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The effectiveness of enzymatic
transesterification of animal tallow using the enzyme Candida antarctica Novozyme 435 was studied. The effects of oil :
alcohol molar ratio (1:1, 1:2, 1:3, 1:4 and 1:5), reaction temperature (35, 40,
45 and 50°C) and reaction time (4, 8, 12 and 16 h) on the biodiesel conversion
yield were evaluated in a solvent system using n-hexane. The highest conversion
yield of biodiesel was obtained at the 1:4 oil : alcohol molar ratio, 16 h
reaction time and 45°C reaction temperature. Increasing the oil : alcohol molar
ratio from 1:1 to 1:4 increased the conversion yield of biodiesel by
21.24-82.11%, depending on the reaction temperature and time. The rate of
conversion of fatty acid esters to biodiesel increased with increases in reaction
time. The reaction proceeds slowly at the beginning and then rapidly due to the
initial mixing and dispersion of alcohol into the oil substrate and the
activation of enzyme. Increasing the reaction time from 4 to 16 h increased the
conversion yield of biodiesel by 13.51-34.07%, depending on the oil : alcohol
molar ratio and reaction temperature. The interactions between enzyme polymer
surface and substrate appears to be dependent on reaction temperature due to
hydrogen bonding and ionic interactions which play important roles in
maintaining the thermostability of lipase in the system. The high temperature
of 50°C denatured the specific structure of enzymes and resulted in a decrease
in methyl esters formation. Increasing the reaction temperature from 40 to 45°C
increased the biodiesel conversion yield by 8.48-33.95%. Using n-hexane in the
reaction helped to stabilize the enzyme and minimize the toxicity of alcohol.
The activity of the enzyme catalyst Candida
antarctica Novozyme 435 in the presence of 2-butanol and n-hexane remained
relatively stable for 10 cycles and then decreased rapidly reaching 11% after
50 cycles.
INTRODUCTION
The high
demand for fossil fuels, their limited and unsecure supply and high cost have
prompted the search for alternative energy sources such as biofuels, solar,
wind, wave, hydro, geothermal and nuclear. However, renewable biofuels such as
biodiesel, bioethanol and biogas from biomass materials are more economical,
and environmentally friendly [1-3]. Biodiesel has several qualities over diesel
including: being sulfur free, non-toxic, biodegradable and non-carcinogenic [4].
These characteristics make it greener and more eco-friendly than diesel [5-9].
In addition, biodiesel can be used in compression-ignition engines instead of
petroleum diesel [10-11].
Biodiesel
can be produced from many raw materials including: plant oils, animal fats,
microbial mass and waste materials ([6]. Popular plants used as a feedstock are
jatropha, canola, coconut, cottonseed, groundnut, karanj, olive, palm, peanut,
rapeseed, safflower, soybean and sunflower [6,9-10].
The main
component of fats and oils are triacylglycerols (triglycerides) which are made
of different types of fatty acids with one glycerol (glycerine) being the
backbone. The types of fatty acids present in the triglycerides determine the fatty
acids profile. Fatty acid profiles from plants and animal sources are different
and each fatty acid has its own chemical and physical properties which can be a
major factor influencing the properties of biodiesel [6,15-16].
Vegetable
oils and animal fats can be transformed into biodiesel by the
transesterification process. Transesterification is a classic chemical process
used to convert the triglycerides (vegetable oils and animal fats) to
biodiesel. A short chain alcohol is used to convert the feedstock to methyl
esters and glycerin which reduces the viscosity of oil by turning it into
biodiesel [17]. Transesterification can proceed with one of three catalysts:
acid, alkali and enzyme. With the acid catalyst, the proton is donated to the
carbonyl group which makes it more reactive. A base catalyst is used to remove
the proton from alcohol which makes the reactants more reactive [18]. However,
using acid and alkali requires more energy and a downstream processing step is
required for removing the by-product (glycerin) [19]. An enzymatic catalyst can
be used to cleave the backbone of the glycerol which makes the reactants more
reactive, giving the product without the need for a downstream processing step.
The glycerol can be extracted easily and the energy required for the process is
minimal.
Objectives
The main aim of this study was to optimize the enzymatic transesterification process for the production of biodiesel from animal tallow obtained from rendering. The specific objectives were: (a) to study the effectiveness of the Candida antarctica Novozyme 435 with the long chain alcohol 2-butanol and hexane as a solvent, (b) to evaluate the effects of oil : alcohol molar ratio (1:1,1:2, 1:3, 1:4 and 1:5), reaction temperature (35, 40, 45 and 50°C) and reaction time (4, 8, 12 and 16 h) on the biodiesel yield and (c) to determine the enzyme stability and reusability.
MATERIALS AND METHODS
The rendering waste was obtained as beef
tallow that had been rendered by the Company S.F Rendering, Centreville Nova
Scotia. Samples (10 Kg) were collected and stored at - 20°C in the
Biotechnology Laboratory of Dalhousie University. The collected material was
yellowish in colour.
The immobilized Lipase was an Candida antarctica (Novozyme
435)obtained from Novozyme (Franklinton, North Carolina, USA). The chemicals
used in the study included: methanol,
n-hexane, tertrahydrofuran, N,
O - Bis (Trimethylsilyl)-trifluroacetamide (BSTFA) and hilditch reagent. They
were purchased from Sigma Aldrich (St. Louis, Missouri, USA). The fatty acid
methyl ester (FAME) standards, which included methyl myristate, methyl
pentadecanote, methyl cis-11-eicosenoate, methyl all-cis-5,8,11,14,17-
eicosapentaenoate (EPA), methyl erucate, methyl
all-cis-7,10,13,16,19-docosapentaenoate (DPA) and methyl
all-cis-4,7,10,13,16,19-docosahexenoate (DHA), were purchased from Sigma
Aldrich (St. Louis, Missouri, USA). The other FAME standard, which included
methyl palmitate, methyl palmitoleate, methyl stearate, methyl oleate, methyl
linoleate and methyl linolenate, were
purchased from Alltech Associates Inc. (Deerfield, Illinois, USA). The
FAME standard methyl-stearidonate was purchased from Cayman Chemical (Ann
Arbor, Michigan, USA).
Purification of Crude Animal Tallow
The animal tallow was first heated to
105-110°C with constant stirring at 50 rpm in a 500 ml round bottom flask for
one hour. During the process of melting the fats, the top layer (consisting of
bubbles and impurities) was discarded regularly. Then, the extracted crude
animal tallow oil was filtered four times using vacuum filtration with
ultra-filter paper (Whatman No.40, Fisher Scientific, Toronto, Ontario,
Canada). The oil percentage was calculated as follows
Oil percentage = ((weight of oil)/(Total weight of Tallow))*100 (1)
Experimental Procedure
The enzymatic transesterification was
carried out in order to extract fatty acid methyl esters from the animal tallow
by the immobilized Candida Antarctica Novozyme
435as shown in Figure 1. Five oil : alcohol molar ratios (1:1, 1:2, 1:3, 1:4 or
1:5), four reaction temperatures (35, 40, 45 or 50°C) and four reaction times
(4, 8, 12 or 16 h) were investigated. The homogenized oil (2.3 ml corresponding
to 2 g of fat) was placed into a 50 ml conical flask and heated on a hot plate
(PC-620, Corning, New York, New York, USA). The immobilized Candida antarctica Novozyme 435 was added
to the flask (25% of the oil weight of 0.5 g). The appropriate amount of
alcohol (2-butanol) was added based on the selected oil : alcohol molar ratio
(1:1, 1:2, 1:3, 1:4 or 1:5). The solution was mixed using a reciprocal shaking
bath (2850 Series, Fisher Scientific, Toronto, Ontario, Canada) at 200 rpm. The
desired temperature (35, 40, 45 or 50°C) was selected. After the desired
reaction time was completed (4, 8, 12 or 16 h), the enzyme was filtered by
vacuum filtration as recommended by Nelson et al.
(1998). Samples (100µl) were taken from the mixture and analyzed using a gas
chromatograph (Hewlett Packard 5890 series II, Agilent, Mississauga, Ontario,
Canada).The same procedure was repeated with all oil:alcohol molar ratios,
reaction temperatures and reaction times.
Determination of Biodiesel Conversion Yield
The procedure used to prepare the biodiesel sample for gas chromatography analysis is shown in Figure 2. A 100 µL aliquot was taken from the transesterification process and flushed with nitrogen in a microprocessor-controlled water bath (280 series, Fisher Scientific, Toronto, Ontario, Canada) at 45°C in order to evaporate the hexane. A 10 mg portion of the residue was dissolved in 100 µL of tertrahydrofuran and 200 µL of BSTFA. Then, the mixture to room temperature (20°C) for few minutes after which 5 mL of hexane was added. An aliquot of 1.5 ml mixture was transferred to the GC crimp vials and capped tightly for further analysis using Gas Chromatography.
A gas chromatograph, equipped with an AT-FAME capillary column that is 30 m in length and of 0.32 mm internal diameter and 0.25 µm film thicknesses (Alltech Associates Inc., Deerfield, Illinois, USA), was used for the analyses. The column was a highly polar and stable bonded polyethylene glycol phase, coupled with flame ionization detector (FID) (HP5890 Series II, Agilent Technologies, Mississauga, Ontario, Canada). An aliquot of 10μl of the mixture was injected directly into the column with the initial oven temperature of 60°C, followed by a flow rate of 20°C/min. A final temperature of 280°C was held for 10 minutes. The detection system was equipped with a flame ionization detector (FID) operating at 275°C with helium as a carrier gas at a flow rate of 0.6 mL/min. The total run time was 40 minutes. The yield was calculated as follows:
yield of Peak = (Peak area A x 100)/(∑(Peak area A+Peak area B+⋯+Peak area N)) (2)
area A (wt%)
Where:
Peak area A = Methyl Oleate
Peak area B = Methyl Palmitate
Peak area N = no. of unknown
peaks
Statistical
Analyses
Statistical analyses were performed on
biodiesel results using Minitab Statistics Software (Ver 16.2.2, Minitab Inc.,
State College, Pennsylvania, USA).
Both analyses of variance (ANOVA) and Tukey's grouping were carried out.
Results
Characterization of Animal Tallow
Table 1 shows the composition of the
animal tallow used in this study. The filtration process removed about 7.5 % of
the total weight of tallow as impurities present in the animal fats. The
homogenized oil was characterized by gas chromatography to identify and
quantify the fatty acid composition of the tallow. Five fatty acids were
identified in the animal tallow: oleic acid (44%), palmitic acids (28%),
stearic acid (26%), linoleic acid (1%), and myristic acid (1%).
Enzymatic Transesterification
Enzymatic transesterification using the Candida antarctica Novozyme 435 was carried out to investigate the
effects of reaction time (4, 8, 12 and 16h), oil : alcohol molar ratios (1:1,
1:2, 1:3, 1:4 and 1:5) and reaction temperature (35, 40, 45 and 50°C) on
biodiesel yield in a solvent system (hexane). The results are shown in Table 2.
Table 3 shows the Analysis of Variance performed on the oil yield data.
The effect of oil:alcohol molar ratio, reaction time and reaction temperature
were highly significant at the 0.001 level. All levels of interactions between the parameters were also highly significant at the 0.001
level.
The results obtained from Tukey's Grouping (Table 4) indicated
that the five levels of oil : alcohol molar ratio (1:1, 1:2, 1:3, 1:4 and 1:5)
were significantly different from one another at the 0.05 level. The highest
mean yield of 84.29% was obtained with the 1:4 oil:alcohol molar ratio. The
four reaction times (4, 8, 12 and 16 h) were significantly different from one
another at the 0.05 level. The highest mean yield of 83.54% was achieved with
the 16h reaction time. The reaction temperatures 40 and 50°C were not
significantly different from each other but were significantly different from
the reaction temperature 45°C at the 0.05 level. The highest mean yield of
82.39% was obtained at the reaction temperature 45°C.
Effect of Oil:Alcohol Molar Ratio
The biodiesel conversion yield at the 4
h increased from 42.87 to 78.07% (82.13%), from 58.9 to 84.35% (43.20%) and
from 55.20 to 73.24% (31.49%) when the oil : alcohol molar ratio was increased
from 1:1 to 1:4 for the reaction temperatures of 40, 45 and 50°C, respectively.
A further increase in the oil : alcohol molar ratio from 1:4 to 1:5 decreased
the biodiesel conversion yield from 78.07 to 51.31% (34.27%), from 84.35 to
69.2% (17.96%) and from 73.24 to 46.73% (36.19%) for the reaction temperatures
of 40, 45 and 50°C, respectively. Similar trends were observed with the 8, 12
and 16 h reaction times at all reaction temperatures (40, 45 and 50°C).
Effect of Reaction Time
Figure 4 shows the effect of reaction
time on the biodiesel conversion yield using the Candida antarctica Novozyme 435 at different reaction temperatures
and oil: alcohol molar ratios. Generally, there was an initial rapid increase
in the biodiesel conversion yield with increases in reaction time during the
first 4 hours which was then followed by a gradual increase till the end of the
experiment (16 h) for all reaction temperatures (40, 45 and 50°C) and oil :
alcohol molar ratios (1:1, 1:2, 1:3, 1:4 and 1:5).
The biodiesel conversion yield at the
40°C reached 42.87%, 46.51%, 58.3%, 78.07% and 51.31% for the oil:alcohol molar
ratios of 1:1, 1:2, 1:3, 1:4 and 1:5, respectively. Further increases in the
reaction time from 4 h to 16 h, increased the biodiesel conversion yield from
42.87 to 66.82% (55.86%), from 46.51 to 79.97% (71.94%), from 58.39 to 88.23%
(51.10%), from 78.07 to 88.26% (13.05%) and from 51.31 to 75.10% (46.36%) for
the oil : alcohol molar ratios of 1:1, 1:2, 1:3, 1:4 and 1:5, respectively.
Similar trends were observed at the 45 and 50°C reaction temperature and with
the oil : alcohol molar ratios (1:1, 1:2, 1:3, 1:4 and 1:5).
Effect of Reaction Temperature
The effect of the number of enzyme cycles on the biodiesel conversion yield using Candida antarctica Novozyme 435 with 2-butanol and hexane at the optimum conditions (45°C reaction temperature, 1:3 oil : alcohol molar ratio and 8 h reaction time) is shown in Figure 6. There was a gradual decrease in conversion yield by the enzyme catalyst Candida antarctica Novozyme 435 with increases in the number of cycles beyond 10 cycles. When the number of cycles was increased to 10, the biodiesel conversion yield slightly decreased from 86.12 to 85.6% (0.6%). A further increase in the number of cycles from 10 to 50 decreased the biodiesel conversion yield gradually from 85.6 to 15.2% (82.24%).
DISCUSSION
Extraction Profiles of the Raw Material
After melting and homogenizing the animal tallow, the impurities (7.5%) were removed by filtration. The fatty acids analysis indicated that the homogenized oil contained high percentages of oleic acid (44%), palmitic acid (28%) and stearic acid (26%) as well as lower percentages of myristic acid (1%) and linoleic acid (1%). A high concentration of oleic acid improves thecharacteristics of biodiesel resulting in high cetane index and combustion temperature [9]. Biodiesel produced from feed stocks containing a high level of oleic acid showed similar characteristics to these of conventional diesel [9,11].Therefore, the biodiesel produced for oil extracted from animal tallow is expected to have good characteristics as a biofuel.The extracted oil can be transformed to biodiesel by chemical or enzymatic transesterification. Watanabe et al. [20], Dorado et al. [21] and Kulkarni and Dalai [22] reported that oxidized oil can inhibit the chemical transesterification process and increase the oxidation of methyl esters. Kulkarni and Dalai [22] stated that an increase in the oxidation of methyl esters might increase the cetane number which tends to delay the ignition time in the engine. Nelson et al. [23] and Watanabe et al. [20] reported that oxidation in crude tallow oroil containing high free fatty acids is a common problem and no negative effects of the oxidized oil substrate on the enzymatic transesterification process was observed. Watanabe et al. [20] stated that in the enzymatic transesterification process, the oxidized substrate becomes a non-recognition site for the enzyme to bind and the process continues with the substrates which are not oxidized. However, the authors stated that using oxidized oil might reduce the biodiesel stability. Nelson et al. [23] reported that the stability of biodiesel can be increased by blending the biodiesel with conventional diesel especially in a cold environment.
In this study, enzymatic
transesterification was carried out and no oxidation stability test was
performed on crude tallow or oil nor were antioxidants used. A high biodiesel
conversion yield of 95.75% was achieved using the Candida antarctica Novozyme 435 at 25% enzyme concentration. Nelson et al. [23] reported biodiesel
conversion yield of 94.5% using immobilized Mucormiehei
with tallow, ethanol at 25% of enzyme concentration. Kumariet al. [24]
reported biodiesel conversion yield of 96% using immobilized Pseudomonas cepaciawith mahua oil,
ethanol at 25% of enzyme concentration. Kumar et al. [3] reported
biodiesel conversion yield of 95.75%) using the experimental enzyme NS88001with
beef tallow oil, methanol at 25% of enzyme concentration.
Effect of Oil: Alcohol Molar Ratios
Increasing the oil:alcohol molar ratio from 1:1 to 1:4 at the 4 h reaction time increased the biodiesel conversion yield by 82.13, 43.20 and 31.49% and a further increase in the oil : alcohol molar from 1:4 to 1:5 at the 4 h reaction time decreased the biodiesel conversion yield by 34.27, 17.96 and 36.19% at the reaction temperatures of 40, 45 and 50°C, respectively.
Kumariet al. [25] noted that the biodiesel conversion yield increased when the oil:alcohol molar ratio was increased up to 1:4 and then decreased when the oil : alcohol molar ratio was further increased to 1:5. Chen et al. [26] reported that increasing the oil : alcohol molar ratio from 1:1 to 1:4 promoted the methanolysis reaction with waste cooking oil, but the formation of methyl esters decreased when the oil : alcohol molar ratios was increased from 1:4 to 1:5 due to the excess of methanol in the system.
The decrease in conversion yield of methyl esters from the oil substrate at higher oil : alcohol molar ratios might be due to the presence of insoluble methanol in the reaction system. Tamalampudi et al. [27] suggested that excess alcohol would cause the active site on the surface of the lipase to be blocked resulting in the surface of the oil substrate being less accessible to the enzyme. Dizge and Keskinler [28] also reported that the use of excessive amount of alcohol might deactivate the lipase in the reaction. Chen et al. [26] suggested that the excess alcohol might distort the essential water layer needed to stabilize the structure of the enzyme.
In this study, increasing the oil:alcohol molar ratio from 1:4 to 1:5 deactivated the lipase catalyst and resulted in low conversion yield. It is likely that once the maximum level of esters was formed, a further increase in number of moles of alcohol decreased the formation of methyl esters in the reaction due to enzyme inactivation [23,26-30].
Several authors [31-34] reported
that deactivation of enzyme occurred by the insoluble alcohol present in the
reaction due to its tendency to be absorbed by the surface support matrix.
However, theoretical 1:3 stoichiometric oil : alcohol molar ratio is needed to
complete the reaction in the following continuous steps (a) the conversion of
triglycerides to diglycerides, (b) the conversion of diglycerides to
monoglycerides and (c) the conversion of monoglycerides to methyl esters and
glycerol [12,35-36].
In this study, the optimum
conversion yield was achieved at the 1:4 oil:alcohol molar ratio in a solvent
system using Candida antarcticaNovozyme
435. Increases in the oil : alcohol molar ratio from 1:3 to 1:4 at 4 h
increased the conversion yield of biodiesel for Candida antarctica Novozyme 435 by 82.13, 43.20 and 31.49% at the
reaction temperatures of 40, 45 and 50°C, respectively. The lipase catalyst Candida antarctica Novozyme 435 used in
this study, showed different activity due to the mass transferconditions, use
of alcohol and solvent system. Based on the stoichiometric reaction, using an
amount of alcohol equal to the number of fatty acids residues was sufficient to
complete the conversion reactions.
Effect of Reaction Time
At the initial phase of the reaction, the enzyme, oil and alcohol appeared to be static and the reaction started when the stirring speed reached 200 rpm which promoted the initial mixing and increased the mass transfer between substrate and enzyme catalyst. Formation of esters increased with increases in reaction time from 1 to 4h. When the reaction time was increased from 4 to 16 h at the 40°C reaction temperature, the biodiesel conversion yield was increased by 55.86, 71.94, 51.10, 13.05 and 46.36% for the oil:alcohol molar ratios of 1:1, 1:2, 1:3, 1:4 and 1:5, respectively. The maximum conversion yield of 95.75 % was obtained at the 16 h. Nelson et al. [23], Chen et al.([26] and Modi et al. [37] observed similar trends for crude tallow, waste cooking oil and vegetable oil, respectively.
Freedman et al. [35], Ma et al. [38], Leung and Guo, [39], Meter et al. [40] and Eeveraet al. [41] reported that the rate of conversion of fatty acid esters increased with increases in reaction time and the reaction proceeded rapidly with initial mixing which caused dispersion of alcohol into the oil substrate and the activation of enzyme. Chen et al. [26] reported that after alcohol was dispersed, it rapidly interacted with fatty acids giving a maximum conversion yield. However, a further increase in the reaction time decreased the conversion yield due to the backward reaction of transesterification.
Nelson et al. [23] reported a maximum biodiesel conversion yield of 83.8 % at a 16 h reaction time with 1:3 oil:alcohol molar ratio using 25% concentration of the enzyme Candida antarctica(SP 435) with hexane and 2-butanol alcohol in the system. Chen [26] achieved a maximum biodiesel conversion yield of 85.12% after 30 h with 1:4 oil:alcohol molar ratio using 30% concentration of the immobilized enzyme Rhizopusoryzae and waste cooking oil as substrate. Modi et al. [37] reported a maximum biodiesel conversion yield of 93.4% after 8 h with 1:4 oil:alcohol molar ratio using the enzyme Candida antarctica Novozyme 435 with vegetable oil. The biodiesel conversion yield obtained in this study was slightly higher than those reported in the literature and were achieved in a shorter time.
Effect of Reaction Temperature
When the reaction temperature was increased from 40 to 45°C with 4 h reaction time, the biodiesel conversion yield increased by 37.39, 58.78, 33.39, 8.04 and 34.86 % and a further increase in the reaction temperature from 45 to 50°C decreased the biodiesel conversion yield by 5.43, 14.55, 11.05, 13.17 and 32.47% for the 1:1, 1:2, 1:3, 1:4 and 1:5 oil:alcohol molar ratios, respectively. Chen et al [26], Dizge and Keskinler. [28], Rodrigues et al. [42] and Nieet al. [43] observed similar trends from waste cooking oil, canola oil, vegetable oil and salad oil, respectively.
Chen et al [26] reported that the biodiesel conversion yield increased (reaching a maximum of 87%) when the reaction temperature was increased from 30 to 40°C and then decreased when the reaction temperature was further increased from 40 to 70°C during conversion of waste cooking oil to methyl esters using Lipozyme RM IM. Dizge and Keskinler [28] reported that the biodiesel conversion yield increased (reaching a maximum of 85.8%) when the reaction temperature was increased from 30 to 40°C and then decreased when the reaction temperature was further increased from 40 to 70°C while converting canola oil to methyl esters using Lipozyme TL. Rodrigues et al. [42] reported a maximum biodiesel conversion yield of 53% at 35°C which then decreased with increases in reaction temperature above 35°C during the conversion of soybean oil to methyl esters using Novozyme 435. Nieet al. [43] reported a maximum biodiesel conversion yield of 90% at 40°C which then decreased when increasing the reaction temperature above 40°C. In this study, the highest conversion yield were obtained at 45°C which was higher than those reported in the literature.
Increasing the reaction temperature reduces the viscosity of the oil and enhances the mass transfer between the substrate and enzyme catalyst which results in higher conversion yield of biodiesel [3]. Kumari et al. ([24] and Antczaket al. [14] reported that the interactions between enzyme and substrate appears to be dependent on reaction temperature due to hydrogen bonding and ionic interactions which play an important role in maintaining the thermostability of lipase in the system. However, very high temperature may denature the specific structure of enzymes which results in a decrease in methyl esters formation. Denaturation of enzyme the support matrix may also promote the enzyme to leak from the outer layer of the support matrix [43]. However, the optimum reaction temperature is dependent on other parameters such as oil:alcohol molar ratio, enzyme activity, stability and type of system used.
Use of Solvent
In this study, a maximum biodiesel conversion yield of 95.75% was obtained by the Candida antarctica Novozyme 435 in the solvent system using hexane at 45°C and 1:4 oil : alcohol molar ratio. Mittelbach [44], Soumanou and Bornscheuer [45], Kumariet al. [24] and Kumar et al. [3] reported similar biodiesel conversion yields with solvent systems.
Xu et al. [46] and Shimada et al. [47] reported that the decreases in biodiesel conversion yield in solvent-free system was due to the inactivation of lipase in the presence of insoluble methanol in reaction. Fjerbaeket al. ([48] and Antczaket al. [14] reported that the use of organic non polar solvents for the transesterification process help to reduce the viscosity of the oil substrate, increase the mass transfer between the enzyme and the substrate and also helps to stabilize the enzyme in the reaction and reduce the toxicity of alcohol. Nieet al. [43] reported that the organic non-polar solvents with a log P value greater than 2 are considered to be suitable in the transesterification reaction due to their hydrophobic property so that water cannot be stripped from the enzyme and the spatial conformation of the active site of the enzyme is maintained. The authors suggested that n-hexane (log P = 3.5) can preserve the catalytic reaction, thus increasing the biodiesel conversion yield. Lu et al. [49] suggested that the n-hexane increased the biodiesel conversion yield with less water residue in the reaction and the non-polar solvent which promotes the usage of short chained alcohols like methanol (a polar alcohol).
Enzyme Reusability
In this study, the activity of Candida antarctica Novozyme 435 in the presence of 2-butanol and hexane at the optimum conditions (a reaction temperature of 45°C, an oil: alcohol molar ratio of 1:4 and a reaction time of 16 h) remained relatively constant for 10 cycles and then decreased gradually reaching 11% after 50 cycles. Ghamguiet al. [50], Xu et al. [51], Bernardeset al. [29] and Kumar et al. [3] obtained similar results from immobilized LipozymeThermomyceslanuginosus, immobilized Lipozyme Rhizomucormiehei, immobilized Rhizopusoryzae and experimental enzyme NS88001, respectively.
Several researchers [45,50,52] stated that repeated use of enzyme in the reaction without removing glycerol from the system might inhibit the interaction between the substrate and lipase. Xu et al. [46] reported that while using methyl acetate as acyl acceptor, no glycerol was produced in the reaction with no loss of enzyme activity for 10 cycles in the reaction. The byproduct from the reaction was triacetylglycerol instead of glycerol which did not affect the product quality.
Conclusions
The effectiveness of enzymatic transesterification of animal tallow using the Candida antarctica Novozyme 435 was studied. The effects of oil: alcohol molar ratio (1:1, 1:2, 1:3, 1:4 and 1:5), reaction temperature (35, 40, 45 and 50°C) and reaction time (4, 8, 12 and 16 h) on the biodiesel conversion yield were evaluated using n-hexane as a solvent. The highest conversion yield of biodiesel was obtained at the 1:4 oil:alcohol molar ratio, 16 h reaction time and 45°C reaction temperature. Increasing the oil : alcohol molar ratio from 1:1 to 1:4 increased the conversion yield of biodiesel by 21.24%. The rate of conversion of fatty acid esters increased with increases in reaction time. The reaction proceeds slowly at the beginning and then rapidly due to the initial mixing and dispersion of alcohol into the oil substrate and activation of enzyme. Increasing the reaction time from 4 to 16 h increased the conversion yield of biodiesel by 13.51%. The interactions between enzyme and substrate appears to be dependent on reaction temperature due to hydrogen bonding and ionic interactions which play important roles in maintaining the thermostability of lipase in the system. The higher temperature denatured the specific structure of enzymes and resulted in a decrease in methyl esters formation. Increasing the reaction temperature from 40 to 45°C increased the biodiesel conversion yield by 8.48%. Using n-hexane in the reaction helped to stabilize the enzyme and minimize the toxicity of alcohol. The activity of experimental enzyme catalyst in the presence n-hexane was slightly reduced after 10 cycles, it then decreased rapidly and stopped after 50 cycles.
acknowledgement
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