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Article 1
Biodiesel production (FAEEs) by heterogeneous 2
combi-lipase biocatalysis using wet extracted lipids 3
from microalgae 4
Alejandra Sánchez-Bayo 1, Victoria Morales 2, Rosalía Rodríguez 1, Gemma Vicente 1 and Luis 5
Fernando Bautista 2,* 6
1 Department of Chemical, Energy and Mechanical Technology. ESCET, Universidad Rey Juan Carlos, 28933, 7
Móstoles, Madrid, Spain; alejandra.sanchezbayo@urjc.es (ASB), rosalia.rodriguez@urjc.es (RR), 8
gemma.vicente@urjc.es (GV) 9
2 Department of Chemical and Environmental Technology. ESCET, Universidad Rey Juan Carlos, 28933, 10
Móstoles, Madrid, Spain; victoria.morales@urjc.es (VM), fernando.bautista@urjc.es (FB) 11
* Correspondence: fernando.bautista@urjc.es (FB); Tel.: +34-914-888-501 12
13
Abstract: The production of fatty acids ethyl esters (FAEEs) to be used as biodiesel from oleaginous 14
microalgae shows great opportunities as an attractive source for the production of renewable fuels 15
without competing with human food. To ensure the economic viability and environmental 16
sustainability of the microbial biomass as a raw material, the integration of its production and 17
transformation into the biorefinery concept is required. In the present work, lipids from wet Isochrysis 18
galbana microalga were extracted with ethyl acetate with and without drying the microalgal biomass 19
(dry and wet extraction method, respectively). Then, FAEEs were produced by lipase-catalyzed 20
transesterification and esterification of the extracted lipids with ethanol using lipase B from Candida 21
antarctica (CALB) and Pseudomonas cepacia (PC) lipase supported on SBA-15 mesoporous silica 22
functionalized with amino groups. The conversion to FAEEs with CALB (97 and 85.5 mol% for dry 23
and wet extraction, respectively) and PS (91 and 87 mol%) biocatalysts reached higher values than 24
those obtained with commercial Novozym 435 (75 and 69.5 mol%). Due to the heterogeneous nature 25
of the composition of microalgae lipids, mixtures with different CALB:PC biocatalyst ratio were used 26
to improve conversion of wet-extracted lipids. The results showed that a 25:75 combi-lipase produced 27
a significantly higher conversion to FAEEs (97.2 mol%) than those produced by each biocatalyst 28
independently from wet-extracted lipids and similar ones than those obtained by each lipase from 29
the dry extraction method. Therefore, that optimised combi-lipase biocatalyst, along with achieving 30
the highest conversion to FAEEs, would allow improving viability of a biorefinery since biodiesel 31
production could be performed without the energy-intensive step of biomass drying. 32
Keywords: FAEEs, biodiesel, mixed biocatalysts, lipases, microalgae. 33
34
1. Introduction 35
Nowadays, we are facing a major energy crisis not only caused by the decline of fossil reserves 36
but also by the problems caused by their use. Therefore, there is a need to look for new sources of 37
cleaner, safer and renewable energy. Currently, the research is focuses in obtaining advanced biofuels 38
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to ensure the economic viability and environmental sustainability of biomass as a raw material1. 39
Oleaginous microalga are promising species that can accumulate large amount of lipids (> 20 wt% of 40
their biomass). They constitute an attractive source for the production of biofuels such as biodiesel 41
without competing with human food2. 42
Biodiesel can be generated from microalga oil that is esterified or transesterified with methanol 43
or ethanol into methyl or ethyl esters of fatty acids (FAMEs or FAEEs, respectively) in the presence 44
of a catalyst. Due to its high content of free fatty acids, the most common way to carry out the 45
transesterification and esterification reactions of the lipids extracted from microalgae is by using acid 46
catalysts. This generates certain disadvantages not only in the process, such as high energy 47
consumption, corrosion of materials and the difficulty of transesterifying triglycerides, but also in the 48
post-reaction treatments, such as the recovery of the catalyst 3. In recent years, new catalysts based 49
on enzymes (biocatalysts) are being developed to avoid these problems4, achieving better selectivity 50
and specificity. Immobilised lipases can be an alternative because they are capable of carrying out the 51
transesterification and esterification reactions at lower temperatures energy cost, facilitating the 52
recovery of the catalyst and the purification of glycerol5. However, the main problem of biocatalysts 53
is the presence of water in the reaction medium that can cause the hydrolysis of esters. In addition, 54
the use of certain solvents such as methanol can lead to inactivation of the catalyst6,7. 55
Due to the high cost of enzymes, the use of heterogeneous biocatalysts is required for the 56
economic viability of the whole process since they can be reused. In addition, the immobilization of 57
enzymes in solid carriers can increase thermal and chemical stability and protects enzyme molecules 58
from denaturation. There are different techniques of enzymatic immobilization: binding to the 59
support, confinement or encapsulation and cross-linking8. The most common technique is to bind the 60
enzyme onto the support by covalent or ionic attachment or by physical adsorption9. To carry out 61
this immobilization, materials such as mesoporous silicas (SBA-15, SBA-16, MCM-41, FDU-12)10–14 or 62
carbon nanotubes15,16 have been used. The above materials have large pores or cavities where the 63
enzymes can be housed. However, it is usually necessary to modify the surface of support to achieve 64
a better anchorage by functionalization with amine, chloride, sulfur or phenol groups10,14,17–20. 65
Because of the high specificity of lipases for different substrates, catalytic activity of lipases 66
depends on the source organism producing the enzyme. Most of biodiesel production from 67
microalgal oil has been done with lipases from Pseudomonas cepacia16,21, P. fluorescens16,22, Thermomyces 68
lanuginosus16,23, Candida rugosa16,24 and C. antarctica11,16. The last one is frequently used in the form of 69
the commercial catalyst Novozym 4356,16,25,26. Although most of studies reported the use of single 70
lipases, some authors showed that the yield of FAEE could increase when some lipases are used in 71
combination5,21. Therefore, the concept of combi-lipase biocatalysts have been recently developed to 72
improve the biodiesel production processes27–29. However, the previous studies using combi-lipase 73
systems used oils from vegetable crops such as palm, soybean or coffee, but they did not used the 74
more complex microalgal oil as a source for biodiesel. 75
In this work, biodiesel (FAEEs) were produced using enzymatic catalysts from lipids extracted 76
with ethyl acetate from both wet and dry biomass from microalgae. For this purpose, two enzymes 77
from different origin were assessed: lipase B from the fungus Candida antarctica (CalB) and lipase 78
from the bacterium Pseudomonas cepacia (PC) which were supported on amino-functionalized SBA-79
15. The results were compared to those obtained with the commercial Novozym 435® catalyst. In 80
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addition, both enzymes were combined in different proportions to evaluate the synergistic activity 81
and selectivity towards biodiesel production. 82
2. Results and Discussions 83
2.1. Characterization of the synthesized enzymatic catalyst 84
This section contains the most relevant results of the synthesis of the mesoporous silica material 85
SBA-15 as well as the modifications thereof when incorporating amino groups, glutaraldehyde and 86
the enzymes. 87
The textural properties are shown in Table 1. The pore surface and diameter are reduced slightly 88
after functionalization of the surface with amino groups and further glutaraldehyde linkage as 89
spacer-arm to favor the subsequent binding of the enzyme. Before functionalization, pore size of SBA-90
15 was 59.6 Å, and the surface area was the largest, 847.2 m2/g, which decreased to 46.3 Å and 841.8 91
m2/g, respectively, as chain length increased after amine group and glutaraldehyde introduction 30. 92
However, pore volume and surface area were drastically reduced with the introduction of enzymes, 93
both properties decreasing by ~70% with respect to the original SBA-15. 94
Table 1. Textural properties and enzyme fixed with synthesized mesoporous materials. 95
Material SBET (m2/g) 1 DP (Å) 2 VP (cm3/g) 3 Protein/material (mg/g)
SBA-15 847.3 59.6 1.307 -
SBA-15-NH2 843.2 58.5 1.233 -
SBA-15-NH2G 841.8 56.3 1.186 -
SBA-15-NH2G-PC 275.4 55.0 0.378 35
SBA-15-NH2G-CalB 262.3 56.2 0.393 33
1 BET surface area, 2 BJH pore diameter, 3 Pore volume at P/P0 = 0.95. 96
The amount of enzyme loaded in each biocatalyst was determined by mass balance measuring 97
the enzyme concentration in the supernatant solution using the Bradford´s assay. The immobilization 98
values were 35 and 33 mg/g for SBA-15-NH2G-PC and SBA-15-NH2G-CalB, respectively, similar to 99
those obtained by Bautista et al.4 (36.1 mg/g) and slightly lower than that obtained by Serra et al. (44 100
mg/g) 31, both using CalB as enzyme. The slighting higher incorporation enzyme in the case of PC 101
may be due to the fact that its size is somewhat smaller (3 x 3.2 x 6.6 nm)32 and can enter into the pores 102
more easily than CalB (3 x 4 x 6 nm)33. 103
Regarding the structure, XRD (Figure 1) shows three reflection peaks at 2θ=0.97°, 1.6° and 1.9° 104
corresponding to p6mm mesoporous hexagonal symmetry (planes (100), (110) and (200), 105
respectively)34. Based on the results obtained, the immobilization of enzymes covalently bound to the 106
material does not modify its structure10. 107
Figure 2 shows TEM images obtained from SBA-15 (a) and modified material with anchored 108
lipases from PC (b) and CalB (c). It is clearly noted that both samples show the arrays of long-range 109
mesopore channels, which are similar to the image of SBA-15 reported in the literature20. When the 110
SBA-15 contains enzymes partially occupying the interior of its pores, it can appreciated with the 111
different degrees of sharpness of the channels, a morphology similar to that observed by Mohammadi 112
et al.35. However, as explained by Abdullah et al.12, the fact that the modification of the surface of the 113
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SBA-15 material was made by grafting, a posteriori, prevents major structural changes from taking 114
place and only intervening in the formation of new links with Si-O-Si that are on the surface. 115
116
Figure 1. X-ray diffractograms of the mesoporous materials. 117
118
(a) (b) (c)
Figure 2. TEM micrographs: (a) SBA-15, (b) SBA-15-NH2G-PC, (c) SBA-15-NH2G-CalB. 119
The 29Si NMR spectrum (Figure 3) of pure silica shows one wide signal within the range -90 to -110 120
ppm that correspond to the Si(OSi)4 (Q4), HOSi(OSi)3 (Q3) and (HO)2Si(OSi)2 (Q2) sites of the silica 121
framework36. Around a displacement -60 to -78 ppm, a broad signal can be seen that corresponds to 122
two signals created by the Si-C links corresponding to RSi(OSi)3 (T3) and RSi(HO)(OSi)2 (T2)37. These 123
last two signals are due to the incorporation of the organic part (CH3CH2CH2NH2) by linking on the 124
surface of the material. The peak areas were calculated after Gaussian deconvolution of the spectra 125
considering the associated species and the respective intensities. The results of the integrations 126
(Table 2) show that the greatest contribution to the areas is given by the peak corresponding to a Si 127
atom linked to four Si-O structures, i.e., corresponding to the Q4 signal. Overlapped with this peak, 128
two signals of smaller area corresponding to the formation of one or two Si-OH bonds, signals Q3 129
and Q2 respectively. The T signals appear when the surface of the material is modified with the 130
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previously mentioned organic group and can be observed in the spectrum into overlapping signals 131
of areas similar one each other. These results confirm that the structure is not modified when both 132
organic modifiers and lipases are introduced38. 133
134
Figure 3. 29Si NMR spectra of the different materials. 135
Table 2. Relative peak area in the NMR-29Si spectra 136
Material Q4 (%) Q3 (%) Q2 (%) T3 (%) T2 (%)
SBA-15 83.8 13.4 2.77 - -
SBA-15-NH2 66 10.2 3.42 10.32 10.23
SBA-15-NH2G 64.5 13.4 2.41 9.78 9.93
SBA-15-NH2G-PC 68.5 7.9 2.8 10.76 9.96
SBA-15-NH2G-CalB 63.9 7.8 2.49 11.87 12.21
137
The elemental analysis shows how the composition of hydrogen, carbon, nitrogen and oxygen 138
varies as new functional groups, amino and aldehyde are introduced into the structure, as well as 139
with the incorporation of enzymes. The results shown in Table 3 are coherent since the initial 140
mesoporous structure contains only silicon and hydrogen. The presence of nitrogen, carbon and 141
oxygen are due to the incorporation of the different functional groups in the SBA-15. The introduction 142
of the amino group from APTES increases the nitrogen concentration to ~2.8%, but the incorporation 143
of glutaraldehyde decreases the nitrogen content to 2.2%, due to the presence of carbon, hydrogen 144
and oxygen into the structure. Similar results are reported previously for different functionalization 145
of SBA-15 materials19. Conversely, an increase of all the elements is observed after lipase 146
immobilisation in SBA-15NH2G-PC and SBA-15NH2G-CalB because the incorporation of the lipase 147
molecules added N, C and H, as expected. 148
149
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Table 3. Elemental analysis of synthesized mesoporous materials 150
Material N (%) C (%) H (%) S (%)
SBA-15 0.00±0.00 0.00±0.00 0.19±0.01 n.d.
SBA-15NH2 2.77±0.01 8.01±0.04 1.97±0.02 n.d.
SBA-15NH2G 2.2±0.1 15.1±0.5 2.23±0.09 n.d.
SBA-15NH2G-PC 2.59±0.02 16.8±0.2 2.71±0.04 n.d.
SBA-15NH2G-CalB 2.49±0.01 21.07±0.02 3.28±0.02 0.01±0.01
n.d: not detected (lower than the detection limit) 151
After water removal at T<100°C, thermogravimetric analysis shows a weight loss between 200 152
and 300°C that corresponds to glutaraldehyde followed by the loss of the amino group at 153
temperatures between 300 and 600°C (Figure 4). The results of this analysis show an organic loss, 154
corresponding to propylamine (CH3CH2CH2NH2), of 11.8 wt% in all the materials. The nitrogen 155
content present in the material SBA-15-NH2 according to this test (2.8 wt%) corroborates the results 156
of elemental analysis obtained (Table 3). 157
A weight loss of 7.48% is observed in the material SBA-15-NH2G, which implies an incorporation 158
of glutaraldehyde in 37 wt% with respect to the amount of available amino groups. This indicates 159
that part of amino groups incorporated into the material did not form bonds with glutaraldehyde. In 160
addition, this test has allowed to corroborate the results of nitrogen content (2.35 wt%) with that 161
obtained by elemental analysis (2.23 wt%). 162
163
Figure 4. Thermogravimetric analysis of SBA-15 and functionalized materials. 164
2.2. Production of FAEEs using single lipase biocatalysts 165
The activity of both synthesized biocatalysts (SBA-15-NH2G-CalB and SBA-15-NH2G-PC) 166
towards FAEEs production from wet and dry extracted lipids from I. galbana was evaluated and 167
compared with the commercial Novozym 435. The results were assessed based on the molar 168
conversion of saponifiable lipids measured by 1H NMR (Table 4). As it can be seen, the lipid extraction 169
method had a large impact on lipase-catalysed conversion to FAEEs. When the microalgal oil, used 170
as feedstock, was extracted by the wet method, the results showed a reduction in FAEEs production 171
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from 97 to 85.5 mol% and from 91 to 87 mol% using CalB and PC lipase-based biocatalysts, 172
respectively. The commercial Novozym 435 also showed a negative effect when lipids extracted by 173
the wet method was used. Microalgal oils from both the wet and dry extraction methods did not 174
contain water. Therefore, the decrease in conversion may be caused by the extraction of water-soluble 175
lipase-inhibitor compounds during the wet route that are not present in the oil extracted from dry 176
biomass, because lipase activity it is negatively affected by polar compounds that can cause inhibition 177
or denaturation 39. 178
Table 4. Conversion to FAEEs using single lipases 179
Lipid extraction method Catalyst Conversion (mol%)
Dry
CalB 97±1
PC 91±2
N435 75±2
Wet
CalB 85.5±0.8
PC 87±2
N435 69.5±0.6
180
The highest conversion to FAEEs (97 mol%) using dry extracted lipids with ethyl acetate from I. 181
galbana microalgae was achieved using SBA-15-NH2G-CalB as biocatalyst. For the wet extraction 182
route, both CalB and PC lipase-based biocatalysts showed similar activity (85.5 and 87 mol%, 183
respectively) so that SBA-15-NH2G-CalB seems to be more sensitive to the presence of water or other 184
polar compounds likely extracted during the wet process. However, in all cases, the synthesised 185
biocatalysts SBA-15-NH2G-CalB and SBA-15-NH2G-PC were significantly more active towards 186
FAEEs production than the commercial Novozym 435, proving the better performance of both 187
biocatalysts synthesised in the present work. 188
2.3. Production of FAEEs using combi-lipase biocatalysts 189
In recent years, the search for new biocatalysts calls for the combination of different enzymes of 190
different specificity to produce a higher production yield of FAEEs27,40. Consequently, a biocatalyst 191
formed by the combination in different proportions of CalB and PC supported on SBA-15 modified 192
with amino groups and glutaraldehyde is used in order to evaluate the FAEEs production in the same 193
conditions used previously, using the lipids extracted by wet route with ethyl acetate as raw material. 194
Table 5. Conversion to FAEEs using combi-lipases. 195
CalB:PC lipase ratio Conversion (mol%)
0:100 87±2
25:75 97.2±0.5
50:50 81±2
75:25 89.7±0.3
100:0 85.0±0.8
196
Table 5 shows the synergistic effect of different combi-lipases on the conversion to FAEEs, along 197
with those values achieves with the corresponding single-lipase biocatalysts It is observed that the 198
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combi-lipase 25:75 produced the highest conversion to FAEEs (97.2 mol%) which is more than 10% 199
higher than biocatalysts formed by CalB or PC alone. This is a remarkable result because it 200
demonstrates that it is possible to design a catalyst that contains an optimized mixture of different 201
lipases that maximizes the conversion to FAEEs. 202
Table 6. Fatty acid profile of FAEEs obtained with combi-lipase 25:75 SBA-15-NH2G-CalB:SBA-15-NH2G-PC. 203
Fatty acid Composition (%)
Myristic C14:0 9.0
Palmitic C16:0 11.3
Palmitoleic C16:1 3.6
Stearic C18:0 0.6
Oleic C18:1 14.5
Linoleic C18:2 5.1
Linolenic C18:3 20.2
Arachidic C20:0 0.5
Behenic C22:0 18.2
Erucic C22:1 0.2
Lignoceric C24:0 16.8
Saturated 56.3
Monounsaturated 18.3
Polyunsaturated 25.4
204
The fatty acid profile of FAEEs produced by the optimised 25:75 SBA-15-NH2G-CalB:SBA-15-205
NH2G-PC is shown in Table 6. The major saturated fatty acids are myristic (C14:0) and palmitic 206
(C16:0) while palmitoleic (C16:1) and oleic (C18:1) acids represented the main monounsaturated and 207
linolenic (C18:3) the most abundant polyunsaturated fatty acid. It is important to highlight the value 208
of linolenic acid (20.2%), a concentration about 10% higher than that regulated by EN 14214 standard 209
(Table 7). This would require further actions after biodiesel production, such as mixing with biodiesel 210
from other lipid sources whose linolenic acid content is lower than that of the microalgae used in this 211
work, to meet the required specifications 212
Table 7. Biodiesel properties obtained by the combi-lipase 25:75 SBA-15-NH2G-CalB:SBA-15-NH2G-PC 213
Property EN 14214 Combi-lipase 25:75
Iodine value (g I2/ 100 g) <120 72
Group I metals (Na, K) (mg/kg) <5 179.1
Group II metals (Ca, Mg) (mg/kg) <5 205.1
Phosphorous (mg/kg) <10.0 250.6
Sulfur (mg/kg) <10 0
Monoglycerides (%) <0.7 0.6
Diglycerides (%) <0.2 <0.1
Triglycerides (%) <0.2 <0.1
Linolenic esters (%) <12 20.2
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Other key properties of the biodiesel produced by the combi-lipase biocatalyst 25:75 SBA-15-214
NH2G-CalB:SBA-15-NH2G-PC using the lipids obtained after wet extraction with ethyl acetate are 215
shown in Table 7. Iodine value as well as mono, di and triglyceride content and sulfur content fulfill 216
the European standard EN 14214. However, metals and phosphorus content are higher than those 217
regulated by the above standard, which would require a further purification stage. 218
2.4. Study of the reuse of the heterogeneous enzymatic catalyst 219
As previously mentioned, one of the key features of heterogeneous catalysts for their economic 220
viability is their possible recovery and reuse in order to reduce the costs of the biodiesel production 221
process41. Therefore, after each reaction, the catalyst was washed with 3 mL of ethanol and dried in 222
order to remove possible impurities that could affect the conversion42. The combi-lipase 25:75 SBA-223
15-NH2G-CalB:SBA-15-NH2G-PC was reused for 10 consecutive cycles under the same operating 224
conditions (Figure 6). 225
226
Figure 6. Reuse of combi-lipase 25:75 SBA-15-NH2G-CalB:SBA-15-NH2G-PC. 227
The results showed that the conversion was above 80 mol% during the first five cycles, 228
decreasing by 10% with respect to the first reaction. Subsequently, the conversion gradually 229
decreased, reaching a conversion of around 70 mol% after the tenth reuse These results are 230
comparable although better than others reported in the literature for the lipase from P. cepacia where 231
after the first use, 10% of the activity was lost and in the fourth cycle the activity was reduced by 232
53%43. This loss of activity can be caused by a possible deterioration of the biocatalyst by enzyme 233
poisoning44. 234
235
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3. Materials and methods 236
3.1. Microalga 237
The microalga used in this work was Isochrysis galbana and it was supplied by AlgaEnergy S.A. 238
(Spain). This strain has been selected due to its high lipid content (33.5 wt%), which makes it favorable 239
for the production of FAEEs. 240
3.2. Lipid extraction 241
The extraction of lipids from the microalga, in the dry biomass procedure, was carried out with 242
a dry microalga:solvent ratio of 1:20, using ethyl acetate as solvent. The samples were stirrer 243
vigorously with the help of a vortex (Finecorp, Korea) for 5 min at 11000 rpm at room temperature to 244
obtain the lipids contained in the microalgae. In the case of the extraction of lipids from wet biomass, 245
the same procedure was carried out starting from a suspension of microalga in water with a 246
concentration of 50 g/L. 247
3.3. Synthesis of lipase-based biocatalysts 248
The assays for the production of FAEEs were carried out using lipase B from Candida antarctica 249
(CalB) and lipase from Pseudomonas cepacia (PC), both from Sigma-Aldrich (USA), supported on SBA-250
15 modified with amino groups and glutaraldehyde following protocols previously described45–47 251
(Figure 7). 252
In a typical synthesis for SBA-15, 8 g of block copolymer surfactant Pluronic 123 (Sigma-Aldrich) 253
were dissolved at room temperature under stirring in 250 mL of 1.9 M HCl (Scharlab. Spain). The 254
solution was heated up to 40 °C and 17.8 g of TEOS (tetraethyl orthosilicate, Sigma-Aldrich) were 255
added to the solution. The resultant mixture was then stirred at that temperature for 20 h and 256
hydrothermally aged at 110 °C for further 24 h. The template was removed by calcination at 550 °C 257
for 5 h at a heating rate of 1.8 °C/min46. 258
The protocol for covalent immobilization of lipases (Fig. 7) was adapted from Wang’s 259
procedure37. Thus, 2 g of calcined mesoporous material SBA-15 were immersed into a solution of 1 g 260
of APTES (n-aminopropyltriethoxysilane, Sigma-Aldrich) in 30 mL of anhydrous toluene (Sigma-261
Aldrich). The mixture was refluxed at 110 °C for 24 h under inert nitrogen atmosphere. Then, the 262
suspension was filtered and washed 3 times with anhydrous toluene. The solid was placed in glass 263
vials and vacuum dried for 24 h at 110 °C, yielding the amine-functionalized support named SBA-15-264
NH2. Then, 1 g of SBA-15-NH2 was blended with 1 mL of 25 vol% aqueous glutaraldehyde (Sigma-265
Aldrich) and 9 mL of 0.1 M phosphate buffer (Sigma-Aldrich) for 2 h. The solid material (SBA-15-266
NH2G) was filtered and washed with phosphate buffer. 267
The functionalized materials were used as carriers for lipase immobilization. 100 mg of amino-268
functionalized and glutaraldehyde linked mesoporous material was blended with 1 mL of lipase in 269
4 mL of 0.1 M phosphate buffer at pH=7.0 and the mixture was shaken at 200 rpm and 25°C for 3 h. 270
Then, the material was filtered and washed three times with 5 mL phosphate buffer. The final 271
biocatalysts produced were named as SBA-15-NH2G-CalB and SBA-15-NH2G-PC, containing 272
immobilized CalB and PC lipase, respectively. 273
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274
Figure 7. Synthesis of the enzymatic catalyst supported on SBA-15 275
3.4. Characterization of biocatalysts 276
The evaluation of the textural properties of SBA-15 mesoporous materials was performed by N2 277
adsorption/desorption isotherms in a Tristar model 3000 equipment (Micromeritics. USA). Prior to 278
the analysis, a degasification stage is carried out based on the controlled heating of the samples up to 279
250°C and 120°C, for inorganic and organically functionalized samples, respectively, and the use of 280
a nitrogen current in a SmartPrep type degasser. The specific surface area was calculated by the 281
Brunauer-Emmett-Teller (BET) method, the pore diameter was measured through the Barret-Joyner-282
Halenda technique (BJH) and pore volume was determined by using the Harkins-Jura technique. 283
X-ray diffraction experiments were performed in a X’Pert MPD (Philips. The Netherlands) using 284
monochromatic Cu Kα radiation, with a wavelength of 1.54 Å. The step size was 0.02° with an 285
accumulation time per step of 5 seconds for a sweep of angles 2θ of 5–50°. 286
Transmission electron microscopy (TEM) analysis was performed with a Tecnai 20 microscope 287
(Philips. The Netherlands) with a resolution of 0.27 nm and ±70° inclination of the sample, equipped 288
with an EDX detector. 289
29Si NMR spectroscopy allow to determine the relationship between condensed and non-290
condensed silicon species, obtaining structural information about the mesoporous siliceous 291
material38. These experiments were performed in an Infinity AS400 NMR spectrometer (Varian. USA) 292
operating at 9.4 Tesla. 293
The content of hydrogen, carbon, nitrogen, sulfur and oxygen was measured in a Vario EL III 294
element analyzer (Elementar Analysensysteme GmbH. Germany) using sulphanilic acid as standard. 295
To evaluate the thermal stability of the SBA-15 materials, thermogravimetric analysis (TGA) 296
were carried out under inert atmosphere (nitrogen) in an 1100 TGA/DSC1 model (Mettler Toledo. 297
USA). The thermal analysis ranged from 40°C to 800°C with a ramp of 10 °C/min. 298
Finally, the amount of enzyme immobilized was calculated by mass balance measuring the 299
enzyme concentration in the supernatant solution using the Bradford´s assay with bovine serum 300
albumin (BSA) as standard48. All the analyses were performed in triplicate. 301
3.5. FAEEs production 302
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FAEE production experiments were carried out in 12 mL glass pressure tubes (Sigma-Aldrich). 303
0.1 g of extracted lipids , 2 mL of ethanol as solvent and 15 mg of the synthesized biocatalyst were 304
employed11. The reaction mixture was maintained at 50°C and 300 rpm for 24 h. After the reaction 305
time, the mixture was cooled to room temperature generating two liquid phases. The upper phase 306
containing FAEE was washed twice with 10 mL of water and then with 10 mL of hexane:diethylether 307
mixture (80:20). To carry out a comparative study, the commercial biocatalysts Novozym 435® (N345) 308
was used. Conversion of saponifiable lipids into FAEEs was measured by 1H-NMR analyses 309
performed in a Varian Mercury Plus 400 unit, following the procedure reported in the literature49. 310
FAEE production reactions were also carried out using combi-lipase biocatalysts with the 311
following SBA15-NH2G-CalB:SBA15-NH2G-PC ratios: 25:75, 50:50 and 75:25 wt%. Finally, successive 312
reactions were carried out under the same conditions to assess the reuse capacity. 313
3.6. FAEE characterization 314
The characteristics of the FAEE to be used as biodiesel have to be evaluated based on the 315
standard EN 14214, in which the protocol for determining the iodine value of the biodiesel obtained 316
is included. In addition, the content of mono-, di- and triglycerides was determined by thin layer 317
chromatography (TLC) following the method described by Vicente et al.2, where the sample was 318
diluted with hexane. The TLC plate was developed, stained with iodine and digitized. The software 319
Un-Scan-It Gel 6.1 (Silk Scientific Inc. USA) was used for the quantification of each lipid fraction using 320
the corresponding standards. The non-saponifiable extracted matter was determined by a 321
gravimetric procedure described elsewhere50. 322
The sulfur content was measured by elemental analysis in a Vario EL III elemental analyzer and 323
metals were determined by inductively coupled plasma atomic emission spectroscopy in a Vista AX 324
CCD simultaneous ICP-AES equipment coupled to a spectrophotometer (Varian Inc. Germany) 325
following EN 14538 (for Na, K, Ca and Mg) and EN 14107 (for P) standards, respectively. 326
4. Conclusions 327
Lipids from Isochrysis galbana extracted with ethyl acetate using both dry and wet biomass were 328
used to produce biodiesel (FAEEs) with heterogeneous lipase-based biocatalysts. Lipase B from 329
Candida antarctica (CalB) and lipase from Pseudomonas cepacia (PC) were covalently immobilised on 330
amino-functionalised SBA-15. Their catalytic activity was compared with that of the commercial 331
catalyst Novozym 435. The conversion to FAEEs using the synthesized biocatalysts SBA-15-NH2G-332
CalB (97 and 85.5 mol% for dry and wet extraction, respectively) and SBA-15-NH2G-PC (91 and 87 333
mol%) resulted in higher conversions for both dry and wet ones than those obtained with the 334
commercial catalyst N435 (75 and 69.5 mol%). Due to the heterogeneous nature of the lipid 335
composition of the microalgae, mixtures with different proportions of CALB:PC biocatalysts were 336
used to improve the conversion of wet extracted lipids. The results showed that a combi-lipase 337
biocatalyst 25:75 SBA-15-NH2G-CalB:SBA-15-NH2G-PC produced a conversion of FAEE (97.2 mol%) 338
significantly higher than those produced from wet extracted oil by biocatalysts containing a single 339
lipase and similar to the maximum conversion using oils from wet extraction. Therefore, the combi-340
lipase presented can help to improve the viability of a microalgal biorefinery since the biomass-341
drying step can be avoided for biodiesel production. 342
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Author Contributions: ASB performed the experimental work (synthesis of biocatalysts, catalytic experiments 343
and sample characterization) and made a first draft of the manuscript. VM and RR analyzed and discussed the 344
results of biocatalysts characterization and VM collaborated in the writing of the first draft of the manuscript. 345
FB and GV devised the experimental work, analyzed the catalytic tests and wrote the final version of the 346
manuscript. The listed authors have contributed substantially to this work. 347
Funding: The authors acknowledge the financial support from the projects S2013/ABI-2783 (Comunidad de 348
Madrid y FEDER “Una manera de hacer Europa”) and CTQ2013-44447-R (Ministerio de Economía y 349
Competitividad). 350
Conflicts of Interest: The authors declare no conflict of interest. 351
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