Production and characterization of biodiesel from oil of fish waste by enzymatic catalysis

Journal Pre-proof Production and characterization of biodiesel from oil of fish waste by enzymatic catalysis Jonny Ching-Velasquez, Roberto Fernández-Lafuente, Rafael C. Rodrigues, Vladimir Plata, Arnulfo Rosales-Quintero, Beatriz Torrestiana-Sánchez, Veymar G. TaciasPascacio PII:

S0960-1481(20)30295-0

DOI:

https://doi.org/10.1016/j.renene.2020.02.100

Reference:

RENE 13121

To appear in:

Renewable Energy

Received Date: 6 November 2019 Revised Date:

4 February 2020

Accepted Date: 25 February 2020

Please cite this article as: Ching-Velasquez J, Fernández-Lafuente R, Rodrigues RC, Plata V, RosalesQuintero A, Torrestiana-Sánchez B, Tacias-Pascacio VG, Production and characterization of biodiesel from oil of fish waste by enzymatic catalysis, Renewable Energy (2020), doi: https://doi.org/10.1016/ j.renene.2020.02.100. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.

CRediT author statement

Jonny Ching-Velasquez: Investigation. Roberto Fernández-Lafuente: Writing - Review & Editing. Rafael C. Rodrigues: Formal analysis. Vladimir Plata: Formal analysis: Arnulfo Rosales-Quintero: Resources. Beatriz Torrestiana-Sánchez: Conceptualization, Supervision. Veymar G. Tacias-Pascacio: Conceptualization, Supervision, Writing Original Draft.

Optimal conditions: 35 °C, 10 % w/w of biocatalyst content and 216 rpm. Oil from waste.

fish 75.3% of Biodiesel yield Methanol

Transesterification reaction Operating condition according to composite central design-response surface methodology. Lipase from Thermomyces lanuginosus immobilized on octadecyl methacrylate beads.

Fuel properties: density, viscosity, calorific value and cloud point meet the recommendations of the ASTM D6751.

1

Production and characterization of biodiesel from oil of fish waste by

2

enzymatic catalysis

3

Jonny Ching-Velasquez a, Roberto Fernández-Lafuente b, Rafael C. Rodrigues c, Vladimir

4

Plata a, Arnulfo Rosales-Quintero d, Beatriz Torrestiana-Sánchez

5

Tacias-Pascacio d, f,*

6

a

INTERFASE, Universidad Industrial de Santander, Calle 9 Carrera 27, Bucaramanga, Colombia.

7

b

Departamento de Biocatálisis. ICP-CSIC. Campus UAM-CSIC. Madrid. Spain.

8

c

Biocatalysis and Enzyme Technology Lab, Institute of Food Science and Technology, Federal

9

University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090, Porto Alegre, RS,

e,

* and Veymar G.

10

Brazil.

11

d

12

Panamericana Km. 1080, 29050 Tuxtla Gutiérrez, Chiapas, México.

13

e

14

Miguel Ángel de Quevedo 2779, 91897 Veracruz, México.

15

f

16

Lib. Norte Pte. 1150, 29039 Tuxtla Gutiérrez, Chiapas, México.

17

* Co-corresponding authors.

18

E-mail addresses: [email protected] (B. Torrestiana-Sánchez), [email protected]

19

(V.G. Tacias-Pascacio)

Tecnológico Nacional de México/Instituto Tecnológico de Tuxtla Gutiérrez, Carretera

Unidad de Investigación y Desarrollo en Alimentos, Instituto Tecnológico de Veracruz, Calzada

Facultad de Ciencias de la Nutrición y Alimentos, Universidad de Ciencias y Artes de Chiapas,

20 21

1

22

Abstract

23

The objective of this paper was to optimize, by composite central design coupled to response

24

surface methodology, the enzymatic biodiesel production from oil coming from fish waste, and to

25

characterize the obtained product. The lipase from Thermomyces lanuginosus immobilized on

26

octadecyl metacrylate beads was used as biocatalyst. Optimal conditions were temperature of 35 °C,

27

10 % w/w of biocatalyst content and 216 rpm of agitation rate. Under optimal conditions, an

28

experimental biodiesel yield of 75.3 % was obtained after 24 h of reaction time. The biodiesel

29

presented an acid value (0.9 ± 0.28 mg KOH/g) that was higher than the established limits, while

30

other parameters like density (0.89 ± 0.01 g/mL), viscosity (5.3 ± 0.004 mm2/s), calorific value

31

(38.1 ± 0.21 MJ/kg) and cloud point (10.5 ± 0.47 °C), complied with the recommendations of the

32

ASTM D6751 standard.

33 34 35 36 37 38

Keywords: oil from fish waste, biodiesel, lipase, transesterification, fuel properties, response

39

surface methodology.

40 41

Introduction

42

Biodiesel is defined as mono alkyl esters of long chain fatty acids derived from natural renewable

43

materials, such as vegetable oils or animal fats [1-5], which are used as fuel for vehicle engines [6-

44

9]. Biodiesel has a lower toxicity, a higher biodegradability, it poses an inherent lubricant potential, 2

45

higher flash points, negligible sulfur content and produces lower polluting emissions than diesel

46

from petroleum [10-15].

47

Transesterification is the most used reaction in biodiesel production; in this reaction an oil or fat

48

reacts with a short chain alcohol (methanol or ethanol) in the presence of a catalyst which can be

49

acid, basic or enzymatic [16-21]. Alkaline catalysts (KOH, NaOH or NaOCH3) are the most utilized

50

for biodiesel production [22-27]; however, these catalysts are highly sensitivity to the free fatty acid

51

(FFA) and water content of the oil. The presence of these impurities can neutralize the catalyst and

52

lead to the formation of soap, decreasing the reaction yields and complicating the subsequent

53

purification processes [19, 28].

54

The problems derived from the FFA content can be overcome by using acid catalysts (e.g., sulfuric

55

acid, hydrochloric acid, or sulfonic acid) [29-31]. However, the use of acid catalysts generates much

56

lower reaction rates and they are very sensitive to water content [32, 33]. In this sense, biocatalysis

57

emerges as a good alternative, because enzymes are more tolerant to variations in oil quality, their

58

energy demand is lower, and they can catalyze esterification and transesterification reactions. In

59

addition, their high selectivity avoids the generation of by-products, and product recovery is easier,

60

with a low amount of effluents in the purification process [34-38]. However, the use of enzymatic

61

catalysis is limited, mainly due to their higher cost compared to chemical catalysts, slower reaction

62

rate than that of the alkaline catalysts, and enzyme inhibition, and inactivation by the alcohols used

63

as substrates (mainly methanol) and by the glycerol produced in the reaction [39-42].

64

Among the enzymes, lipases are the most widely used in biodiesel production, either in solvent-free

65

systems [43-47] or using different co-solvents such as organic solvents (n-hexane, tert-butanol),

66

ionic liquids or supercritical fluids [48-51]. Enzyme immobilization has many advantages, among

67

these to solve the problem of the enzyme water-solubility and make feasible their use in successive

68

batches or in continuous processes [52, 53]. Nowadays, a proper immobilization should permit to

69

solve other enzymes limitations, e.g. increasing enzyme activity or enzymatic stability [54-57]; 3

70

purifying the enzyme and reducing inhibition, etc. [58-61]. In the specific case of lipases,

71

immobilization in very hydrophobic supports has proved to prevent the glycerol adsorption that can

72

make the biocatalysts inactive [54-57].

73

Lipase from Thermomyces lanuginosus (TLL) is one of the most used enzymes [62]. Tacias et al.

74

(2016), reported that the TLL immobilization on hydrophobic octadecyl methacrylate support

75

(LifetechTM ECR8806M from Purolite®) [63] greatly improves its performance in biodiesel

76

production from used cooking oils, generating higher yields than those obtained with preparations

77

of this and other enzymes (home-made or commercial) [64], and its performance is near to that of

78

homogeneous alkaline catalysis [65].

79

Biodiesel is usually produced from high cost edible vegetable oils such as sunflower, soya, rapeseed

80

or canola oils [65], which make it economically non-competitive when compared to petroleum

81

diesel. In addition, the use of edible raw materials in the production of fuels could generate

82

economic, social and environmental negative impacts, mainly in developing countries [16, 66-71].

83

This situation led to the search and use of alternative raw materials for the biodiesel production,

84

such as non-edible vegetable oils (castor oil, Jatropha, Karanja, cotton seed, neem, rubber seed and

85

others) [4, 72-78], used cooking oils [5, 79-81], fatty acids from algae [82-84] and animal fats [85-

86

89]. Animal fats are obtained mainly as by-products from meat animal processing facilities and by

87

the rendering process, and among these fish oil from fish industry is included [26, 30, 90-92].

88

Fish industry generates a huge amount of waste and their indiscriminate disposal represents a threat

89

to the environment [73, 93, 94]. In 2016 world fish production was around 171 million tons, of

90

which 88% was destined for human consumption and the rest was used for non-food products

91

(FAO, 2018). Approximately 76 million tons of fish waste (heads, tail, fins, viscera and skin) were

92

generated in that year. These residues of the fish industry contain approximately 40% - 65% of oil,

93

which can be converted into biodiesel by chemical catalysis [4, 92, 95, 96]. As far as we know, the

94

production of biodiesel from oil fish waste by enzymatic catalysis has not been reported to date. 4

95

This paper analyzes the feasibility of the new TLL biocatalyst, that have shown excellent properties

96

with other materials [64, 65], in the production of biodiesel utilizing methanol and oil from fish

97

waste as raw materials. The optimization of this kind of reactions must consider some covariance

98

between different variables, and thus the response surface methodology (RSM) in the reaction

99

design to increase the possibilities of success. The factors studied were temperature, biocatalyst

100

content and agitation rate, while the response was the FAME yield. Moreover, the properties of the

101

biodiesel obtained were determined and compared to UNE-EN 14214 and ASTM D6751 standards.

102

The success of this process would have a double environmental impact: reduction of the

103

contamination caused by the disposal of fish waste in nature, second, to produce a sustainable

104

combustible.

105 106

Materials and Methods

107

Materials

108

Lipase from T. lanuginosus (TLL) was purchased from Novozyme (México) and octadecyl

109

methacrylate support was kindly donated by Purolite ® ECR Enzyme Immobilization Resins

110

(Wales, UK). TLL was immobilized on octadecyl methacrylate following the method described

111

elsewhere [64]. The employed feedstock was a mixture of oils obtained from fish viscera as

112

described below. Methyl heptadecanoate from Sigma-Aldrich (St. Louis, MO, USA) has been used

113

as an internal standard and FAME mix (C4-C24) analytical standard from Supelco was used to

114

identify the peaks at different retention times. Methanol, heptane and other chemicals were of

115

analytical or HPLC grade supplied from Sigma-Aldrich (St. Louis, MO, USA).

116

Extraction and characterization of the oil from fish waste

117

The oil from fish waste was obtained from viscera of Mexican snook (Centropomus Poeyi), black

118

seabream (Spondyliosoma cantharus), king mackerel (Scomberomorus cavalla) and striped mojarra 5

119

(Eugerres plumieri) collected in a fish market located in Veracruz, Veracruz, México. Oil extraction

120

was carried out using 1 kg of fish viscera which was incubated with two volumes of water in a

121

metal container at high temperature (97 °C) for 1.5 h and 400 rpm. After this treatment, the mixture

122

was filtered in a polystyrene strainer and the filtrate was centrifuged at 4800 rpm for 15 min [95].

123

After centrifugation, the upper layer containing the oily phase was withdrawn with a micropipette

124

and finally, the oil obtained was dried in a vacuum oven at 60 ° C for 24 h, and then stored in an

125

amber flask at 4 ° C until its use. The fish oil was obtained with a yield of 75 % with respect to the

126

viscera fish weight.

127

The fish oil was analyzed in terms of some physicochemical properties. Moisture content, acid

128

value and saponification number were determined by AOAC (Association of Official Analytical

129

Chemist) methods [97]. In addition, density and viscosity determinations were carried out in a

130

Stabinger (model SVM 300) viscometer.

131 132

Fatty acid composition of oil from fish waste

133

Fatty acid composition was determined by gas chromatography-mass spectrometry (GC-MS) in an

134

Agilent Technologies chromatograph model 5975 inert XL Net Work GC system equipped with a

135

DB-WAX capillary column (60 m x 250 mm x 0.25 mm). Prior to injection, the oil from fish

136

waste was converted to its corresponding fatty acid methyl esters (FAME); briefly, 5 mg of each

137

sample was mixed with 0.2 mL of toluene and 0.4 mL of 1% H2SO4 in methanol. The samples were

138

heated at 80 ° C for 30 min and allowed to cool to room temperature. The FAME produced were

139

extracted by adding 1 mL of hexane [98], and then 1 µL of FAME samples were injected using a

140

1:100 split ratio. The oven temperature was programmed as follows: an initial temperature of 60°C

141

was maintained for 5 minutes and increased to 210 °C at 20°C per min, then increased to 213 °C at

142

1°C/min and finally increased to 225 °C at 20°C per min up to 225°C. The carrier gas was helium

6

143

with a constant flow rate of 1 mL/min, and the injector temperature was 250°C. Fatty acid

144

composition of fish oil were identified by comparing their mass spectral fragmentation patterns with

145

those of the similar compounds stored in the GC-MS system software database (NIST

146

spectral

147

expressed as the average percentage (%) of individual fatty acids with respect to the total

148

determined fatty acids [99].

149

Enzymatic transesterification reaction

150

Enzymatic transesterification reactions were carried out in 25-mL Erlenmeyer flasks in a New

151

BrunswickTM Excella® E24 – Incubator Shaker. The reaction mixture consisted of 2 g oil from

152

fish waste, 1% of distilled water (w/w of fish oil) and 3:1 methanol to oil from fish waste molar

153

ratio. Amounts of biocatalyst (previously dried at 40 °C for 24 h), reaction temperature and

154

agitation rate, were varied according to the experimental design (Table 1). After 24 h of reaction

155

time the reaction mixture was centrifuged in a Labnet Z306 A centrifuge at 4000 rpm at 4°C during

156

15 min. The upper phase, containing the methyl esters, was analyzed by gas chromatography as

157

described below.

Finder

2.0

Mass

Library, NIST/EPA/NIH). Relative oil composition percentages were

158 159 160

Determination of fatty acid methyl esters by gas chromatography

161

Fatty acid methyl esters content was determined by gas chromatography in an Agilent Technologies

162

gas chromatograph (model 5975C) coupled to a flame ionization detector (FID) and equipped with a

163

BD-EN14103 capillary column (30 m x 320 µm x 0.25 µm). 100 mg of each sample was accurately

164

weighed and mixed with 2 mL of an internal standard stock solution of methyl heptadecanoate in

165

heptane (10 mg/mL), and then 1 µL of sample was injected using a 50:1 split ratio. The oven

166

temperature was programmed as follows: starting at 120 °C and then increased to 220 °C at 4 °C 7

167

min-1 and held at 220 °C for 2 min, with a final increase up to 250 °C at 15 °C min-1 and held for

168

10 min. Total analysis time was 39 min and nitrogen was used as carrier gas at a flow rate of 2.0 mL

169

min- 1. Injector and detector temperatures were 250 °C and 300 °C, respectively [99]. The FAME

170

content was calculated using the compensated normalization method with internal standardization,

171

based on the European standard EN 14103.

172

Physicochemical properties of fatty acid methyl esters

173

Physicochemical properties of the FAME were measured according to the methods described in the

174

international standards. Density (EN ISO 3675), kinematic viscosity (EN ISO 3104), acid value (EN

175

14014), cloud point (ASTM D2500) and calorific value (ASTM D240) were determined.

176

Experimental design

177

A five-level three-factor Central Composite Design (CCD) was employed to optimize the reaction

178

conditions for biodiesel synthesis. Table 1 shows 17 runs of the three variables. The design was

179

made up of 8 factorial points, 6 axial points (two axial points on the axis of design variable) and 3

180

replications at the central point. In each case, the FAME content was determined. A second order

181

polynomial model was adjusted to the experimental data according to equation 1.

= 182 183 184

+

+

+

(1)

where Y represents the predicted response, βo represents the constant coefficient, represents the linear effect, squared effect, and

and

represents the interaction effect,

represents the

represent the independent factors.

185 186

Table 1. Matrix of Central Composite Design (CCD) experiments and the results of reaction yield.

8

187

Treatment

X1

X2

X3

FAME Content (%w/w)

1

30

5

250

72 ± 1.78

2

40

5

250

20 ± 1.24

3

30

10

250

76 ± 1.77

4

40

10

250

74 ± 1.06

5

30

5

350

70 ± 2.86

6

40

5

350

16 ± 0.05

7

30

10

350

76 ± 1.63

8

40

10

350

24 ± 2.16

9

27

7.5

300

61 ± 0.22

10

43

7.5

300

12 ± 2.90

11

35

3

300

22 ± 1.54

12

35

12

300

49 ± 0.21

13

35

7.5

216

66 ± 3.24

14

35

7.5

384

30 ± 0.77

15

35

7.5

300

65 ± 1.35

16

35

7.5

300

70 ± 1.45

17

35

7.5

300

71 ± 2.01

X1: Temperature; X2: Biocatalyst content; X3: Agitation rate

188 189

Statistical analysis

9

190

The experimental design and results analysis were carried out using Statistica 13.5 (Statsoft, Tulsa,

191

OK, USA). The statistical analysis of the model was made by the analysis of variance (ANOVA).

192

The significance of the regression coefficients and the associated probabilities, p(t), were

193

determined by Student’s t-test; the second-order model equation significance was determined by

194

Fisher’s F-test. The variance explained by the model is given by the multiple determination

195

coefficients, R2. For each variable, the quadratic models were represented as contour plots (2D).

196 197 198

Results and discussion

199

Extraction and characterization of the oil from fish waste

200

Physicochemical characteristics of oil from fish waste are presented in Table 2. As it can be seen,

201

density and saponification number are very similar to the ones reported by other authors; however,

202

water content and acid value are much higher than those found in another paper [7]. This can be due

203

to the high temperature in the presence of water used in this paper, which can promote the

204

hydrolysis of the triglycerides, increasing the free fatty acids content and in consequence the acid

205

value. It is known that the presence of high water contents and high acid values seriously affect the

206

alkaline transesterification of the oil, severely reducing the yield of methyl esters by soap

207

production. However, since enzymatic transesterification is used in this work, and thus this situation

208

does not represent any inconvenience.

209 210

Table 2. Physicochemical properties of oil from fish waste used in this study in comparison with

211

Nile tilapia and Hybrid Sorubim oils.

10

Property

This study

Nile tilapia [7]

Hybrid Sorubim [7]

Density (g/mL)

0.92 ± 0.001

0.927

0.910

Viscosity (mm2/s) (40°C)

31.76 ± 0.01

-

-

Acid value (mg KOH/g)

3.17 ± 0.04

0.412

0.306

197.76 ± 1.99

194.99

212.29

0.08 ± 0.01

0.02

0.016

Saponification number (mg KOH/g) Moisture content (%w/w) 212 213

Fatty acid composition of oil from fish waste

214

The fatty acid composition of the oil from fish waste used in this study is presented in Table 3. The

215

main fatty acids found were myristic (C14:0, 4.0 % w/w), pentadecanoic (C15:0, 2.6 % w/w),

216

palmitic (C16:0, 25.4 % w/w), palmitoleic (C16:1, 8.2 % w/w),

217

w/w), stearic (C18:0, 8.6 % w/w), oleic (C18:1, 25.4 % w/w), linoleic (C18:2, 6.6 % w/w),

218

linolenic (C18:3, 1.7 % w/w), eicosatetraenoic (C20:4, 2.0 % w/w), eicosapentaenoic (C20:5, 2.1

219

% w/w), docosatetraenoic (C22:4, 2.6 % w/w), and docosahexaenoic (C22:6, 5.5 % w/w). This

220

composition is similar to the most common fatty acids found in fish oils, which are myristic (C14:0,

221

up to 7 % w/w), palmitic (C16:0, up to 20 % w/w), palmitoleic (C16:1, up to 28 % w/w), stearic

222

(C18:0, up to 7 % w/w), oleic (C18:1, up to 42 % w/w) and also significant amounts of

223

polyunsaturated fatty acids (PUFA) such as arachidonic (C20:4, up to 3 % w/w), eicosapentaenoic

224

(C20:5, up to 11 % w/w), docosapentaenoic acid (C22:5, up to 15 % w/w), and docosahexaenoic

225

(C22:6, up to 39 % w/w) [6, 100, 101]. Saturated fatty acid, monounsaturated fatty acid and PUFA

226

fractions of the oil from fish waste were 42.1 % w/w, 37.4 % w/w and 20.5 % w/w, respectively. In

227

this study, the percentage of PUFAs (20.5 % w/w) found was lower than the one reported by Costa

228

et al., (2013), Behçet, (2011) and Lyn and Li, (2009), who report 34.4% w/w, 38.1 % w/w and

229

28.99 % w/w of PUFAs, respectively; this can be due to the differences in the type of fish or parts

230

of fish used, and the obtaining conditions of the oil. The presence of PUFA in the fish oil when it is

heptadecanoic (C17:0, 1.7 %

11

231

used for biodiesel production have the advantage to improve the viscosity and in general fluidity

232

properties of the biofuel; however, it is know that PUFA decreases the oxidative stability of

233

biodiesel since these fatty acids are more susceptible to oxidation reactions [6]. It is important to

234

note that PUFA are considered beneficial for human health, because of that the use of this waste fish

235

oil for feed o nutritional purposes could be considered, but we have focused in the use in biodiesel

236

production.

237 238

Table 3. Comparison as percentage of total fatty acids of the oil from fish waste in this study with

239

the waste fish oil, anchovy oil and marine fish oil.

Fatty acid

Fatty acid composition of different fish oil

Chemical structure

This study

Waste fish oil [101]

Anchovy oil [100]

Marine fish oil [6]

Myristic

C14:0

4.0

6.6

6.71

3.16

Pentadecanoic

C15:0

2.6

-

-

-

Palmitic

C16:0

25.4

21.6

20.20

19.61

Palmitoleic

C16:1

8.2

8.0

6.59

5.16

Heptadecanoic

C17:0

1.7

-

0.23

1.82

Stearic

C18:0

8.6

4.1

4.2

3.77

Oleic

C18:1

25.4

17.3

19.71

20.94

Linoleic

C18:2

6.6

1.7

2.63

2.69

Linolenic

C18:3

1.7

2.9

1.64

0.9

Arachidic

C20:0

-

-

-

4.75

Eicosenoic

C20:1

-

4.2

-

-

Eicosadienoic

C20:2

-

-

0.23

0.81

Eicosatetraenoic

C20:4

2.0

-

0.79

2.54

Eicosapentaenoic

C20:5

2.1

13.3

10.41

3.7

Behenic

C22:0

-

-

-

1.55

Docosenoic

C22:1

-

3.8

-

12

Docosadienoic

C22:2

-

1.7

-

-

Docosatetraenoic

C22:4

2.6

-

-

-

Docosapentaenoic

C22:5

-

-

0.82

2.44

Docosahexaenoic

C22:6

5.5

-

21.58

15.91

Saturated

-

42.1

32.3

31.34

34.66

Monounsaturated

-

37.4

33.3

26.3

26.1

Polyunsaturated

-

20.5

34.4

38.1

28.99

240 241

Enzymatic transesterification reaction

242

Model fitting and ANOVA

243

The response values from the Central Composite Design (CCD) experiments conducted to examine

244

the combined effect of temperature, biocatalyst content and agitation rate on the FAME are shown

245

in Table 1. The content FAME ranged from 12 to 76 %. This result is similar to that found by

246

Marín-Suárez et al., (2019), who studied the enzymatic production of biodiesel from fish oil. These

247

authors reported a maximum biodiesel yield of 75 % after 8 h of reaction time and 50 % w/w of

248

enzyme loading, using Lipozyme RM IM, Novozym 435 or Lipozyme TL IM (the commercial

249

version of our biocatalyst). Other authors reported a slightly higher yield of 83 % for biodiesel

250

produced from waste fish oil using 20 % w/w of Carica papaya lipase with a methanol to oil molar

251

ratio of 1:4, water activity of lipase at 0.23 at 40 °C and 18 h of reaction time, but using 20 %

252

(based on oil weight) of tert-butanol as co-solvent [102]. It is important to mention that the amount

253

of biocatalyst used in the aforementioned works is much higher than that used in this study (10 %

254

w/w of biocatalyst content for 76 % of biodiesel yield).

255

The model was tested using Fisher’s statistical test for analysis of variance (ANOVA). The

256

computed F-value (5.38) was significant (p = 0.0185). The goodness of a model can be checked by

257

the determination coefficient (R2) and correlation coefficient (R). The determination coefficient (R2

258

= 0.87) indicates that the sample variation of 87 % for FAME synthesis is attributed to the 13

259

independent variables, and explained by the model. The closer the value of R (correlation

260

coefficient) to 1, the better the correlation between the experimental and predicted values are. The

261

calculated value of R (0.93) suggests that the model is a good representation of the process. Linear,

262

quadratic and interaction terms were significant at the 5% level. Thus, the second-order polynomial

263

model is represented by equation 2:

264

Y = 67.78 – 17.76*X1 – 8.06*X1X1 + 8.68*X2 – 8.43*X2X2 – 8.64*X3 – 4.08*X3X3 + 6.54*X1X2 –

265

6.52*X1X3

266

(2)

267

where Y is the FAME content, and X1, X2 and X3 are the coded values of temperature, biocatalyst

268

content and agitation rate, respectively.

–

5.35*X2X3

269 270

Effect of reaction parameters on FAME content

271

The linear, quadratic and interactions effects of the studied variables are presented in Table 4. All

272

parameters were statistically significant at 5 % level. The linear effects of temperature and agitation

273

rate were negative, which represents that the increase in the level of these variables from -1 to 1 the

274

response was reduced. It can be observed when comparing runs 1 and 2 or 4 and 8. In the first case

275

the only change is in the reaction temperature and FAME content was lower at the higher

276

temperature, perhaps due to a distortion of the enzyme at too high a temperature. In the second one,

277

the change is in the agitation rate, and as well as observed for the temperature, the FAME content

278

was lower at the higher level of agitation rate. The agitation rate permits a better mixing of the

279

different reaction medium and should reduce the external diffusion limitations, the negative effect

280

suggests that this homogenization of the reactants has a negative effect on enzyme performance

281

(e.g., perhaps favoring the entry of more oil to the biocatalysts).

14

282

On the other hand, the biocatalyst content presented a positive effect in the response. The increase

283

in the biocatalyst content leads to a higher FAME content. It can be confirmed comparing

284

experiments 2 and 4, where the only change in reaction conditions was the increase in biocatalyst

285

content. This result fits the expectations and suggests that the biocatalysts did not aggregate forming

286

large particles where diffusion limitations may become a problem.

287

Table 4. Linear, quadratic and interaction effects estimated for the reaction parameters Variable

Effect

Standard error

p- value

X1

-35.52

1.62

0.0020

X1X1

-16.12

1.79

0.0121

X2

17.36

1.62

0.0086

X2X2

-16.87

1.79

0.0111

X3

-17.29

1.62

0.0087

X3X3

-8.17

1.79

0.0449

X1X2

13.08

2.12

0.0254

X1X3

-13.04

2.12

0.0255

X2X3

-10.71

2.12

0.0372

288 289

The relationship between reaction parameters and response can be better understood by examining

290

the contour plots presented in Figure 1. In each plot, the missing variable was fixed at the central

291

point. In Figures 1a and 1b, it is clearly noted that at the lowest temperatures, the FAME content

292

increased, as well as the fact that the effect of temperature was higher that the effects of biocatalyst

293

content and agitation rate. In Figure 1c, the combined effect of biocatalyst content and agitation rate

294

can be observed. Increasing the biocatalyst content and reducing the agitation rate increased the

295

FAME content.

15

296

a)

297

16

298

b)

299

17

300

c)

301 302

Fig. 1. Contour plots of FAME content in methanolysis reaction of oil from fish waste catalyzed

303

TLL immobilized on octadecyl methacrylate. (a) Temperature vs biocatalyst content; (b)

304

Temperature vs agitation rate; (c) Biocatalyst content vs agitation rate.

305 306

Optimal conditions and model validation

307

The optimal conditions for FAME synthesis were determined using the response desirability

308

profiling of Statistica 13.5, presented in Figure 2. The response desirability profiling combines the

309

optimal value for each variable that maximizes the response. The coded optimal values were: X1 =

310

0; X2 = 1.07; X3 = −1.68. The uncoded optimal conditions for FAME synthesis were: temperature =

18

311

35 °C; biocatalyst content = 10 % w/w; agitation rate = 216 rpm. Under the optimum conditions, the

312

predicted FAME content was 80.04 %. In order to validate the model, experiments were performed

313

under the optimal conditions. The experimental result was 75.3 ± 0.12 %, close to the predicted by

314

the model.

315 316

Fig. 2. Profiles for predicted values and desirability for the variables.

317 318

Fuel properties of fatty acid methyl esters

319

The fuel properties of the FAME from oil of fish waste in comparison with FAME from marine fish

320

oil and waste cooking oil, commercial diesel fuel and ASTM D6751 standard are presented in Table

321

5. As it can be seen, the density value found in this study (0.89 ± 0.01 g/mL) was very similar to the

322

reported for FAME from marine fish oil and waste cooking oil, and is within the limits established

323

for ASTM D6751 standard; however, with respect to commercial diesel, the density of biodiesel are

324

really higher. In terms of engine performance, fuels with higher densities provide greater amount of

325

energy. In this sense, biodiesel can potentially provide more power per liter than that commercial 19

326

diesel [103]. Density is related to the fatty acid composition, and it has been shown that the higher

327

the degree of unsaturation, the greater the density of the oil, and therefore of the biodiesel [83].

328

The kinematic viscosity of biodiesel from oil from fish waste (5.3 ± 0.004 mm2/s) was slightly

329

higher than biodiesel from marine fish oil and WCO. However, it meets the value established by

330

ASTM D6751, from which we can infer that in a fuel injection system, biodiesel should be easy to

331

atomize, pump and achieve fine droplets [46]. Higher viscosity is undesirable since it can affect the

332

atomization and cold flow properties of the fuel (like Cold Filter Plugging Point), and promote the

333

formation of deposits inside the engine [83]. The viscosity is also related to the fatty acid

334

composition, and it is well known that its value increases with the increase in the length of the fatty

335

acid chain, and decreases with the increase in the amount of unsaturations [104].

336

The acid value is related to the free fatty acid content of the fuel [6], and with the long term stability

337

of biodiesel against corrosiveness, so that the lower its value the better the biodiesel quality [82].

338

The biodiesel of oil from fish waste in this study had a higher acid value than the biodiesel from

339

waste cooking oil, and exceeds the limits allowed by ASTM D6751 for this parameter. This result is

340

due to the presence of water in the oil from fish waste; since a high water content in the raw oil

341

generates a higher acid value in the oil and in consequence in the biodiesel produced. In fact, it has

342

been shown that the acid value of a biodiesel increases 3 mg of KOH / g / 1 % w/w water content

343

in its raw oil [105]. To avoid this situation the obvious alternative is to completely dry the oil before

344

the transesterification reaction and, adding, for example molecular sieves during the reaction.

345

However, the increase in the acidity of the substrate can occur during the extraction process of the

346

oil, especially if it is extracted using water at high temperatures (as in the present study). In this

347

case, a solvent extraction method ( for example Soxhlet extraction) or supercritical extraction using

348

carbon dioxide could be used to avoid the presence of water and thereby reduce the possibility of

349

hydrolysis of the oil [106].

20

350

The high acid value in the oil (and biodiesel) from fish waste is also due to the presence of 20.5%

351

polyunsaturated fatty acids, which are more susceptible to oxidation and free fatty acid formation,

352

resulting in a biodiesel with higher acid values than biodiesel produced from other raw oils with

353

lower content of PUFA´s [6]. Another alternative to reduce the acid value of biodiesel is the

354

pretreating of the oil from fish waste by acidic esterification as reported by El-Mashad et al., who

355

performed an acid esterification using 1% w/w of H2SO4 and molar ratio methanol to oil of 6: 1,

356

with agitation of 600 rpm at 52°C for 1 h, to reduce the acid value of a salmon oil (3.5 or 12 mg

357

KOH/ g) at acceptable values for alkaline transesterification (2 mg of KOH/ g) [107].

358

Cloud point indicates the minimum temperature at which a fuel can ignite efficiently; it is defined

359

as the temperature at which crystal formation begins to form precipitates, and it is the most used

360

parameter to measure the cold flow properties of a fuel [108]. The cloud point of biodiesel from oil

361

of fish waste was found to be 10.5 ° C, which is under the ASTM D6751 recommended limits.

362

Below this value, the formation of crystals that then precipitate into bottom of the storage tank

363

begins, resulting in the clogging of fuel filters and engine injectors [109].

364

Finally, calorific value of biodiesel from oil of fish waste in this study (38.1 MJ/kg) was similar to

365

the reported for biodiesel from marine fish oil (41.37 MJ/kg) [6] and WCO (38.67 MJ/kg) [110],

366

and it is slightly lower than the commercial diesel fuel (45.62-46.48 MJ/kg). This value is within

367

the limits established for ASTM D6751 standard and it can be considered acceptable for smooth

368

engine performance [108, 111]. A low calorific value (low energy contents) of biofuels affects

369

parameters such as horsepower and torque, which are key in fuel performance [112], and it also

370

affects the break specific fuel consumption (BSFC) because a larger amount of fuel with lower

371

calorific value is needed to maintain the specified power [82].

372

Table 5. Fuel properties of fatty acid methyl esters from oil of fish waste (this study) in comparison

373

with FAME from marine fish oil and waste cooking oil, commercial diesel fuel and ASTM D6751

374

standard. 21

FAME’s from different feedstock Marine

Waste

Commercial

ASTM

fish oil

cooking oil

diesel fuel [1]

D6751 [1]

[6]

[110]

0.89 ± 0.01

0.86

0.873

0.075-0.084

0.86-0.90

5.3 ± 0.004

4.4

3.7

1.9-4.1

1.9-6.0

0.9 ± 0.28

1.17

0.15

-

0.5

10.5 ± 0.47

-

-1

-

-3 to 12

38.1 ± 0.21

41.37

38.67

45.62-46.48

>35

Properties This study

Density at 15°C (g/mL) Viscosity at 40°C 2

(mm /s) Acid value (mg KOH/g) Cloud point (°C) Calorific value (MJ/kg) 375 376

Conclusion

377

This paper shows that oil from viscera fish (oil from fish waste) can be successfully converted into

378

fatty acid methyl ester by enzymatic catalysis, and that TLL-octadecyl methacrylate produce similar

379

biodiesel yield with less amount of biocatalyst to the obtained using commercial biocatalyst like

380

Lipozyme RM IM, Novozym 435 or Lipozyme TL IM, which have been used to obtain biodiesel

381

from fish oil. Optimal conditions for enzymatic catalysis were a temperature of 35 °C, a biocatalyst

382

content of 10 % w/w and an agitation rate of 216 rpm. Under optimal conditions an experimental

383

biodiesel yield of 75.3 % was obtained; this result was close to the yield of 80 % predicted by the

22

384

statistical model. It was found that temperature has a negative effect in FAME production so that

385

FAME content was lower at the higher temperature. The same behavior was found for the agitation

386

rate, that is, the FAME content was lower at the highest level of the agitation rate. Only biocatalyst

387

content presented a positive effect in the response. The increase in the biocatalyst content leads to a

388

higher FAME content. The fuel properties of the biodiesel obtained were determined, finding that

389

most of the parameters (density, viscosity, calorific value and cloud point) studied complied with

390

the recommendations of the ASTM D6751. Only the acid value was higher than the established

391

limits, which could be avoided using another method of extracting fish oil with lower temperatures,

392

and also, taking care of the amount of water present in the raw oil.

393

The use of oil from fish waste for biodiesel production is a good alternative to mitigate

394

environmental pollution since it reduces the contamination caused by the disposal of these materials

395

in nature and produces a sustainable fuel, especially if enzymatic catalysis is used.

396 397

Acknowledgements

398

Dr. Tacias-Pascacio expresses her gratitude to CONACYT Mexico for her Postdoctoral fellowship

399

(No. 005126). Dr. Roberto Fernández-Lafuente gratefully recognizes the support from MICIU from

400

Spanish Government (project number CTQ2017-86170-R). The help and suggestions from Dr.

401

Ángel Berenguer (Departamento de Química Inorgánica, Universidad de Alicante) are gratefully

402

recognized.

403

23

404

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Highlights •

Oil from viscera fish was used for biodiesel production.

•

The use of this material solves two environmental problems.

•

Enzymatic transesterification with methanol was performed.

•

Optimization was performed using a central composite design (RSM).

•

Fuel properties were determined and only acidity was under regulation values.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Sincerely, Jonny Ching-Velasquez, Roberto Fernández-Lafuente, Rafael C. Rodrigues, Vladimir Plata, Arnulfo Rosales-Quintero, Beatriz Torrestiana-Sánchez, Veymar Guadalupe Tacias Pascacio.