Synthesis of fatty acids methyl esters (FAMEs) from Nannochloropsis gaditana microalga using heterogeneous acid catalysts

Accepted Manuscript Title: Synthesis of fatty acids methyl esters (FAMEs) from Nannochloropsis gaditana microalga using heterogeneous acid catalysts Author: Alicia Carrero Gemma Vicente Rosal´ıa Rodr´ıguez Gonzalo L.del Peso Cleis Santos PII: DOI: Reference:

S1369-703X(15)00037-6 http://dx.doi.org/doi:10.1016/j.bej.2015.02.003 BEJ 6115

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

22-8-2014 30-1-2015 3-2-2015

Please cite this article as: Alicia Carrero, Gemma Vicente, Rosal´ia Rodr´iguez, Gonzalo L.del Peso, Cleis Santos, Synthesis of fatty acids methyl esters (FAMEs) from Nannochloropsis gaditana microalga using heterogeneous acid catalysts, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2015.02.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of fatty acids methyl esters (FAMEs) from Nannochloropsis gaditana microalga using heterogeneous acid catalysts.

Alicia Carrero*, Gemma Vicente, Rosalía Rodríguez, Gonzalo L. del Peso, Cleis Santos

Department of Chemical and Energy Technology, ESCET, Universidad Rey Juan Carlos, c/ Tulipán s/n, 28933, Móstoles (Madrid), Spain

*Corresponding author: Tel.: 34-91-4888088; fax: 34-91-4887068. E-mail address: [email protected] (A. Carrero).

Highlights

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The oil fraction (42 wt %) of Nannochloropsis gaditana was extracted with methanol

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Oil fraction contains 86.7 wt % of saponifiable lipids mainly polar lipids and FFAs

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The conversion of lipids with ion-exchange resins gave FAMEs yields above 90 mol %

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In comparison, the direct conversion of microalga gave lower FAMEs yield (80 mol %)

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Ion-exchange resin catalysts were regenerated by washing with methanol and HCl

Abstract

Oleaginous microorganisms like microalgae have emerged as a promising alternative feedstock in the production of fatty acid methyl esters (FAMEs) since they can accumulate high levels of lipids without competing with food production and having oil productivity values higher than oilseed crops. The lipids of Nannochloropsis gaditana microalga were extracted with methanol and analysed to determine its chemical composition. Since typical homogenous catalysis requires additional purification units and extracted oil presented many free fatty acids (FFAs) (~22 wt%), FAMEs were synthesized using solid acid catalysts like ion-exchange resins (Amberlite-15, CT-275, CT-269), KSF clay and silica-alumina. Despite their high surface area, the lower acidity of silica-alumina led to a FAME yield lower than the ones obtained using KSF clay and ion-exchange resins. The good results obtained with these catalysts discard diffusion limitations when resins or KSF clay are used as catalysts. FAME synthesis through an indirect method with a previous lipid extraction was compared with the direct reaction of dry microalga biomass. Better results (FAME yields above 90 mol%) were obtained in the two-step method using ion-exchange resins. However, these catalysts lost their activity, so they were regenerated by washing with methanol and HCl.

Keywords: fatty acid methyl esters, Nannochloropsis gaditana, microalgae, transesterification, heterogeneous reaction, solid acid catalysts.

1. Introduction It is widely accepted that there is a need to reduce CO2 emissions from human activities that contribute to the greenhouse effect and thus to global warming. Clean and renewable energy sources appear as the solution to replace the use of fossil fuel in transport and in industry (the main anthropogenic sources of greenhouse gas emissions), as well as dealing with the problem of crude oil exhaustion in the future [1, 2]. Biodiesel is an attractive alternative to fossil fuel, as it is compatible with current commercial diesel engines and has clear benefits compared to diesel fuel including enhanced biodegradation, reduced toxicity and lower emission profile [3]. However, biodiesel presents some disadvantages: a high manufacturing cost and the majority of raw materials for biodiesel production are vegetables oils which compete with the food industry and need a very large percentage of the current available arable land [4]. Therefore, it is necessary to explore new sources to face up to these problems. In this context, oil from microorganisms such as microalgae has emerged as one of the most promising alternative sources of lipids to be used in biodiesel production because of high microalgal growth rates and productivity compared to conventional vegetable oil crops [5]. Also, microalgae contain high amounts of lipids (> 20 wt%) which can provide sufficient feedstock for large-scale biodiesel production [6, 7]. In this sense, nitrogen concentration in the culture medium is known as the main factor upsetting lipid synthesis from microalgae [8]. Furthermore, microalga cells have photosynthetic mechanisms similar to those of higher plants to fix CO2 in air and convert the C to carbohydrates and lipids, contributing the reduction of CO2 emissions [6, 7]. As an additional advantage, microalgae may be obtained on lands unsuitable for conventional agriculture such as desert areas or in large saline water ponds [9, 10]. However, for a cost-effective biodiesel production from microalgae, the complete lipid extraction should be achieved and the resultant residual biomass should be processed too (e.g. by anaerobic digestion, etc.) [11]. Some of the autotrophic microalgae that are used to produce biodiesel are Chlorella vulgaris, Chlorella emersonii, Chlorella protothecoides, Nannochloris oculata, Nannochloropsis gaditana, Nannochloropsis oceanica, Neochloris oleoabundans, Phaeodactylum tricornutum and Tetraselmis sueica [6-8, 10-14]. Biodiesel is commonly produced by transesterification of triglycerides (usually from vegetable oils) with methanol, obtaining fatty acid methyl esters (FAMEs). Although triglycerides are one of the main components of microbial oil, other lipids with fatty acid ester linkage (saponifiable lipids), free fatty acids (FFAs) and non-saponifiable lipids are also present in its composition. However, only saponifiable lipids and FFAs can produce FAMEs through catalysed transesterification and esterification reactions, respectively [14,15]. Acid and basic catalysts have been widely investigated in the transesterification of triglycerides from conventional feedstocks like vegetable oils. Despite basic catalysed transesterification is faster than acid catalysed reaction (about 4000 times), acid catalysts can simultaneously promote esterification of FFAs and transesterification of triglycerides, so they are recommended when the content of FFA in

the oil is high (> 3 wt%) [16], such as microbial oil [14, 17]. Sulphuric acid has been one of the main acid catalysts used for FAME synthesis; however the most important drawback of the homogenous processes is catalyst separation, requiring additional purification units, which will result in higher biodiesel production costs. Thus, large amounts of waste water are generated during the separation and cleaning steps. For these reasons, the use of solid catalysts seems to be an appropriate solution to overcome problems associated with homogeneous catalysts [18-20].Many works have been published dealing with FAME synthesis from conventional vegetable oil using heterogeneous basic catalysts such as CaO, MgO [21-25], zinc aluminate [26], and hydrotalcites [27-30], together with solid acid catalysts like zeolites [31, 32], acid clays [33-35], mesostructured materials [36,37] and ion-exchange resins [33, 38,39]. The production of biodiesel from microalga oil has been previously reported in the literature using the conventional route [40]. This process requires an initial extraction of the lipids from the microalga biomass followed by their conversion to FAMEs. One alternative to the conventional process, which reduces the processing units and costs, is the direct conversion of the microalga biomass, without previous extraction, to produce FAMEs. In this method, only the solvent elimination is required to obtain a high quality biodiesel [41-42]. Based on the above premises, this work is focused in the use of lipids from Nannochloropsis gaditana microalga to produce FAMEs by solid acid catalysts like ion-exchange resins (Amberlyst-15, CT-269 and CT- 275), KSF clay and silica-alumina since these materials have been scarcely used for FAME production from microalgae [43-46]. Esterification/transesterification reactions were carried out in two different ways: a two-step process (lipid extraction followed by reaction), and direct microalga transformation without a previous extraction step. 2. Materials and methods 2.1. Oil extraction and characterisation Lipids from the dry cell biomass of Nannochloropsis gaditana (Easy algae, Spain) were extracted according to the procedure previously reported by Carrero et al. [14], using methanol (99%, Scharlab, Spain) under reflux during 20 min, with a methanol/microalga ratio of 13/1 wt/wt. FFAs, triglycerides, diglycerides, monoglycerides, carotenoids, sterol esters, sterols, tocoferols, polar lipids (phospholipids, glycolipids and sphingolipids) and a small fraction of non-lipidic compounds were identified in the extracted oil and quantified by Thin Layer Chromatography (TLC). Chromatographic separation was developed in 20 cm x 20 cm silica-coated aluminium plates (Alugram Sil G/UV. Macherey-Nagel GmbH, Düren, Germany) using a solvent mixture of 88 % vol n-hexane, 11 % vol diethyl ether and 1 % vol glacial acetic acid. Visualisation was carried out by staining with iodine. Digital image analyses of staining plates were performed with Un-Scan-It Gel 6.1 software (Silk Scientific Inc. Orem, UT, USA) and the lipid compositions were quantified by the corresponding calibration curves. The saponification value was calculated according to ASTM D1962 – 85 standard. In addition, the non-saponifiable matter (non-saponifiable lipids and non-lipidic

compounds) in the extracted oil were determined by a gravimetric procedure [47]. The fatty acid profile of microbial oil was performed by gas chromatography (GC) in a CP-3800 gas chromatograph (Varian Inc.) fitted with an FID detector and a ZB-WAX capillary column (30 m length, 0.32 mm internal diameter; 0.25 µm film thickness), from Phenomenex, USA. Prior to GC analysis, the oil sample was transformed into the corresponding methyl esters using the borum triflouride method described in the EN ISO 5509 standard. Finally, 1 µl of this sample containing FAMEs was injected into the capillary column where the separation was achieved using a heating rate of 1 ºC/min from 150 ºC to 240 ºC at a flow rate of 1 ml/min (injector temperature: 180 ºC, detector temperature: 280 ºC, injection mode: splitless). Identification of chromatographic peaks was carried out by comparison with a FAME standard mixture (Sigma-Aldrich) and quantification by means of external standards and their corresponding calibration curve. The acid and iodine values of extracted oil were determined according to European standard methods EN 14101, and EN 14111, respectively. Measurements of total nitrogen, carbon, and hydrogen content of the extracted oil were determined by elemental analysis in an Elementar Vario EL III CHNS instrument. Oxygen flow of 65 mlN/min was used in the combustion of the sample. Combustion gases were selectively separated by flowing through different columns and detected by thermal conductivity. Metal contents of the oil (Na, Mg, Ca, K) and P were determined by Inductively Coupled Plasma (ICP-AES) on a VARIAN Vista AX Axial CCD Simultaneous ICP-AES spectrometer. Previously, the sample was digested by acid treatment with H2SO4 and HF. 2.2. Catalysts characterisation Acid resins were provided by Purolite whereas KSF clay and silica-alumina were provided by SigmaAldrich. These solids were characterised using different techniques. Nitrogen adsorption-desorption isotherms at 77 K were performed in a Micromeritics Tristar 3000 apparatus. The samples were previously outgassed during 6 hours under vacuum at 80 ºC for ionexchange resins, at 150 ºC for KSF clay and at 210 ºC for silica-alumina. The surface areas were calculated by means of the BET equation, whereas the pore size distributions were determined by the BJH method applied to the adsorption branch of the isotherms. Mean pore size was obtained from the maximum of the BJH pore size distribution. Pore volumes were calculated from the nitrogen adsorbed volume at P/P0 = 0.95. The acid capacity of the catalysts was determined by a titration procedure as follows: a solution of NaCl (2M) is added to the sample and the mixture was stirred during 30 min to exchange Na+-H+ cations, and these protons were potentiometrically titrated [48,49] 2.3. FAME production The catalytic experiments were carried out at 100 ºC and autogenous pressure under stirring (1000 rpm) during 4 hours in a 0.1 l batch autoclave reactor, equipped with a temperature controller and a pressure gauge. Microalga oil, methanol and catalyst were introduced into the glass reactor

(methanol/oil = 40 wt/wt and catalyst/oil = 0.8 wt/wt). Direct reaction (one-step) was made by using the same reactor, solvent and operating conditions, feeding the solid microalgae biomass directly to obtain the same methanol/oil mass ratio. Then, the FAME layer was collected, washed with a mixture of n-hexane and diethyl ether, and water and dried. The yield of FAMEs was determined through 1H NMR analyses performed in a Varian Mercury Plus 400 unit in a similar way to that described by Gelbard [50]. 2.4. Catalyst regeneration Exhaust ion exchange resins were washed with 20 ml/g of methanol and (methanol + HCl). The concentration of HCl solution was 37 w/w. In the second case, additional washings with methanol were done to remove residual HCl . Finally, in both cases, resins were dried at 80 ºC during 12 h. 2.5. Statistical analysis All the experiments were carried out three times in order to determine the variability of the results and to assess the experimental errors. In this way, the arithmetical averages and the standard deviations were calculated for all the results. 3. Results and discussion 3.1. Microbial oil extraction and characterisation A previous work [14] demonstrated that methanol is a suitable solvent for the extraction of the lipids from this microalga and it is an interesting alternative to avoid the use of chlorinated solvents (e.g. chloroform), because of the adverse effect of these extraction solvents on the environment. Following this method, the extracted oil reached a high yield (47.1 ± 0.6 wt%), which is consistent with the habitual oil content of this microalga (12- 56 % dry wt.) [51]. The analysis of microalgal oil is summarized in Table 1. Furthermore, the saponifiable value measured by ASTM D 1962 – 85 is set out in Table 2. According to this oil fraction composition 40.82 wt % corresponds to saponifiable lipids, 1.02 wt % to non-saponifiable lipids and 5.26 wt % to non lipidic compounds. So, based on dry weight of biomass, the amount of total lipids is 41.84 wt % being 40.82 wt % saponifiable lipids. Non-saponifiable lipids and non-lipidic compounds cannot be converted into FAMEs, but most of these compounds are separated during the FAME purification step. This oil did not contain a significant amount of triglycerides because they had probably been hydrolysed to FFAs during the biomass liophylization process. The high saponifiable lipids and FFAs concentration in the extracted oil denotes the high quantity of compounds that could be transformed into FAMEs. Table 2 shows the acid and iodine values together with the fatty acid profile for the saponifiable lipids extracted from Nannochloropsis gaditana. The concentration of FFAs found in the lipidic fraction of this microalga was measured through the acid value (44 mg KOH/g) and is in agreement with the FFAs content calculated by TLC (Table 1). This value determines an acid-catalysed process in FAME production, because it would avoid the yield losses produced from FFAs neutralisation using a basic catalyst [52]. On the other hand, iodine value of saponifiable lipids determined according to the EN ISO 3961 standard was 161 g I2/100 g, which is higher than the limit of 120 g I2/100g specified in the

EN 14214 standard. This parameter is a measure of the unsaturation level providing high values because of the high concentration of eicosapentaenoic acid (20:5) in the microbial oil. As previously reported [7], it is remarkable that microalgae oils usually differ from most vegetable oils in being quite rich in polyunsaturated fatty acids. The high degree of unsaturation inherent to the FAMEs derived from these fatty acids would evidence lower oxidative stability but excellent fuel properties at low temperatures like cold-filter plugging point, which is an advantage in winter operation [3]. In our case, the content of polyunsaturated, monounsaturated and saturated fatty acids was 23.8 %, 46.5% and 29.8%, respectively. So, this microalgal oil contains high concentrations of monounsaturated and saturated fatty acids. These values are similar to those corresponding to Nannochloropsis gaditana microalga previously published [14]. Oil measurements of total nitrogen, carbon, and hydrogen quantities by elemental analysis gave 0.5 wt%, 72.3 wt%, and 17.4 wt%, respectively. The nitrogen value depends on the amount of proteins in the oil (amount of nitrogen multiplied by an average factor of 6.5 [53]) and the sphingolipids contained in the oil, around 10 wt% in this sample of Nannochloropsis gaditana. Besides, microalgal oil contains Ca: 0.01 wt%, Mg: 0.02 wt%, Na: 2.3 wt%, and K: 2.0 wt%. 3.2. Catalyst characterisation Different acid catalysts were tested in the transesterification and esterification reactions with microalgal oil: three ionic exchange resins (Amberlite-15, designed as A-15, CT-275 and CT-269), KSF clay and silica alumina (designed as SiAl). The physicochemical properties of these materials, BET area, pore volume, pore diameter and acidity, are shown in Table 3. BET surface area and pore volume of the silica alumina were very high (370 m2/g and 1.29 cm3/g, respectively), however poor acid properties were found to be 0.5 mmol H+/g. Protonic resins showed wider pores due to the macro-reticular structure and very high acidity values, being CT-269 the sample with slightly higher acidity value (6.3 mmol H+/g). On the other hand, KSF clay belongs to the monmorillonite family and it is constituted by laminar and bidimensional aluminosilicates [54]. The superposition of these laminar layers leads to a compacted structure with low pore volume (0.02 cm3/g) and BET surface area (8 m2/g). However, KSF clay has an intermediate number of acid sites (3.6 mm H+/g) between resins and silica alumina. 3.3. FAMEs production Figure 1 represents FAME yield analysed by 1H NMR (mol %) obtained without and with each catalyst after 4 hours of reaction. It is interesting to note that the best catalytic behaviour was found with ion-exchange resins, which present FAME yield above 90 ± 0.8 mol%, while silica-alumina showed worse catalytic behaviour with only 18.2 ± 0.3 mol% of FAME yield. This value was higher than the corresponding one (9 ± 0.1 mol%) obtained in absence of catalyst. The low activity obtained with silica-alumina was also reported by Zanette et al in the transesterification of Jatropha curcas oil [33].

The reactions with KSF clay also presented a high FAME yield (67 ± 0.7 mol%), although slightly lower than the ones achieved with the ion- exchange resins. These results are a consequence of the great acidity and pore diameter exhibited by protonic resins making the lipid diffusion through their structure and subsequent reaction easier. Tesser et al. [38] reported similar results in the FAME production using artificially acidified soybean oil and A-15 as catalyst. However, Zanette et al. [33] obtained lower FAME yield using the same resin and Jatropha curcas oil, because this oil contains mainly triglycerides unlike microalgal oil used in this work which contain mainly FFAs and polar lipids. Thus, the acid resin is capable of converting a feedstock with high FFAs and polar lipids content, but the efficiency of the triglyceride acid-catalysed transesterification is much lower. The relatively high yield achieved in the reaction with the KSF clay was due to its high acidity value being insignificant the lack of porous structure. This result is in agreement with those obtained by Neji et al. [34] in esterification of stearic acid with methanol using KSF clay as a catalyst. Zanette et al. [33] reported lower FAME yield in the transesterification of Jatropha curcas oil with KSF, in the same way as with A-15 resin. So, KSF clay and A-15 resin provide higher FAME yields in the esterification of FFAs than in the transesterification of triglycerides. The product distribution after transesterification and esterification reactions is shown in Table 4. The reactions with the resins A-15 and CT-269 presented the highest content of FAMEs (85.2 ± 1.1 wt% and 86.2 ± 1.3 wt%, respectively) with the lowest values (≈ 10 wt%) of FFAs and non-converted polar lipids. However, the FAME concentrations were lower than the 96.5 wt% included in the European standard EN14214 because thermodynamic equilibrium was reached during the reaction. Therefore, a purification process is necessary to eliminate all the compounds that contaminate the final product to be used as biodiesel. The silica-alumina showed the highest quantity of non-reacted FFAs and polar lipids leading to the low FAME yield. Also the product obtained with KSF contains nonreacted FFAs and polar lipids. Based on the above results, the study continued only with ion-exchange resins, to study the influence of the (catalyst/microalga) mass ratio (Figure 2). This parameter was analysed to test its influence on the FAME yield, denoting that (catalyst/microalga) mass ratios around 1 were enough to obtain FAME yields of 90 ± 0.5 mol%. Good results were obtained too by Tesser et al. [38] in the fatty acids esterification but lower (catalyst/oil) ratio was required. As explained before, FAME production was carried out with and without previous lipid extraction following the two-step process (extraction + reaction) and the direct biomass transformation, respectively. In both cases, esterification/transesterification reactions were done with ion exchange resins as catalysts. Figure 3 shows the results achieved with both methods, in-situ reactions provided a FAME yield around 80 mol %, slightly lower than previous values reached with the two-step method. This is a very interesting result since the one-step method is a very promising alternative for the synthesis of FAMEs from microalga biomass, avoiding the previous costly solvent extraction step. In addition, these catalysts are commercial ones, easily obtained, and present good results in the FAME production under mild reaction conditions.

After the reactions, a study of the ion-exchange resin reutilization and regeneration was carried out. These results are illustrated in Figure 4. The FAME yield decreased significantly in the second use of these resins after 4 hours of reaction (FAME yield = 10 ± 0.4 wt%). Thus, a washing with methanol was done, but catalysts did not recover their activity. Therefore, catalyst deactivation is not due to organic fouling inside the resin structure. A second washing adding HCl was necessary to regenerate resins acid sites and recover initial FAME yield. Thus, resins deactivation may be caused by the exchange between the H+ and metals like Na and K contained into the microalga as evidenced in the lipid characterisation section.

4. Conclusions The oil fraction (42 wt %) of Nannochloropsis gaditana microalga has been extracted with methanol containing mainly saponifiable lipids (86.7 wt %), with a high concentration of polar lipids and FFAs (~22 wt%). The fatty acid profile evidenced the presence of polyunsaturated fatty acids like eicosapentaenoic (20:5) frequently found in microalgae oils. The conversion of extracted lipids with ion-exchange resins (Amberlite-15, CT-275 and CT-269) gave FAME yields above 90 mol % because of their high number of acid sites. On line with this, KSF clay only reached FAME yields of 67 mol % and the lower acidity of silica-alumina does not allow to get FAME yields above 18.2 mol%. So, ion exchange resins presented high activity in the esterification/transesterification of Nannochloropsis gaditana lipids. Despite this, lower FAME yields were achieved (around 80 mol %) in the direct biomass conversion. However, this one-step method is a very promising alternative for the synthesis of FAMEs from microalga biomass, avoiding the costly solvent extraction and recovery steps. Unfortunately, ion-exchange resin catalysts were deactivated after the reaction and it was necessary to wash them with methanol and hydrochloric acid to regenerate their acidity and recover the activity of these resins.

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Table captions.

Table 1. Composition of the lipids extracted from N. gaditana. Table 2. Fatty acid composition in the saponifiable lipids and fatty acids extracted from N. gaditana. Table 3. Physicochemical properties of the catalysts. Table 4. Product distribution obtained with KSF clay, ion exchange resins ( A-15, CT-275 and CT-269), and the SiAl catalyst. FFAs: free fatty acids; FAMEs: fatty acid methyl esters; PL: polar lipids; OT: others (carotenoids, sterols, sterol esters and non-lipid compounds).

Table 1 Compounds

Type of lipid

FFAs and saponifiable lipids

FFAs

22.00 ± 2.4

Monoglycerides

0.40 ± 0.01

Diglycerides

0.55 ± 0.03

Triglycerides

0.24 ± 0.01

Sterol esters

4.57

Polar lipids

58.92 ± 1.2

Total FFAs and saponifiable lipids

Non-saponifiable lipids

Concentration (wt%)

0.2

86.68 ± 3,85

Carotenoids

1.10 ± 0.04

Sterols and tocopherols

1.08 ± 0.02

Retinoids Total non-saponifiable lipids

Non-lipidic compounds

n.d. 2.18 ± 0.06 11.14 ± 0.6

Table 2

Acidic Value (mg KOH/g)

43.7

Saponifiable Value (mg KOH/g)

106.8

Iodine Value (g I2/100 g)a

161*

Fatty Acid Composition Fatty acid

Concentration (wt%)

Myristic

14:0

4.4 ± 0.5

Myristoleic

14:1

2.1 ± 0.6

Palmitic

16:0

24.6 ± 0.9

Palmitoleic

16:1

32.4 ± 1.1

Stearic

18:0

0.8 ± 0.1

Oleic

18:1

3.9 ± 0.1

Linoleic

18:2

2.3 ± 0.5

Linolenic

18:3

2.1 ± 0.6

Arachidonic

20:4

2.4 ± 0.2

Eicosapentaenoic

20:5

16.9 ± 0.9

Erucic

22:1

8.0 ± 0.3

a

Measured only from saponifiable lipids

Table 3

Catalyst

SBET (m2/g)

Vpore (cm3/g)

D pore (nm)

Acidity (mmol H+ /g)

A-15

42

0.35

33.5

5.6

CT-275

23

0.19

32.2

5.7

CT-269

64

0.57

58.5

6.3

KSF

8

0.02

5.5

3.6

Silica-alumina (SiAl)

370

1.29

19.4

0.5

Table 4. FFAs

PL

FAMEs

OT

(wt %)

(wt %)

(wt %)

(wt %)

Catalyst

A-15

1.7 ±0.1

8.5 ±0.8 85.2 ±1.3 4.6 ±0.5

CT-269

1.2 ±0.2

8.7 ±0.6 86.2 ±1.3 3.9 ±0.3

CT-275

1.9 ±0.4 10.9 ±1.0 84.2 ±1.8 3.0 ±0.5

KSF

20.2 ±0.7 16.8 ±0.6 57.9 ±2.4 5.1 ±0.2

SiAl

43.6 ±1.5 20.1 ±1.0 17.5 ±0.9 18.8 ±1.2

Figure captions

Figure 1. FAMEs yield obtained in the esterification of Nannochoropsis gaditana microalgal oil with A-15, CT-275, CT-269 resins, KSF clay and SiAl catalysts. T = 100 ºC, methanol/oil = 40 w/w, catalyst/oil = 0.8 w/w, t = 4 h and 1000 rpm. Figure 2. Influence of the catalyst/oil ratio in the FAMEs yield obtained in the esterification of Nannochoropsis gaditana microalgal oil with ion-exchange resins CT-275, CT- 269 and A-15. T = 100 ºC, methanol/oil = 40 w/w, t = 4 h and 1000 rpm. Figure 3. FAMEs yield obtained in the esterification of Nannochoropsis gaditana microalgal oil with ion-exchange resins CT-275, CT- 269 and A-15. Comparison between indirect (extraction + reaction) and direct (in-situ reaction) methods. T = 100 ºC, methanol/oil = 40 w/w, catalyst/oil = 0.8 w/w, t = 4 h and 1000 rpm.

Figure 4. FAMEs yield obtained in the in-situ reaction of Nannochoropsis gaditana microalgae biomass with CT-275, CT-269 and A-15 resins. Study of catalysts reutilization. T = methanol/oil = 40 w/w, catalyst/oil = 0.8 w/w, t = 4 h and 1000 rpm.

Fig. 1

Fig. 2

100 ºC,

Fig. 3

Fig. 4