesel due to its high resistance to be grown in outdoor

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conditions under adverse conditions of temperature and irradiance, capability to accumulate lipids under nutrient-deprived conditions and high lipid content. Many studies have demonstrated that this microalga adapts to change in the culture conditions, modifying its metabolism and enhance the synthesis of compounds, such as lipids. Factors including nutrient limitation, temperature, salinity and irradiance have been highlighted as elements affecting the molecular composition of this microalga (San Pedro et al., 2014). Thus for example San Pedro et al. (2013) chose N. gaditana to produce biodiesel between five species, because this microalga gives the maximum biomass productivities in continuous cultures at different dilution rates and culture media. There are different methods to transform the microalgal oils into biodiesel: (i) saponifiable lipid (SL) extraction and subsequent transesterification to fatty acid methyl esters (FAMEs, biodiesel) (Wang et al., 2014; Tran et al., 2012; Da Rós et al., 2012; Li et al., 2007), (ii) direct saponification of SLs in the biomass to extract free fatty acids (FFAs) and esterification of FFAs to FAMEs (Hita et al., 2015; Castillo-López et al., 2015), and (iii) direct transesterification of microalgal SLs in the biomass and extraction of FAMEs (Tran et al., 2012, 2013). Currently, practically 100% of biodiesel is produced using alkalis as catalysts. However, if the starting oil contains a small amount of FFAs (>0.5%), as occurs with microalgal oils, soaps are formed, which leads to a reduction in yield and, above all, an increase in the downstream processing of the biodiesel produced (RoblesMedina et al., 2009). Microalgae oils with high FFA contents can be transesterified by acid and enzymatic catalysts, but lipases work at lower temperatures (25–50 °C), which implies less energy consumption, and if the lipase is immobilized it can be easily separated from the reaction mixture by filtration. Moreover, the subsequent separation and purification of biodiesel and glycerol is easier than with acid catalysts. This last catalyst needs to be eliminated, which implies using a great amount of water and consequently makes it a less environment-friendly process (Khor et al., 2010). The major disadvantages of enzymatic processes are the risk of enzyme inactivation due to the short-chain alcohols (methanol and ethanol) and the high cost of lipases, although this cost could be reduced if enzymatic process were implemented on an industrial scale. Several alternatives have been proposed to avoid or decrease enzyme inactivation due to methanol or ethanol, such as addition of the alcohol by steps (Shimada et al., 1999), immobilization of lipase (Ma and Hanna, 1999) and utilization of a solvent as reaction medium (Khor et al., 2010). The objective of this work was to produce biodiesel from the SLs extracted from the wet biomass of the microalga N. gaditana. First SLs were extracted using the ethanol–hexane system and then these SLs were transformed to FAMEs by enzymatic transesterification. The intention was to attain high FAME yields, but taking into account both biodiesel purity and lipase stability.

acid composition of the wet paste biomass from N. gaditana used in this study. The transesterification reactions were catalyzed by lipases Novozym 435Ò from Candida antárctica, LipozymeÒTL IM from Thermomyces lanuginosus, LipozymeÒRM IM from Rhizomucor miehei (all three kindly donated by Novozymes A/S, Bagsvaerd, Denmark) and lipase DFÒ from Rhizopus oryzae (Amano Pharmaceutical Co., Nagoya, Japan). Novozym 435 (N435) is supplied immobilized on a macroporous acrylic resin and usually this lipase does not show positional specificity. LipozymeÒTL IM is immobilized on silica gel and LipozymeÒRM IM on an anionic exchange resin. Lipase DF was immobilized on Accurel MP 1000 (Membrana GmbH, Oberburg, Germany) following the procedure described in Hita et al. (2007). These last three lipases show sn1,3 positional specificity. The chemicals used were analytical grade ethanol (96% v/v), hexane (95% purity, synthesis quality), acetone (analytical quality) (all three from Panreac S.A., Barcelona, Spain), methanol (99.9% purity, Carlo Erba Reagents, Rodano, Italy) and tert-butanol (analytical grade, Fluka, Barcelona, Spain). All reagents used in the analytical determinations were of analytical grade. Standards were obtained from Sigma–Aldrich (St. Louis, MO, USA) and used without further purification. 2.2. Lipid extraction from wet microalgal biomass and purification by crystallization in acetone Lipid extraction from the wet biomass of microalga N. gaditana was performed using a procedure based on a similar method tuned for the microalga Phaeodactylum tricornutum (Ramírez Fajardo et al., 2007). The extraction was carried out treating 442 g of wet biomass (26.4 wt% of biomass) with 3.50 L of ethanol (96% v/v) (30 mL ethanol (96%)/g dry biomass), in a 5 L stirred tank which was jacketed for temperature control. Extraction was carried out at 60 °C for 30 min with constant agitation at 150 rpm with a propeller stirrer (Eurostar digital, IKA Staufen, Germany). The biomass residue was then separated from the hydroalcoholic solution by centrifugation at 7000 rpm for 13 min (centrifuge Brand Supelco 4–15, Germany). Water and hexane were then added to the hydroalcoholic solution to attain the proportion ethanol:water:hexane 1:0.6:0.3 v/v/v. The mixture was stirred for 10 min at 150 rpm

Table 1 (A) Fatty acid composition (percentage with respect to the total fatty acids of the biomass) of the wet paste biomass used from N. gaditana. (B) Percentages of neutral lipids (NLs), glycolipids (GLs) and phospholipids (PLs) obtained by lipid fractionation (Section 2.5) of microalgal lipids before (31% SLs) and after (95% SLs) the acetone purification treatment, and percentages of triacylglycerols (TAGs), diacylglycerols (DAGs) and free fatty acids (FFAs) of the NL fraction. (A)

2. Methods 2.1. Microalga, lipases and chemicals Wet paste biomass from the marine microalga N. gaditana was used as an oil-rich substrate. Cells were grown in an outdoor tubular photobioreactor in ‘‘Las Palmerillas, Cajamar’’ research centre (El Ejido, Almería, Spain). This wet paste biomass was centrifuged at 7000 rpm (Centrifuge model Brand Supelco 4–15, Germany) for 13 min, and then stored at 24 °C until the time of use. This wet biomass contained 26.4 wt% of dry biomass and 31.1 ± 0.1 wt% of total lipids (TLs) in the dry biomass. The total fatty acid content (or saponifiable lipids, SLs, as equivalent fatty acids) in the biomass was 12.0 ± 0.1 wt% in the dry biomass. Table 1A shows the fatty

Fatty acids

wt%

14:00 16:00 16:1n7 18:1n9 18:2n6 20:4n6 20:5n3

7.3 ± 0.9 23.3 ± 1.6 24.8 ± 1.7 3.9 ± 0.4 3.8 ± 0.7 6.8 ± 0.5 29.8 ± 2.8

(B)

a b

Lipid species

Microalgal lipids 31% SLs

Microalgal lipids 95% SLs

NLsa TAGsb DAGsb FFAsb GLsa PLsa

26.9 ± 1.3 74.8 ± 1.3 8.1 ± 1.0 17.1 ± 1.4 61.6 ± 1.7 11.5 ± 0.7

51.0 ± 1.8 64.3 ± 0.2 8.7 ± 0.5 27.1 ± 0.3 43.5 ± 0.9 5.5 ± 1.9

Percentages on TLs (NLs + GLs + PLs). Percentages on neutral saponifiable lipids (TAGs + DAGs + FFAs).

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and then allowed to settle for 10 min. After this time, the appearance of two phases was clearly visible, a lower hydroalcoholic phase and an upper hexanic phase which contains the lipids. This extraction of the SL contained in the hydroalcoholic phase with hexane was carried out once again to increase the SL extraction yield. The two hexanic extracts were mixed and analyzed by gas chromatography (GC, Section 2.4) to determine the SL extraction yield. All the hexanic extracts were evaporated in a rotary evaporator (Buchi, R210, with a vacuum pump V-700, Switzerland) to recover the hexane. The purity of extracted SLs was increased following a procedure of crystallization in acetone based on the methods described by Rajam et al. (2005) and Vandana et al. (2001). 1.4 g of extracted microalgal lipids was mixed with 30.8 mL of acetone in topaz flasks hermetically closed with screw caps. These flasks were placed in an orbital shaker (Inkubator 1000, Unimax 1010 Heidolph, Klein, Germany) at 50 °C and agitated at 200 rpm for 2 h. They were then maintained in a cold room at 4 °C for 24 h, after which supernatant and precipitate were separated by decantation and a supernatant sample was taken for analysis to determine the SL recovery yield and purity. 2.3. Transesterification of microalgal saponifiable lipids (SLs) Microalgal SLs were transformed to fatty acid methyl esters (FAMEs) by lipase catalyzed methanolysis. In a typical experiment 1.4 g of extracted microalgal lipids (31 or 95 wt% SL purity) was mixed with 4.3 or 13.3 mL of solvent (10 mL/g SLs) and 0.01 or 0.30 g of immobilized lipase (0.225 g immobilized lipase/g SL). 0.30 mL of methanol (7.42 mmol) was added in four steps at the reaction times of 0, 10, 24 and 48 h, adding 62.7 lL in the first step and 78.4 lL in the others. In the experiments with 31% SLs this total methanol amount corresponds to a 4.75/1 methanol/SL molar ratio, because SLs are expressed as equivalent fatty acids (mean molecular weight 277 g/mole) and therefore the molar ratios are expressed as moles of methanol per mole of fatty acid in the microalgal SLs. The esterification reaction was carried out in 50 mL Erlenmeyer flasks with silicone-capped stoppers. The mixture was incubated at 40 °C and stirred in an orbital shaking airbath (Inkubator 1000, Unimax 1010 Heidolph, Klein, Germany) at 200 rpm for 72 h. The reactions were stopped by separation of the lipase by filtration (glass plate of porosity 3). The final reaction mixture was conserved at 24 °C until analysis. All reactions were carried out in duplicate and their corresponding analyses in triplicate, and each value recorded is therefore the arithmetic mean of six experimental data (data shown as mean value ± standard deviation). 2.4. Determination of total lipids (TLs), SLs and conversion of the transesterification reaction TLs comprise both SLs and unsaponifiable lipids. The former can be transformed to fatty acid methyl esters (FAMEs, biodiesel), while the latter cannot. SLs comprise neutral saponifiable lipids (NSLs, such as acylglycerols and free fatty acids) and polar lipids, such as glucolipids (GLs) and phospholipids (PLs). The TL content of N. gaditana biomass was determined by the method of Kochet (1978) (Hita et al., 2015). The SL content of the microalgal biomass was quantified by direct transesterification of microalgal biomass to transform all SLs into FAMEs, which were then analyzed by gas chromatography (GC). This SL content and the fatty acid profile of biomass samples were determined following the procedure described in JiménezCallejón et al. (2014). The SL yield (wt%) in the crude lipidic extracts from microalgal biomass is the percentage of extracted SLs with respect to the total

amount of the SLs contained in the original biomass. The amount of extracted SLs was also determined by methylation and GC analysis, following the procedure described in Jiménez-Callejón et al. (2014). The SL purity (wt%) is the weight percentage of SLs (determined by quantitative GC) with respect to the total amount of extracted lipids, determined by weighing after complete removal of the solvent contained in the lipid extract. In the experiments of SL purification by acetone crystallization, supernatant samples were taken and acetone was removed in N2 stream. Then 1 mL of hexane and the internal standard (nonadecanoic acid, 19:0) were added. Samples were methylated also by the procedure described in Jiménez-Callejón et al. (2014). Conversion to FAMEs by transesterification of SLs catalyzed by lipases (Section 2.4) was determined following the procedure described in Martín et al. (2012). This conversion was calculated by the equation:

FAME conversion ð%Þ ¼ 100

SL amount transformed to FAMEs Total SL amount convertible to FAMEs ð1Þ

The SLs transformed to FAMEs (numerator of Eq. (1)) were determined mixing 20 lL samples from the transesterification reaction (or a volume of sample containing about 1 mg of fatty acids), 50 lL of internal standard (prepared dissolving 25 mg of 19:0 methyl ester in 10 mL of hexane) and 1 mL of hexane. This mixture was analyzed directly by GC. To determine the total SL amount as FAMEs (denominator of Eq. (1)) the mixture of sample (20 lL), internal standard solution and hexane was methylated by direct transesterification with acetyl chloride/methanol and analyzed again by GC (Jiménez-Callejón et al., 2014). 2.5. Fractionation of TLs The lipid extracts obtained from microalgal biomass by the procedures described in Section 2.2 were fractionated into neutral lipids (NLs), glycolipids (GLs) and phospholipids (PLs) following the procedure described in Jiménez-Callejón et al., 2014, which is based on the elution of the microalgal TLs in Sep-pack classic cartridges (Waters Corporation, Milford, MA) with chloroform, acetone along with chloroform:methanol 85:15 v/v and methanol to collect from each of these mobile phases the NLs, GLs and PLs, respectively (Kates, 1986). The solvents were evaporated in a rotary evaporator (Buchi, R210, with vacuum pump V-700 and controller V-850, Switzerland) and the percentage of each fraction was determined by weighing. Moreover, the fraction of neutral lipids was fractionated by thin layer chromatography (TLC) to identify and quantify by GC the neutral saponifiable lipids, monoacylglycerols (MAGs), diacylglycerols (DAGs), triacylglycerols (TAGs) and free fatty acids (FFAs), following the procedure described in Hita et al. (2007). 3. Results and discussion 3.1. Extraction of saponifiable lipids (SLs) from microalga N. gaditana The wet biomass used in this work contained 26.4 wt% of dry biomass, 31.1 ± 0.1 wt% of total lipids (TLs) and 12.0 ± 0.1 wt% of saponifiable lipids (SLs) in the dry biomass. It is therefore a microalgal biomass with a low content of SLs and a high content of unsaponifiable lipids (19.1 wt%), which are not convertible to fatty acids methyl esters (FAMEs, biodiesel). This biomass was cultivated in continuous mode, with standard algal medium (8.0 mM nitrate) at a dilution rate of 0.3 day1. In these conditions maximum biomass productivity was achieved, but this biomass has a low SL content (San Pedro et al., 2013). To increase this SL content,

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3.2. Synthesis of methyl esters by enzymatic transesterification The objective was to optimize the transesterification conditions to maximize the conversion of the microalgal SLs to FAMEs, aiming for a conversion rate of over 90%, to minimize the acylglycerol content in the final biodiesel. This optimization was also carried out taking into account that it is necessary to preserve lipase stability. For this reason, experiments were carried out using immobilized lipases, solvents and adding the methanol by steps to reduce its concentration and avoid or decrease lipase deactivation caused by methanol. The first solvent used was hexane since the SLs were already dissolved in this solvent after the extraction process. Moreover, hexane is commonly used in the production of biodiesel by enzymatic transesterification (Soumanou and Bornscheuer, 2003; Tran et al., 2012, 2013) because it is a non-polar solvent (log P = 3.5) which can trap the water around the enzyme and create a microaqueous layer which can help maintain the active conformation of the lipase and preserve the catalytic activity (Nie et al., 2006; Li et al., 2007). The organic solvent also acts reducing the high viscosity of microalgal lipids. In this respect the hexane/ total lipids (TLs) ratio used (3.1 mL/g TLs or 10 mL/g SLs) was established by adding increasing amounts of hexane until the total microalgal lipids were dissolved and the viscosity of the reaction

mixture was low enough to allow easy homogenization. In this way Li et al. (2007) found an optimal hexane/oil ratio of 2.5 in the transesterification of lipids from Chlorella protothecoides. 3.2.1. Influence of lipase type Fig. 1 shows the conversions to FAMEs attained between 10 and 48 h with the four lipases tested. The highest initial reaction velocity and conversion were attained with lipase N435 (85% conversion at 48 h), followed by Lipozyme RM IM (70%), TL IM (67%) and lipase DF (50%). Lipase N435 is one of the most widely used lipases to produce biodiesel due to its high activity and stability (Shimada et al., 1999; Li et al., 2006; Hernández-Martín and Otero, 2008; Rodrigues et al., 2008; Da Rós et al., 2012). In this respect Rodrigues et al. (2008) proved that N435 was more stable than Lipozyme RM IM and TL IM, since the former maintained 90% of its activity after seven uses of the same lipase batch, while Lipozyme RM IM and TL IM maintained 75 and 80%, respectively, of the initial activity in the same conditions. Therefore, N435 was chosen to optimize the transesterification reaction conditions of microalgal SLs. 3.2.2. Influence of the lipase/SL ratio The previous experiments were carried out using 0.4 g N435/g SLs. In order to reduce this lipase amount, experiments were carried out with 0.15 and 0.30 g N435/g SLs. Fig. 2 shows that as the lipase amount decreased, so did the reaction velocity and SL conversion to FAMEs. Maximal conversions of around 71%, 81% and 89% were attained with 0.15, 0.30 and 0.40 N435/SL ratios, respectively. Since a conversion rate of over 95% is important to produce biodiesel with low acylglycerol content, a high amount of lipase should be used. Although authors have tried using low lipase amounts, in many cases large amounts must be used to attain high conversions. For instance, Hernández-Martín and Otero (2008) used up to 50% (w/w) N435 (based on weight of oil) in the ethanolysis of soybean oil to obtain 100% conversion in 7 h. In previous works the lipase amount  reaction time/substrate amount was used as a variable that represents the intensity of treatment (IOT) of lipase catalyzed reactions. It was proved that experiments carried out maintaining this variable constant (although the reaction time and the lipase/substrate ratio were different) lead to similar conversions, which also indicated that no lipase deactivation occurred. It was even proved that maintaining this variable constant is a useful criterion for scaling up the lipase catalyzed reaction or changing the operation mode, e.g. operating

100

80

Conversion (%wt)

these authors carried out a culture in two steps, firstly in continuous mode, in the conditions previously indicated, and then in nitrogen starvation conditions, which implies centrifuging the continuous culture and re-suspending the pellet in nitrate-free culture medium to operate in batch mode for 12 days. This microalgal biomass contained 21.6 wt% of SL and only 3.8 wt% of unsaponifiable lipids and produce purer biodiesel with higher yields (JiménezCallejón et al., 2014). However, the biomass productivity is low in nitrogen starvation conditions and these culture conditions are much more laborious (San Pedro et al., 2013) and difficult to implement at industrial scale. With the goal of attaining high SL yields, lipids were extracted from N. gaditana wet microalgal biomass using ethanol (96% v/v) instead of hexane. Using hexane as solvent only 57% of SLs were extracted from the same microalgal biomass, after subjecting the biomass to a homogenization pretreatment at 1700 bar (JiménezCallejón et al., 2014). The procedure applied in the present work is an adaptation to this microalga of a similar process applied to the microalga P. tricornutum (Ramírez Fajardo et al., 2007), although several differences exist between the procedures applied. Firstly, in that work lyophilized biomass was used, while in this case the extraction was applied to wet biomass (73.6 wt% water), in order to suppress the drying step, and so a higher ethanol (96%)/dry biomass ratio was required (30 as opposed to 10 mL/g). Ramírez Fajardo et al. (2007) carried out two extraction steps of 10 and 1.25 h at room temperature, but to simplify this process in the present work a single extraction step of 30 min was carried out at 60 °C. Similar SL yields were attained by both procedures: 85 wt% in this work and 82 wt% by Ramírez Fajardo et al., 2007. However the SL purity attained in this work was low (31 wt%) (Ramírez Fajardo et al., 2007, did not report data on SL purity), which indicates that high amounts of lipids and non-lipid contaminants were extracted from the microalgal biomass. This is because the microalgal biomass contains a high percentage of unsaponifiable lipids and because extraction was carried out with ethanol at high temperature. Ramluckan et al. (2014) extracted lipids from microalga Chlorella sp. using ethanol by the soxhlet extraction method (at ethanol boiling temperature) and only 52% of extracted lipids were SLs. In any case the extraction procedures must be optimized for each microalga species bearing in mind that it is difficult to obtain simultaneously high SL yields and purities (JiménezCallejón et al., 2014).

60

40 Novozym 435 Lipozyme RM IM Lipozyme TL IM Lipase DF

20

0 0

10

20

30

40

50

60

Reaction time(h) Fig. 1. Influence of lipase type and reaction time on the conversion of microalgal SLs to FAMEs. Conditions: 1.4 g lipids (31% SLs), lipase/SL ratio 0.4 w/w, 10 mL hexane/g SL, methanol/SL molar ratio 3.5:1, 3 additions of methanol at 0, 10, 24 h.

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Conversion (% wt)

80

60

40

0.15 gN435/gSL 0.30 gN435/gSL 0.40 gN435/gSL

20

0 0

20

40

60

80

Reaction time (h) Fig. 2. Influence of the N435/SL (w/w) ratio and reaction time on the conversion of microalgal SLs to FAMEs. Conditions: 1.4 g lipids (31% SLs), 10 mL hexane/g SL, methanol/SL molar ratio 4.75:1, 72 h, 4 additions of methanol at 0, 10, 24, 48 h.

in discontinuous or continuous mode or in a stirred tank or packed bed reactor (Hita et al., 2007). Fig. 2 shows that very similar conversions were attained (around 71%) with the N435/SL ratios of 0.15, 0.30 and 0.40, at the reaction times of 72, 34 and 24 h, respectively. These conditions correspond to IOTs of 10.8, 10.2 and 9.6 g N435  h/g SL, respectively, i.e., once again the same conversions were attained using similar IOTs. This shows that also in this case the IOT could be used as a criterion to scale up the reaction or to predict whether lipase loses activity throughout the reaction. In this regard, the slight IOT diminution from 10.8 to 9.6 g h/g could mean a slight lipase deactivation during the reaction.

3.2.3. Lipase stability in the operational conditions For biodiesel production by enzymatic catalysis to be economically feasible it is essential that the catalyst can be used repeatedly to catalyze successive reactions. For this reason experiments were carried out using the same N435 batch to catalyze three consecutive transesterification reactions. These experiments were carried out with microalgal SLs and sunflower oil. Methanol was added in four steps and hexane was used with both oils. Fig. 3 shows that the conversion attained in the first use of lipase with sunflower oil was higher than with microalgal SLs. Moreover, with microalgal SLs the conversion decreased by 23% after three transesterification

cycles, while with sunflower oil this diminution was only 6%. Therefore, this result shows that the low SL purity (31%) or the microalgal lipid composition (Table 1B) affects the transesterification conversion and possibly increases the lipase inactivation velocity. These results are qualitatively similar to the obtained by Watanabe et al. (2002) in the methanolysis of soybean oil catalyzed by N435. These authors found that the conversion to FAMEs obtained from crude oil was significantly lower than the one obtained from degummed and refined oil. They also demonstrate that the reaction velocity decreased as the phospholipid (PL) content increased. Table 1B shows that this microalgal biomass contains 61.6% of galactolipids (GLs) and 11.5% of PLs, and therefore the lower reaction velocities and conversions attained with microalgal lipids could be because polar lipids bound on the immobilized lipase preparation and interfered the interaction of the lipase molecule with substrates (Watanabe et al., 2002). On the other hand, it also must be taken into account that methanol is poorly soluble in hexane and can deactivate the lipase (Shimada et al., 1999). Along these lines, Tran et al. (2013) also observed that the FAME yield decreased in the second, third and fourth uses of a lipase batch (83%, 76% and 68%, respectively) in the direct transesterification of Chlorella vulgaris lipids catalyzed by the immobilized Burkholderia lipase, using a very high methanol/oil molar ratio (66.9) and hexane as reaction medium. 3.2.4. Influence of the microalgal SL purity and solvent type To check whether the microalgal SL purity influences the conversion to FAMEs, 31% pure microalgal SLs were purified by crystallization in acetone following the procedure described in Section 2.2. This procedure is based on the insolubility of gums and waxes on acetone at low temperature, and therefore the SLs remain dissolved in acetone, while the insoluble fraction is rich in PLs and waxes (Rajam et al., 2005; Huynh et al., 2010). In this way, 90–95% of the SLs contained in the 31% pure SLs were recovered with 95% purity. Table 1B shows that following this procedure the NL content increased from 26.9% to 51%, while the polar lipid content (GLs and PLs) decreased from 73.1% to 49.0%. GLs and PLs are SLs and these 95% pure SLs were used to produce biodiesel without additional purification steps in order not to decrease too much the FAME yield with respect to total microalgal SLs. Fig. 4 compares the conversions attained using microalgal lipids without purifying (31% SLs) and purified (95% SLs), and using

100

100 90

Conversion (%wt)

80 70

88.8

95.4

93.4

74.9 68.2

60

Cycle 1

50

Conversion (%wt)

80

99.4

60

31%SL-hexane 31%SL-t-butanol 95%SL-hexane 95%SL-t-butanol

40

Cycle 2

40

Cycle 3

30

20

20 10

0 0

0 Microalgae oil

Sunflower oil

Fig. 3. Influence of the number of uses of a same N435 batch on the conversion, using microalgal SLs and sunflower oil. Conditions: 1.4 g microalgal lipids (31% SLs) or sunflower oil, 10 mL hexane/g SL, N435/SL ratio 0.4 w/w, 4.75/1 methanol/SL molar ratio, 72 h, 4 additions of methanol at 0, 10, 24, 48 h.

20

40

60

80

Reaction time (h) Fig. 4. Influence of the microalgal SL purity (31 and 95%), solvent type (hexane and t-butanol) and reaction time on the reaction conversion. Conditions: 1.4 g lipids, 10 mL solvent/g SL, N435/SL ratio 0.15 w/w, 4.75/1 methanol/SL molar ratio, 72 h, 4 additions of methanol at 0, 10, 24, 48 h.

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hexane and t-butanol as reaction media. In these experiments the N435/SL ratio used was 0.15 instead of 0.40 to observe better the difference between the conversions attained. t-Butanol was tested because it is a solvent of intermediate polarity which dissolves neutral and polar lipids (Table 1B), methanol and the glycerol produced in the transesterification reaction, which is a hydrophilic product with poor solubility in hydrophobic solvents, such as hexane. Thus, the use of t-butanol allows the use of higher methanol concentrations without causing lipase deactivation by the undissolved methanol (Shimada et al., 1999), and it avoids deposits of glycerol and polar lipids on the lipase immobilization support which block the lipase active sites (Li et al., 2006; Séverac et al., 2011; Watanabe et al., 2002). Fig. 4 shows that appreciable lower reaction velocity and conversions were attained using 31% pure SLs and hexane, and similar reaction velocities and conversions were attained with 31% SL–t-butanol, 95% SL–hexane and 95% SL–t-butanol. These results may be due to the lower polar lipid content of 95% pure SL (Table 1B) and because t-butanol dissolves both the polar lipids and glycerol formed, which are not adsorbed on the lipase immobilization support, thus decreasing the lipase inhibition effect. With 95% pure SL, hexane can also solve the polar lipids at the hexane/TL ratio used (9.5 mL/g TL). With 31% pure SLs the hexane/TL ratio was lower (3.2 mL/g TL) because in this work the solvent/SL ratio was maintained constant at 10 mL/g SL (Section 3.2). Therefore, from these comparisons it can be concluded that purifying SLs from 31% to 95% and using t-butanol instead of hexane improve both the reaction velocity and the conversion. On the other hand, both lipids contain a high percentage of FFAs (Table 1B), and as Castillo-López et al. (2015) found, esterification of FFAs is much faster than transesterification. This may well explain, therefore, the relatively high initial reaction velocities observed in Figs. 2 and 4. A review of the literature regarding the FAME yields and conditions required to obtain biodiesel from microalgal lipids and vegetable oils indicates that higher IOTs and methanol/oil molar ratios are required with the former, due to the lower purity of microalgal lipids and, above all, to the different lipid composition. In this respect Wang et al. (2014) compared the transesterification efficiency attained using different microalgal oils and soy oil and found that oil from microalga Nannochloropsis oceanica, cultivated under nitrogen-depleted conditions (and therefore highly rich in neutral lipids), and soy oil (also rich in neutral lipids) gave higher transesterification efficiencies than oils from microalga N. oceanica obtained under nitrogen-replete conditions (and therefore rich in polar lipids), like the microalgal lipids used in this work. All these results confirm the importance of using microalgal oils with low polar lipid contents to achieve high FAME conversions, as was observed by Jiménez-Callejón et al. (2014). 3.2.5. Influence of substrate concentrations and methanol/SL molar ratio In previous experiments the conversion to FAMEs was always below 90%, even when using a 0.4 lipase/SL ratio and 72 h reaction time (IOT = 28.8 g lipase h/g SL). In an attempt to increase the substrate conversions, firstly experiments were carried out reducing the t-butanol amount to increase the substrate concentrations. In these experiments t-butanol/SL ratios of 10, 6.4, 5.8 and 3.4 mL/g were tested. No lower t-butanol/SL ratios could be tested due to the high viscosity of the reaction mixture and, therefore, to the difficulty of attaining correct homogenization of the reaction mixture. These experiments shown that the initial reaction velocities only increase slightly with the substrate concentration and similar conversions were attained at the t-butanol/SL ratios tested. This result seems to indicate that, while the reaction velocities should increase with the substrate concentrations, the reaction mixture viscosity also increases, and consequently the higher the substrate

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concentration the more the reaction velocity is controlled by the mass transfer resistance. Subsequently the methanol/SL molar ratio was increased from 4.75:1 to 11:1 (Fig. 5A). In these experiments a t-butanol/SL ratio of 10 mL/g was used to decrease the reaction mixture viscosity and preserve the lipase stability at the high methanol/SL molar ratios used. Fig. 5A shows that both the reaction velocity and the final conversion increased with the methanol/SL molar ratio. A conversion rate of 93.5% was attained with 11/1 methanol/SL molar ratio at a reaction time of 72 h. To reduce this long reaction time to at least 48 h, experiments were carried out where the IOT was kept constant (IOT = 0.15 g Novozym/g SL  72 h = 10.8 g N435 h/g SL = 0.225 g Novozym/g SL  48 h). Therefore, in these experiments a 0.225 N435/SL weight ratio was used. Fig. 5A shows that effectively the same conversion to FAMEs (93.5% and 93.4%) was obtained at 72 h with 0.15 g N435/g SL, and at 48 h with 0.225 g N435/g SL. This result indicates that additional reduction in the reaction time could be obtained working with larger lipase amounts and maintaining the IOT constant. Then, the reduction of the methanol/SL molar ratio from 11/1 to 8/1 was checked (Fig. 5B), but in these new experiments three additions of methanol at 0, 10 and 24 h were carried out instead of four additions. Fig. 5B shows that both the reaction velocity and the final conversion were appreciably higher using an 11/1 methanol/SL ratio (94.7% and 90.0% using an 8/1 molar ratio). It also can be observed that with 11/1 methanol/SL molar ratio and carrying out three methanol additions were obtained slightly higher reaction rates and final conversions (94.7%, Fig. 5B) than with four methanol additions (93.4%, Fig. 5A). This result is because the methanol concentration, and therefore the reaction velocity increase adding the same methanol amount in fewer steps. Therefore, the conditions for the transesterification of SLs (95% pure) from microalga N. gaditana at which the maximum FAME yield was attained were: 10 mL t-butanol/g SL, 11/1 methanol/SL molar ratio, IOT = 10.8 g N435  h/g SL (for example 0.225 g N435/ g SLs and 48 h), three additions of methanol at 0, 10 and 24 h, 40 °C and stirring at 200 rpm. This yield (94.7%) is higher than or similar to those obtained by other authors who transform microalgal SLs into biodiesel by enzymatic catalysis in similar conditions. For example, Da Rós et al. (2012) attained 80% ester yield in the transesterification of lipids from the cyanobacterium Microcystis aeruginosa with ethanol in similar conditions to those in this work. Tran et al. (2013) attained FAME conversion of between 91% and 96% by direct transesterification of C. vulgaris lipids, catalyzed by immobilized Burkoholderia lipase, but using methanol/oil molar ratios of over 67/1, between 2 and 16 g lipase/g oil and 48 h reaction time. 3.2.6. Reuse of lipase in the optimal transesterification conditions Fig. 6 shows the conversion attained in three consecutive transesterification reactions catalyzed by the same N435 batch, using the experimental conditions at which the maximum FAME conversion was attained. After each use the N435 lipase was washed with t-butanol. This figure shows that both reaction velocity (lipase activity) and final conversion decrease with the number of uses of lipase. The conversion attained at 48 h decreased about 5% in each reaction cycle (maximum conversions of 94.7, 90.1 and 85.4% were attained in the first, second and third uses, respectively), i.e., a decrease of 9.8% was observed after maintaining the lipase 144 h in the reaction conditions. This result seems to indicate that in these experimental conditions there is a constant loss of lipase activity, which contrasts with the results of other authors who observed that N435 does not deactivate in presence of t-butanol, even though methanol is used. However, those research works used refined vegetable oils as substrates rather than microalgal oils (Li et al., 2006; Royon et al., 2007). However,

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Fig. 5. Influence of the methanol/LS molar ratio, lipase/SL ratio and reaction time on the FAME conversion. Conditions: 1.4 g lipids (95% SLs), 10 mL t-butanol/g SL. A) (.) 15% N435, 72 h, 4.75:1 methanol/SL molar ratio, 4 methanol additions at 0, 10, 24, 48 h. (d) 15% N435, 72 h, 11/1 methanol/SL molar ratio, 4 methanol additions at 0, 10, 24, 48 h with methanol/SL molar ratios of 2:1, 3:1, 3:1 and 3:1. (s) 22.5% N435, 48 h, 11/1 methanol/SL molar ratio, 4 methanol additions at 0, 7, 16, 32 h. B) (s) 22.5% N435, 48 h, 11/ 1 methanol/SL molar ratio, 3 methanol additions at 0, 10, 24 h with methanol/SL molar ratios of 3:1, 4:1 and 4:1. (d) 22.5% N435, 48 h, 8/1 methanol/SL molar ratio, 3 methanol additions at 0, 10, 24 h with methanol/SL molar ratios of 2:1, 3:1 and 3:1.

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with the number of uses. Jiménez-Callejón et al. (2014) show that microalgal biomass with low lipid content also has low acylglycerol and high polar and unsaponifiable lipid percentages. These results mean that polar or unsaponifiable lipids could also be the cause of the lipase deactivation. The results obtained in this work and its comparison with the obtained by other authors allow to show the influence of the microalgal lipid composition and the importance of polar lipids on the transesterification velocity, biodiesel yield and lipase stability.

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Wang et al. (2014) treated the lipase N435 for 165 h with t-butanol and methanol in the transesterification of lipids extracted from microalga N. oceanica and found that the conversion efficiency remained greater than 90% after 165 h (a similar result to the one obtained in this work). These authors used crude algal oils extracted from microalgal biomass cultivated under nitrogen-depleted conditions, and therefore with higher contents in triacylglycerols (neutral lipids) than in polar lipids. The results obtained in the present work are also related to those obtained by Tran et al. (2013), who observed that the immobilized Burkholderia lipase maintained different activities over repeated reaction cycles, depending on the lipid content of C. vulgaris ESP-31 microalga. They found that the higher the lipid content, the higher the activity of the recycled lipase, although these activities always decreased

The high polar lipid content of microalgal SLs affects the conversion to FAMEs, because when using hexane as reaction medium polar lipids adsorb on the lipase immobilization support. As a result, both the use of partially degummed SL (95% purity) and the use of t-butanol, which dissolves polar lipids, increased both the reaction velocity and FAME conversion. 94.7% of microalgal SLs were transformed to FAMEs, although relatively high IOTs, t-butanol/SL and methanol/SL ratios were used. The FAME conversion decreased by 9.8% when a lipase batch was used to catalyze three transesterification reactions. Acknowledgements This research was supported by grants from the Ministerio de Educación y Ciencia (Spain), Project CTQ2010-16931. This project was co-funded by the FEDER (European Fund for Regional Development). References Castillo-López, B., Esteban-Cerdán, L., Robles-Medina, A., Navarro-López, E., MartínValverde, L., Hita-Peña, E., González-Moreno, P.A., Molina-Grima, E., 2015. J. Biosci. Biotechnol. Biochem. http://dx.doi.org/10.1016/j.jbiosc.2014.11.002. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306.

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