Au nanoparticles supporting lipase catalyst for biodiesel production from waste tomato

Applied Surface Science xxx (2018) xxx–xxx

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Highly active and stable Fe3O4/Au nanoparticles supporting lipase catalyst for biodiesel production from waste tomato Maria Sarno a,b,⇑, Mariagrazia Iuliano a a b

Department of Industrial Engineering, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy NANO_MATES Research Centre, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy

a r t i c l e

i n f o

Article history: Received 30 November 2017 Revised 23 March 2018 Accepted 6 April 2018 Available online xxxx Keywords: Magnetic nanoparticle supported lipase Tomato seeds recycling High activity recovery Biodiesel production

a b s t r a c t Snowman-like Fe3O4/Au nanoparticles (NPs), made up of small Au nanoparticles grown on larger magnetite NPs, were used to directly bond lipase (E.C.3.1.1.3) from Thermomyces lanuginosus (TL), through physical interactions including interfacial activation. Immobilized lipase was able to hydrolyse olive oil triglycerides with high activity (up to 109%, activity of the immobilized enzyme compared to that of its free form, at pH = 7, standard deviation (SD) of 2.1). Improved stability, derived from gold based NPs, was observed with temperature and pH (e.g. 334.9% at pH = 8.5, . . .). Specifically, snowman-like NPs were used to catalyse the transesterification of tomato seed oil and their activity was compared with that obtained with the free enzyme and the enzyme immobilized on Fe3O4 NPs alone. At a lipase concentration of 20%, reaction temperature of 45 °C, oil/methanol ratio 1:6 obtained by adding two times methanol to a 1:1 M initial solution, with a reaction time of 24 h, immobilized lipase exhibits a remarkable biodiesel yield of 98.5%. In particular, it shows the following: fatty acid methyl ester (FAME) composition in accordance to that of the extracted tomato seed oil; ester (97.2% ± 0.26) and linolenic methyl ester (4.3% ± 0.22 contents) in compliance with EN14103 methods and in agreement with EN14124 requirements. The highest activity was observed at a molar ratio oil/methanol of 1:6 M. It is worth noting, that in transesterification reactions, in contrast with the behaviour shown for hydrolysis, lipase anchored on snowman-like NPs results more active than on magnetite alone. The immobilized lipase activity stays above 84% after three cycles of use, showing an excellent reusability also due to the stabilizing effect of gold. Ó 2018 Published by Elsevier B.V.

1. Introduction Today there is an urgent need for sustainable, clean and efficient sources of energy. Biodiesel as fatty acid methyl esters has many advantages such as biodegradability, no toxicity and a favourable combustion-emission profile, except for NOx emission, compared to petroleum-based diesel [1]. Biodiesel is an attractive substitute to conventional diesel, conveying significant environmental advantages for its renewable feedstock. It can be derived from animal fats, from vegetable oils and also as valuable product by recycling of wastes. The use of vegetable oil for biodiesel production as a renewable energy source can have five main goals: reduction of dependence on fossil energy; increase of the economic value of vegetable oils; increase of the economic value of fruits; reduction of total CO2 emissions and wastes [2,3]. Tomato is one of the most widely cultivated vegetable crops in the world, approximately one third of the tomatoes is used to ⇑ Corresponding author at: Department of Industrial Engineering, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy. E-mail address: [email protected] (M. Sarno).

produce tomato juice, paste, puree, ketchup, sauce and salsa. Tomato pomace as a processing by product consists mainly of peel and seed [4]. Nearly 60% of the total waste is tomato seed, which is usually disposed as livestock feed with low value or is otherwise dumped in landfills causing environmental problems [4]. To take into account environmental concerns and to maximize the economic potential of tomatoes, industries search for alternative uses of tomato seeds. On the other hand, oil content in tomato seeds accounts for 20–40% of seeds (dry basis), while each year 0.20 million tons of oil are extracted in the world [4,5]. Because of the high content of unsaturated fatty acids, especially linoleic acid, tomato seed oil should be a good source of oil, e.g. for cosmetics, energy production [6] that is the simplest route for a profitable reuse, among other uses. There are several methods [7–9] to produce oil from the seeds, including: mechanical pressing; supercritical fluid extraction; and solvent extraction that is, for its simplicity and good yield, the primary method currently used. Production of biodiesel to replace petroleum-refined diesel and with purposes of sustainability and pollution reduction is a known

https://doi.org/10.1016/j.apsusc.2018.04.060 0169-4332/Ó 2018 Published by Elsevier B.V.

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and good technique. The most established method for biodiesel production is chemically catalyzed transesterification. However, the complexity and cost of production and sustainability concerns push towards alternative methods for biodiesel production [10– 12]. Enzyme-catalysed transesterification, that draws high attention because the process is ‘‘greener”, is a relatively new method for biodiesel production [13]. In this method, enzymes which have the capability to catalyze transesterification of oils/fats (lipids) commonly known as lipases are used. Lipases transesterification processes do not have the typical disadvantages of the chemical transesterification, as they are: insensitive to free fatty acid (FFA); the oil does not require a pre-treatment; both the esterification and transesterification take place simultaneously to produce biodiesel directly; and moisture content in the raw oil does not hinder the reaction [10], although above certain values a reduction of lipase activity can occurs [11]. On the other hand, the extended period of reaction and cost of enzymes restrict the wide acceptance of enzymatic transesterification for biodiesel production. Lipase immobilization technology provides a number of important benefits, including: (a) easy separation of product from enzyme; (b) enzyme reusability; and (c) decrease in the inhibition rate. It has been used to obtain biodiesel from soybean oil [1,13–15], cotton seed oil [16], and waste products such as pomace olive oil [17] and cooking oil [18–20]. In these papers covalent immobilization was chosen to anchor enzyme on the nanoparticles, resulting in good activity but lower than that of the free counterpart. On the other hand, new ways of immobilization for more active and stable enzymes, can further reduce biodiesel production costs [13]. Here we report, for the first time, to the best of our knowledge, biodiesel production from tomato waste, by Thermomyces lanuginosus (TL) lipase directly bonded on citric acid modified snowman-like Fe3O4/Au nanoparticles. In particular, the nanoparticles surface easily coupled with lipase, without requiring complex procedures and reagents, through physical adsorption [21]. The advantages of physical adsorption are: (i) reversibility; (ii) possibility to be an easy process; and (iii) enzyme stability preserving activity [21,22]. Interfacial activation, that takes advantage of small amount of residual oleic acid chains from synthesis, also occurs, leading to higher enzymatic activity than that of the native counterpart [23,24]. The lipase from TL has found applications in many different industrial areas, from biodiesel production to fine chemicals [25– 27]. In the present work, lipases have been employed for biodiesel synthesis in extracted pomace seed oil. The nanosupport typically used for enzymes immobilization is inexpensive magnetite [21]. Moreover, to take advantage from gold induced enhanced stability, which has been supposed due to favourable conformational changes and electrostatic interaction [28], small Au NPs were directly grown on larger magnetite nanoparticles. Therefore, in the present study TL lipase immobilized through multiple physical interaction with the support including interfacial interaction, on Au [29–32] inducing magnetite surface polarity modification (Fe3O4/Au NPs), was analysed, in comparison with free enzyme, for its activity and stability with temperature, pH and time. The conversion to biodiesel, at a low lipase concentration, and the activity maintenance, depending on the molar ratio oil/methanol, were also explored.

tonate (Fe(acac)3) (97%), gold(III) chloride trihydrate (HauCl4), citric acid (CA), Thermomyces lanuginosus (TL) lipase (solution
100,000 U/g), bovine serum albumin (BSA), polyvinyl alcohol, potassium hydroxide, olive oil, heptane, methyl heptadecanoate of known purity (99%) and F.A.M.E. Mix (C14-C24) was acquired from Aldrich Chemical Co. All chemicals were of analytical grade. Tomato seeds were from local San Marzano tomatoes.

2.2. Synthesis of snowman-like Fe3O4/Au The synthesis process was modified based on the Hao et al. method [33]. Magnetic Fe3O4/Au NPs (Fe3O4/Au@OA) was prepared in an experimental apparatus as previously described [34]. 1-Octadecene 20 mL, Fe(acac)3 2 mmol, HauCl4 0.1 mmol, 1,2hexadecanediol 10 mmol, oleic acid 12 mmol were mixed in a reaction flask [36] and magnetically stirred under heating from 25 °C to 200 °C for 2 h and then to 285 °C for 1 h. For the washing, centrifugation (7500 rpm; 0.5 h) in ethanol and then in a mixture of 2-propanol and hexane (2:1 v/v), were performed.

2.3. Ligand exchange to obtain hydrophilic NPs The Fe3O4/Au@OA was modified through a ligand exchange with citric acid (CA) at room temperature. 30 g of CA and 30 mL of water (3 M) were first injected into a 10 mL vessel and stirred for 5 min. Then 30 mL of Fe3O4/Au@OA-hexane solution (100 mg of Fe3O4/Au@OA) was loaded into a 50 mL syringe and slowly added to the vessel without stirring. Within 5 min white precipitate was suspended at the interface. A slow transfer of the Fe3O4/ Au NPs into the CA solution occurred in 24 h. Citric acid covering Fe3O4/Au, named Fe3O4/Au@CA, was separated just applying an external magnetic field and finally treated with water [21].

2.4. Lipase immobilization The above modified NPs were mixed with 10 mL of buffer solution (phosphate buffer 0.1 M, to give pH = 3.0, pH = 4.0 and pH = 7.0) comprehending 2 mg of TL (solution
100,000 U/g from Sigma Aldrich), further shaken at 200 rpm, at a temperature of 4 °C, for 180 min. Magnetic Fe3O4/Au@CA _L was separated taking advantage of a magnetic field (the perfect separation of the modified NPs was proved testing the supernatant under a magnetic field). They were treated with phosphate buffer to remove free enzyme, and finally used for the activity tests. Moreover, in the same operating conditions but 10–20–40 mL of buffer solution (phosphate buffer 0.1 M; pH = 7.0) with 2–4–8 mg of lipase (solution
100,000 U/g form Sigma Aldrich) we tested the influence of three different lipase loading.

2.5. Bradford method to estimate lipase immobilized amount Bradford method [35], was used to evaluate the lipase protein concentration. The lipase immobilization efficiency was evaluated from:

ie ¼ ðCi  Cf ÞV1 =Ci V2 ; 2. Experimental 2.1. Materials 2-Propanol and hexane were used as received. 1-Octadecene (90%), oleic acid (OA), 1,2-hexadecanediol (90%), iron(III) acetylace-

where: ie is the immobilization efficiency (%); Ci and Cf are the TL concentrations, in mg mL1, measured in the supernatant at the starting and final point, respectively; V1 and V2 the volumes of solution in mL. The data used were averages of two different tests. BSA was used for calibration.

Please cite this article in press as: M. Sarno, M. Iuliano, Highly active and stable Fe3O4/Au nanoparticles supporting lipase catalyst for biodiesel production from waste tomato, Appl. Surf. Sci. (2018), https://doi.org/10.1016/j.apsusc.2018.04.060

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2.6. Lipase assay Hydrolytic activities tests for immobilized and free lipase were performed in 5.6 mL of olive oil, Polyvinyl alcohol (PVA) 1 w/v% [21], free (0.2 mg/mL) or immobilized lipase (2 mg of lipase loaded on the nanoparticles) and 7 mL 0.025 M of buffer (pH = 7.0). Lipase assays were obtained at 35, 45, 55 and 65 °C during 0.5 h; at 40 °C during 0.5 h, 1 h, 1.5 h and 2 h for pH = 7; and at 40 °C from pH = 4 to pH = 8.5. The activities of free and immobilized lipase, stored for 4 months at temperature of 4 °C, were determined after 1, 2 and 4 months. 0.1 M potassium hydroxide solution was used for titration to measure the total hydrolysed fatty acid. One unit (1 U) of catalyst activity corresponds to the amount of TL able to hydrolyse 1 lmol/min of C18H34O2. Potassium hydroxide 0.1 M solution was also used to titrate olive oil hydrolysis in presence of washed NPs, for this reference experiments no free acids were detected. The ratio: immobilized lipase activity on free lipase activity is, in the following, the activity recovery (%). Measurements were performed in triplicate, and the mean values as well as standard deviations and coefficients of variation are reported.

Tomato seeds, separated from pulp and cleaned in water, was dried for 3 h in an oven at 50 °C. Hexane at 50 °C was used to extract tomato oil from the seeds in a batch reactor. After extraction, the tomato oil was separated from residue by means of centrifugation at 7500 rpm for 10 min at 4 °C [4]. The hexane was further removed [36] using a rotary vacuum evaporator (Across International SE05.110). The mass of oil extracted was calculated as a percentage by the following formula [37]:

ðwt: of predried sample  wt: of extracted sampleÞ ðwt: of predried sampleÞ  100

where: wt. predried sample is the weight of the seed after drying, wt. of extracted sample is the weight of seed after extraction and drying in the vacuum evaporator. 2.8. Synthesis of methyl ester Methyl ester production (see Scheme 1) was carried out in 25 ml vessel reactor under continuous stirring (200 rpm) at 45 °C. Three different experiments were performed starting from 1 g of tomato seed oil (reddish orange colour in the Scheme 1), in the presence of free or immobilized lipase (10% g of enzyme/g of tomato oil), and adding: 1st experiment, oil:methanol molar ratio 1:1; 2nd experiment, oil:methanol molar ratio 1:2 M; and 3rd experiment, oil:methanol molar ratio 1:6 M. Then, for each experiments new methanol was added, two times, to reach 1:3 M, 1:6 M and 1:18 M oil/methanol ratio, respectively. This is to give the right amount of methanol, hindering possible catalyst deactivation [17,38]. The mixtures were allowed to react for 24 h typically giving a two phase separation, with the reaction product (transparent

CH2—OCOR1 | CH—OCOR2 | CH2—OCOR3 Tomato seed Oil

conv ersion ð%Þ ¼

nester mester  100 ¼  100 moil 3  noil 3  MW MW ester oil

mester MW oil ¼   100 moil 3  MW ester where mester: weight of ester collected (g); moil: weight of the oil sample (g); MWoil: averaged molecular weight of oil sample.

2.7. Tomato seeds oil extraction

Oil ð%Þ ¼

light yellow phase) on the top and an unreacted product + glycerol + excess methanol mixture (dense brown colour phase) on the bottom [38] (see Scheme 1). Furthermore, starting form the best results in terms of conversion obtained, two new experiments, with 1:6 oil/methanol molar ratio, were performed, analysing different amount of lipase, e.g. 5 and 20% g of enzyme for grams of tomato oil. Effect of cycle number were finally examined at 20% of enzyme (grams of enzyme per grams of tomato oil). After the transesterification reactions immobilized enzyme was magnetically separated and the sample was centrifuged for 5 min at 5000 rpm. The upper layer was wet washed with hot distilled water at 60 °C and then dried to obtain tomato oil biodiesel. The oil conversion to methyl ester [39] (washer and dried upper layer after reaction), that is the yield to methyl ester (%), named conversion in the following, was determined as follows:

i

MWi: molecular weight of fatty acid i; %mi: percentage of fatty acid i in the raw material. MWester: averaged molecular weight of fatty acid ester.

MW ester ¼

Methanol

X ðMW i  %mi Þ þ 14 i

The FAME analysis was carried out with a split injection onto an analytical column and a FID detector. In particular, they were analysed by GC–MS (Thermo Fischer Scientific) by using TraceGOLDTM TG-WaxMS GC capillary column (0.25 mm  0.25 mm  30 m) from 50 to 250 °C, 6 °C/min [40]. Injector and detector temperatures were set at 200 and 250 °C, respectively. Carrier gas helium, column flow 1 mL/min, split flow 50 mL/min, split ratio 50:1; injection volume 1 lL; FID: temperature 250 °C. To determine the retention times of the fatty acid methyl esters, an available commercially FAME standard has been used. In particular, the FAME mixture with the known concentration and derivatised tomato seed oil, obtained by a methanol:BF3 method [40], have been tested to determine the retention time of each component experimentally, to be compared with the methyl esters produced. It has been observed that the retention times were almost similar. The methyl ester content has been evaluated in compliance with EN14214. Heptadecanoic acid methyl ester was used as an internal standard. In particular, total methyl ester and linolenic methyl ester contents were evaluated following the procedures described in EN14103. Methyl heptadecanoate, 10 mg/ml solution was obtained accurately weighing 500 mg of methyl heptadecanoate in a 50 ml volumetric flask and make up to mark with heptane. For sample preparation 250 mg of sample was loaded in a 10 ml vial, then 5 ml of methyl heptadecanoate solution was slowly

Fe3O4/Au@CA_L

3CH3OH

X ðMW i  %mi Þ þ 38

MW oil ¼ 3 

CH2OH | CHOH | CH2OH

R1COOCH3

Glycerol

Methyl ester

R2COOCH3 R3COOCH3

Scheme 1. Transesterification of Tomato seed oil.

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added. Duplicated of the biodiesel sample were prepared to measure the reproducibility of the analysis. The ester C content, expressed as a mass fraction in percent, is calculated according with EN14103. In particular, we have used the modified method reported in [41], as indicated in note b of EN14214 Table 1, for the determination. Measurements were performed in triplicate. 2.9. Characterization of catalyst Transmission electron microscopy (TEM) (FEI-Tecnai; 200 kV) was used to analyse NPs after sonication in hexane for few minutes. Thermogravimetric analyses (SDTQ 600-TA Instruments) were obtained in air (10 °C/min). FT-IR spectra (Vertex 70 apparatus (Bruker Corporation)) by applying KBr technique, X-ray diffraction measurements (Bruker D8 X-ray diffractometer using CuKa radiation) were also performed. 3. Results and discussion 3.1. Lipase nanoparticles production

nanoparticles are characterized by a quasi-spherical Fe3O4 NPs (about 8 nm diameter) supporting faceted Au NPs exposing their square section (<2 nm) (see Fig. 2 insert, for which, in the sample preparation, a more diluted solution was used). 3.3. Thermogravimetric analysis (TG-DTG) In Fig. 3a the thermogravimetric profiles of oleic acid, citric acid, Fe3O4/Au@OA and Fe3O4/Au@CA NPs are shown for comparison. The profiles of OA and CA highlight the different decomposition/ oxidation temperatures of the two acids chains. CA decomposition mainly occurs at temperatures lower than those shown by OA. In particular, the TG curve of Fe3O4/Au@OA displays the following: (i) a weight loss of about 3 wt% at the beginning; followed by (ii) the oxidation-decomposition of OA (18 wt% total based). A lower organic content was observed for Fe3O4/Au@CA, likely due to the inferior molecular weight of CA. The downshift of the DTG peak (Fig. 3b) highlights the successful ligand exchange. On the other hand, the occurrence of a considerable weight loss in the temperature range between 225 °C and 425 °C suggests OA residual presence. It is worth noting, that the organic content in Fe3O4/Au@CA (7 wt%) was lower than the organic content measured (13 wt %) on magnetite alone after ligand exchange to replace oleic acid with citric acid [21], suggesting an increased replacement of the OA chains in the presence of Au NPs. The successful lipase immobilization in Fe3O4/Au@CA_L, TG profile in Fig. 3c, is highlighted by the two lipase typical peaks at temperatures lower than 225 °C (see Fig. 3d).

The mixture of Fe3O4/Au@OA in hexane (30 mL) and 30 mL of CA in water undergoes a phase separation: hexane containing Fe3O4/Au@OA at the top and CA in water at the bottom (Fig. 1). After precipitation of Fe3O4/Au@OA at the interface, the ligand exchange process took place, followed by a Fe3O4 transfer into the water ligand solution, which requires approximately 24 h (see the photo in Fig. 1). The binding of COOA groups of CA on the Fe3O4/Au surface changes the colour of CA solution to yellow. In Fig. 1a scheme of the surface modification is shown. The bimetallic nanoparticles at the end of the synthesis result capped (see Fig. 1 insert a) with a hydrophobic layer of oleic acid. This shows more affinity with the magnetite surface, which results typically capped with the more polar ligand in snowman-like Fe3O4/ Au NPs [42,43]. Oleic acid substitution on Fe3O4, with the citric acid chains, proceeds through a ligand exchange process and with a higher efficiency, due to the gold NPs, than that observed on magnetite alone [21]. This is probably due to a gold induced Fe3O4 surface polarity modification, enhancing affinity with polar molecules. In particular, due to the heterojunction between magnetite and gold, and the difference in their work function [29–31], electrons flow from the Fe3O4 to Au [32]. In the final step, physical adsorption through polar interaction between ACOO moieties of CA and the positive charges on lipase [44] and hydrogen bonds between the enzyme ANH groups and carboxyl groups on the NPs surface [45–49] permits the lipase direct immobilization on the NPs. Interfacial activation, due to OA survived ligand molecules [23,24] also occurs, as discussed in more detail below, while covalent bonds appear difficult to occur, as previously reported [45,46].

The typical bands at 645 cm1 (m1 (FeAO)) and 590 cm1 (m2 (FeAO)) for Fe3O4/Au@OA can be seen in Fig. 4 [50]. The peaks at 1532 cm1 and 1645 cm1, asymmetric and symmetric COOA stretch, in the Fe3O4/Au@OA spectrum, mean that ACOOH groups are bound to the NPs surface [51]. CA coated NPs (cyan) and CA (blue) FTIR spectra are shown in Fig. 4, too. The three bands at 1705 cm1, 1740 cm1 and 1755 cm1 are the carbonyl vibration bands of the CA carboxyl groups [52]. After CA adsorption on NPs, a new large band at about 1600 cm1 [53–55] appears in place of the first two C@O bands of free CA, due to complexation with the Fe3O4 surface [53]. On the other hand, the typical bands due to the oleic acid bonding on the surface of the nanoparticles [56] are suggested by the sawtooth profiles in the corresponding IR regions. Fe3O4/Au@CA_L FTIR spectrum exhibited the bands of both lipase and Fe3O4/Au. After immobilization the spectrum is dominated by the typical lipase bands also due to the interaction with the nano-support. The change in the wavelength around 1700 cm1 is due to lipase-CA complexation [46]. The vibrational bands of amide I and amide II [21] result shifted to higher wavelength (1673 cm1 and 1556 cm1), suggesting a protein conformational change and formation of hydrogen bonds [45,46].

3.2. Transmission electron microscopy (TEM) analysis

3.5. XRD studies on Fe3O4 NPs

The images, obtained by TEM, at lower magnification revealed the formation of nanoparticles (Fig. 2). The snowman-like

X-ray diffraction analysis of Fe3O4/Au NPs is shown in Fig. 5. The magnetite typical peaks [21,56] together with the peaks at

3.4. FT-IR spectroscopy analysis

Table 1 Immobilization efficiency (%) and activity recovery (%) at different pH, T = 40 °C. pH

Immobilization efficiency (%)

Activity recovery (%) pH 3

3 4 7 7

84.3 74.4 61.8 61.8

42.1

Activity recovery (%) pH 4

Activity recovery (%) pH 7

59.6

108.7 106.2 100.6

Activity recovery (%) pH 8.5

334.9

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5

Fig. 1. Schematic representation of the ligand exchange process of Fe3O4/Au@OA with CA and Lipase immobilization on the surface of Fe3O4/Au@CA.

38.2° (1 1 1), 44.4° (2 0 0) and 64.6° (2 20) ascribed to Au positions [57] can be observed in Fig. 5.

3.6. Hydrolytic activity evaluation

Fig. 2. TEM images of Fe3O4/Au@OA nanoparticles.

Fe3O4/Au based catalyst was analysed in comparison with the catalyst immobilized on magnetite NPs alone [21] and the free enzyme. Considering that the pI (isoelectric point) of TL is 4.4, immobilization efficiency (IE) has been obtained at pH 3 (before pI), pH 4 (close to pI) and pH 7 (optimum pH for free TL [11]). Immobilization efficiencies of 85%, 74% and 62% were found, respectively, see Table 1, indicating that higher lipase loading is obtained, as expected, in the range of pH in which the polar interactions are favoured. This is due to Au NPs on Fe3O4, changing the surface electronic behaviour of the nanoparticles and increasing the tendency to exchange ligand with CA, unlike on magnetite alone [21]. The ratio between the activity of immobilized TL and that of its free counterpart, named the activity recovery % (AR %), measured at pH 7, results almost the same for the different immobilization

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120

80

Transmittance (%)

Weight (%)

a

40

0 25

225

425 Temperature (°C)

625

Universal V4.5A TA Instruments

OA CA Fe3O4/Au@OA Fe3O4/Au@CA

3

1

Deriv. Weight (%/min)

2

L Fe3O4/Au@CA_L

b 25

225

425 Temperature (°C)

625

2000

1500

1000

500

-1

Wavelenght (cm ) Fig. 4. FT-IR spectra in the range of wavenumber 2000–400 cm1 of nanoparticles synthesized in the presence of surfactants (Fe3O4/Au@OA), nanoparticles after the ligand exchange (Fe3O4/Au@CA), nanoparticles after lipase immobilization (Fe3O4/ Au@CA_L), free citric acid, oleic acid and lipase.

0

Universal V4.5A TA Instruments

Fe3O4/Au

111

Intensity

120

80

311 220

511

200

400

440 220 422

40

0 25

10 225 Temperature (°C)

425 Universal V4.5A TA Instruments

3

2

1

d

25

225 Temperature (°C)

20

30

40

50

60

70

2 Theta Fig. 5. XRD spectra of Fe3O4/Au.

Deriv. Weight (%/min)

Weight (%)

c

0 425

Universal V4.5A TA Instruments

Fig. 3. Fe3O4/Au@OA, OA, Fe3O4/Au@CA, CA: TG analyses (a); and DTG analyses in the range 0–3%/min to magnified the smaller weight losses (b). Fe3O4/Au@CA, lipase solution (Sigma Aldrich), Fe3O4/Au@CA_L: TG analyses (c); and DTG analyses in the range 0–3%/min to magnified the smaller weight losses (d).

pH. This result suggests an increased stability induced by the enzyme-NPs interaction in the presence of gold NPs, e.g. strong ionic interaction [58] . . ., able to compensate for the pH variations between the immobilization and the operating phases. On the other hand at higher pH (pH 8.5) the activity recovery grows up, showing an increased stability for the immobilized enzyme relative to its soluble form. Furthermore, at pH 7 the effect of lipase amount during immobilization was analysed, see Fig. 6a. The IE and AR% decrease from 2 mg to 8 mg of lipase amount, this is probably due to diffusion limitations caused by the high activity of the catalyst [27,59]. The IE and AR % were also evaluated under immobilizing time increase. Fig. 6b evidences that under coupling time increase from 1 to 3 h, the immobilized lipase amount increase, likely due to residual carboxyl groups able to bind lipase. Improved conformation flexibility determines the AR% increase with time [21,60].

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7

80

80

40

40 Immobilization Efficiency (%) Activity Recovery (%)

0

2,5

5,0

80

40

40

0

60

80

100

140

160

80 60 40

Free Lipase Immobilized Lipase

100 Relative Activity (%)

Relative Activity (%)

Free Lipase Immobilized Lipase

c

120

180

0

Time (min)

Amounts of lipase (mg)

100

120

80

0 10,0

7,5

Immobilization Efficiency (%) Activity Recovery (%)

b

120

Activity Recovery (%)

120

Immobilization Efficiency (%)

a

120

Activity Recovery (%)

Immobilization Efficiency (%)

M. Sarno, M. Iuliano / Applied Surface Science xxx (2018) xxx–xxx

d

80 60 40 20

20 35

40

45

50

55

60

0

65

4

5

6

Temperature (°C) Free Lipase Immobilized Lipase

80 60 40 20 0 20

100 Relative activity (%)

Relative Activity (%)

100

e 40

60

80

7

8

9

pH

100

120

Time (min)

Free Lipase Immobilized Lipase

80 60 40 20 0

f 0

20

40

60

80

100

120

Storage time (days)

Fig. 6. Immobilization efficiency and activity recovery (reaction conditions: temperature, 40 °C; pH, 7; time, 30 min): (a) effect of lipase amounts (immobilization conditions: coupling temperature, 4 °C; coupling pH, 7; coupling time, 3 h); (b) effect of coupling time (immobilization conditions: coupling temperature, 4 °C; coupling pH, 7; lipase amount, 2 mg). Hydrolytic activities* of the free and immobilized lipases (immobilization conditions: coupling temperature, 4 °C; coupling pH, 7; coupling time: 3 h; lipase amount, 2 mg): (c) effect of temperature (reaction conditions: pH, 7; time, 30 min); (d) effect of pH (reaction conditions: time, 30 min; temperature 40 °C); (e) effect of time (reaction conditions: temperature, 40 °C; pH, 7); (f) effect of storage time (reaction conditions: time 30 min; temperature, 40 °C; pH, 7). *The maxima were defined as 100% activity.

The activity recovery higher than that of the free enzyme can be attributed to lipase conformational structure modifications, mainly interfacial activation [23,24] and higher availability of the active sites [29]. Indeed, Au, in the reaction media, can act as conduction centres to facilitate transfer of electrons, helping enzymes to assume a favourable orientation. Gold and silver have been found able to absorb protein and anchor enzyme retaining their activity [61]. A comparison between the activity recovery obtained by using magnetite alone [21] and magnetite/gold NPs as support, must take into account the lower amounts of enzyme loaded on Fe3O4/Au NPs (e.g. different space availability affecting bond formation). Indeed, although under the same operating conditions (Immobilization conditions: coupling temperature, 4 °C; coupling

pH, 7; lipase amount, 2 mg. Reaction conditions: temperature, 40 °C; pH, 7; time, 30 min) the amount of lipase on the two supports results different. On the other hand, the different interaction of the lipase with the support, e.g. in presence of gold prevails ionic interaction and probably the lower amount of residual oleic acid chains, must be taken into account as well. This can also explain the small differences between the activities of free and immobilized lipase at pH = 7, here observed (see Table 1). The relative activity, the ratio between the activity of immobilized or free enzyme respect to their highest activity, varies at different temperatures and pH. Free lipase shows its highest activity at a temperature of 40 °C, that increases until 45 °C for the immobilized enzyme, see Fig. 6c. Moreover, in the temperature range

Please cite this article in press as: M. Sarno, M. Iuliano, Highly active and stable Fe3O4/Au nanoparticles supporting lipase catalyst for biodiesel production from waste tomato, Appl. Surf. Sci. (2018), https://doi.org/10.1016/j.apsusc.2018.04.060

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45–65 °C the lipase immobilized on snowman-like Fe3O4/Au NPs, due to a support inducing stability, results less sensitive to the temperature change than free counterpart and lipase immobilized on Fe3O4 NPs alone [21]. The same behaviour is observed under pH increase from 4 to 8, induced by the enzyme affinity with the support surface [11] and amplified by the gold NPs grown on Fe3O4 [21]. In particular, the free enzyme activity has a maximum at pH = 7 and then fell drastically. On the contrary, immobilized enzyme, due to the support induced protons partitioning between the bulk phase and the enzyme near surface [62] and the induced stability [11], retains a rather high activity as the pH increases, resulting in an AR% after immobilization of 334.9% at pH = 8.5. The immobilized enzyme retained 82% of its activity after 2 h in comparison with 74% for the free enzyme, showing higher thermostability, also if compared with the results obtained using Fe3O4 NPs alone, Fig. 6e. After 30 days of storage 88% of initial activity was observed, Fig. 6d. This is a considerable result also in comparison with that obtained by covalent immobilization [63] and even more improved by the gold NPs.

The UV–Vis spectrum of extracted tomato seed oil was obtained in cyclohexane (1% wt./V g/m3) (see Fig. 7). The presence of a two conjugated double bond system (e.g. linoleic acid) gives rise to a UV absorption at 233 nm. A three double bond conjugated system (e.g. linolenic acid) gives rise to a triplet with absorptions at 270 nm [64]. The effect of oil/methanol molar ratio on biodiesel yield is reported in Fig. 8, together with activity recovery data. Immobilized lipase shows at each molar ratio a conversion higher than that of the free counterpart. At 1:6 M highest conversion of 97.4% is reached, though at a lower molar ratio of 1:3 the conversion results as high as 93.2%. These values obtained at a low lipase concentration, are considerably high even when compared to previous papers reporting transesterification by using immobilized lipase [11,13,65,66]. For both free and immobilized lipase, the conversion decreases when the molar ratio is increased to M > 1:6. This is probably due to methanol inducing catalyst deactivation, that may occur in the presence of excess methanol [17,38] and can also depends on

100

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Fig. 8. Effect of oil to methanol molar ratio on the free and immobilized lipase for biodiesel production. Immobilization conditions: coupling temperature, 4 °C; coupling pH, 7; coupling time: 3 h; lipase amount, 2 mg. Reaction conditions: reaction time 24 h; reaction temperature, 45 °C; lipase concentration, 10%.

Fig. 10. Effect of cycle number on biodiesel production for immobilized lipase, at two different oil/methanol molar ratio. Immobilization conditions: coupling temperature, 4 °C; coupling pH, 7; coupling time, 3 h; lipase amount, 2 mg. Reaction conditions: reaction time 24 h; reaction temperature, 45 °C; lipase concentration, 20%.

Please cite this article in press as: M. Sarno, M. Iuliano, Highly active and stable Fe3O4/Au nanoparticles supporting lipase catalyst for biodiesel production from waste tomato, Appl. Surf. Sci. (2018), https://doi.org/10.1016/j.apsusc.2018.04.060

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how methanol was added [17]. It results more pronounced for free enzyme, as immobilization slows down the substrate inhibition phenomenon (see Fig. 8). The data of activity recovery, in this case the ratio between the conversion achieved by immobilized and free lipase, highlight the catalytic performance of the lipase anchored on the Fe3O4/Au NPs in promoting transesterification of tomato seed oil. As a reference test the experiment at 1:6 oil/ methanol molar ratio has been duplicated in the same operating conditions but magnetite support alone [21], reaching, with a higher lipase loading, 88.1% conversion. It is worth noting, that in contrast with the behaviour shown for hydrolysis, lipase anchored on snowman-like NPs results more active than on magnetite alone in transesterification reactions. This is likely due to specific physical interactions with the support [67], inducing a different nucleophilic selectivity [68].

In Fig. 9 the effect of immobilized lipase concentration at 1:6 M ratio was reported. During the enzymatic transesterification for biodiesel production, the oil conversion was enhanced with the increase in the lipase loading, from 75% conversion at 5% immobilized lipase concentration, to 97.4% and 98.5% conversions at 10% and 20% immobilized lipase concentration, respectively. This is really a remarkable result, especially in view of the limited lipase content used [11,13,65,66], enjoying the multiple interaction of the enzyme with the support including interfacial interaction. Although the increase in conversion is contained, especially from 10% to 20% (g lipase/g oil), the possibility to easily recover the catalyst, thanks to its magnetic component, can justify the use of amount of lipase larger than 5%. The key aspect of the enzyme recycling and reuse was investigated under cycles of 24 h without interruption, and the results

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Fig. 11. GC–MS of biodiesel from tomato seed oil (Immobilization conditions: coupling temperature, 4 °C; coupling pH, 7; coupling time, 3 h; lipase amount, 2 mg. Reaction conditions: reaction time, 24 h; reaction temperature, 45 °C; lipase concentration, 20%; Gas chromatography spectrum of FAMEs (a); mass spectra of Linoleic acid methyl ester (b), Palmitic acid methyl ester (c), and Oleic acid methyl ester (d).

Please cite this article in press as: M. Sarno, M. Iuliano, Highly active and stable Fe3O4/Au nanoparticles supporting lipase catalyst for biodiesel production from waste tomato, Appl. Surf. Sci. (2018), https://doi.org/10.1016/j.apsusc.2018.04.060

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M. Sarno, M. Iuliano / Applied Surface Science xxx (2018) xxx–xxx

are shown in Fig. 10. Catalytic activity of the immobilized lipase was measured five times over a period of 5 days, for the two oil/ methanol molar ratio 1:3 and 1:6 in Fig. 10. Before each cycle the immobilized lipase was washed with sodium phosphate buffer solution (0.1 M, pH 7), after reactions easily separated from product by a magnet, and next reused in a new experiment. The immobilized lipase retains 70% of its initial activity after five cycle, showing an excellent reusability at the molar ratio oil/methanol of 1:3. Moreover, the immobilized lipase activity stays above 70% at the 3°cycle and at about 40% of its initial activity after five successive reuse, showing also at 1:6 M ratio, at which catalyst poisoning phenomena may occur, a good stability, even higher than that shown by TL anchored on Fe3O4 alone (about 25% after five cycles). Finally, a typical chromatogram of biodiesel from tomato seed oil (Fig. 11a) showed that there were nine fatty acid methyl esters (FAMEs), which were further analysed with mass spectrometer (MS). The composition of the biodiesel analysed by GC–MS suggested that tomato seed oil biodiesel (see Table 2) was composed Table 2 The retention times and content of each fatty acid methyl ester in biodiesel from tomato seed oil. FAMEs

Retention time (min)

Area (%)

Heptadecanoic acid, methyl ester Eicosanoic acid, methyl ester Myristic acid, methyl ester Palmitic acid, methyl ester Palmitoelic acid, methyl ester Linoleic acid, methyl ester Oleic acid, methyl ester Linolenic acid, methyl ester Stearic acid, methyl ester

32.90 34.80 35.70 38.24 39.06 42.29 42.43 43.04 43.11

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Fig. 12. TG-DTG profiles of biodiesel and tomato seed oil (Immobilization conditions: coupling temperature, 4 °C; coupling pH, 7; coupling time, 3 h; lipase amount, 2 mg. Reaction conditions: reaction time, 24 h; reaction temperature, 45 °C; lipase concentration, 20%.

of: palmitic acid methyl ester, oleic acid methyl ester, linoleic acid methyl ester, linolenic acid methyl ester, stearic acid methyl ester, heptadecanoic acid methyl ester, eicosanoic acid methyl ester, myristic acid methyl ester, palmitoelic acid methyl ester. The spectra of three main fatty acid methyl ester are reported in Fig. 11b–d. The three main components account for more than 94% of the total biodiesel. The determined components were in accordance with the composition of the extracted tomato seed oil, as previously reported in other cases [69]. Because in our FAME heptadecanoic methyl ester naturally appears and this can result in a lower measured fatty acid methyl ester content (EN14214), we have used the modified method reported in [41], as indicated in note b of EN14214 Table 1, for the determination. Ester content and linolenic methyl ester amount, that are in agreement with the European Standard, result equal to 97.2% ± 0.26 and 4.3% ± 0.22, respectively. For the sample obtained under the same conditions, but using a 10% lipase concentration, the results are almost the same. Thermogravimetric analysis in a useful way for quantitative evaluation of the produced biodiesel, indeed the thermogravimetric profiles of oil and biodiesel are largely different (Fig. 12). The DTG profiles evidence highlight the differences, biodiesel loses 96% of its weight in the temperature range 50–210 °C, while the decomposition of the tomato seed oil is centred at 270 °C. This result is in a good agreement with the previous GC–MS results.

4. Conclusion Uniform size snowman-like NPs, constituted of a quasispherical Fe3O4 NPs (about 8 nm diameter) supporting faceted Au NPs exposing their square section (<2 nm), were used to prepare a new magnetic lipase catalyst. The significant stability and activity of the bio-catalyst (activity recovery more than 100%; activity retention of 87% and 69% after 30 and 120 days, respectively) can be ascribed to the surface polarity of the support and the resulting interactions. Indeed, the different work function of magnetite and gold NPs enhance the affinity of the Fe3O4 surface with polar molecules, favouring strong ionic interaction. Moreover, in the reaction media, Au NPs can act as conduction centres to facilitate transfer of electron, helping enzymes to assume a favourable orientation. A very high conversion yield, e.g. up to 97.4%, was reached at low lipase loading (10% immobilized lipase concentration), thanks to the multiple interaction of the enzyme with the support, including interfacial interaction. In contrast with the behaviour shown for hydrolysis, lipase anchored on snowman-like NPs results more active than on magnetite alone in transesterification reactions, likely due to specific physical links with the support, inducing a different nucleophilic selectivity. The MS analysis of biodiesel from tomato seed oil showed that three main components (palmitic acid methyl ester, oleic acid methyl ester, linoleic acid methyl ester) account for more than 94% of the total biodiesel, in accordance with the oil composition. For the biodiesel prepared at lipase concentration of 20%, reaction temperature 45 °C, oil/methanol ratio 1:6 M, reaction time 24 h, total FAME and linolenic methyl ester of 97.2% ± 0.26 and 4.3% ± 0.22, were obtained, respectively, in accordance with the specification reported in EN14124. References [1] M.C. Andrade, A.L.A. Parussulo, C.G.C.M. Netto, L.H. Andrade, H.E. Toma, Lipase immobilized on polydopamine-coated magnetite nanoparticles for biodiesel production from soybean oil, Biofuel Res. J. 10 (2016) 403–409. [2] A.M. Giuffre, M. Capocasale, C. Zappia, V. Sicari, T.M. Pellicano, M. Poiana, G. Panzera, Tomato seed oil for biodiesel production, Eur. J. Lipid Sci. Technol. 118 (2016) 640–650. [3] A.M. Giuffrè, V. Sicari, M. Capocasale, C. Zappia, T.M. Pellicanò, M. Poiana, Physico-chemical properties of tomato seed oil (Solanum lycopersicum L.) for biodiesel production, Acta Hortic. 1081 (2015) 237–244.

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M. Sarno, M. Iuliano / Applied Surface Science xxx (2018) xxx–xxx [4] D. Shao, G.G. Atungulu, Z. Pan, T. Yue, A. Zhang, X. Li, Study of optimal extraction conditions for achieving high yield and antioxidant activity of tomato seed, J. Food Sci. 77 (2012) E202–E208. [5] F.J. Eller, J.K. Moser, J.A. Kenar, S.L. Taylor, Extraction and analysis of tomato seed oil, J. Am. Oil Chem. Soc. 87 (2010) 755–762. [6] V. Oreopoulou, C. Tzia, Utilization of plant by-products for the recovery of proteins, dietary fibers, antioxidants, and colorants, Springer Verlag 3 (2007) 209–232. [7] C.H. Tan, H.M. Ghazali, A. Kuntom, C.P. Tan, A.A. Ariffin, Extraction and physicochemical properties of low free fatty acid crude palm oil, Food Chem. 113 (2009) 645–650. [8] M.J. Cocero, L. Calvo, Supercritical fluid extraction of sunflower seed oil with CO2-ethanol mixtures, J. Am. Oil Chem. Soc. 73 (1996) 1573–1578. [9] A. Marasabessy, R. Maelita, P.M. Moeis, J. Sanders, R.A. Weusthuis, Coconut oil extraction by the traditional Java method: an investigation of its potential application in aqueous jatropha oil extraction, Biomass Bioenergy 34 (2010) 1141–1148. [10] J. Sebastian, C. Muraleedharan, A. Santhiagu, A comparative study between chemical and enzymatic transesterification of high free fatty acid contained rubber seed oil for biodiesel production, Cogent Eng. 3 (2016) 1178370. [11] W. Xie, N. Ma, Immobilized lipase on Fe3O4 nanoparticles as biocatalyst for biodiesel production, Energy Fuels 23 (2009) 1347–1353. [12] A.A. Deba, H.I. Tijani, A.I. Galadima, B.S. Mienda, F.A. Deb, L.M. Zargoun, Waste cooking oil: a resourceful waste for lipase catalyzed biodiesel production, IJSRP 4 (2014) 1–6. [13] T. Tan, J. Lu, K. Nie, L. Deng, F. Wang, Biodiesel production with immobilized lipase: a review, Biotechnol. Adv. 28 (2010) 628–634. [14] B. Thangaraj, Z. Jia, L. Dai, D. Liu, W. Du, Lipase NS81006 immobilized on Fe3O4 magnetic nanoparticles for biodiesel production, Ovidius Uni. An. Chem. 27 (2016) 13–21. [15] J. Mukherjea, M.N. Gupta, Lipase coated clusters of iron oxide nanoparticle for biodiesel synthesis in a solvent free medium, Bioresour. Technol. 209 (2016) 166–171. [16] Y.X. Li, B.X. Dong, Optimization of lipase-catalysed transesterification of cotton seed oil for biodiesel production using response surface methodology, Braz. Arch. Biol. Technol. 59 (2016) e16150357. [17] Y. Yücel, Biodiesel production from pomace oil by using lipase immobilized onto olive pomace, Bioresour. Technol. 102 (2011) 3977–3980. [18] X. Wang, X. Qin, D. Li, B. Yang, Y. Wang, One step synthesis of high yield biodiesel form waste cooking oils by a novel and highly methanol-tolerant immobilized lipase, Bioresour. Technol. 235 (2017) 18–24. [19] V.G.T. Pascacio, J.J.V. Ortíz, M.J. Pérez, M. Yates, B.T. Sanchez, A.R. Quintero, R.F. Lafuente, Evaluation of different lipase biocatalysts in the production of biodiesel from used cooking oil: critical role of the immobilization support, Fuel 200 (2017) 1–10. [20] M.R. Mehrasbi, J. Mohammadi, M. Peyda, M. Mohammadi, Covalent immobilization of Candida antarctica lipase on core-shell magnetic nanoparticle for production of biodiesel form waste, RE 101 (2017) 593–602. [21] M. Sarno, M. Iuliano, M. Polichetti, P. Ciambelli, High activity and selectivity immobilized lipase on Fe3O4 nanoparticles for banana flavour synthesis, Process Biochem. 56 (2017) 98–108. [22] V.R. Murty, J. Bhat, P.K.A. Muniswaran, Hydrolysis of oils by using immobilized lipase enzyme: a review, Biotechnol. Bioprocess Eng. 7 (2002) 57–66. [23] J.C.J. Quilles, R.R. Brito, J.P. Borges, C.C. Aragon, G.F. Lorente, D.A.B. Martins, E. Gomes, R. Da Silva, M. Boscolo, J.M. Guisan, Modulation of the activity and selectivity of the immobilized lipases by surfactants and solvents, Biochem. Eng. J. 93 (2015) 274–280. [24] J. Cui, Y. Zhao, R. Liu, C. Zhong, S. Jia, Surfactant-activated lipase hybridnanoflowers with enhanced enzymatic performance, Sci. Rep. 6 (2016) e27928. [25] R.F. Lafuente, Lipase from Thermomyces lanuginosus: uses and prospects as an industrial biocatalyst, J. Mol. Catal. B-Enzym. 62 (2010) 197–212. [26] A.B. Martins, J.L.R. Friedrich, J.C. Cavalheiro, C.G. Galan, O. Barbosa, M.A.Z. Ayub, R.F. Lafuente, R.C. Rodrigues, Improved production of butyl butyrate with lipase from Thermomyces lanuginosus immobilized on styrenedivinylbenzene beads, Bioresour. Technol. 134 (2013) 417–422. [27] F.A.P. Lage, J.J. Bassi, M.C.C. Corradini, L.M. Todero, J.H.H. Luiz, A.A. Mendes, Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system, Enzyme Microb. Tech. 84 (2016) 56–67. [28] C.S. Wu, C.T. Wu, Y.S. Yang, F.H. Ko, An enzymatic kinetics investigation into the significant enhanced activity of functionalized gold nanoparticles, Chem. Commun. 5327–5329 (2008). [29] T. Pabisiak, M.J. Winiarski, T. Ossowski, A. Kiejna, Adsorption of gold subnanostructures on a magnetite(111) surface and their interaction with CO, PCCP 18 (2016) 18169–18179. [30] N.A. Frey, M.H. Phan, H. Srikanth, S. Srinath, C. Wang, S. Sun, Interparticle interactions in coupled Au-Fe3O4Au-Fe3O4 nanoparticles, J. Appl. Phys. 105 (2009) 07B5022009. [31] J. Noh, O.I. Osman, S.G. Aziz, P. Winget, J.L. Brédas, Magnetite Fe3O4 (111) surfaces: impact of defects on structure, stability, and electronic properties, Chem. Mater. 27 (2015) 5856–5867. [32] B. Xu, G. Zhou, X. Wang, Rational synthesis and the structure-property relationships of nanoheterostructures: a combinative study of experiments and theory, NPG Asia Mater. 7 (2015) e164.

11

[33] D. Ha, S.C. Min, H. Chao, X.Z. Chuan, L. Chen, T. Yuan, S.X. Zhao, G.H. Jun, Synthesis and properties of Au Fe3O4 and Ag Fe3O4 heterodimeric nanoparticles, Chin. Phys. B 19 (2010) 066102. [34] M. Sarno, E. Ponticorvo, Much enhanced electrocatalysis of Pt/PtO2 and low platinum loading Pt/PtO2-Fe3O4 dumbbell nanoparticles, Int. J. Hydrog. Energy 42 (2017) 23631–23638. [35] M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, J. Anal. Biochem. 72 (1976) 248–254. [36] P.N. Giannelos, S. Sxizas, E. Lois, F. Zannikos, G. Anastopoulos, Physical, chemical and fuel related properties of tomato seed oil for evaluating its direct use in diesel engines, Ind. Crops Prod. 22 (2005) 193–199. [37] F. Jamil, A.H.A. Muhtaseb, L.A. Haj, M.A.A. Hinai, P. Hellier, U. Rashid, Optimization of oil extraction from waste ‘‘Date pits” for biodiesel production, Energy Convers. Manage. 117 (2016) 264–272. [38] J.K. Lu, K.L. Nie, F. Xie, F. Wang, T.W. Tan, Enzymatic synthesis of fatty acid methyl esters from lard with immobilized Candida sp. 99–125, Process Biochem. 42 (2007) 1367–1370. [39] A.N. Phan, T.M. Phan, Biodiesel production from waste cooking oils, Fuel 87 (2008) 3490–3496. [40] D.I. Ha˘da˘ruga˘, N.G. Ha˘da˘ruga˘, A. Hermenean, A. Riviș, V. Pâslaru, G. Codina, Bionanomaterials: thermal stability of the oleic acid/a- and b-cyclodextrin complexes, Rev. Chim. 59 (2008) 994–998. [41] S. Schober, I. Seidl, M. Mittelbach, Ester content evaluation in biodiesel from animal fats and lauric oils, Eur. J. Lipid Sci. Technol. 108 (2006) 309–314. [42] F. Lin, R. Doong, Highly efficient reduction of 4-nitrophenol by heterostructured gold-magnetite nanocatalysts, Appl. Catal. A 486 (2014) 32–41. [43] Y. Zhang, Y. Zhao, Y. Yang, J. Shen, H. Yang, Z. Zhou, S. Yang, A bifunctional sensor based on Au-Fe3O4 nanoparticle for the detection of Cd+2, Sens. Actuat. B 220 (2015) 622–626. [44] M. Ramanathan, L.K. Shrestha, T. Mori, Q. Ji, J.P. Hill, K. Ariga, Amphiphile nanoarchitectonics: from basic physical chemistry to advanced applications, PCCP 15 (2013) 10580–10611. [45] A. Bahrami, P. Hejazi, Electrostatic immobilization of pectinase on negatively charged AOT-Fe3O4 nanoparticles, J. Mol. Catal. B-Enzym. 93 (2013) 1–7. [46] B. Sahoo, S.K. Sahu, D. Bhattacharya, D. Dhara, P. Pramanika, A novel approach for efficient immobilization and stabilization of papain on magnetic gold nanocomposites, Colloid Surf. B 101 (2013) 280–289. [47] K. Atacan, M. Özacar, Characterization and immobilization of trypsin on tannic acid modified Fe3O4 nanoparticles, Colloids Surf. B 128 (2015) 227–236. [48] K. Atacan, B. Çakırog˘lu, M. Özacar, Improvement of the stability and activity of immobilized trypsin on modified Fe3O4 magnetic nanoparticles for hydrolysis of bovine serum albumin and its application in the bovine milk, Food Chem. 212 (2016) 460–468. [49] E. Agostinelli, F. Belli, G. Tempera, A. Mura, G. Floris, L. Toniolo, A. Vavasori, S. Fabris, F. Momo, R. Stevanato, Polyketone polymer: a new support for direct enzyme immobilization, J. Biotechnol. 127 (2007) 670–678. [50] M. Ma, Y. Zhang, W. Yu, H.-Y. Shen, H.-Q. Zhang, N. Gu, Preparation and characterization of magnetite nanoparticles coated by amino silane, Colloids Surf. A 212 (2003) 219–226. [51] L. Zhang, R. He, H.C. Gu, Oleic acid coating on the monodisperse magnetite nanoparticles, Appl. Surf. Sci. 253 (2006) 2611–2617. [52] E. Cheraghipour, S. Javadpour, A.R. Mehdizadeh, Citrate capped superparamagnetic iron oxide nanoparticles used for hyperthermia therapy, J. Biomed. Sci. Eng. 5 (2012) 715–719. [53] D. Singh, R.K. Gautam, R. Kumar, B.K. Shukla, V. Shankar, V. Krishna, Citric acid coated magnetic nanoparticles: synthesis, characterization and application in removal of Cd(II) ions from aqueous solution, J. Water Process Eng. 4 (2014) 233–241. [54] S. Nigam, K.C. Barick, D. Bahadur, Development of citrate-stabilizer Fe3O4 nanoparticles: conjugation and release of doxorubicin for therapeutic applications, J. Magn. Magn. Mater. 323 (2011) 237–243. [55] M. Sarno, E. Ponticorvo, C. Cirillo, High surface area monodispersed Fe3O4 nanoparticles alone and on physical exfoliated graphite for improved supercapacitors, J. Phys. Chem. Solids 99 (2016) 138–147. [56] S. Sun, H. Zeng, D.B. Robinson, S. Raoux, P.M. Rice, S.X. Wang, G. Li, Monodisperse MFe2O4(M = Fe Co, Mn) nanoparticles, J. Am. Chem. Soc. 126 (2004) 273–279. [57] Y. Xing, Y.Y. Jin, J.C. Si, M.L. Peng, X.F. Wang, C. Chen, Y.L. Cui, Controllable synthesis and characterization of Fe3O4/Au composite nanoparticles, J. Magn. Magn. Mater. 380 (2015) 150–156. [58] N.R. Mohamad, N.H.C. Marzuki, N.A. Buang, F. Huyop, R.A. Waha, An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes, Biotechnol. Biotechnol. Equip. 29 (2015) 205–220. [59] K. Hernandez, C.G. Galan, R.F. Lafuente, Simple and efficient immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads, Enzyme Microb. Technol. 49 (2011) 72–78. [60] S.H. Huang, M.H. Liao, D.H. Chen, Direct binding and characterization of lipase onto magnetic nanoparticles, Biotechnol. Prog. 19 (2003) 1095–1100. [61] S. Ma, J. Mu, Y. Qu, L. Jiang, Effect of refluxed silver nanoparticles on inhibition and enhancement of enzymatic activity of glucose oxidase, Colloids Surf. A: Physicochem. Eng. Aspects 345 (2009) 101–105. [62] G. Bayramog˘lu, A.U. Metin, B. Altintas, M.Y. Arica, Reversible immobilization of glucose oxidase on polyaniline grafted polyacrylonitrile conductive composite membrane, Bioresour. Technol. 101 (2010) 6881–6887.

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M. Sarno, M. Iuliano / Applied Surface Science xxx (2018) xxx–xxx

[63] C.L. Wu, Y.P. Chen, J.C. Yang, H.F. Lo, L.L. Lin, Characterization of lysine-tagged Bacillus stearothermophilus leucine aminopeptidase II immobilized onto carboxylated gold nanoparticles, J. Mol. Catal. B: Enzym. 54 (2008) 83–89. [64] B. Sels, A. Philippaerts, J. Spivey, K. Shingfield, J. Buyse, Y. Park, J. Ogawa, R. Quirino, K. Belkacemi, J. Kramer, Conjugated linoleic acids and conjugated vegetable oils, RSC (2014). [65] W. Xie, N. Ma, Enzymatic transesterificationm of soybean oil by using immobilized lipase on magnetic nano-particles, Biomass Bioenergy 34 (2010) 890–896. [66] Y. Chen, b. Xiao, J. Chang, Y. Fu, P. Lv, X. Wang, Synthesis of biodiesel from waste cooking oil using immobilized lipase in fixed bed reactor, Energy Convers. Manage. 50 (2009) 668–673.

[67] C. Jiang, C. Cheng, M. Hao, H. Wang, Z. Wang, C. Shen, L.Z. Cheong, Enhanced catalytic stability of lipase immobilized on oxidized and disulfide-rich eggshell membrane for esters hydrolysis and transesterification, Int. J. Biol. Macromol. 105 (2017) 1328–1336. [68] L. Ma, M. Persson, P. Adlercreutz, Water activity dependence of lipase catalysis in organic media explains successful transesterification reactions, Enzyme Microb. Technol. 31 (2002) 1024–1029. [69] M. Tariq, S. Ali, F. Ahmad, M. Ahmad, M. Zafar, N. Khalid, M. AjabKhan, Identification, FT-IR, NMR (1H and 13C) and GC/MS studies of fatty acid methyl esters in biodiesel from rocket seed oil, Fuel Sci. Technol. 92 (2011) 336–341.

Please cite this article in press as: M. Sarno, M. Iuliano, Highly active and stable Fe3O4/Au nanoparticles supporting lipase catalyst for biodiesel production from waste tomato, Appl. Surf. Sci. (2018), https://doi.org/10.1016/j.apsusc.2018.04.060