Fuel 202 (2017) 196–205

Contents lists available at ScienceDirect

Fuel
journal homepage: www.elsevier.com/locate/fuel

Full Length Article

Two-step enzymatic production of environmentally friendly

biolubricants using castor oil: Enzyme selection and product
characterization
J. Greco-Duarte a, E.D. Cavalcanti-Oliveira a, J.A.C. Da Silva b, R. Fernandez-Lafuente c,⇑, D.M.G. Freire a,⇑
a
Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Cidade Universitária, Centro de Tecnologia, Bl. A, Sl. 549, Ilha do Fundão, 21949-900 Rio
de Janeiro, Brazil
b
Cenpes, Centro de Pesquisas e Desenvolvimento Leopoldo Américo Miguez de Mello, Petrobras, 21941-970 Rio de Janeiro, Brazil
c
Departamento de Biocatalisis, ICP-CSIC, C/ Marie Curie 2, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain

h i g h l i g h t s

Castor oil hydrolysis using castor-seed lipases gives a high yield of unmodified COFFA.

Molecules with good lubricant characteristics are produced by the esterification of COFFA.

Estolides from COFFA esterification by CRL are the main reaction product.

CRL prefers ricinoleic acid as a nucleophile compared to polyols.

Estolide biolubricant properties are outstanding except for high acidity.

a r t i c l e i n f o a b s t r a c t

Article history: We performed two-step enzymatic biolubricant production: castor oil hydrolysis and esterification of
Received 20 February 2017 castor oil free fatty acids (COFFA) rich in ricinoleic acid. The hydrolysis step utilized castor seeds as cat-
Received in revised form 5 April 2017 alyst yielding 93.13 ± 5.9% COFFA in only 1 h. In the esterification step, C. rugosa lipase (CRL) decreased
Accepted 8 April 2017
the acidity by over 80% after 96 h, utilizing as polyols neopenthylglicol (NPG), trimethylolpropane
(TMP) and pentaerytritol (PE) and showed about 70% acidity decrease without polyols, indicating the pro-
duction of COFFA polymers (estolides). Nuclear magnetic resonance (NMR) confirmed estolides as the
Keywords:
main product, reaching a polymerization degree of 6/7 without polyols, 4 in the presence of PE and rici-
Biolubricant
Estolide
noleic acid dimers in the presence of NPG and TMP. Analyses of biolubricant properties showed that the
Castor oil best product was the estolide with a polymerization degree of 6/7: viscosity index of 162, oxidation sta-
Ricinoleic acid bility of 51 min and pour point of 42 °C.
Candida rugosa lipase Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Currently used mineral lubricants derived from petroleum

sources are clear examples of useful but environmentally danger-
The 21st century is marked by environmental degradation and ous products [4]. The current legislative environmental standards
grave changes in the natural functioning of the biosphere [1]. It have led to the replacement of mineral oils with more ecologically
is essential to develop green technologies that allow economic benign synthetic oils, usually based on esters of long-chain fatty
growth without environmental disturbance [2]. Environmentally acids from vegetable oils and polyols [5].
friendly processes that are comparably efficient to those already Vegetable oils are the mainly raw material for this purpose due
in use are being studied in order to develop technologies that allow to its renewable character and the quality of the final product. Biol-
gradual replacement of conventional chemical processes by biolog- ubricants may be produced from chemical modification of veg-
ical processes [3]. etable oils that spare the structural problems of vegetable oils
making them able for the application as lubricant [4] and this pro-
cess can be made by chemical [6] or enzymatic catalysis [7–10].
⇑ Corresponding author. Also vegetable oils present good viscosity index, good pour point
E-mail addresses: [email protected] (R. Fernandez-Lafuente), [email protected] and good evaporation loss, but their wider application is limited
(D.M.G. Freire). by the thermal and oxidative instabilities [11].

http://dx.doi.org/10.1016/j.fuel.2017.04.036
0016-2361/Ó 2017 Elsevier Ltd. All rights reserved.
 J. Greco-Duarte et al. / Fuel 202 (2017) 196–205 197

Moreover the production by green enzymatic routes together An esterification reaction is a thermodynamically controlled
with their biodegradability constitutes a clear example of green process [35–37] Thus, the thermodynamic constant of the process
chemistry, to be considered biodegradable, biolubricants must and therefore the water activity (product of the reaction), concen-
decompose within one year through natural degradation [12]. trations and molar ratios of the substrates, pH, temperature, etc.
Vegetable oils are known as typically 99% biodegradable, usually will determine the yield of the process, whereas the catalyst only
falling to 90–98% after mixing with additives, while mineral oils determines the reaction rate [35–37]. However, using this hydroxy
are only 20% biodegradable [13]. This is more one between the fac- fatty acid, it is possible that, depending on the conditions and
tors described here that make vegetable oils a promising substrate enzyme employed, some estolide (where the hydroxyl groups of
for biolubricant production. one ricinoleic molecule can be esterified with the carboxylic group
One of the ways to produce biolubricants is through the esteri- of another molecule) can be produced [38], and this could depend
fication of the free fatty acids [9,10] that can be obtained by the on the enzyme employed, affecting the overall ester yield. In addi-
vegetable oil hydrolysis. Ricinoleic acid esters have been suggested tion to being a complication in the reaction design, the presence of
as potential biolubricants, due to their structure. Ricinoleic acid is this estolide may be considered as merely a problem, or on the
the main component of the castor oil plant Ricinus communis L., contrary, may be interesting as a biolubricant (e.g., hydroxyl group
reaching 70–90% of the total FFA content [14]. Castor-oil plant oxidation is no longer a problem).
seeds, in addition to their high oil content [15,16], contain certain We analyzed the properties of the several biolubricants pro-
enzymes [17] of which lipases were used in this study [18,19]. duced: oxidation stability, pour point, viscosity at 40 and 100 °C,
Currently, the industrial hydrolysis of this oil to produce free viscosity index (VI), total acidity index (TAI) and water index. As
fatty acids is performed by thermal or chemical hydrolysis (usually catalysts, we utilized some of the most frequently used enzymes:
using alkaline catalysts) [20], which can cause oxidation of the lipase B from Candida antarctica (NovozymÒ 435) [39,40], lipases
final product (see below). Ricinoleic acid can also be produced by from Rhizomucor miehei [41,42] and commercial extracts from Can-
saponification followed by acidification [21]. This procedure, dida rugosa lipases [43]. This last has been shown by zymography
besides the mild temperatures compared to thermal hydrolysis, to produce some esterification using castor oil biodiesel [44].
gives a product with color and odor degradations and a large quan- Here we present a new strategy to produce biolubricant mole-
tity of an acid sludge and hard water in the downstream process cules based on ricinoleic acid, using castor oil as the raw material
[22], making it desirable to hydrolyze the oil using lipases [23] to in a solvent-free reaction medium, to avoid the use of any solvent
obtain ricinoleic acid. that could be considered a contaminant. The purpose is to obtain
Ricinoleic acid, formally known as 12-hydroxy-9-cis- ricinoleic acid esters through hydroesterification [45,46,33] in a
octadecenoic acid [24], is a fatty acid with specific physical proper- two-step enzymatic process: enzymatic hydrolysis of vegetable
ties due to the hydroxyl group in carbon 12 and the double bond oils and subsequent esterification of the free fatty acids obtained
between carbons 9 and 10. This rigid configuration offers several by the previous step with the alcohol selected. Direct use of castor
advantages, making this compound interesting for different appli- oil as a substrate would eliminate the chemical or thermal catalysis
cations such as cosmetics (emulsifiers), paper (as anti-foam), food stage, making the process more ecologically benign and avoiding
(as an additive), etc. [25]. However, the oxidative stability of rici- undesired modifications.
noleic acid is questioned due to the hydroxyl group in its structure
[26]. The preservation of this hydroxyl group makes it unsuitable 2. Materials and methods
to use chemical catalysis to produce the esters, and reinforces
interest in enzymatic catalysis for the modification of this acid, 2.1. Materials
which can be produced under milder conditions. However, the very
rigid structure of the acid may make it difficult to find a lipase that Castor oil was purchased from Campestre (São Bernardo do
can recognize it as a substrate. On the other hand, considering that Campo, Brazil); Castor seeds and ricinoleic acid were kindly
lubricants may be used at high temperatures, this instability of donated by Bioóleo Bahia (Feira de Santana, Brazil) and
ricinoleic acid could be a problem for their applications. One likely Miracema-Nuodex (Jardim São José, Brazil) respectively. The poly-
solution for this is esterification of the hydroxyl group [27]. ols neopentylglycol (NPG), trimethylolpropane (TMP) and pen-
Enzymatic catalysis, mainly using lipases (triacylglycerol ester taerythritol (PE) were purchased from Sigma-Aldrich (Jurubatuba,
hydrolases, E.C. 3.1.1.3 - IUPAC), has been used to successfully pro- Brazil). The lyophilized commercial lipase from Candida rugosa,
duce a variety of fuels like biodiesel and biolubricants [7–10,23]. In LipomodTM 34 MDP, was obtained from Biocatalysts Inc. (USA).
vivo, lipases generally act at a substrate/ aqueous interface, The immobilized enzymes from Rhizomucor miehei, LipozymeÒ
catalyzing the hydrolysis of triacylglycerol ester linkages, RM-IM, and Candida antarctica, NovozymÒ 435, were obtained
producing free fatty acids and glycerol [28,29]. To fulfill this from Novozymes (Araucária, Brazil).
function, lipases have a peculiar catalytic mechanism, called
interfacial activation, which involves the movement of an 2.2. Methods
oligopeptide that blocks the active center and the lipase adsorption
on the hydrophobic surface of the oil drop [30,31]. In vitro, lipases 2.2.1. Determination of enzymatic activity
can be used to catalyze several synthetic reactions such as 2.2.1.1. Activity determination of lipases from beans from castor-oil
esterification and transesterification [32,33], which increases their plant. The hydrolytic activity of the lipases present in the dormant
industrial importance. castor beans was quantified by titration of butyric acid released in
According to [34], the thermal stability of the esters of ricinoleic the hydrolysis of tributyrin (5% w/v) in Triton X-100 (25% w/v) in
acid is directly related to the presence or absence of the hydrogen 0.1 M sodium acetate buffer pH 4.0 at 30 °C using the fat-free ace-
in the b-carbon for the alcohol function. The presence of the hydro- tone preparation of lipases from the castor beans. This preparation
xyl group could cause decomposition of the ester molecule, pro- was made as described in [47]. One unit (U) of hydrolytic activity
ducing an acid and an alkene with little energy input. Thus, the was defined as the amount of enzyme that releases 1 mmole of
selected alcohols for this study were neopenthylglicol (NPG), butyric acid per minute under the assay conditions.
trimethylolpropane for (TMP) and pentaerytrithol (PE); the
absence of the b-hydrogen confers steric protection of the ester 2.2.1.2. Hydrolytic activity determination of commercial enzymes.
linkage and improved thermal and oxidative stability. The hydrolytic activity of the commercial lipases was measured
198 J. Greco-Duarte et al. / Fuel 202 (2017) 196–205

using 0.025 M p-nitrophenyl laurate in acetonitrile: DMSO [1:1(v/ %Acidity reduction ¼ 100  ðCOFFAi  COFFAf Þ=COFFAi ð1Þ
v)] (pNP-laurate) as substrate dissolved in 0.025 M phosphate buf-
fer at pH 7.0 as described in [48]. One unit (U) of hydrolytic activity where:
was defined as the amount of enzyme that releases 1 mmole of p- COFFAf: COFFA remaining in the system.
nitrophenol per minute under the assay conditions. COFFAi: COFFA fed to the system.

2.2.2. Castor oil hydrolysis and recovery of COFFA 2.2.3.2. Analysis of the enzymatic production of estolides. After the
2.2.2.1. Hydrolysis. Castor oil was hydrolyzed using 30 g of castor most suitable enzyme was selected, new reactions were performed
seeds and 150 mL of 0.1 M sodium acetate buffer pH 4, maintaining under the same conditions mentioned above in Section 2.2.3.1,
a ratio (w/v) of 1:5. Castor seeds were blended together with the however performing the reaction without added polyols in order
buffer serving as a biocatalyst for hydrolyzing the endogenous oil to investigate the production of estolides from ricinoleic acid.
present in the seeds. This suspension was transferred to a jacketed The reactions were also performed in biological duplicates and
reactor where it was maintained at 30 °C under mechanical stirring analytical triplicates.
at 200 rpm.
Castor oil was also hydrolyzed with extra castor oil added to the 2.2.4. Polyol solubilization
mixture of 0.1 M sodium acetate buffer pH 4.0 and seeds, before The temperature of the mixture between COFFA and NPG, TMP
blended, in the same volume as the buffer (150 mL). The ratio of or PE at 2.5:1, 3.75:1 and 5:1 molar ratios, respectively, was slowly
seeds/buffer/castor oil was 1:5:5 (w/v/v). The reactions of both sys- increased from 40 °C, under constant magnetic stirring, until
tems formed an emulsion system and were performed in biological reaching the melting point of the polyols and/or 150 °C (at this
duplicates and analytical triplicates. temperature the color of the COFFA changed, indicating thermal
This biocatalyst proposed in this paper is a new, green and low degradation). After melting, the mixture was maintained under
cost method to hydrolyze castor oil by crushing castor bean seeds constant magnetic stirring until the temperature decreased to
in the reaction medium. 40 °C, which was the enzymatic reaction temperature. Using PE,
the solid was heated only until reaching 260 °C (its melting point)
2.2.2.2. Recovery of COFFA. Both reactions described above (Sec- and the liquefied polyol was added to the COFFA solution at 40 °C.
tion 2.2.2.1) were stopped by extracting the free fatty acids with Because the polyol solidified at mild temperature and for the rea-
ethyl acetate - used in the ratio 1:2 (v/v) of reaction mixture. The sons discussed below, PE could not be used to characterize the
mixture then formed two layers: 1) the upper layer containing sol- reaction product.
vent and COFFA solved, and 2) the lower layer formed an emulsion
containing crushed seeds, buffer ethyl acetate and glycerol. The
upper layer (1) was recovered and stored and the lower layer (2) 2.2.5. Scale-up of biolubricant ester production
was centrifuged for 5 min at 10,000 rpm to break up the emulsion The reactions for the characterization of the biolubricant prop-
recovering the solvent with COFFA and to separate the biocatalyst. erties were scaled up for reactors with 210 g COFFA with the fol-
After this procedure, the biocatalyst was washed with more lowing reaction composition:
300 mL of ethyl acetate to recover the still remaining COFFA. The
solvent stored was weighed, and to it, 10% (w/w) of anhydrous a) Home-made COFFA + Lipomod 34 MDP (4% w/w) + NPG
sodium sulfate was added and maintained under constant mag- (molar ratio 2.5:1);
netic stirring for 30 min to absorb the remaining water in the sol- b) Home-made COFFA + Lipomod 34 MDP (4% w/w) + TMP
vent. Afterward, the anhydrous sodium sulfate was decanted and (molar ratio 3.75:1);
the COFFA-rich solvent was filtered with filter paper and then fully c) Home-made COFFA + Lipomod 34 MDP (4% w/w);
evaporated in a rotary evaporator to recover the COFFA. d) Commercial COFFA + Lipomod 34 MDP (4% w/w);
The COFFA content was determined by titration in an automatic
titrator (Mettler Toledo). At fixed intervals, the increase in acidity As both NPG and TMP are solids, the temperature of the mixture
was analyzed by titrating triplicate samples of 100 mL COFFA dis- was raised until the polyol crystals were completely solubilized,
solved in acetone and ethanol (45 mL) 1:1 (v/v) with 0.4 M NaOH and then reduced to the operating temperature of 40 °C before
until the end point at pH 11. the enzyme was added. All reactions were performed under con-
stant mechanical stirring at 200 rpm, for 24 h in reactions ‘‘a”
2.2.3. COFFA enzymatic esterification and ‘‘b”, and for 96 h in reactions ‘‘c” and ‘‘d”. At the end of all reac-
2.2.3.1. Evaluation of different enzymes in esterification reac- tions, the reaction mixture was centrifuged (10,000 rpm, 5 min)
tions. COFFA were used as substrates in the esterification reactions and the supernatant was stored at 4 °C until use.
catalyzed by the different lipases with the polyols NPG, TMP and PE
at 2.5:1, 3.75:1 and 5:1 molar ratios, respectively. These ratios 2.2.6. Biolubricant characterization
were selected to maintain the stoichiometry between the hydrox- The biolubricants produced were characterized in terms of pour
yls of the polyol and COFFA molecules for the three cases. The point, kinematic viscosity, viscosity index (VI), oxidative stability
esterification reactions were conducted in thermostated reactors by the rotating pressure vessel method (RPVOT) and total acidity
(maximum volume of 30 mL) at 40 °C, under constant magnetic index (TAI).
stirring (200 rpm). The reaction medium consisted of 12g of COFFA Pour point was analyzed according to the ASTM D97 method.
and 0.48g (4% w/w COFFA) of Lipomod 34 MDP, Lipozyme RM-IM Kinematic viscosity at 40 and 100 °C was determined according
or Novozym 435 as biocatalysts. After 24 and 96 h, the mixture to ASTM D445 and the viscosity index was determined through
was centrifuged (10,000 rpm for 5 min) and the supernatant was calculation from the ASTM D2270 method, which takes into
stored at 4 °C until analysis of the COFFA by the titration method, account the product viscosities at 40 and 100 °C. The oxidative sta-
using an automatic titrator (Mettler Toledo) with 0.04 M NaOH as bility was determined using the RPVOT according to ASTM D2272
the titrating reagent until the end point of pH 11 was reached. The as described in [9].
reactions were performed in biological duplicates and analytical Also, the TAI was determined through potentiometric titration,
triplicates. The percentage of acidity reduction (conversion to using KOH (0.1 mol l–1) and titrating until all acids were neutral-
ester) was calculated as in Eq. (1). ized. The end point is expressed in mg KOH g–1 sample.
 J. Greco-Duarte et al. / Fuel 202 (2017) 196–205 199

2.2.7. Nuclear magnetic Resonance analysis 100

The Nuclear Magnetic Resonance (NMR) analysis was per-

Acidity (g/100g sample)

formed with the products from the reactions without solubiliza- 80
tion of the polyols (96 h), Section 2.2.3.2; and afterward for
reactions ‘‘a” and ‘‘b”, Section 2.2.5. Before the analysis, the reac- 60
tion medium was centrifuged at 10,000 rpm for 10 min in order
40
to obtain only the medium-soluble particles. Afterward, the reac-
tion samples from the bench-scale reactions were submitted to 20
1
H and 13C NMR for structural identification of the reaction prod-
ucts. An Agilent INOVA-300 (7.05T) spectrometer was employed. 0
Samples were prepared in deuterated chloroform (CDCl3) at 0 4 8 12 16 20 24
25 °C, at 5 (1H) and 20% 13C concentration, and compared with Time (h)
pure chemical compounds present in the spectra library [44]. Endogenous oil hydrolysis Castor oil hydrolysis

2.2.8. Gas chromatography Fig. 1. Comparison between hydrolysis using only crushed seeds or with castor oil
The free fatty acids present in the home-made and commercial added in the process. For both hydrolysis procedures, castor seeds (with a lipase
activity of 226 U/g) were crushed in a mixer together with 0.1 M buffer pH 4.0 in a
COFFA were methylated according to the method adapted by [49]
seed:buffer ratio of 1:5 (w/v) in the absence or presence of additional castor oil
The fatty acid methyl esters were analyzed in a gas chromatograph [1:5:5(w/v/v)seed/buffer/castor oil]. After trituration of the seeds, both mixtures
(CG2010; Shimadzu, Japan) equipped with an Omegawax 320 cap- were incubated at 30 °C under magnetic stirring. The oil was extracted and its
illary column (30 m  0.32 mm  0.25 lm; Sigma, São Paulo, Bra- acidity was determined by titration with 0.04 M NaOH.
zil). The following conditions were used: injector temperature:
260 °C; carrier gas: helium at 4.0 mLmin1; sample injection vol- out through the noncatalytic hydrolysis of triglycerides using
ume of 1 lL with split ratio: 1:20. The column temperature gradi- superheated steam in large reactors made of expensive
ent was 150 °C for 5 min, increased at 2 °C min1 to 210 °C, and corrosion-resistant materials, making the process costly and
remained constant for 30 min. The flame ionization detector tem- energy-intensive [54].
perature was 280 °C [50]. The results of the biological triplicates Enzymatic hydrolysis of castor oil has been described previ-
were compared with each other, and the two samples were used ously. Khaskheli et al. [55], studying optimization of the hydrolysis
for the t-test with 95% confidence in order to confirm that they of castor oil, employed 0.01% of Rhizopus oryzae lipase in an oil-
were equivalent. water emulsion system in a 1:4 oil:water ratio, in which the reac-
tion was conducted for 12 h, at 37 °C in order to obtain 90% free
3. Results and discussion fatty acids. Gomes et al. [56], with the main objective of using
the hydrolyzed castor oil in producing aromas, developed an enzy-
3.1. Castor oil hydrolysis using endogenous seed enzymes matic hydrolysis procedure using commercial lipases. They
obtained the best result with Lipozyme TL-IM, achieving approxi-
A strategy for the hydrolysis of castor oil was developed using mately 95% free fatty acids at 25 h, at pH 8 and 27 °C. Usually,
the dormant lipases present in the castor beans themselves the enzymatic processes reported in the literature require longer
[18,19]. However, the COFFA was extracted after hydrolysis using times to obtain a high level of COFFA, and use commercial
ethyl acetate. This solvent is described in the ‘‘Guide for industry” enzymes, which increases the final cost and requires a solvent to
[51] in Class 3, which includes solvents that pose no risk to human recover the COFFA from the medium.
health and have low toxicity. This strategy aimed to produce a biol- In the present study, however, it was possible to perform a
ubricant obtained completely by enzymatic routes and fully envi- hydrolysis with a high reaction rate, using lipases with only the
ronmentally benign. cost of seed crushing, very mild reaction conditions, and using a
The product of the hydrolysis of only the oil contained in the low-toxicity solvent to recover the COFFA, so that this process con-
seeds (COFFA) by the endogenous lipases with activity of forms to the aims of green chemistry.
225.7 ± 0.5 U/g (endogenous oil hydrolysis) reached 93.13 ± 5.9% Thus, hydrolysis of the castor oil using endogenous enzymes
acidity in 45 min of reaction, finally reaching 94.62 ± 0.84% in 4 h from the seeds as catalysts was chosen as a first step in the new
(Fig. 1). Despite the rapid hydrolysis of castor oil, the oil content route of biolubricant production using castor oil.
in the seeds varied from 45 to 50% of the total mass [52], generat- The FFA compositions of the COFFA produced by our methodol-
ing only a small amount of COFFA. Thus, to increase the amount of ogy and a commercial COFFA supplied by Miracema are very sim-
COFFA, additional castor oil was added in a ratio of 1:5:5 (w/v/v) ilar (Table 1). However, commercial samples have more than a 4-
seeds: buffer: castor oil. Then, in comparison to the previous reac- fold higher percentage of undetermined compounds. This index
tion, hydrolysis with the added oil was also performed and its of non-identified fatty acids of the commercial COFFA may be
kinetic hydrolysis (castor oil hydrolysis) is shown in Fig. 1. related to products of thermal degradation of the castor oil and
The reaction with additional castor oil produced 87.83 ± 3.67% COFFA. This difference in the quality of the samples from the two
acidity in 4 h, reaching 91.9 ± 0.65% acidity in 6 h of reaction. Com- processes is perceptible not only from the lack of odor but also
parison with the amount obtained from the endogenous oil hydrol- from the appearance of the two samples at similar concentrations,
ysis (94.62 ± 0.84%) shows a decrease in productivity from as seen in Fig. 2. Commercial COFFA (B) is reddish, while home-
approximately 94 g (endogenous oil hydrolysis) to 15.33 g (castor made COFFA (A) is yellowish, closer to the color of castor oil (C)
oil hydrolysis) of free fatty acids h–1, which was expected due to This suggests that the commercial hydrolysis process partially
the addition of 6-fold more oil for the same amount of enzyme. degrades the acids of the castor oil.
High-temperature hydrolysis of oils containing hydroxy fatty
acids, such as castor oil, is not recommended. These conditions 3.2. Production of biolubricants by COFFA esterification
generate undesirable changes in the free fatty acids from thermal
decomposition of triacylglycerol, which leads to changes in color 3.2.1. Evaluation of different enzymes in esterification reactions
or odor and also reduces the content of free fatty acids [53]. The COFFA obtained in step 3.1 was used for the esterification
Besides, the commercial production of free fatty acids is carried reactions to obtain esters from NPG, TMP and PE, catalyzed by
200 J. Greco-Duarte et al. / Fuel 202 (2017) 196–205

Table 1 acid. This fatty acid has a hydroxyl group in position 12, which
Comparison between the fatty acid composition of home-made and commercial forms an ‘‘L” shape that probably fits better in the active site of
COFFA.
CRL [43].
Free fatty acids (FFA) Home-made (g fatty acid/ Commercial(g fatty acid/
100 g of sample) 100 g of sample)
3.2.2. Analysis of the enzymatic production of estolides
C16:0 1.02 ± 0.01 1.01 ± 0.05 Some reports have described the formation of oligomeric polye-
C18:0 0.70 ± 0.01 0.39 ± 0.03
sters of hydroxylated fatty acids known as estolides [57]. Bódalo
C18:1n9 2.73 ± 0.02 2.82 ± 0.15
C18:2n6 4.96 ± 0.06 3.48 ± 1.18 et al. [38] described the use of CRL to produce estolides from rici-
C18:3n3 0.47 ± 0.03 0.43 ± 0.02 noleic fatty acid. This acid is the majority component of COFFA,
C22:0 1.43 ± 0.31 2.17 ± 0.89 comprising about 88% of its fatty acid composition (see Table 1).
C18:1-OH 87.57 ± 0.15 85.61 ± 2.75 This acid has a hydroxyl group at carbon 12 that enables the ester-
Non-identified 0.95 ± 0.25 4.308 ± 2.7
ification reaction between this hydroxyl and the carboxyl of differ-
ent molecules, forming oligomeric polyesters of ricinoleic acid [58],
as seen in Fig. 3 showing the estolide formation catalyzed by CRL
the commercial lipases Lipomod 34 MDP, Lipozyme RM-IM and [58].
Novozym 435. The results of the acidity reduction (%) in these reac- In view of the possibility that the reduction in acidity of the
tions are shown in Table 2. reaction samples could be related not only to the production of
All enzymes evaluated catalyzed some degree of conversion to polyol derivatives but also to the formation of estolides, a
esters using NPG and TMP. However, using PE, the reaction pro- reaction was performed using only COFFA and CRL (Lipomod 34
gressed only partly when CRL Lipomod 34 MDP was used (Table 2). MDP) as the biocatalyst under similar reaction conditions to those
The use of NPG reached different conversions by the use of the dif- used in the presence of polyols (see Section 2.2.3.2). Fig. 4 shows
ferent lipases. Lipozyme RM-IM showed 24.8 ± 0.3% conversion at the high percentage of acidity reduction in this reaction, using
24 h and reached 61.0 ± 0.6% in 96 h of reaction; Novozymes 435 Lipomod 34 MDP as biocatalysts. The result was similar to that
achieved in 24 h of reaction 23.0 ± 0.2% reaching 55.1 ± 0.2% at obtained in the presence of polyols, and suggested that the
96 h; and, over all the enzymes, Lipomod 34 MDP reached estolide formation may be the main reaction using the lipases
80.6 ± 1.4% at 24 h and 90.2 ± 0.9% in 96 h of reaction. The use of Lipomod 34 MDP, since the other two enzymes produced no
TMP show results quite inferiors for all lipases: Lipozyme RM-IM reduction in the acidity in this reaction medium without polyols
which the conversion to esters at 24 h was almost negligible (data not shown).
(3.5 ± 1.5%), reaching 27.8 ± 0.3% in 96 h and Novozym 435 that Using NPG or TMP, an acidity reduction of about 90% after 96 h
performed slightly better with the use of TMP, reaching 13 ± 0.5% of reaction was observed, and using PE this reduction reached 80%
in 24 h; however, this conversion was statistically the same at in 96 h. Without polyols, the initial acidity decreased to approxi-
96 h – 16.4 ± 0.4% –, Lipomod 34 MDP, however, presents mately 40% in 24 h and 70% in 96 h of reaction. This result not only
80.6 ± 1.4% conversion in only 24 h and 90.7 ± 1.5% conversion at showed that the lipase Lipomod 34 MDP is capable of catalyzing
96 h. The use of PE, however, did not show any result when it used the esterification between the ricinoleic acid, forming estolides,
Lipomod RM-IM nor Novozyme 435 but for Lipomod 34 MDP the but also suggested that estolides may be also a main reaction pro-
acidity reduction was approximately 74.9 ± 0.3% in 96 h. duct even in the presence of polyols.
The commercial enzyme from Candida rugosa has been reported
as a specific catalyst for the modification of castor bean derivatives, 3.2.3. NMR analyses of the reaction products
to produce either ricinoleic acid oligomers known as estolides [36], NMR analyses were performed for the final reaction products
or biolubricants from castor biodiesel [9]. According to Da Silva (96 h) obtained from the reactions in Section 2.2.3.2. Table 3 sum-
et al. [9], better results were obtained for the reaction between cas- marizes the composition of the different final reaction media.
tor biodiesel and TMP with the use of the C. rugosa lipase Lipomod The data in Table 3 show that the reactions performed without
34 MDP than with the lipases Lipozyme RM-IM and Novozym 435. polyol and with PE produced only estolide molecules. It is possible
The enzymatic reaction produced a higher conversion (approxi- that the PE was not properly dissolved during the reaction. This
mately 98%) compared to chemical catalysis (60%). polyol did not appear in the NMR analyses, as occurred with the
The better performance of this enzyme for castor oil free fatty other polyols (Table 3). As the NMR analyses were performed with
acids may be related to the active site architecture of CRL. Some- only the supernatant from the reaction-medium centrifugation
how, this singular architecture favors the recognition of ricinoleic (section 2.2.7), this result confirms that the PE concentration in

Fig. 2. Color comparison between home-made COFFA (A), commercial COFFA (B) and castor oil (C).
 J. Greco-Duarte et al. / Fuel 202 (2017) 196–205 201

Table 2
Acidity reduction (%) or conversion to esters from the ester synthesis catalyzed by lipases Novozym 435, Lipozyme RM-IM and Lipomod 34 MDP at 4% (w/w of COFFA). The
reactions, described in the subsection 2.2.3.1, were conducted at 40 °C in a stirred-bed reactor using COFFA and NPG, TMP or PE polyols as the substrate in COFFA:polyol molar
ratios of 2.5:1, 3.75:1 and 5:1, respectively.

NPG TMP PE
24 h RM-IM 24.8 ± 0.3 3.5 ± 1.5 0
Novozymes 435 23.0 ± 0.2 13 ± 0.5 0
Lipomod 34 MDP 80.6 ± 1.4 65.2 ± 0.3 48.9 ± 0.8
96 h RM-IM 61.0 ± 0.6 27.8 ± 0.3 0
Novozymes 435 55.1 ± 0.2 16.4 ± 0.4 0
Lipomod 34 MDP 90.2 ± 0.9 90.7 ± 1.5 74.9 ± 0.3

Fig. 3. Estolide formation from the polymerization of ricinoleic acid.

100 result shows the inability of these enzymes to produce estolides

from ricinoleic acids. The other two polyols dissolved, although
Acidity reducon (%)

80 not immediately, in the reaction medium, allowing some ester

formation.
60 The presence of polyols using Lipomod 34 MDP reduced the
degree of polymerization of the estolide. The reaction performed
40 in absence of polyols produced hexameter or heptamer molecules.
However, in the presence of PE, the maximum degree of estolide
20 polymerization was tetramers, showing that the presence of a solid
powder of this polyol hindered the formation of larger estolide
0 molecules, even without being soluble, perhaps by somehow
24 h 96 h affecting the aggregates of the enzyme (Table 3).
NPG TMP PE Without polyols Although the production of esters from NPG and TMP was
observed, the desired peracylated product (dineopentyl rici-
Fig. 4. Acidity reduction (%) from the ester synthesis and control reactions
noleate) was obtained only with NPG. Using TMP, mono- and
catalyzed by the commercial lipase Lipomod 34 MDP at 4% (w/w of COFFA). The
reactions were conducted at 40 °C and aliquots for titration analysis are taken in 24 dimethylpropyl ricinoleates were observed, but no trimethylpropyl
and 96 h. The reaction were conducted in a stirred-bed reactor using COFFA and ricinoleate was detected (Table 3). However, all free fatty acids had
NPG, TMP or PE polyols as substrates in COFFA:polyol molar ratios of 2.5:1; 3.75:1 been consumed to produce mainly dimers of ricinoleic acid (com-
and 5:1, respectively. The control reaction that was performed under the same prising 70.7 and 65.9% of the total weight, respectively).
conditions, in the absence of polyols.
A likely explanation for these results is that the delay in the sol-
ubilization of the polyols may permit the formation of dimers of
the reaction medium was negligible. On the other hand, the utiliza- ricinoleic acid before the polyol solubilization, and this prevents
tion of PE as a substrate in the reactions catalyzed by Lipozyme the synthesis of polyol-peracylated compounds. Therefore, solubi-
RM-IM and Novozym 435 did not produce any ester (Table 2). This lization of the polyols was optimized before starting the reaction.
202 J. Greco-Duarte et al. / Fuel 202 (2017) 196–205

Table 3
NMR analysis of the supernatant obtained by centrifugation of the reactions between home-made COFFA with and without polyols NPG, TMP and PE (molar ratios 2.5:1, 3.75:1
and 5:1, respectively) the reactions are described in the Section 2.2.3.2.

Substrates Compound Molar (%) Weight (%)

COFFA + NPG (molar ratio 2,5:1) 6,7 1,3
NEOPENTHYLGLYCOL (NPG)

13,6 17,1

DINEOPENTHYL RICINOLEATE
15 10,9
MONONEOPENTHYL RICINOLEATE

64,7 70,7

RICINOLEIC ACID DIMER

COFFA + TMP(molar ratio 3,75:1) 9,8 2,5
TRIMETHYLOLPROPANE (TMP)

15 19,7

DIMETHYLOLPROPYL RICINOLEATE

15,1 11,9

MONOMETHYLPROPYL RICINOLEATE
60,1 65,9
RICINOLEIC ACID DIMER

COFFA + PE(molar ratio 5:1) 100 100

RICINOLEIC ACID OLIGOMERS (n = 2)

COFFA 100 100

RICINOLEIC ACID OLIGOMERS (n = 4 or 5)

3.2.4. Effect of polyol solubilization

100
Before the enzyme addition, the reaction medium was heated
Acidity reducon (%)

until complete solubilization of the different polyols as described 80

in Section 2.2.4.
Using PE, although the temperature of the reaction medium was 60
increased to 150 °C it remained fully insoluble. To solve this prob-
lem, PE was incubated at 260 °C (the melting point) and was incor- 40
porated as a liquid in the COFFA sample preheated to 40 °C.
However, the PE solidified when the temperature decreased. 20
Therefore we discarded the use of this polyol in this medium. PE
has been used in chemical catalysis in similar reactions, but at 0
higher temperatures, for example in the transesterification with 16 h 24 h 48 h 96 h
castor oil at 200 °C [59] and with palm oil at 158 °C [60]; however, TMP NPG
the oxidative stability of the products obtained was not reported.
Both NPG and TMP were solubilized by heating the reaction Fig. 5. Acidity reduction (%) from the ester synthesis, with fully solubilized polyols,
catalyzed by the commercial lipase Lipomod 34 MDP at 4% (w/w of COFFA). The
medium to between 56 and 58 °C and they remained in solution
biolubricant synthesis reactions were conducted at 40 °C for 96 h in a stirred-bed
even when the temperature was reduced again to 40 °C. Thus, it reactor using COFFA and NPG or TMP polyols as substrates in COFFA:polyol molar
was possible to have these two polyols soluble before starting ratios of 2.5:1 and 3.75:1 respectively.
the reaction.
Solubilizing the polyols allowed us to reduce the time required
to obtain an approximately 90% reduction of acidity from 96 h to final reaction mixtures are shown in Table 4. Using pre-
24 h, suggesting that the lack of polyol solubility hindered the solubilized alcohols, the polyol esters increased while the forma-
esterification of the COFFA (Fig. 5). tion of ricinoleic acid dimers decreased. However, the dimer of rici-
NMR analyses of these new reaction products (‘‘a” and ‘‘b” of noleic acid remained the main component (53.3% using NPG and
Section 2.2.5) were performed, and the main components of the 65.4% using TMP of the total weight), while the peracylated polyols
 J. Greco-Duarte et al. / Fuel 202 (2017) 196–205 203

Table 4
NMR analysis of the supernatant obtained by centrifugation of the reactions ‘‘a” and ‘‘b” between home-made COFFA and the fully solubilized polyols NPG and TMP (molar ratios
2.5:1 and 3.75:1 respectively). The reactions are described in the Section 2.2.5.

Substrates Compounds Molar (%) Weight (%)

COFFA + NPG (molar ratio 2,5:1) 17,4 3,9
NEOPENTHYLGLYCOL (NPG)

15,1 21,8
DINEOPENTHYL RICINOLEATE

25,1 20,9
MONONEOPENTHYL RICINOLEATE

42,4 53,3
RICINOLEIC ACID DIMER

COFFA + TMP (molar ratio 3,75:1) 4,4 1

TRIMETHYLOLPROPANE

15,3 18,1

DIMETHYLOLPROPYL RICINOLEATE

7,4 5,5

MONOMETHYLPROPYL RICINOLEATE

6 10
TRIMETHYLOLPROPYL RICINOLEATE

66,4 65,4
RICINOLEIC ACID DIMER

were a minority product (21.8% dineopenthyl ricinoleate and only Viscosity is the most important index of these products, since it
10% trimethylolpropyl ricinoleate of the total weight) relates to the formation of a protective film on the metal, which
This suggests that Lipomod 34 MDP lipase has a preferential protects against friction [62]. A high viscosity index such as this
affinity for ricinoleic acid as a nucleophile reactant compared to means that the viscosity of the fluid does not change with
the polyols used, even when these polyols are present in molar increased temperature [9,62], which is desirable for any lubricant
excess. application. However, the IAT was higher using only estolides
(40.4 and 58.4 mg KOH g–1 of home-made and commercial COFFA,
3.2.5. Product characterization as lubricants respectively) than the NPG and TMP products (7.3 and 4.3 mg KOH
Table 5 shows the main features of the products obtained using g–1 of sample, respectively). This can be explained by the acidic
COFFA produced by our methodology (home-made COFFA) with characteristic of the estolide molecule from the carboxylic tip of
previously solubilized polyols and with the products obtained ricinoleic acid. The acidity of the final product might be decreased
without polyols using a commercial COFFA or our home-made using different strategies presently under study in our laboratory,
COFFA e.g., esterification of estolides with a simple alcohol.
The lubricating properties of all samples were satisfactory. Both On the other hand, the use of home-made or commercial COFFA
the pour point and oxidative stability were similar and higher than has some clear differences. The home-made product had a lower
those previously reported for biolubricants [9,10,25,61]. Surpris- pour point (–42 °C versus –36 °C) and a 1.4-fold increase in the
ingly, when only long-chain estolides were present, the viscosity oxidative stability (51 min versus 37 min). These differences must
index (162 and 154 for home-made and commercial COFFA, be attributed to the different hydrolysis processes: the enzymatic
respectively) was higher than the results for the other products. process preserved the ricinoleic acid structure, while the thermal

Table 5
Biolubricant properties of the products from the ester reactions between home-made COFFA and NPG and TMP (molar ratios 2.5:1 and 3.75:1, respectively) and without polyols
using commercial and home-made COFFA. All reactions were catalyzed by Lipomod 34 MDP at 4% (w/w of COFFA) and are described in the Section 2.2.5.

Assay Reaction substrates

COFFA + NPG COFFA + TMP Home-made COFFA Commercial COFFA
2
Viscosity 40 °C (mm /s) 172.0 278.1 379.9 408.18
Viscosity 100 °C (mm2/s) 17.60 24.49 41.43 41.54
Viscosity index 111 112 162 154
Oxidative estability (min) 50 54 51 37
Pour point (°C) 42 39 -42 -36
IAT potentiometric (mg KOH/g of sample) 7.3 4.3 40.4 58.4
204 J. Greco-Duarte et al. / Fuel 202 (2017) 196–205

process produced byproducts that affected the biolubricant fea- [14] Goswami D, Sen R, Basu JK, De S. Maximization of bioconversion of castor oil
into ricinoleic acid by response surface methodology. Bioresour Technol
tures. The product obtained from the esterification of ricinoleic
2009;100(18):4067–73.
acid obtained using enzymatically produced COFFA is better than [15] Scarpa A, Guerci A. Various uses of the castor oil plant (Ricinus communis L.) a
any other biolubricant molecules described in the literature, and review. J Ethnopharmacol 1982;5:117–37.
is produced in an eco-friendly way. [16] Bale AT, Adebayo RT, Ogundele DT, Bodunde VT. Fatty acid composition and
physicochemical properties of castor (Ricinus Communis L.) seed obtained
from malete, moro local government area, kwara state. Nigeria. Chem Mater
Res 2013;3(12).
4. Conclusion [17] Kermode AR, Bewley JD. The role of maturation drying in the transition from
seed development to germination: II. Post-germinative enzyme production
and soluble protein synthetic pattern changes within the endosperm of
The new two-step enzymatic process described here permits
Ricinus communis L. seeds. J Exp Bot 1985;36(12):1916–27.
one to obtain, in a green and very efficient way, new products with [18] Avelar MHM, Cassimiro DMJ, Santos KC, Domingues RCC, De Castro HF,
properties that surpass those of other biolubricants reported in the Mendes AA. Hydrolysis of vegetable oils catalyzed by lipase extract powder
from dormant castor bean seeds. Ind Crops Prod 2013;44:452–8.
literature. The use of endogenous lipases from castor-oil seed is a
[19] Fuchs C, Vine N, Hills MJ. Purification and characterization of the acid
low-cost and efficient method to obtain intact COFFA, and the fur- lipase from the endosperm of castor oil seeds. J Plant Physiol 1996;149(1–2):
ther estolide formation catalyzed by the enzymes from C. rugosa 23–9.
allows the formation of hexamers/heptamer estolides of ricinoleic [20] Maher KD, Bressler DC. Review Pyrolysis of triglyceride materials for the
production of renewable fuels and chemicals. Bioresour Technol 2007;98
acid. Ricinoleic acid is a better nucleophile for this enzyme than the (12):2351–68.
polyols employed, whose reactions always generate estolides as [21] Satyarthi JK, Srinivas D, Ratnasamy P. Hydrolysis of vegetable oils and fats to
the main product. These estolides have promising lubricant charac- fatty acids over solid acid catalysts. Appl Catal A 2011;391(1–2):427–35.
[22] Noor IM, Hasan M, Ramchandran KB. Effect of operating variables on
teristics (better than the products obtained using the polyols) for thehydrolysis rate of palm by lipase. Process Biochem 2003;39:13–20.
industrial use. They also improve the properties of the product [23] Hermansyah H, Wijanarko A, Dianursanti D, Gozan M, Praswasti PDKW,
derived from commercial COFFA obtained by thermal treatment, Arbianti R, et al. Kinetic model for triglyceride hydrolysis using lipase: review.
Makara J Technol 2007;11(1):30–5.
confirming that the new route is not only more sustainable, but [24] Goodrum JW, Geller DP. Influence of fatty acid methyl esters from
also more efficient. However, some properties such as excess acid- hydroxylated vegetable oils on diesel fuel lubricity. Bioresour Technol
ity need to be improved, and are presently a focus of our research 2005;96(7):851–5.
[25] Beopoulos A, Verbeke J, Bordes F, Guicherd M, Bressy M, Marty A, et al.
group.
Metabolic engineering for ricinoleic acid production in the oleaginous yeast
Yarrowia lipolytica. Appl Microbiol Biotechnol 2014;98:251–62.
[26] Azevedo DMP, Lima EF. O Agronegócio da Mamona no Brasil. 21ª
Acknowledgements ed. Embrapa: Brasília; 2001.
[27] Yao L, Hammond EG, Wang T, Bhuyan S, Sundararajan S. Synthesis and
The authors are grateful to CNPq (Brazil, Conselho Nacional de physical properties of potential biolubricants based on ricinoleic acid. J Am Oil
Chemist’s Soc 2010;87(8):937–46.
Desenvolvimento Científico e Tecnológico), PVE Project from CNPq, [28] Angajala G, Pavan P, Subashini R. Lipases: an overview of its current challenges
CAPES (Brazil, Coordenação de Aperfeiçoamento de Pessoal de and prospectives in the revolution of biocatalysis. Biocatal Agric Biotechnol
Nível Superior), FAPERJ (Brazil, Fundação Carlos Chagas Filho de 2016;7:257–70.
[29] Sharma R, Chisti Y, Banerjee UC. Production, purification, characterization and
Apoio à Pesquisa do Estado do Rio de Janeiro), MINECO (Spain, pro- applications of lipases. Biotechnol Adv 2001;19:627–62.
ject number CTQ2013-41507-R) and Petrobras, ANP, MCTI and [30] Derewenda ZS. Structure and function of lipases. Adv Protein Chem
FINEP for scholarships and financial support. 1994;45:1–52.
[31] Verger R. ‘‘Interfacial activation” of lipases: facts and artifacts. Trends
Biotechnol 1997;15:32–8.
References [32] Aarthy M, Saravanan P, Gowthaman MK, Rose C, Kamini NR. Enzymatic
transesterification for production of biodiesel using yeast lipases: an overview.
Chem Eng Res Des 2014;92:1591–601.
[1] Tollefson J. How much longer can Antarctica’s hostile ocean delay global
[33] Aguieiras ECG, Cavalcanti-Oliveira ED, Freire DMG. Current status and new
warming? Nature 2016;539(7629):346–8.
developments of biodiesel production using fungal lipases. Fuel
[2] Anastas P, Eghbali N. Green chemistry: principles and practice. Chem Soc Rev
2015;159:52–67.
2010;39(1):301–12.
[34] Mouloungui Z, Pelet S. Study of theacyl transfer reaction: structure and
[3] Ferreira-Leitão VS, Cammarota MC, Aguieiras ECG, de Sá LRV, Fernandez-
properties of glycerol carbonate esters. Eur J Lipid Sci Technol
Lafuente R, Freire DMG. The protagonism of biocatalysis in green chemistry
2001;103:216–22.
and its environmental benefits. Catalysts 2017;7:9. http://dx.doi.org/
[35] Kasche V. Mechanism and yields in enzyme catalysed equilibrium and
10.3390/catal7010009.
kinetically controlled synthesis of b-lactam antibiotics, peptides and other
[4] Panchal TM, Patel A, Chauhan DD, Thomas M, Patel JV. A methodological
condensation products. Enzyme Microb Technol 1986;8(1):4–16.
review on bio-lubricants from vegetable oil based resources. Renew Sustain
[36] Kasche V, Haufler U, Riechmann L. Equilibrium and kinetically controlled
Energy Rev 2017;70:65–70.
synthesis with enzymes: semisynthesis of penicillins and peptides. Methods
[5] Kania D, Yunus R, Omar R, Rashid SA, Jan BM. Review of biolubricants in
Enzymol 1987;136:280–92.
drilling fluids: Recent research, performance, and applications. J Petrol Sci Eng
[37] Švedas VK, Margolin AL, Berezin IV. Enzymatic synthesis of b-lactam
2015;135:177–84.
antibiotics: a thermodynamic background. Enzyme Microb Technol 1980;2
[6] McNutt J, He SQ. Development of biolubricants from vegetable oils via
(2):138–44.
chemical modification. J Ind Eng Chem 2016;36:1–12.
[38] Bódalo-Santoyo A, Bastida-Rodríguez J, Máximo-Martín MF, Montiel-Morte
[7] Nagendrama P, Kaul S. Development of ecofriendly/biodegradable lubricants:
MC, Murcia-Almagro MD. Enzymatic biosynthesis of ricinoleic acid estolídeos.
an overview. Renew Sustain Energy Rev 2012;16:764–74.
Biochem Eng J 2005;26:155–8.
[8] Dormo N, Belafi-Bako K, Bartha L, Ehrenstein U, Gubicza L. Manufacture of an
[39] Haigha KF, Vladisavljevića GT, Reynoldsb JC, Nagya Z, Sahac B. Kinetics of the
environmental-safe biolubricant from fusel oil by enzymatic esterification in
pre-treatment of used cooking oil using Novozyme 435 for biodiesel
solvent-free system. Biochem Eng J 2004;21:229–34.
production. Chem Eng Res Des 2014;92(4):713–9.
[9] Da Silva JAC, Soares VF, Fernandez-Lafuente R, Habert AC, Freire DMG.
[40] Anderson EM, Larsson KM, Kirk O. One biocatalyst - many applications: the use
Enzymatic production and characterization of potential biolubricants from
of Candida antarctica B-lipase in organic synthesis. Biocatal Biotransform
castor bean biodiesel. J Mol Catal B Enzym 2015;122:323–9.
1998;16(3):181–204.
[10] Åkerman CO, Hagströma AEV, Mollaahmada MA, Karlssonb S, Hatti-Kaul R.
[41] Andualema B, Gessesse B. Microbial lipases and their industrial applications:
Biolubricant synthesis using immobilised lipase: process optimisation of
review. Biotechnology 2012;11(3):100–18.
trimethylolpropane oleate production. Process Biochem 2011;46:2225–31.
[42] Rodrigues RC, Fernandez-Lafuente R. Lipase from Rhizomucor miehei as an
[11] Bart JCJ, Gucciardi E, Cavallaro S. Renewable feedstocks for lubricant
industrial biocatalyst in chemical process. J Mol Catal B Enzym 2010;64(1–2):
production. In: Biolubricants Science and technology. Woodhead Publishing
1–22.
Limited; 2013.
[43] De María PD, Sánchez-Montero JM, Sinisterra JV, Alcántara AR. Understanding
[12] Whitby RD. Understanding the global lubricants business-regional markets,
Candida rugosa lipases: an overview. Biotechnol Adv 2006;24(2):180–96.
economic issues and profitability. In: Course notes. Oxford, England: The
[44] Duarte JG, Leone-Ignacio K, Da Silva JAC, Fernandez-Lafuente R, Freire DMG.
Oxford Princeton Programme; 2005.
Rapid determination of the synthetic activity of lipases/esterases via
[13] Makkonen I. Environmentally compatible oils, Field Note No. General, Pointe-
transesterification and esterification zymography. Fuel 2016;177:123–9.
Claire, 39. Que: Forest Engineering Research Institute of Canada (FERIC); 1994.
 J. Greco-Duarte et al. / Fuel 202 (2017) 196–205 205

[45] Pourzolfaghar H, Abnisa F, Daud WMAW, Aroua MK. A review of the enzymatic [54] Ngaosuwan K, Lotero E, Suwannakarn K, Goodwin Jr JG, Praserthdam P.
hydroesterification process for biodiesel production. Renew Sustain Energy Hydrolysis of triglycerides using solid acid catalysts. Ind Eng Chem Res
Rev 2016;61:245–57. 2009;48(10):4757–67. http://dx.doi.org/10.1021/ie8013988.
[46] De Sousa JS, Cavalcanti-Oliveira EA, Aranda DAG, Freire DMG. Application of [55] Khaskheli AA, Talpur FN, Ashraf MA, Cebeci A, Jawaid S, Afridi HI. Monitoring
lipase from the physic nut (Jatropha curcas L.) to a new hybrid the Rhizopus oryzae lipase catalyzed hydrolysis of castor oil by ATR-FTIR
(enzyme/chemical) hydroesterification process for biodiesel production. J spectroscopy. J Mol Catal B Enzym 2015;113:56–61.
Mol Catal B Enzym 2010;65:133–7. [56] Gomes N, Braga A, Teixeira JA, Belo I. Impact of lipase-mediated hydrolysis of
[47] Cavalcanti EDC, Maciel FM, Villeneuve P, Lago RCA, Machado OLT, Freire DMG. castor oil on c-decalactone production by yarrowia lipolytica. J Am Oil Chem
Acetona powder from dormant seeds of Ricinus communis L. Appl Biochem Soc 2013;90(8):1131–7.
Biotechnol 2007;136–140:57–66. [57] Pelaez M, Orellana C, Marqués A, Busquets M, Guerrero A, Manresa A. Natural
[48] Gutarra MLE, Godoy MG, Maugeri F, Rodrigues MI, Freire DMG, Castilho LR. estolides produced by Pseudomonas sp. 42A2 grown on oleic acid: Production
Production of an acidic and thermostable lipase of the mesophilic fungus and characterization. JAOCS 2003;80(9):859–66.
Penicillium simplicissimum by solid-state fermentation. Bioresour Technol [58] Bódalo A, Bastida J, Máximo MF, Montiel MC, Murcia MD, Ortega S. Influence of
2009;100:5249–54. the operating conditions on lipase-catalysed synthesis of ricinoleic acid
[49] Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step estolídeos in solvente-free systems. Biochem Eng J 2009;44:214–9.
reaction. J Lipids Res 1986;27:114–20. [59] Valero MF, Gonzalez A. Polyurethane adhesive system from castor oil modified
[50] Sousa JS, Torres AG, Freire DMG. Nutritional enrichment of vegetable oils with by a transesterification reaction. J Elastomers Plast 2012;44(5):433–42.
long-chain n-3 fatty acids through enzymatic interesterification with a new [60] Aziz NAM, Yunus R, Rashid U, Syam AM. Application of response surface
vegetable lipase. Grasas y Aceites Int J Fats Oils 2015;66:2. methodology (RSM) for optimizing the palm-based pentaerythritol ester
[51] Food and drug administration. Guide for industry Q3C – Tables and list synthesis. Ind Crops Prod 2014;62:305–12.
2012. In: http://www.fda.gov/downloads/drugs/guidancecomplianceregulatory- [61] Cavalcante IG, Rocha NRC, Maier ME, Lima APD, Neto DMA, Brito DHA, et al.
information/guidances/ucm073395.pdf [access 02.01.17]. Synthesis and characterization of new esters of oleic acid and glycerol
[52] Tambascia MB, Teixeira JPF. Mamona: determinação quantitativa do teor de analogues as potential lubricants. Ind Crops Prod 2014;62:453–9.
óleo. Bragantia 1986;45(1):23–7. [62] Bart JCJ, Gucciardi E, Cavallaro S. Principals of lubrication. In: Biolubricants
[53] Holliday RL, King JW, List GR. Hydrolysis of vegetable oils in sub- and Science and Technology. Woodhead publishing Series in Energy; 2013. 10-22.
supercritical water. Ind Eng Chem Res 1997;36(3):932–5. http://dx.doi.org/
10.1021/ie960668f.