Accepted Manuscript

Optimized procedure for the preparation of an enzymatic nanocatalyst to pro-

duce a bio-lubricant from waste cooking oil

Maria Sarno, Mariagrazia Iuliano, Claudia Cirillo

PII: S1385-8947(18)32171-5
DOI: https://doi.org/10.1016/j.cej.2018.10.210
Reference: CEJ 20273

To appear in: Chemical Engineering Journal

Received Date: 20 June 2018

Revised Date: 17 September 2018
Accepted Date: 28 October 2018

Please cite this article as: M. Sarno, M. Iuliano, C. Cirillo, Optimized procedure for the preparation of an enzymatic
nanocatalyst to produce a bio-lubricant from waste cooking oil, Chemical Engineering Journal (2018), doi: https://
doi.org/10.1016/j.cej.2018.10.210

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 Optimized procedure for the preparation of an enzymatic nanocatalyst to produce a bio-
lubricant from waste cooking oil
Maria Sarno1,2*, Mariagrazia Iuliano1, Claudia Cirillo1
1 Department of Industrial Engineering, University of Salerno, via Giovanni Paolo II, 132 - 84084 Fisciano (SA), Italy
2 NANO_MATES Research Centre, University of Salerno, via Giovanni Paolo II, 132 - 84084 Fisciano (SA), Italy

ABSTRACT
Bio-lubricant was obtained through esterification of WCOs free fatty acids with neopentyl glycol by

using Thermomyces lanuginosus lipase (TL) immobilized on Fe3O4-CA (citric acid modified

magnetite nanoparticles) catalyst. An optimized eco-friendly procedure was used to synthesize

Fe3O4-CA nanosupport for direct immobilization of the enzyme. The experimental results evidence

the successful immobilization of lipase on the as produced nanoparticles with high efficiency >96

%. The immobilized TL shows very high activity (activity recovery ~ 99 %). The immobilized

lipase was used for the bio-lubricant synthesis from WCOs in a solvent-free system. The bio-

lubricant conversion has been monitored for 24 h. High conversion of ~ 45 % and ~ 68 % were

observed already after 3 hours and 6 hours of synthesis, respectively. No significant reduction,

during ten cycles of use, of the lipase activity occurs. An increased conversion was obtained with

the addition of molecular sieves, which permits to achieve a maximum conversion of 94 % after 24

h. Analyses of the bio-lubricant properties showed a viscosity index of ~179 and a pour point of ~ -

8 °C.

Keywords: one-step immobilization, waste cooking oil reuse, bio-lubricant, high activity and

stability.
*Corresponding authors: Tel.: +39 089 963460; fax: +39 089 964057; E-mail address:

msarno@unisa.it (M. Sarno).

1. Introduction
In different industry segments lubricants are used for the mechanical equipment. Indeed, lubricants

are substances which provide a protective thin layer between two moving surfaces [1] to reduce

friction and wear, avoid oxidation, protect metal surfaces from corrosion, act as an insulator and
sealing agent against dust and water [2]. Nowadays, the majority of the lubricants come from

petroleum oils [3]. These types of oils present serious environmental concerns because of their poor

degradability and contaminant behaviour [4]. Furthermore, the increase in the price of petroleum oil

has, also, pushed in the direction of developing sustainable and eco-friendly products [5]. In

particular, the increasing environmental pollution concerns and diminishing petroleum reserves has

brought into attention the use of vegetable oils for production of bio-lubricants. Compared to

petroleum-derived lubricants, vegetable oils have some intrinsic advantages, such as good viscosity

index, high flash point, low volatility and good metal adherence [6,7]. Indeed, the amphiphilic

nature of the vegetable oils allow them to be used as lubricant in the boundary regime [8-11].

On the other hand, the use of vegetable oils for bio-lubricant synthesis results expensive, accounting

for 70÷80% of total production cost [12]. The valorisation of wastes and biomass-derived

substances is, of course, an attracting alternative. In this regard, WCOs (waste cooking oils) acts as

an inexhaustible renewable raw material. In the world, indeed, every day large quantities of WCOs

are generated by food processing industries, fast foods, restaurants and families. WCOs removal is

an environmental challenge, which can be overcome using them for energy (e.g. biodiesel, ..) or

bio-lubricants production. In this way, bio-refineries hold the promise of “zero waste” and “green

chemistry”, and encourage waste and side-product recycle/utilization by eco-friendly procedures

[13,14]. The use of WCOs for bio-lubricant base-stock synthesis can significantly reduce the overall

cost of the final products and prevent the illicit recycling of WCOs as fake edible vegetable oils, as

well [15]. Transesterification/esterification are the general procedures to convert vegetable oils in

new esters with refined physical properties. These processes are typically multi-steps, the first step

is the triglycerides reaction with methanol to produced methyl esters (MEs) or, the triglycerides

hydrolysis to obtained free fatty acids (FFAs). The resulting ME or FFA can react with various

types of alcohols, in the presence of a catalyst, to yield a bio-lubricant. Due to the wide array of

possible reactants, the resulting bio-lubricants can have different properties. The
transesterification/esterification of ME or FFA leads to increased thermo-oxidative stability and to

decreased pour points, preserving the viscosity and lubricity characteristics of the base oils [16-20].

For these processes, base catalysts are usually the preferential industrial choice [2]. On the other

hand, enzymatic catalysts have many advantages over acid/base catalysts such as: (i) higher

selectivity, (ii) lower reaction temperatures, (iii) reduced energy demand, (iv) less expensive waste

treatment, (v) no need of harmful reagents and (vi) ability to use cheap bio-based raw materials

[21,22,23]. Enzymes are a good choice in terms of sustainability. On the other hand, enzymes are

still underused today, e.g. only 3 % of industrial processes use enzymes over a decade ago [24] and

this is mainly for cost reasons, e.g. the enzyme cost ranges from 22 % to 50 % of the raw material

cost [25]. On the other hand, immobilization, together with process design and optimization, is a

process intensification strategy [26]. Enzyme immobilization can led to a significant process cost

saving [27]. Moreover, the improved enzyme performance yield a further increase in productivity,

up to 15-fold as reported in some cases [27], resulting in a major reduction of costs and waste. On

the other hand, in some cases the cost of immobilization has been reported competitive with the

enzyme cost itself [28], but this is overcome if the support can be reutilized and for easy

immobilization procedure.

In particular the application of lipases in a large scale process was often limited by their high cost

and sensitivity to high temperature and organic solvents. Moreover, it is difficult to separate

enzymes from the reaction system, which limits its recovery and may lead to contamination of the

final product [29-31]. In order to overcome these problems, lipases have been immobilized by

several methods [29,32,33]. Indeed, immobilization of an enzyme can: (i) reduce the operational

process cost and (ii) enhance the reusability. Immobilized enzymes are often more stable with pH

than free enzymes [34]. Nanomaterials can serve as promising supporting materials for

immobilization because of their specific surface area, low mass transfer resistance and effective

enzyme loading [35]. The literature presents different types of nanomaterials for enzyme

immobilization. Examples of nanomaterials used for enzyme immobilization include: carbon

nanotubes (CNTs), nanoparticles, magnetic nanoparticles (MNPs), nanocomposites, nanofibers,

nanorods and mesoporous media [36].

Among nanosupports, magnetic nanoparticles (MNPs) offer additional advantages of: easy

separation, just applying a magnetic field; minor leaching problems; and, possible higher selectivity

[34]. Furthermore, Fe3O4 magnetic nanoparticles have been regarded as promising supports because

of biocompatibility and low cost [33]. Immobilization can basically occur in different ways: by

adsorption, covalent attachment, and encapsulation [37].

Physical adsorption is an attractive protocol from the industrial point of view, because of it allows

the reuse of the support, by desorption of inactive enzyme molecules from the biocatalyst surface,

and preserves activity [33,38,39].

The aim of this work was to synthesize an environmentally friendly lubricant of neopentyl glycol

fatty acid esters. Particular attention was devoted to the optimization of the preparation of the

enzymatic catalyst, which consists of nanoparticles directly functionalized in a single step of

synthesis to anchor the enzyme through physical bonds. The synthesis of mono-dispersed metal

nanoparticles is of essential importance for the development of a novel technology. In particular,

significant progress has been made in the magnetic nanoparticles synthesis using different

techniques [40,41]. Nowadays, the synthesis of nanoparticles using non-aqueous process is

preferred. Thermal decomposition process is very often used for the synthesis of metal and alloy

nanoparticles, but, the coproduction of highly toxic carbon monoxide and the difference between

the decomposition temperatures of the metallic compounds, in the cases of alloy particles, make

these processes environmentally unfriendly and complex. On the other hand, environmentally

friendly synthesis can be obtained using polyols, that can act as solvent, reducing and protecting

agents [42]. Another important aspect is the choice of the surfactant, which can provide

coordination with the nanoparticle stabilizing them. Surfactants with –COOH group, because of

their strong affinity to FeIII ions, were often chosen [43]. In particular, here for the first time, citric

acid, with three different carboxyl groups, was chosen to directly bond with the enzyme. A new
experimental apparatus, consisting in an autoclave, which permits to work in an inert atmosphere

and to easily carry out spillages during the synthesis to monitor the process, has been designed and

implemented for the nanoparticles synthesis.

In particular, the bio-lubricant was obtained thought esterification of WCOs free fatty acids with

neopentyl glycol by using Thermomyces lanuginosus lipase (TL) immobilized on Fe3O4-CA (citric

acid modified magnetite nanoparticles) catalyst, and in a solvent-free system. Thermomyces

lanuginosus lipase, a commercial and quite stable enzyme has been usually used in oils and fats

modification, following very different strategies [44]. In literature, enzymatic approaches, to

produce bio-lubricant from WCOs, have been little or nothing explored up to now [45,46]. Our

experimental results evidence the one-step successfully immobilization of lipase on the as produced

nanoparticles. The immobilized enzyme shows very high activity and stability during bio-lubricant

synthesis. Furthermore, we analysed the properties of the produced bio-lubricant: pour point,

viscosity at 40-100 °C, viscosity index and acid value, according to the Standards.

2. Materials & Method

2.1 Materials
The waste cooking oil (WCO) was collected, from food restaurants, and purified by a collection

centre (Consortium in Benevento, South of Italy) to remove water and dispersed solids [46]. The

property of WCO was reported in Table 1. Ferric chloride hexahydrate (FeCl3·6H2O, 98%), urea

(100.5%), ethylene glycol (EG), citric acid (99.9%), ethanol, lipase from Thermomyces

lanuginousus (TLL) (solution ≥100,000 U/g), bovine serum albumin (BSA), polyvinyl alcohol

(PVA), potassium hydroxide (KOH), olive oil (highly refined-low acidity, CAS 8001-25-0),

heptane, methyl heptadecanoate of known purity (99 %) (C:17:0), boron trifluoride-methanol

solution (BF3-Methanol) and neopentyl glycol (NPG) were acquired from Aldrich Chemical Co. All

chemicals were of analytical grade.

2.2 Method
2.2.1 Hydrophilic magnetic Fe3O4 synthesis
FeCl3·6H2O 3 mmol, citric acid 1 mmol and urea 30 mmol were completely mixed in 30 ml of

ethylene glycol and sonicated, at a fixed frequency of 100 kHz, for 5 min. The orange solution was

located in an autoclave with 50 mL of capacity (see Figure 1). The reaction starts when the

temperature was 200 °C and continuous for 4 h. The inner pressure of the autoclave was measured

as approximately 8 bar. After 4 h the solution was cooled at room temperature and the black

material was washed with ethanol and deionized water for several times and finally dried at 60° C

for 12 h.

2.2.2 Immobilization of Thermomyces lanuginosus lipase.

50 mg of nanoparticles were added to 10 ml of 1 M buffer solution, pH 3.0, containing 0.1 mg/ml,

0.2 mg/ml and 0.3 mg/ml of Thermomyces lanuginosus lipase. The nanoparticles were sonicated

with the enzyme (Ultrasonic Cleaning machines CP104) at a fixed frequency and at 50 W power

rating, for 3 min. The mixtures were kept at 4 °C under constant shaking (200 rpm) for 3 h. In the

case of the mixture with 0.1 mg/ml enzyme concentration, two addition samples were obtained after

1 or 2 h of shaking.

2.2.3 Lipase estimation

The protein concentration was determined using the Coomassie brilliant blue reagent according to

the Bradford method using bovine serum albumin as the standard protein [33, 48,49].

2.2.4 Lipase activity assay

The activities of the soluble and immobilized lipase were determined by hydrolysis of olive oil

emulsion, according with the methodology described by Sarno et al. [33, 48]. The substrate was

prepared mixing 0.5 g of olive oil with 1 % of PVA (wt. PVA / wt. olive oil). Later, 1 mg of free

and immobilized lipase was added to 0.5 mL of buffer solution pH=7 (optimum pH of free lipase

[50]) and incubated for 15 min at 40 °C under agitation at 200 rpm. The free fatty acids were then

titrated with a standard 0.1 M KOH solution using phenolphthalein as indicator. The experiments

were triplicated.
2.2.5 Determination of immobilization parameters

Immobilization efficiency was determined according to the following equation Eq. (1):
(𝐶0 ‒ 𝐶𝑓)𝑉1
𝐼𝑚𝑚𝑜𝑏𝑖𝑙𝑖𝑧𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 𝐶𝑖𝑉2
(%) (1)

where: C0 and Cf (mg/mL) are initial and final concentration; V1 and V2 (mL) the volumes of the

two solutions.

Lipase loading was calculated as follows Eq. (2):

𝑉 (𝐶𝑜 ‒ 𝐶𝑓)
𝐿𝑖𝑝𝑎𝑠𝑒 𝑙𝑜𝑎𝑑𝑖𝑛𝑔 = 𝑚𝑁𝑃𝑠
(%) (2)

where V is the volume of enzyme solution (mL), and mNPs is the mass of support (g).

Activity recovery was calculated as shown in Eq. (3):

𝑆𝐴𝑖𝑚𝑚𝑜𝑏𝑖𝑙𝑖𝑧𝑒𝑑 𝑇𝐿
𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦 (%) = 𝑆𝐴𝑠𝑜𝑙𝑢𝑏𝑙𝑒
(%) (3)

where SA is the specific activity of the immobilized and soluble TL (U/mg).

2.2.6 FFA Preparation

Enzymatic hydrolysis for FFA preparation was conducted in a 250 mL-scale reaction using a

mechanically stirred reactor. WCO (10 g), 1 % of PVA (wt. PVA / wt. WCO), 0.1 M sodium

phosphate buffer at pH 7.0 (50 ml) and immobilized TL (1 % w/w of oil) were incubated at 40 °C

for 4 h. After 4 h the FFA was recovered using hexane. Finally, the product was purified using a

rotary evaporator and the FFA content was determined by the acidity using KOH 0.1 M.

2.2.7 Esterification process

A mixture of FFA:NPG (2:1 and 4:1 molar ratio) was stirred in the autoclave (see Figure 1) in

presence of 5 % wt. /wt. (enzyme mg per mg of FFA) of immobilized TL on Fe3O4-CA. The

reaction was carried out at 45 °C under mechanical stirring at 250 rpm and for 24 h, see Figure 2 for

the reaction scheme. After reaction, immobilized TL was recovered by an external magnetic field

and the product was dried in sodium phosphate. The progress of the reaction was monitored

measuring the acid value on the upper layer obtained after centrifugation (5,000 rpm for 10 min) of

the different samplings.

The conversion to bio-lubricant was calculated by Eq. (4):
100 ∗ (Ao ‒ At ) ∗ 𝑀
𝐶𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 (%) = Ao ∗ nOH
(%) (4)

where: Ao, were the initial acidity and At the acidity at a time t; M the FFA:NPG molar ratio; and

nOH the number of -OH groups in the NPG.

Furthermore, the effect of a molecular sieve, 50 % wt./wt. molecular sieve/FFA, on the

esterification, was investigated. The stability of TL immobilized in repeated uses was analysed, too.

For each use, the reaction conditions were: temperature 45°C, time 24 h. Finally, the physico-

chemical properties of the product were determined following standard methods, to evaluate its

usability as a lubricant. In particular, acid value (ASTM D 664), viscosity at 40°C-100°C (ASTM D

445), viscosity index (ASTM D 2270) and pour point (ASTM D 97) were determined.

2.2.8 Analysis of WCO

The GC-MS analysis of WCO was performed using Thermo-Fischer gas chromatography

equipment, capillary column (Trace-GOLD TG-POLAR GC Columns 0.25 µm×0.25 mm×60 m).

The carrier gas used was helium and the flow rate was maintained at 1.5 ml/min. Inlet and detector

temperature were kept at 250 °C and the initial temperature was 150 °C up to 190 °C, 15 °C/min

and 5 min at 190 °C. It was followed by a further temperature increase up to 230 °C, 4 °C/min and

10 min at 230 °C.

2.2.9 Characterization

The characterization was obtained by: Transmission electron microscopy (TEM) (FEI Tecnai

electron microscope operating at 200 KV), for the measurement the nanoparticles was sonicated in

water for 5 min; Thermogravimetric analysis (TG-DTG) (SDTQ 600 Analyser (TA Instruments) in

flowing air at a 10 K/min heating rate; FT-IR spectra (Vertex 70apparatus (Bruker Corporation) )

by applying KBr technique; XRD measurements (Bruker D8 X-ray diffractometer using CuKα

radiation). For SEM images a thin layer of gold was deposited for 5 min by sputtering (FESEM
LEO1525). Agilent INOVA-300 (7.05 T) spectrometer was used to analyse the produced samples,

which were treated with deuterated chloroform before measurements.

3. Results and Discussion

3.1 Characterization of Support and Biocatalyst

The XRD profile of Fe3O4-CA (Figure 3a) NPs evidences the typical peaks of the magnetite at

30.1° (220), 35.6° (311), 43.1° (400), 53.4° (422), 57.2° (511) and 62.5° (440) [51]. The particle

size of the sample, estimated from the SEM picture (Figure 3b), is a little larger than that obtained

from TEM analysis (Figure 4), because of the surfactant covering the nanoparticles and the gold

metallization for SEM measurements. TEM images in Figure 4 show that all of the magnetite

particles obtained have nearly square shape and uniform size (mean diameter 16 nm, standard

deviation 3 nm).

TG-DTG profiles of CA and Fe3O4-CA NPs after synthesis, are shown in Figure 5a. The weight

losses due to CA in Fe3O4-CA was slight shifted to higher temperatures likely due to the bond with

the nanoparticles. The amount of CA in samples is about 18 % wt./wt. After the immobilization,

due to the stirring [33], the amount of CA results reduced, see Figure 5b. Indeed, the total organic

content in Fe3O4-CA_TL, is about 10 % (wt./wt.). On the other hand, the occurrence of the new

weight loss at 530 °C suggests the presence of lipase. FT-IR spectra (Figure 6) were used to

confirm the immobilization of TL on the surface of the nanoparticles. For Fe3O4-CA a strong band

at 596 cm-1 was present due to the Fe–O stretching vibration of magnetite [52-54]. Vibrational

bands observed at ~1601 cm−1 and ~1645 cm−1 can be attributed to the –COO- stretching bands of

the carboxyl groups of CA interacting with the nanoparticles surface [33,48,55-57]. The band at

~1400 cm−1 can be assigned to the asymmetric stretching of CO form COOH groups [58]. The band

at ~1732 cm−1 is due to the free –COOH of CA. After immobilization of the lipase on NPs the FT-

IR spectrum displays the characteristic bands of the TL lipase [33, 48], whereas the band of free –

COOH is no more visible due to the interaction with the enzyme. After immobilization the
vibrational bands of amide I and amide II of lipase are no longer distinguishable, indicating the

possibility of hydrogen bonds formation [33].

The NPs are stable for different months [59], also after enzyme immobilization. However, as far as

Fe3O4 NPs stability is concerned, it is known that NPs colloidal stability and magnetic separation

have a conflicting behaviour [60]. On the other hand, the time required for NPs separation, in our

laboratory tests, is much more less than one minute. Moreover, the separation can be favored by the

right pH [60, 61].

3.2 Factor affecting TL immobilization on NPs

In Figure 7a the lipase loading on NPs, as a function of the initial lipase concentration, was shown.

Lipase loading on the nanoparticles increases with increasing initial lipase concentration up to 0.3

mg/ml. Under lipase concentration increase diffusion limitations can been observed [62,63], which

exert further strain on the enzyme catalysis, making product release difficult [33, 48, 64] and

enzyme activity decrease. The data shown in Figure 7b represent the relationships between lipase

loading, immobilization efficiency and activity recovery under coupling time increase from 1 h to 4

h. The maximum activity recovery was 99 % ± 1 SD at 0.1 mg/ml, corresponding to a high level of

protein loading of more than 19 mg (mg lipase /g NPs). The results indicate that support saturation

occurred within 3 h.

3.3 Effect of reaction parameters on bio-lubricant synthesis

Figure 8 shows the conversion of the –OH groups of the NPG under time increase and as a function

of FFA:NPG molar ratio, at a temperature of 45 °C. The conversion increases rapidly during the

first 6 h for both molar ratio. After the first 6 h, the rate of conversion decreases because of the

reduced concentration of -OH groups. The inhibition of the enzyme by the high acidic concentration

could be a contributing factor [65, 66]. The highest -OH conversion was achieved at a FFA:NPG

molar ratio of 4:1. The higher amount of acid favours the thermodynamic of equilibrium and faster

kinetic, magnifying the ester yield [66, 67]. A maximum conversion of 88 % was found at the

FFA:NPG molar ratio of 4:1, after 24 h of synthesis. Moreover, it is also to take into account that
the esterification of -OH promotes a water formation, which negatively affects the conversion.

Indeed, too high water content retards the reaction rate. The decrease in the conversion rate with

increasing water content may be attributed to the fact that a water content may favour the backward

hydrolysis reaction resulting in reduced ester conversion rate or it promote enzyme particles

aggregation leading to increased diffusional limitation [68]. For this reason the effect of the

presence of a molecular sieve during the reaction, for adsorbing water, was investigated. The use of

molecular sieves is a convenient way to capture and reduce the water molecules in the biocatalyst

microenvironment [69,70]. The effect of silica gel is shown in Figure 9. In particular, the blue

profile was obtained in the presence of 50 % wt./wt. (weight of the molecular sieve per weight of

the FFA), to be compared with the red profile obtained in the absence of molecular sieve. A

maximum conversion of 94 % was found after 24 h, showing that the addition of a molecular sieve

in the reaction mixture exhibits significant influence on the reaction rates and the conversion of –

OH group.

3.4 Reusability of Bio-catalyst

The very key aspect of reusability has been also explored, see the experimental part for details, and

the results reported in Figure 10. The activity of the immobilized enzyme stays almost constant after

7 cycles and still results higher than 75 % after 10 cycles of reuse, likely due to the physical

interaction with the support [33, 48]. As it can be observed in Figure 10, the biocatalyst almost

retains its initial activity up to ten cycles, showing an excellent reusability.

3.5 Physiochemical property of Bio-lubricant

The nuclear magnetic resonance (NMR) analysis (1H NMR, Figure S1) evidences the formation of

ester group CH2OOR at 3.96 ppm. The lack of the typical peaks of NPG at 3.45 ppm and 3.65 ppm

confirms the absence of partially modified NPG. Table 2 reports the properties evaluated according

to the standards, see column 3, of the synthesized NPG fatty acids esters. The results of the

characterization evidence that the lubricant properties are satisfactory [22]. Indeed, important

properties of a bio-lubricant are the viscosities at 40 °C and 100 °C that are useful in determining
the fluidity at low and high temperatures. The viscosity index obtained for our NPG fatty acids

esters is 179.5, which is comparable to other plant based bio-lubricant and typical lubricant base oil.

Pour point, that is the lowest temperature at which oil flows is crucial factor at low temperatures.

The pour point of the synthesized bio-lubricant results improved when compared to that of the

starting WCO and it is also comparable to the pour point of based oils [22, 71]. FFA content in both

starting oil and produced bio-lubricant [72] leads to saponification during synthesis or lubrication

uses. The amount of free fatty acids in the produced sample is very low (0.6 wt.%). On the other

hand, it means no-linked acid to the polyols. While, it is not expected a negative effect on the

tribological performance because of polar functionalities easily link to the tribopairs in contact

improving tribofilm formation. The composition of the WCO used in this work is similar to that of

a peanut oil. The starting oil composition obviously affects the characteristics of the bio-lubricant

produced (e.g. oxidative stability increases under unsaturated fatty acids concentration increase). On

the other hand, the oil cannot be chosen only on the basis of this consideration because other

equally fundamental properties would be too disadvantaged [73], while the replacement of the

glycerol moiety with polyols returns a lubricant [73] and thermal and oxidative properties can be

also improved by additional antioxidant [74].

3.6 Experimental design and Statistical analysis

A preliminary D-optimal design of experiments was applied in this study in order to reduce the

number and cost of experiments and obtain more information per experiment. 8 runs were

conducted for different levels of two independent variables; x1 reaction time (h) and x2 FFA:NPG

(molar ratio), to study their effect on the –OH conversion, yield of the produced bio-lubricant at the

constant temperature of 45 °C. Independent variables and their levels are presented in Table S1 and

Table S2.

The following interaction model permits to predict yields with a R2 coefficient of 0.8997:

𝒀 = 𝒃𝟎 + 𝒃𝟏𝒃𝒙𝟏 + 𝒃𝟐𝒙𝟐 + 𝒃𝟏𝟐 𝒙𝟐𝟏𝒙𝟎.𝟎𝟓

𝟐
where: x1 and x2 are the levels of the factors under study; Y is the predicted response of the process

(conversion %); b0 equal to 0.1 is the intercept term; b1, b2 and b12 coefficients are equal to 8.5813,

6.02, -0.244, respectively.

4. Conclusion

TEM images show that all of the magnetite particles obtained have nearly square shape and uniform

size (mean diameter 16 nm, standard deviation of 3 nm). The TL was immobilized through a simple

and eco-friendly procedure. The particle sizes of the samples estimated from the SEM picture is a

little larger than that obtained from TEM analysis, because of the surfactant covering the

nanoparticles and the gold metallization for SEM measurements. As prepared citric acid modified

nanoparticles results an excellent support for lipase immobilization, permitting to achieve through

electrostatic interaction an immobilization efficiency of more than 96 %, at pH 3. The immobilized

enzyme shows a very high activity recovery, 99 % ± 1 at 0.1 mg/ml, corresponding to a high level

of protein loading of more than 19 mg (mg lipase /g NPs). In the bio-lubricant synthesis, the

conversion of the –OH groups of the NPG, at a temperature of 45 ◦C, rapidly increases during the

first 6 h, reaching a maximum value of 88 %, after 24 h of synthesis. A maximum conversion of 94

% was found after 24 h, showing that the addition of molecular sieves significantly influenced the

reaction rates and the conversion of –OH group.

This work demonstrates the feasibility of the enzymatic approach used for the production of bio-

lubricants. The value of the viscosity index and the lowering of the pour point indicate the

efficiency of the approach for the production of base oils with improved properties.

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Table 1. Physicochemical properties of WCO. Fatty acid composition of the WCO. Each value represents the
mean of three replicates ± SD.
Property Value
Acid value (mgKOH/g) 1.80±0.02
Free fatty acid content (%) 0.90±0.01 Table 2.
Moisture (%) 0.02±0.03 Physico
Saponification Index (mgKOH/g) 189±0.04 chemica
Iodine Value (gI2/100g oil) 93±0.02 l
Fatty Acids composition % properti
es of the
Tetradecanoic acid 0.16
WCO
Palmitic acid 8.69 and bio-
Palmitoleic acid 0.40 lubrican
Stearic Acid 5.51 t. Each
value
Oleic acid 55.14 represe
Linoleic Acid 25.53 nts the
Eicosanoic acid 0.67 mean of
three
Linolenic acid 0.34 replicat
Cis 11-eicosanoic acid 0.68 es.
Behenic acid 2.01
Lignoceric acid 0.86
Physicochemical properties
Property Value WCO Value bio- Method
lubricant
Viscosity at 40°C mm2/s 38.1 28.7 ASTM D445
Viscosity at 100°C mm2/s 7.5 6.3 ASTM D445
Viscosity index (mm2/s)
Pour point (°C)
168.4
-6.7
179.5
-8.3
ASTM D2270
ASTM D97 Gas Inlet
Acid Value (mg of 1.80 1.2 ASTM D664
KOH/g)

Thermocou

Figure 1. scheme of the experimental apparatus

.
 O
H2C
O O
HC
O O
H2C
O
Triglyceride

Immobilized TL catalyst Hydrolysis

O
2 H
O
FFA

Esterif ication
Immobilized TL catalyst CH3
H2 H2
HO C C C OH

CH3

CH3 NPG
O
H2
H2C C C O

CH3 + 2 H2O
O
O
Bio-lubricant
Figure 2. Reaction pathway used for bio-lubricant production.
 Fe3O4-CA
a (311)
b

(440)
(511)
(220)

(400)

(422)

20 nm
30 40 50 60 70
2 Theta

Figure 3. (a) XRD spectrum of Fe3O4-CA NPs; (b) SEM image of Fe3O4-CA NPs

Figure 4. TEM images at different magnification of Fe3O4-CA NPs.

120 a 2
––––––– CA
––––––– Fe3O4_CA
Deriv. Weight (%/min)

80
Weight (%)

40

0 0
30 230 430 630
Temperature (°C)
Universal V4.5A TA Instruments
 120 0.5
b ––––––– FE3O4-CA_TL
––––––– TL

Deriv. Weight (%/min)

80
Weight (%)

40

0 0.0
30 230 430 630
Temperature (°C)
Universal V4.5A TA Instruments

Figure 5. TG-DTG profile of: (a) Fe3O4-CA, CA; (b) Fe3O4-CA_TL, lipase solution (TL) (Sigma Aldrich).
Transmittance (%)

Fe3O4-CA
Fe3O4-CA_TL

2000 1000
Wavelenght (cm-1)

Figure 6. FT-IR spectra in the range of wavenumber 2000-500 cm-1 of Fe3O4-CA and Fe3O4-CA_TL.
(a)

lipase loading (mg lipase/g MNPs)

Immobilization efficency (%)

100
Activity recovery (%)

40
80

60

40
20

20
0,1 0,2 0,3
Initial lipase concentration (mg/ml)
(b)
20

lipase loading(mg lipase/g MNPs)

Immobilization efficiency (%)

100
Activity Recovery (%)

80

15
60

40

20 10
1h 2h 3h 4h

Coupling time (h)

Figure 7. Effect of the initial lipase concentration on lipase loading, immobilization efficiency and activity recovery
(the measurements were performed by setting the coupling time to 3 h, coupling pH 3) (a). These parameters were also
measured by varying the coupling time of the immobilization procedure (initial lipase concentration: 0.1 mg/ml) (b).
 100

80

Conversion OH (%) 60

40

20
4:1 FFA:NPG
2:1 FFA:NPG
0
0 6 12 18 24
Time

Figure 8. Effect of the molar ratio on bio-lubricant synthesis, catalyzed by immobilized TL, using FFA from the WCO
and NPG as substrate. Immobilization conditions: coupling temperature, 4 °C; coupling pH, 3; lipase concentration , 0.1
mg/ml. Synthesis conditions: reaction temperature, 45 °C; lipase concentration, 5 %.

100

80
Conversion OH (%)

60

40

20
0 % Molecular sieve
50 % Molecular sieve
0
0 6 12 18 24
Time

Figure 9. Effect of the 50 % w/w molecular sieve/FFA on bio-lubricant synthesis reaction catalyzed by Immobilized
TL using FFA from WCO and NPG as substrate. Immobilization conditions: coupling temperature, 4 °C; coupling pH,
3; lipase concentration , 0.1 mg/ml. Synthesis conditions: reaction temperature, 45 °C; lipase concentration, 5 %, molar
ratio 4:1 FFA:NPG.
 90

Conversion OH (%)
60

30

0
1° 2° 3° 4° 5° 6° 7° 8° 9° 10°
Cycles

Figure 10. Effect of cycle number on bio-lubricant synthesis reaction catalyzed by Immobilized TL using FFA from
WCO and NPG as substrate. Immobilization conditions: coupling temperature, 4 °C; coupling pH, 3; lipase
concentration, 0.1 mg/ml. Synthesis conditions: reaction temperature, 45 °C; lipase concentration, 5 %; molar ratio 4:1
FFA:NPG.
Green and optimized synthesis of a nano bio-catalyst

Easy recovery and reuse of the enzyme

Waste cooking oil reuse

High activity and stability

Bio-lubricant properties according to the Standard