One-step synthesis of high-yield biodiesel from waste cooking oils by a novel and highly methanol-tolerant immobilized lipase

Accepted Manuscript One-step synthesis of high-yield biodiesel from waste cooking oils by a novel and highly methanol-tolerant immobilized lipase Xiumei Wang, Xiaoli Qin, Daoming Li, Bo Yang, Yonghua Wang PII: DOI: Reference:

S0960-8524(17)30358-9 http://dx.doi.org/10.1016/j.biortech.2017.03.086 BITE 17790

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

23 January 2017 13 March 2017 14 March 2017

Please cite this article as: Wang, X., Qin, X., Li, D., Yang, B., Wang, Y., One-step synthesis of high-yield biodiesel from waste cooking oils by a novel and highly methanol-tolerant immobilized lipase, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.03.086

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.

1

One-step synthesis of high-yield biodiesel from waste cooking oils by

2

a novel and highly methanol-tolerant immobilized lipase

3

Xiumei Wanga, Xiaoli Qinb, Daoming Lic, Bo Yanga, Yonghua Wangc*

4

a

5

510006, China.

6

b

College of Food Science, Southwest University, Chongqing 400715, China.

7

c

School of Food Science and Engineering, South China University of Technology,

8

School of Bioscience and Bioengineering, South China University of Technology, Guangzhou

Guangzhou 510640, China.

9

10

*Corresponding author:

11

Prof. Yonghua Wang, School of Food Science and Engineering, South China

12

University of Technology, Guangzhou 510640, P. R. China

13

E-mail：[email protected]

14

Fax: +86-020-87113842

15 16 17 18 19 20

1

21

Abstract

22

This study reported a novel immobilized MAS1 lipase from marine Streptomyces sp.

23

strain W007 for synthesizing high-yield biodiesel from waste cooking oils (WCO)

24

with one-step addition of methanol in a solvent-free system. Immobilized MAS1

25

lipase was selected for the transesterification reactions with one-step addition of

26

methanol due to its much more higher biodiesel yield (89.50%) when compared with

27

the other three commercial immobilized lipases (<10%). The highest biodiesel yield

28

(95.45%) was acquired with one-step addition of methanol under the optimized

29

conditions. Moreover, it was observed that immobilized MAS1 lipase retained

30

approximately 70% of its initial activity after being used for four batch cycles.

31

Finally, the obtained biodiesel was further characterized using FT-IR, 1H and 13C

32

NMR spectroscopy. These findings indicated that immobilized MAS1 lipase is a

33

promising catalyst for biodiesel production from WCO with one-step addition of

34

methanol under high methanol concentration.

35 36

Keywords: Transesterification; Immobilized MAS1 lipase; Waste cooking oils;

37

Biodiesel; Characterization

38 39 40 41 42

2

43 44

1. Introduction In recent years, biodiesel is becoming more and more attractive as a renewable,

45

non-toxic, and biodegradable fuel (Huang et al., 2015). However, the high production

46

cost limits the development and use of biodiesel, of which the cost of raw materials

47

accounts for >85% (Kuo et al., 2015). Thus, researchers are focusing their attention

48

on minimizing feedstock costs such as using microbial oils or waste cooking oils

49

(WCO). A large amount of WCO available around the world are cheaper than refined

50

oils and can do great harm to environment (Yan et al., 2014; Utlu Z., 2007).

51

Therefore, production of biodiesel from WCO could help solve environmental

52

pollution and reduce the production cost (Chen et al., 2005; Farag et al., 2011; Meng

53

et al., 2008).

54

Nowadays, preparation of biodiesel from WCO could be performed by chemical

55

and enzymatic processes. Compared with chemical methods, enzymatic production of

56

biodiesel has drawn great attention due to its moderate reaction conditions, easier

57

recovery of products, more simple purification process, insensitive to water content

58

and acidity value (Singh et al., 2015; Lee et al., 2011; Dizge et al., 2009). Moreover,

59

enzymes in immobilized forms can allow their reuse, and be used in continuous

60

operations (Rodrigues et al., 2016; Juan et al., 2011; Halim et al., 2009; Modi et al.,

61

2007). Enzymatic hydroesterification and transesterification reactions are the most

62

widely used methods for biodiesel production(de Araújo et al., 2013). Nevertheless,

63

enzymatic hydroesterification reactions, including two consecutive steps, are

64

relatively more complicated than transesterification reactions (Aguieiras et al., 2015;

3

65

Haigh et al., 2013). Moreover, the cost of lipases is relatively high and the reaction is

66

slow in the biocatalytic hydroesterification processes (Arumugam and Ponnusami,

67

2014). Therefore, lipase-catalyzed transesterification reaction is considered to be a

68

promising alternative to produce biodiesel.

69

In the transesterification reactions, methanol is the most widely used acyl

70

acceptor for biodiesel production due to its economic feasibility and accessibility

71

compared with other alcohols (Deng et al., 2005; Antczak et al., 2009; Zhao et al.,

72

2014). However, small droplets of methanol could result in the denaturation and

73

inactivation of the lipases compared with longer aliphatic alcohols (Romdhane et al.,

74

2013; Salis et al., 2005; Tan et al., 2010). To overcome this drawback, trials using

75

stepwise addition of methanol to reaction mixtures (Duarte et al., 2015; You et al.,

76

2013), co-solvents like t-butanol (López et al., 2016; Royon et al., 2007; Li et al.,

77

2006), longer-chain alcohols as acyl acceptors (Iso et al., 2001), and methyl or ethyl

78

acetate as acyl acceptors (Goembira and Saka, 2013; Razack and Duraiarasan, 2016)

79

have been performed in previous studies. Nevertheless, these strategies increased the

80

additional processing steps in the production of biodiesel, resulting in a higher

81

production cost (Véras et al., 2011). Therefore, efforts are made to look for novel

82

lipases with the ability of one-step addition of methanol for biodiesel production

83

under high methanol concentration.

84

In the present work, lipase MAS1 from marine Streptomyces sp. strain W007

85

(Yuan et al., 2015), immobilized onto XAD1180 resin, was used as catalyst for

86

biodiesel production through transesterification of WCO with methanol in a solvent-

4

87

free system. Firstly, the catalytic properties of immobilized MAS1 lipase, Novozym

88

435, Lipozyme RM IM, and Lipozyme TL IM were compared in the production of

89

biodiesel. Then, the effects of oil/methanol molar ratio, enzyme loading and

90

temperature on biodiesel production were separately investigated. Furthermore, the

91

reusability of immobilized MAS1 lipase was evaluated. Finally, the obtained biodiesel

92

was further characterized by FT-IR, 1H and 13C NMR spectroscopy.

93

2. Materials and Methods

94

2.1. Materials

95

Waste cooking oils (WCO) were obtained from a local restaurant and were

96

centrifuged before use. The WCO consisted of 92.18% triacylglycerols (TAG), 3.94%

97

free fatty acids (FA), 3.88% diacylglycerols (DAG). The FA composition of WCO

98

was composed of 0.19% lauric acid (C12:0), 1.13% myristic acid (C14:0), 24.3%

99

palmitic acid (C16:0), 2.13% palmitoleic acid (C16:1), 0.33% heptadecanoic acid

100

(C17:0), 6.66% oleic acid (C18:1), 41.11% linoleic acid (C18:2), 21.93% linolenic

101

acid (C18:3n6), 1.48% (eicosatrienoic acid (C20:3n6), 0.21% heneicosanoic acid

102

(C21:0), 0.35% (docosanoic acid C22:0), and 0.18% tetracosanoic acid (C24:0). The

103

oils didn’t contain any n-3 polyunsaturated fatty acids with more than four double

104

bonds that were easily oxidized during storage. Based on the FA composition, WCO

105

had an average molecular weight of 900 g/mol.

106

Lipase MAS1 was produced according to the method described by Lan et al.

107

(2016). Novozym 435, Lipozyme RM IM and Lipozyme TL IM were supplied by

108

Novozymes A/S (Bagsvaerd, Denmark). According to Novozymes propyl laurate unit

5

109

(PLU) method (Basso et al., 2013), the esterification activities of Novozym 435,

110

Lipozyme RM IM and Lipozyme TL IM were 8247, 3200 and 483 U/g, respectively.

111

Standards of monooleoylglycerol, dioleoylglycerol (15% of 1,2-dioleoylglycerol and

112

85% of 1,3-dioleoylglycerol), trioleoylglycerol, methyl oleate, and 37-component

113

FAME mix (C4-C24) were purchased from Sigma-Aldrich. n-Hexane, 2-propanol,

114

formic acid and methanol of chromatographic grade were sourced from Kermel

115

Chemical Reagent Co., Ltd. (Tianjin, China). The Amberlite XAD1180 resin was

116

acquired from Rohm and Haas Company (USA). All other chemicals were of

117

analytical grade unless otherwise stated.

118

2.2. Preparation of immobilized MAS1 lipase

119

The preparation of immobilized MAS1 lipase was carried out under the

120

conditions of 75 mg lipase solution/g XAD1180 resin, and an equal volume of sodium

121

phosphate buffer (0.02 M, pH 8.0) at a temperature of 30 °C and a speed of 200 rpm

122

for 8 h. Subsequently, the obtained immobilized MAS1 lipase was rinsed with sodium

123

phosphate buffer (0.02 M, pH 8.0) repeatedly until no protein was detected in the

124

eluate. Finally, the obtained immobilized MAS1 lipase was dried in a vacuum

125

desiccator at 40 °C for 8 h and stored in closed vials at 4 °C until use. The

126

esterification activity of immobilized MAS1 lipase was 1605±30.7 U/g according to

127

Novozymes propyl laurate unit (PLU) method (Basso et al., 2013).

128

2.3. Transesterification of WCO with methanol

129 130

The enzymatic transesterification reactions were performed in a 50 mL-conical flask containing 10 g substrates and were initiated by the addition of immobilized

6

131

lipases with constant shaking at 200 rpm for 24 h. Novozym 435, Lipozyme RM IM,

132

Lipozyme TL IM and immobilized MAS1 lipase as biocatalysts were compared in the

133

production of biodiesel through transesterification of WCO with methanol under the

134

same conditions in a solvent-free system. Then, the effects of oil/methanol molar ratio

135

(1:1, 1:2, 1:3, 1:4, 1:5, 1:6), enzyme loading (40, 60, 80, 100, 120 U/g substrate) and

136

temperature (25, 30, 35, 40, 45 °C) on biodiesel production were investigated,

137

respectively. Samples were withdrawn periodically for high-performance liquid

138

chromatography (HPLC) analysis.

139

2.4. Reusability of immobilized MAS1 lipase

140

The reusability of immobilized MAS1 lipase was assessed in the

141

transesterification of WCO with methanol for biodiesel production. The reactions

142

were performed under the optimized conditions. After each reaction cycle (24 h),

143

immobilized MAS1 lipase was separated from the reaction mixture by filtration, and

144

then washed with n-hexane for three times. After that, the recovered immobilized

145

MAS1 lipase was placed at room temperature to remove residual n-hexane, and then

146

reused in the next cycle under the same reaction conditions with the introduction of

147

fresh substrates. The reusability of immobilized MAS1 lipase was evaluated by

148

measuring relative biodiesel yield in subsequent reactions compared to that of the first

149

reaction.

150

2.5. Purification of biodiesel

151 152

Separation of FAME from other compositions was performed using thin-layer chromatography method in the literature (Qin et al., 2014). Then, the scraped FAME

7

153

bands were dissolved in 30 mL of chloroform/methanol (2:1, v/v). After that, the

154

mixture was sonicated under the ultrasonic power of 200 W for 20 min and then

155

centrifuged at 3,000×g for 10 min. Subsequently, the supernatant was collected and

156

subjected to evaporation under vacuum at 30 °C to obtain FAME. Finally, the

157

resultant FAME were characterized by FT-IR, 1H and 13C NMR spectroscopy.

158

2.6. FT-IR analysis of the final FAME product

159

One milligram of the purified sample with 100 mg KBr were initially mixed,

160

ground, and mortar. Then, the mixture was pressed into a pellet. Finally, the resultant

161

FAME was analyzed by using a Nicolet 8210E FT-IR spectrometer. The wavelength

162

ranged from 400 to 4,000 cm-1 during 128 scans, with the resolution at 2 cm-1.

163

2.7. 1H and 13C NMR analysis of the final FAME product

164

1

H and 13C NMR analysis of the final FAME product were carried out at 600

165

MHz using a Bruker AVANCE

166

Before the analysis, the obtained FAME (200 mg) were mixed with 1 mL deuterated

167

chloroform (CDCl3). The 1H NMR spectrum was acquired with a recycle delay of 1.0

168

s and 16 scans. The 13C NMR spectrum was obtained with a recycle delay of 2.0 s and

169

160 scans. MestReNova software (Mestrelab Research SL, Santiago de Compostela,

170

Spain) was employed to analyze the spectrum. The peaks were identified from the

171

spectra according to Ullah et al.(2015) and Kumar et al. (2016).

172

2.8. Analysis of the composition of the reaction mixtures by HPLC

173 174

600HD spectrometer with 5 mm BBO probes.

The analysis of the composition of the reaction mixtures was carried out using a normal-phase HPLC equipped with a refractive index detector and a Phenomenex

8

175

Luna column (250 mm×4.6 mm i.d., 5 µm particle size) according to the method

176

previously described (Li et al., 2015) with a minor modification. The mobile phase

177

consisted of n-hexane, isopropanol and formic acid (21:1:0.003, by vol) with a flow

178

rate of 1 mL/min. Peaks in HPLC were evaluated by comparison of their retention

179

times with those known standards. Retention times were 3.07 (TAG), 3.41 (FAME),

180

3.76 (FA), 4.73 (1,3-DAG), 6.31 [1,2(2,3)-DAG], 33.17 [1(3)-MAG], 37.39 (2-

181

MAG). Results of each acylglycerol species, FAME and FA were expressed as weight

182

percentage as calculated from peak areas. Waters 2695 integration software was used

183

to calculate peak-area percentages.

184

In this study, biodiesel yield was calculated as follows: FAME (%) × 100% TAG (%) + DAG (%) + MAG (%) + FAME (%) + FA (%)

185

Biodiesel yield (%) =

186

2.9. Analysis of FA composition of WCO and FAME by GC

187

The substrate (WCO) was initially methylated to FAME according to the method

188

described by Wang et al. (2010). Then, FA composition of WCO and the final FAME

189

product was determined using GC (Agilent 7890A) equipped with a capillary column

190

CP-Sil 88 (60 m× 0.25 mm × 0.2 µm). The detailed analysis was carried out according

191

to the method described by Qin et al. (2011).

192

2.10. Statistical analysis

193

All experiments were repeated in triplicate. The results were presented as the

194

means ± standard deviations (SD). SPSS for Windows 13.0 was employed to perform

195

statistical calculations. Significant differences among mean values were assessed

196

through significant differences test and variance analysis. 9

197

3. Results and discussions

198

3.1. Screening of immobilized lipases

199

Numerous studies have shown that Lipozyme TL IM (Yagiz et al., 2007),

200

Novozym 435 (Modi et al., 2007; Royon et al., 2007), and Lipozyme RM IM

201

(Bernardes et al., 2007) are the most widely used immobilized lipases for biodiesel

202

production. Thus, four immobilized lipases, including immobilized MAS1 lipase,

203

Novozym 435, Lipozyme RM IM and Lipozyme TL IM, were screened for their

204

ability to produce biodiesel from WCO in the solvent-free system. The

205

transesterification reactions were carried out under the conditions of enzyme loading

206

of 80 U/g substrate, oil/methanol molar ratio of 1:3, reaction temperature of 40 °C,

207

one-step addition of methanol at the beginning of the reaction for 24 h. The results are

208

shown in Fig.1. Among the four lipases tested, immobilized MAS1 lipase exhibited

209

the highest catalytic activity. The maximum biodiesel yield reached 89.50%. The

210

results also indicated that among the other three commercial immobilized lipases,

211

Novozym 435 showed better catalytic activity than Lipozyme TL IM and Lipozyme

212

RM IM. The biodiesel yield was 9.32%, 5.8%, and 2.79% in transesterification

213

reactions separately catalyzed by Novozym 435, Lipozyme TL IM, and Lipozyme

214

RM IM. This was because the activities of Novozym 435, Lipozyme RM IM and

215

Lipozyme TL IM were inhibited when more than 1.5 molar equivalents of methanol

216

was presented in the reaction mixture. Similar results were reported by Zhao et al.

217

(2014), who found that less than 10% biodiesel yield was obtained by Novozym 435,

218

Lipozyme TL IM, and Lipozyme RM IM-catalyzed transesterification when the molar

10

219

proportion of methanol in the blended alcohol reached 100% and methanol was added

220

at the beginning of the reaction. It could be concluded that immobilized MAS1 lipase

221

exhibited much more higher catalytic efficiency compared to the other three

222

commercial immobilized lipases when all the methanol was added at the beginning of

223

the reaction. Therefore, immobilized MAS1 lipase was chosen for the further studies.

224

3.2. Effect of oil/ methanol molar ratio

225

The effect of oil/methanol molar ratio on biodiesel yield by immobilized MAS1-

226

catalyzed transesterification reactions is shown in Fig.2a. Biodiesel yield increased

227

when the oil/methanol molar ratio varied from 1:1 to 1:3. The maximum biodiesel

228

yield (87.89%) was obtained at a oil/methanol molar ratio of 1:3. However, further

229

increase in oil/methanol molar ratio beyond 1:3 resulted in a decrease in biodiesel

230

yield. This may be possibly because of the denaturation and inactivation of the lipase

231

by exposure to a high concentration of methanol. When oil/methanol molar ratio

232

increased from 1:4 to 1:5, significant differences (0.002
233

increased. This may probably be the protection of the activity of immobilized MAS1

234

lipase by non-polar XAD1180 macroporous resin, since lipases can be adsorbed at

235

both the outer surface and within the pores of supporting material. Therefore, a

236

oil/methanol molar ratio of 1:3 was used for further experiments.

237

3.3. Effect of enzyme loading

238

Fig.2b shows the effect of enzyme loading on biodiesel yield. Biodiesel yield

239

increased from 44.43% to 88.51% when enzyme loading ranged from 40 U/g to 80

240

U/g. After that, biodiesel yield remained almost constant with an increase in enzyme

11

241

loading beyond 80 U/g. Increasing enzyme loading only affected the time that reached

242

equilibrium but had no significant effect on biodiesel yield. Therefore, a fixed enzyme

243

loading of 80 U/g substrate was used for the subsequent experiments.

244

3.4. Effect of temperature

245

The effect of temperature on biodiesel yield obtained by immobilized MAS1

246

lipase-catalyzed transesterification was investigated in the range from 25 to 45 °C.

247

The results are shown in Fig.2c. After 24 h of reaction, biodiesel yield increased when

248

reaction temperature was varied from 25 to 30 °C. The maximum biodiesel yield

249

(95.12%) was obtained at 30 °C. However, there was a slight decrease in biodiesel

250

yield when reaction temperature was changed from 35 to 40 °C. After that, biodiesel

251

yield decreased rapidly with a further increase in reaction temperature from 40 to 45

252

°C. This may be probably because of the deactivation of lipase at a higher

253

temperature. Therefore, the reaction temperature was fixed at 30 °C in the subsequent

254

experiments.

255

3.5. Reusability of immobilized MAS1 lipase

256

Fig.3 shows the results about the reusability of immobilized MAS1 lipase for

257

biodiesel production in the transesterification reaction. Biodiesel yield after the first

258

reaction was 95.45%. When used in the 3rd run, biodiesel yield was 78.67%, which

259

retained about 82% of its initial activity. After four cycles, biodiesel yield declined to

260

65.24%, which was approximately 68% of its initial yield. Although Lee et al (2013)

261

reported that co-immobilized CRL and ROL lipases still kept 75% of initial activity

262

after 20 cycles and biodiesel conversion was about 70% after 40 cycles, differences in

12

263

stabilities and catalytic effects of immobilized lipases between the report and this

264

study should be pointed out as follows: (1) Methanol feeding method was different

265

(step-wise vs one-step). (2) Oil/methanol molar ratio was different (1:1 vs 1:3). (3)

266

Enzyme loading was different (20% vs 5%, relative to the weight of substrates). (4)

267

Immobilization method was different (cross-linking vs physical absorption). It could

268

be concluded that when compared with the literature, the reasons for the loss of the

269

activity of immobilized MAS1 lipase in repeated use may be caused by the desorption

270

process and the deactivation of the lipases with less amount by high methanol

271

concentration with one-step addition of methanol. Nevertheless, this study still offered

272

considerable advantages such as high catalytic efficiency with one-step addition of

273

methanol, lower production cost, easier operation processes, relatively lower cost of

274

carrier materials, easier immobilization procedure and better resistance to high

275

methanol concentration. Moreover, Yan et al. (2014) reported that the whole cell

276

catalyst was only reused for three times and retained only 78% activity with one-step

277

addition of methanol after three batch cycles. Therefore, these results indicated that

278

immobilized MAS1 lipase had relatively good reusability and is a promising catalyst

279

for biodiesel production with one-step addition of methanol.

280

A comparison of the catalytic abilities of immobilized MAS1 lipase with other

281

lipases in the literatures for biodiesel production with one-step addition of methanol is

282

shown in Table 1. High biodiesel yield (95.45%) was obtained by immobilized MAS1

283

lipase-catalyzed transesterification reactions with one-step addition of methanol in a

284

solvent-free system in this study. However, lower biodiesel yield (≤80%) was

13

285

observed when other lipases were used to catalyze the transesterification reactions

286

with one-step addition of methanol in the presence or absence of solvent (Yan et al.,

287

2014; Zhao et al., 2014; Cerveró et al., 2014; You et al., 2013; Lu et al., 2010; Su et

288

al., 2007). Although t-butanol, a medium polar solvent, could dissolve both methanol

289

and oil, biodiesel yield in the paper of Ji et al. (2010) was also lower than that in this

290

study when all the mathanol was added into the reaction mixture at the beginning.

291

These results indicated that immobilized MAS1 lipase exhibited much higher catalytic

292

efficiency than other lipases when all the methanol was added into the reaction

293

mixtures at the beginning.

294

High biodiesel yield obtained by immobilized MAS1 lipase can mainly be

295

explained as follows: Transesterification of TAG with alcohol contains three

296

consecutive reversible reactions. TAG is firstly converted to DAG, followed by the

297

conversion from DAG to MAG, and finally MAG is converted to glycerol. At each

298

step, one mole of fatty acids ester is produced. When 1,3-regiospecific immobilized

299

lipases are used to catalyze the transesterification reactions, the conversion from DAG

300

to MAG is a rate-limiting step. However, immobilized MAS1 lipase is a non-

301

regiospecific lipase and can migrate acyl groups from positions sn-1, 2, and 3 of TAG

302

to methanol (Wang et al., 2017). Therefore, the conversion from DAG to MAG

303

catalyzed by immobilized MAS1 lipase was spontaneous and fast, resulting in a

304

significantly higher biodiesel yield. These results indicated that non-regiospecific

305

immobilized MAS1 lipase is a promising biocatalyst with high catalytic efficiency

306

and relatively good reusability for production of biodiesel with one-step addition of

14

307

methanol.

308

3.6. Characterization of the final FAME product

309

3.6.1. Analysis of the FA composition and simple formula of FAME As seen from Fig.4, the FA composition of the final FAME product mainly

310 311

consisted of 26.15% C16:0, 6.74% C18:1, 43.83% C18:2, and 18.1% C18:3, which

312

was similar to that of WCO. Thus, it could be concluded that the final FAME product

313

mainly consisted of methyl palmitate, methyl oleate, methyl linoleate, and methyl

314

linolenate.

315

3.6.2. FT-IR analysis of the final FAME product

316

The FT-IR spectrum of the final FAME product obtained is shown in Fig.S1. The

317

strong C=O group stretching band of methoxy ester (-CO-OCH3) appears at 1747 cm-

318

1

319

The peaks at 2854 cm-1 and 2930 cm-1 correspond to the C-H stretching vibration of

320

terminal methyl group (-CH3) of fatty acid chain and the methylene, respectively. The

321

peak at 3015 cm-1 represents the C-H stretching vibration of vinylic/ cis olefinic CH

322

double bond (=C-H). The absorption band at 3472 cm-1 is due to the O-H stretching

323

vibration of carboxylic acids. The C-O stretching vibration peak is found between

324

1015 and 1168 cm-1. The peaks at 1369 cm-1 and 1433 cm-1 correspond to the bending

325

vibrations of CH3 and CH2 aliphatic groups, respectively.

326

3.6.3. 1H and 13C NMR analysis of the final FAME product

. The rocking bending vibration of methylene groups (-CH2-) appears at 723 cm-1.

327

The 1H NMR spectrum of the final FAME product obtained is shown in Fig.S2a.

328

The peak at 3.65 ppm corresponds to the methoxy protons (-OCH3) of methyl ester (-

15

329

COOCH3). The triplet signals of the bis-allylic proton (-C=C-CH2-C=C-) of the

330

polyunsaturated fatty acid chain and α-methylene protons (α-CH2) of ester (-CH2-

331

COO-Me) appear at 2.75 and 2.30 ppm, respectively. The multiplet signal of α-

332

methylene protons (α-CH2) of the double bond (-CH2-C=C-) is observed at 2.03 ppm.

333

The β-methylene protons of ester (-CH2-C-COOMe) appear a triplet signal at 1.60

334

ppm. The multiplet signal at 5.32 ppm is related to olefinic protons (-CH=CH-)

335

bonded to carbon atoms. The trilpet signal at 0.89 ppm indicates the presence of the

336

terminal methyl protons (-C-CH3). The peaks at 1.33 ppm are attributed to the protons

337

of the methylene (-(CH2)n-) backbone of long fatty acid chains.

338

The 13C NMR spectrum of the final FAME product is shown in Fig.S2b. The

339

peak at 174.24 ppm represents the ester carbonyl carbon (-COO-). The doublet signal

340

between 127.87 and 129.96 ppm indicates the presence of olefinic carbons. The

341

methoxy carbons of methyl esters appear a singlet signal at 51.34 ppm. The peaks at

342

14.02-14.06 ppm are due to the presence of the terminal carbons of methyl groups.

343

The methylene carbons of long carbon chains appear in the range of 22.55-34.05 ppm.

344

These results further proved that the successful conversion of triacylglycerols to

345

biodiesel was achieved.

346

4. Conclusions

347

This study is the first investigation of a novel immobilized MAS1 lipase-

348

catalyzed synthesis of biodiesel from WCO with one-step addition of methanol in the

349

solvent-free system. Immobilized MAS1 lipase exhibited much higher catalytic

350

activity than the three commercially available immobilized lipases. The high catalytic

16

351

efficiency (biodiesel yield of 95.45%), good resistance to high methanol concentration

352

and relatively good reusability enable immobilized MAS1 lipase to be a great

353

potential catalyst for biodiesel production.

354

Acknowledgements

355

This work was supported by National High Technology Research and Development

356

Program of China (2014AA093514, 2014AA093601) and Science and Technology

357

Planning project of Guangdong province (2014B020204003, 2015B020231006,

358

2015TX01N207).

359 360

References

361

1. Aguieiras, E. C. G., Cavalcanti-Oliveira, E. D., Freire, D. M. G., 2015. Current status and new

362 363

developments of biodiesel production using fungal lipases. Fuel 159, 52-67. 2. Almeida, J. R., Fávaro, L. C., Quirino, B. F., 2012. Biodiesel biorefinery: opportunities and

364

challenges for microbial production of fuels and chemicals from glycerol waste. Biotechnol.

365

Biofuels 5, 48.

366 367 368

3. Antczak, M. S., Kubiak, A., Antczak, T., Bielecki, S., 2009. Enzymatic biodiesel synthesis-Key factors affecting efficiency of the process. Renew. Energ. 34, 1185-1194. 4. Arumugam, M., Ponnusami, V., 2014. Biodiesel production from Calophyllum inophyllum oil

369

using lipase producing Rhizopus oryzae cells immobilized within reticulated foams. Renew.

370

Energ. 64, 276-282.

371 372

5. Basso, A., Froment, L., Hesseler, M., Serban, S., 2013. New highly robust divinyl benzene/acrylate polymer for immobilization of lipase CALB. Eur. J. Lipid Sci. Technol. 115,

17

373

468-472.

374

6. Bernardes, O. L., Bevilaqua, J. V., Leal, M. C. M. R., Freire, D. M. G., Langone, M. A. P., 2007.

375

Biodiesel fuel production by the transesterification reaction of soybean oil using immobilized

376

lipase. Appl. Biochem. Biotechnol. 105-114.

377 378 379 380

7. Chen, Z. F., Zong, M. H., Wu, H., 2005. Improving enzymatic transformation of waste edible oil to biodiesel by adding organic base. Div. Fuel Chem. 50, 2. 8. de Araújo, C. D. M., de Andrade, C. C., e Silva, E. S., Dupas, F. A, 2013. Biodiesel production from used cooking oil: A review. Renew. Sust. Energ. Rev. 27, 445-452.

381

9. Deng, L., Xu, X. B., Haraldsson, G. G., Tan, T. W., Wang, F., 2005. Enzymatic production of

382

alkyl esters through alcoholysis: A a critical evaluation of lipases and alcohols. J. Am. Oil

383

Chem. Soc. 82, 341-347.

384

10. Dizge, N., Aydiner, C., Imer, D. Y., Bayramoglu, M., Tanriseven, A., Keskinler, B., 2009.

385

Biodiesel production from sunflower, soybean, and waste cooking oils by transesterification

386

using lipase immobilized onto a novel microporous polymer. Bioresour. Technol. 100, 1983-

387

1991.

388

11. Duarte, S. H., Hernández, G. L. P., Canet, A., Benaiges, M. D., Maugeria, F., Valero, F., 2015.

389

Enzymatic biodiesel synthesis from yeast oil using immobilized recombinant Rhizopus

390

oryzae lipase. Bioresour. Technol. 183, 175-180.

391

12. Farag, H., El-Maghraby, A., Taha, N. A., 2011. Optimization of factors affecting esterification

392

of mixed oil with high percentage of free fatty acid. Fuel Process. Technol. 92, 507-510.

393

13. Goembira, F., Saka, S., 2013. Optimization of biodiesel production by supercritical methyl

394

acetate. Bioresour. Technol. 131, 47-52.

18

395

14. Haigh, K. F., Abidin, S. Z., Vladisavljević, G. T., Saha, B., 2013. Comparison of Novozyme

396

435 and Purolite D5081 as heterogeneous catalysts for the pretreatment of used cooking oil

397

for biodiesel production. Fuel 111, 186-193.

398

15. Halim, S. F. A., Kamaruddin, A. H., Fernando, W. J. N., 2009. Continuous biosynthsis of

399

biodiesel from waste cooking palm oil in a packed bed reactor: Optimization using response

400

surface methodology (RSM) and mass transfer studies. Bioresour. Technol. 100, 710-716.

401

16. Huang, J. J., Xia, J., Jiang, W., Li, Y., Li, J. L., 2015. Biodiesel production from microalgae oil

402

catalyzed by a recombinant lipase. Bioresour. Technol. 180, 47-53.

403

17. Iso, M., Chen, B., Eguchi, M., Kudo, T., Shrestha, S., 2001. Production of biodiesel fuel from

404

triglycerides and alcohol using immobilized lipase. J. Mol. Catal. B, Enzym. 16, 53-58.

405

18. Ji, Q. C., Xiao, S. J., He, B. F., Liu, X. N. 2010. Purification and characterization of an organic

406

solvent-tolerant lipase from Pesudomonas aeruginosa LX1 and its application for biodiesel

407

production. J. Mol. Catal. B, Enzym. 66, 264-269.

408

19. Juan, J. C., Kartika, D. A., Wub, T. Y., Hin, T. Y., 2011. Biodiesel production from jatropha oil

409

by catalytic and non-catalytic approaches: an overview. Bioresour. Technol. 102, 452-460.

410

20. Kumar, M., Ghosh, P., Khosla, K., Thakur, I. S., 2016. Biodiesel production from municipal

411 412

secondary sludge. Bioresour. Technol. 216, 165-171. 21. Kuo, T. C., Shaw, J. F., Lee, G. C., 2015. Conversion of crude Jatropha curcas seed oil into

413

biodiesel using liquid recombinant Candida rugosa lipase isozymes. Bioresour. Technol. 192,

414

54-59.

415 416

22. Lan, D. M., Qu, M., Yang, B., Wang, Y. H., 2016. Enhancing production of lipase MAS1 from marine Streptomyces sp. Strain in Pichia pastoris by chaperones co-expression. Electron. J.

19

417 418

Biotechnol. 22, 62-67. 23. Lee, J. H., Kim, S. B., Kang, S. W., Song, Y. S., Park, C., Han, S. O., Kim, S. W., 2011.

419

Biodiesel production by a mixture of Candida rugosa and Rhizopus oryzae lipases using a

420

supercritical carbon dioxide process. Bioresour. Technol. 102, 2105-2108.

421

24. Lee, J. H., Kin, S. B., Yoo, H. Y., Lee, J. H., Han, S. O., Park, C., Kim, S. W., 2013. Co-

422

immobilization of Candida rugosa and Rhyzopus oryzae lipases and biodiesel production. J.

423

Chem. Eng. 30, 1335-1338.

424

25. Li, D. M., Qin, X. L., Wang, J. R., Yang, B., Wang, W. F., Huang, W. L., Wang, Y. H., 2015.

425

Hydrolysis of soybean oil to produce diacylglycerol by a lipase from Rhizopus oryzae. J.

426

Mol. Catal. B, Enzym. 115, 43-50.

427

26. Li, L., Du, W., Liu, D., Wang, L., Li, Z., 2006. Lipase-catalyzed transesterification of rapeseed

428

oils for biodiesel production with a novel organic solvent as the reaction medium. J. Mol.

429

Catal. B, Enzym. 43, 58-62.

430

27. López, E. N., Medina, A. R., Moreno, P. A. G., Cerdán, L. E., Valverde, L. M., Grima, E. M.,

431

2016. Biodiesel production from Nannochloropsis gaditana lipids through transesterification

432

catalyzed by Rhizopus oryzae lipase. Bioresour. Technol. 203, 236-244.

433 434 435

28. Meng, X., Chen, G., Wang, Y., 2008. Biodiesel production from waste cooking oil via alkali catalyst and its engine test. Fuel Process. Technol. 89, 851-857. 29. Modi, M. K., Reddy, J. R. C., Roa, B. V. S. K., Prasad, R. B. N., 2007. Lipase-mediated

436

conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor. Bioresour.

437

Technol. 98, 1260-1264.

438

30. Qin, X. L., Huang, H. H., Lan, D. M., Wang, Y. H., Yang, B., 2014. Typoselectivity of crude

20

439

Geobacillus sp. T1 lipase fused with a cellulose-binding domain and its use in the synthesis

440

of structured lipids. J. Am. Oil Chem. Soc. 91, 55-62.

441 442 443

31. Qin, X. L., Wang, Y. M., Wang, Y. H., Huang, H. H., Yang, B., 2011. Preparation and characterization of 1,3-dioleoyl-2-palmitoylglycerol. J. Agric. Food Chem. 59, 5714-5719. 32. Razack, S. A., Duraiarasan, S., 2016. Response surface methodology assisted biodiesel

444

production from waste cooking oil using encapsulated mixed enzyme. Waste Manage. 47, 98-

445

104.

446

33. Rodrigues, J., Canet, A., Rivera, I., Osório, N. M., Sandoval, G., Valero, F., Ferreira-Dias, S.,

447

2016. Biodiesel production from crude Jatropha oil catalyzed by non-commercial

448

immobilized heterologous Rhizopus oryzae and Carica papaya lipases. Bioresour. Technol.

449

213, 88-95.

450

34. Romdhane, I. B. B., Romdhane, Z. B., Bouzid, M. H., Gargouri, A., Belghith, H., 2013.

451

Application of a chitosan-immobilized Talaromyces thermophilus lipase to a batch biodiesel

452

production from waste frying oils. Appl. Biochem. Biotech. 171, 1986-2002.

453 454 455 456 457

35. Royon, D., Daz, M., Ellenrieder, G., Locatelli, S., 2007. Enzymatic production of biodiesel from cotton seed oil using t-butanol as a solvent. Bioresour. Technol. 98, 648-653. 36. Salis, A., Pinna, M., Monduzzi, M., Solinas, V., 2005. Biodiesel production from triolein and short chain alcohols through biocatalysis. J. Biotechnol. 119, 291-299. 37. Singh, J., Singh, M. K., Kumar, M., Thakur, I. S., 2015. Immobilized lipase from

458

Schizophyllum commune ISTL04 for the production of fatty acids methyl esters from

459

cyanobacterial oil. Bioresour. Technol. 188, 214-218.

460

38. Tan, T. W., Lu, J. K., Nie, K. L., Deng, L., Wang, F., 2010. Biodiesel production with

21

461 462

immobilized lipase: A review. Biotechnol. Adv. 28, 628-634. 39. Ullah, K., Ahmad, M., Sofia, Qureshi, F. A., Qamar, R., Sharma, V. K., Sultana, S., Zafar, M.,

463

2015. Synthesis and characterization of biodiesel from Aamla oil: A promoting non-edible oil

464

source for bioenergy industry. Fuel Process. Technol. 133, 173-182.

465 466

40. Utlu, Z., 2007. Evaluation of biodiesel obtained from waste cooking oil. Energy Sources, Part A 29, 1295-1304.

467

41. Véras, I. C., Silva, F. A. L., Ferrão-Gonzales, A. D., Moreau, V. H., 2011. One-step enzymatic

468

production of fatty acid ethyl ester from high-acidity waste feedstocks in solvent-free media.

469

Bioresour. Technol. 102, 9653-9658.

470

42. Wang, X. M., Li, D. M., Qu, M., Durrani, R., Yang, B., Wang, Y. H., 2017. Immobilized

471

MAS1 lipase showed high esterification activity in the production of triacylglycerols with n-3

472

polyunsaturated fatty acids. Food Chem. 216, 260-267.

473 474 475 476

43. Wang, Y. H., Mai, Q. Y., Qin, X. L., Yang, B., Wang, Z. L., Chen, H. T., 2010. Establishment of an evaluation model for human milk fat substitutes. J. Agric. Food Chem. 58, 642-649. 44. Yagiz, F., Kazan, D., Akin, A. N., 2007. Biodiesel production from waste oils by using lipase immobilized on hydrotalcite and zeolites. Chem. Eng. J. 134, 262-267.

477

45. Yan, J. Y., Zheng, X. L., Li, S. Y., 2014. A novel and robust recombinant Pichia pastoris yeast

478

whole cell biocatalyst with intracellular over expression of a Thermomyces lanuginosus

479

lipase: Preparation, characterization and application in biodiesel production. Bioresour.

480

Technol. 151, 43-48.

481 482

46. You, Q., Yin, X., Zhao, Y., Zhang, Y., 2013. Biodiesel production from jatropha oil catalyzed by immobilized Burkholderia cepacia lipase on modified attapulgite. Bioresour. Technol.

22

483 484

148, 202-207. 47. Yuan, D. J., Lan, D. M., Xin, R. P., Yang B., Wang Y. H., 2015. Screening and characterization

485

of a thermostable lipase from marine Streptomyces sp. strain W007. Biotechnol Appl

486

Biochem. 63, 41-50.

487

48. Zhao, T. T., No, D. S., Kim, Y. H., Kim, Y. S., Kim, I. H., 2014. Novel strategy for lipase-

488

catalyzed synthesis of biodiesel using blended alcohol as an acyl acceptor. J. Mol. Catal., B:

489

Enzym. 107, 17-22.

490 491 492 493 494 495 496 497 498 499 500 501 502 503 504

23

505 506

Figure captions

507

Fig.1. Time course of transesterification of WCO with methanol catalyzed by immobilized MAS1,

508

Novozym 435, Lipozyme RM IM, and Lipozyme TL IM. Reaction conditions: oil/methanol molar

509

ratio of 1:3, enzyme loading of 80 U/g substrate, reaction temperature of 40 °C, one-step addition

510

of methanol at the beginning of the reaction for 24 h.

511

Fig.2. Effects of oil/methanol molar ratio, enzyme loading and reaction temperature on biodiesel

512

yield. (a) Effect of oil/methanol molar ratio on biodiesel yield. Reaction conditions: reaction

513

temperature of 40 °C, enzyme loading of 80 U/g substrate, one-step addition of methanol at the

514

beginning of the reaction for 24 h; (b) Effect of enzyme loading on biodiesel yield. Reaction

515

conditions: reaction temperature of 40 °C, oil/methanol molar ratio of 1:3, one-step addition of

516

methanol at the beginning of the reaction for 24 h; (c) Effect of temperature on biodiesel yield.

517

Reaction conditions: oil/methanol molar ratio of 1:3, enzyme loading 80 U/g substrate, one-step

518

addition of methanol at the beginning of the reaction.

519

Fig.3. Reusability of immobilized MAS1 lipase during transesterification of WCO with methanol.

520

Reaction conditions:oil/methanol molar ratio of 1:3, enzyme loading 80 U/g substrate, reaction

521

temperature of 30 °C, one-step addition of methanol at the beginning of the reaction for 24 h.

522

Fig. 4. The FA composition of the final FAME product.

523

24

524

525 526

Fig.1. Time course of transesterification of WCO with methanol catalyzed by immobilized MAS1,

527

Novozym 435, Lipozyme RM IM, and Lipozyme TL IM. Reaction conditions: oil/methanol molar

528

ratio of 1:3, enzyme loading of 80 U/g substrate, reaction temperature of 40 °C, one-step addition

529

of methanol at the beginning of the reaction for 24 h.

530 531 532

25

533 534 535

536 537

Fig.2. Effects of oil/methanol molar ratio, enzyme loading and reaction temperature on biodiesel

538

yield. (a) Effect of oil/methanol molar ratio on biodiesel yield. Reaction conditions: reaction

539

temperature of 40 °C, enzyme loading of 80 U/g substrate, one-step addition of methanol at the

540

beginning of the reaction for 24 h; (b) Effect of enzyme loading on biodiesel yield. Reaction

541

conditions: reaction temperature of 40 °C, oil/methanol molar ratio of 1:3, one-step addition of

542

methanol at the beginning of the reaction for 24 h; (c) Effect of temperature on biodiesel yield.

543

Reaction conditions: oil/methanol molar ratio of 1:3, enzyme loading 80 U/g substrate, one-step

544

addition of methanol at the beginning of the reaction.

545 546 547

26

548 549 550 551

552 553

Fig.3. Reusability of immobilized MAS1 lipase during transesterification of WCO with methanol.

554

Reaction conditions:oil/methanol molar ratio of 1:3, enzyme loading 80 U/g substrate, reaction

555

temperature of 30 °C, one-step addition of methanol at the beginning of the reaction for 24 h.

556 557 558

27

559 560

561 562

Fig. 4. The FA composition of the final FAME product

563 564

28

565

Table 1 Comparison of the catalytic abilities of different lipases for biodiesel production with one-

566

step addition of methanol in different reaction systems

567

Lipase

Substrate

solvent

Oil/methanol molar ratio

Lipase weight (%)--based on oil weight

Immobilized MAS1 lipase

Waste cooking oils

-

1:3

5

30

24

-

95.45

4

Novozym 435

Soybean oil

-

1:3

5

37

24

-

40

-

TLL whole cell catalyst

Waste cooking oils

-

1:4

6

40

120

5

80

3

Jatropha oil

-

1:6.6

8

30

30

7

62

-

You et al (2013)

Soybean oil

-

1:3

5

40

12

-

-

Zhao et al (2014)

-

Su et al (2007)

Immobilized Burkholderia cepacia lipase Lipozyme RM IM Lipozyme TL IM

T (℃)

t (h)

Water (%)

Biodiesel yield (%)

Number of reuses

Ref

This study

<5 <5

Cotton seed oil Novozym 435

Candida sp. 99-125 Immobilized lipase from Pseudomona s aeruginosa LX1

Cerveró et al (2014) Yan et al (2014)

37.4

Soybean oil Rapesee d oil Soybean oil

nheptane

nhexane

1:1

10

40

12

10

2.44

-

Lu et al (2010)

Soybean oil

tbutanol

1:3

22

30

72

6

80

-

Ji et al (2010)

1:3

10

40

24

-

24.5 19.3

568

29

569 570 571

572 573 574 575 576

30

577 578 579

Highlights

580


Immobilized MAS1 can be efficiently catalytic waste cooking oils into FAME.

581


Immobilized MAS1 showed high tolerance to methanol.

582


More than 95% biodiesel yield was obtained with one-step addition of methanol.

583


Immobilized MAS1 exhibited good reusability during four batch cycles.

584


Characterization of the obtained biodiesel was done.

585 586

31