Bioresource Technology 129 (2013) 315320

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Optimization of biodiesel production from animal fat residue in wastewater

using response surface methodology
Sary Awad , Maria Paraschiv, Edwin Geo Varuvel, Mohand Tazerout
cole des Mines de Nantes, Dpartement Systmes Energtiques et Environnement, (DSEE), GEPEA, CNRS-UMR 6144, 4 rue Alfred Kastler, BP20722, 44307 Nantes Cedex 03, France

h i g h l i g h t s
" Biodiesel was obtained from waste water animal fat residue.
" Two-step acid-catalyzed transesterication process was employed.
" Operating parameters were optimized by response surface methodology.

a r t i c l e

i n f o

Article history:
Received 12 May 2012
Received in revised form 7 November 2012
Accepted 19 November 2012
Available online 28 November 2012
Keywords:
Animal fat residue
Waste valorization
Biodiesel
Acid catalyzed transesterication

a b s t r a c t
Animal fat residues (AFR) from waste water were used as feedstock to produce biodiesel by a two-step
acid-catalyzed process. Treatment of the AFRs with 5.4% (w/w) of 17 M H2SO4 at a methanol/AFR ratio
of 13:1 (50% w/w) at 60 C converted more than 95% of the triglycerides into fatty acid methyl esters
(FAMEs) with an acid value (AV) of 1.3 mgKOH/gbiodiesel. Response surface methodology indicated that a
lower AV cannot be reached using a one-step acid catalyzed process. Thus a two-step acid catalyzed
process was employed using 3.6% catalyst and 30% methanol for 5 h for the rst step and 1.8%
catalyst and 10% methanol for 1 h in the second step, resulting in a yield higher than 98% and an AV of
0.3 mgKOH/gbiodiesel. The product thus conforms to the European norm EN14214 concerning biodiesel.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Biodiesel is a promising biofuel because its physical and chemical properties are similar to those of diesel fuel (Demirbas, 2008;
Balat and Balat, 2008) and because its oxygen content enhances
combustion and reduces hydrocarbon and particulate matter emissions (Demirbas, 2005; Rahimi et al., 2009; Basha et al., 2009).
Most of the produced biodiesel in the world is from vegetable
oils, namely rapeseed oil in Europe and Canada and soybean oil
in USA (Karmakar et al., 2010). Since biodiesel production from
these sources can lead to competition with food production, other
feedstock for biodiesel need to be explored. Animal fat residues
(AFR) collected from fat traps are a cheap source of lipids that
can be used to produce biodiesel. Andersen and Weinbach (2010)
have estimated that in Norway 2.5 tons of AFR per 1000
households per year could be collected. Assuming that the
European Union has the same potential as Norway, with 501
million households, the EU-27 has a potential to collect

Corresponding author. Tel.: +33 (0) 2 51 85 82 88; fax: +33 (0) 2 51 85 82 99.
E-mail address: sary.awad@gmail.com (S. Awad).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.11.086

1.25 million tons of AFR that could be transformed into 1250 million liters of biodiesel. The problem with AFRs is their high content
of free fatty acids (FFA). Using an alkali catalyzed process, the FFA
will be transformed immediately into soap. An acid-catalyzed reaction will be 4000 times slower than an alkali-catalyzed process and
requires higher amounts of alcohol and catalyst and higher temperatures (Sharma and Singh, 2009; Lam et al., 2010; Agarwal,
2007). In order to solve this problem, an acid-catalyzed pre-esterication step is required to decrease the FFA level to 1% (West
et al., 2008; Tiwari et al., 2007; Dias et al., 2009). This process is
usually followed by an alkali-catalyzed process due to its reaction
speed. Optimization of these reactions requires a large number of
experiments and a mathematical tool that can predict the effect
of each parameter of the reaction and their interactions. Response
surface methodology has been successfully applied to the optimization of biodiesel production from different raw materials using
different types of catalysts (Yuan et al., 2008; Bautista et al.,
2009; Tiwari et al., 2007; Ghadge and Raheman, 2005).
In the present study, biodiesel production from AFR using a
two-step acid catalyzed reaction was studied. The rst step of
reaction was optimized using response surface methodology. The
produced biodiesel was analyzed and compared to European norm
EN14214.

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S. Awad et al. / Bioresource Technology 129 (2013) 315320

2. Methods
2.1. Raw materials
Animal fat residues (AFR) were collected and pre-treated in a
wastewater treatment centre, CTMV-CTMA, in Lussac near to Bordeaux, France. In this centre waste water was collected from an
area of 2800 km2 with 250,000 households and a number of food
and meat processing companies. The AFR were separated from
the wastewater as detailed in Supplementary information (Appendix A). Sulfuric acid (17 M) and methanol with a purity of 98% were
purchased from Sigma Aldrich, France. The duck fat was a co-product of the duck meat processing industry. Waste cooking oil (WCO)
was obtained from the Universitys restaurant and was composed
of 45% palm, 45% sunower, and 10% rapeseed oil. Sunower oil
was purchased from a supermarket. The three types of oils and fats
were used to compare their viscosities to that of AFR in order to
demonstrate the effect of degradation on the latter.
2.2. Experimental work
2.2.1. AFR analyses
Elemental composition analysis was performed using a CHNS-O
Analyzer FLASH 1112 series E. The heating value was measured
using an isoperibolic PARR 6200CLEF calorimeter. Acid values were
determined by titrimetry. Viscosities of AFR, vegetable oils and
duck fat were measured using a vibro viscometer with an error
margin of 3%. Samples were heated to 80 C and introduced into
the viscometer equipped with a thermometer. The viscosity was
measured at different temperatures while the liquid was cooling
down until its temperature reached 30 C. Density was measured
with a densimeter having a precision of 0.01 kg/l. Water content
was measured using the xylene vapor method (ASTM D 4006,
ISO 9029).
2.2.2. FAME production
The experimental setup was composed of a 2-l double-necked
ask immerged in an isothermal bath of water at 60 C. A mechanical stirrer was introduced vertically through a rubber plug that
sealed the asks neck. The second neck was also plugged with rubber stopper and was used to introduce reagents at the beginning of
the reaction and to take samples. Each experiment consisted of
mixing 200 g of AFR with a mixture of methanol (CH3OH) and sulfuric acid (H2SO4). The nal H2SO4 and methanol concentrations
were 1.85.4% and 3050% by wt., respectively. Samples were taken after 1 h and 3 h and the reaction was stopped after 5 h. Before
each sampling, the ask was removed from the water bath for
2 min, to minimize the amount of methanol vapors that could escape. The samples and the nal products were separated from glycerol and impurities by decantation using a separatory funnel for
2 h at 30 C, washed with distilled water, dried with calcium chloride and the acid values were measured. After the rst-step reaction, the product was washed with distilled water, the phases
were separated by centrifugation at a relative centrifugal force of
450g for 5 min and ltered through a layer of anhydrous calcium
chloride. The second step was accomplished by reacting the ltered products with methanol via an acid-catalyzed process to
eliminate the FFAs.
2.2.3. FAME characterization
Viscosity, density, water content, elemental composition, heating and acid value were determined as described in Section 2.2.1.
PENSKY MARTENS NPM440 device with a precision of 2 C was
used to determine the ash point of the biodiesel. A Perkin Elmer
Turbo Mass Gold mass spectrometer coupled to a Clarus 500 gas

chromatograph was used to analyze the composition and to

determine the amounts of fatty acid methyl esters (FAMEs),
mono-, di-, and triglycerides, free glycerol and methanol in the
samples. A SBLTM-5 ms capillary column of 30 m length and
0.25 mm diameter with a 0.25-lm lm thickness was used for
this analysis. A FID Perkin Elmer Autosampler XL chromatograph
and an FID Perkin Elmer Clarus 500 gas chromatograph were used
to analyze the reaction products. In both chromatographs a Varian WCOT fused silica column, 30 m in length, 0.39 mm in diameter and with a 0.25-lm lm thickness was used. Based on the
spectrometry analysis of different samples, the predominant components were the methyl esters of myristic, palmitoleic, palmitic,
stearic, oleic and linoleic acids. Thus the following standards were
purchased from Sigma Aldrich laboratories and used to analyze
the reaction products: AOCS N6 analytical standard (FAME
mixture), EN 14105: 2003 Monoglyceride stock solution and
EN14105:2003 Standard kit (diglycerides, triglycerides and free
glycerol mixture).

2.3. Statistical analysis

A three-level-three-factor design requiring 27 experimental
combinations was employed in this optimization study. Each
experiment was repeated three times and the mean acid value
was calculated. The coded and uncoded levels of the independent
variables are given in Table 1. The experimental data obtained by
the procedure described above were analyzed by a second order
polynomial regression described in Eq. (1).

y b0

3
3
2 X
3
X
X
X
bi xi
bii x2i
bij xi xj
i1

i1

i1 ji1

where y is the acid value of the rst step reaction product, xi and xj
are the independent coded variables, b0, bi, bii, bij are the intercept,
linear, quadratic and interaction coefcients, respectively. The
regression analysis and the Analysis of Variance (ANOVA) were performed and the effects of independent variables on the reaction
were evaluated using statistical tools like the Taylor test also called
t-statistic test.

3. Results and discussion

3.1. AFR characteristics
The AFR had a very high acid value of 60 mgKOH/gfat, and a
lower viscosity than usual animal fats and vegetable oils
(Fig. 1). The elemental analysis indicated that the fat did not
have apparent amounts of impurities and has almost same composition as non degraded fat (77% C, 13% H and 10% O). The
density and the higher heating value of AFR were 870 g/l and
38 MJ/kg, respectively. No water was detected in the tested
samples. The high acid value of the AFR required acid-catalyzed
pretreatment.

Table 1
Coded and uncoded levels of the design.
Variable

Reactions duration [h]

Catalyst amount [%]
Methanol amount [%]

Symbol

T
C
M

Coded levels
1

1
1.8
30

3
3.6
40

5
5.4
50

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S. Awad et al. / Bioresource Technology 129 (2013) 315320

Kinematic viscosity [mm/s]

90

Table 3
Student test of signicance of the regression parameters.

Sunflower oil

80

Duck fat
WCO

70

AFR

60
50
40
30
20
10
0
20

30

40

50

60

70

Variable

bj

SE

Signicant at 1% level?

Intercept
T
C
M
T2
C2
M2
TC
TM
MC

46.62
7.82
4.45
0.51
0.49
0.16
0.001
0.067
0.062
0.045

2.87
0.28
0.32
0.03
0.07
0.08
0.00
0.046
0.01
0.01

Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
Yes

80
R2 = 0.988 R2adj = 0.982 F-value = 142 t-Value = 2.92.

Temperature [C]
Fig. 1. Viscosity of AFR compared to conventional fats and oils.

3.2. Pretreatment step

The experimental mean acid values of the product at the design
points are listed in the Table 2. The coefcients of the regression
were determined using the least squares method. At coded levels,
the correlation between acid value of the products and the operational parameters is presented in Eq. (2).

AV 5:47  4:3T  2:2C  2:9M 1:9T 2 0:53C 2

 0:163M2 0:274TC 1:2TM 0:8MC

low levels of each of these parameters the reaction was more

sensitive to the change in other parameters than at high levels
(Fig. 2a and b). This effect can be also seen in Fig. 3, where the acid
value of the product is plotted versus the time of reaction for different amounts of methanol with a catalyst amount of 5.4%. The
acid value of the product decreased strongly with reaction time
and methanol quantities at the beginning, but approached an
asymptotic value that it could not exceed even for reaction times
longer than 5 h or methanol amounts higher than 50%. This nding
can also be explained by water accumulation during the acid esterication reaction which inhibits the conversion of FFA to FAME.
3.3. Biodiesel characteristics

The correlation for uncoded levels is described in Eq. (3)

AV 46:62  7:82T  4:45C  0:51M 0:49T 2 0:16C 2

 0:001M2 0:067TC 0:062TM 0:045MC:

The Fischer test with a very low probability P value indicates a

very high signicance of the model. The coefcient of determination R2 and the adjusted coefcient R2 adj are 0.988 and 0.982,
respectively.
For further accuracy the t-statistic test was used to determine
the signicance of each term of the regression and its signicance
level (Bradley, 2007). A regression coefcient is signicant if its
t-value is higher than the critical t-value. Table 3 illustrates the
t-statistic test results for the ANOVA regression. The results of
t-statistic test show that the coefcients of the regression were
signicant at a 1% level, except the quadratic terms of methanol
(M2) and catalyst (C2) and the interaction between time and
catalyst (TC).
The most signicant term in the regression was time, its negative value indicates that an increase in reaction duration led to a
reduction in acid value. The quadratic term of time was positive,
which means that the acid value reached a minimum after certain
duration; once it is exceeded the inverse reaction took place. The
effect of methanol amount was slightly higher than that of the
catalyst amount; the negative signs of both parameters mean that
increase in these entities promoted FFA esterication.
The interaction term of time and methanol and that of catalyst
and methanol were positive. This positive sign means that at the

The visual aspect of the product produced after 5 h was similar

to biodiesel at ambient temperature. The acid value of the product
was reduced drastically after the rst reaction. The viscosities of
the products obtained after 5 h and their dependence on temperature are presented in Fig 4. The values are very close and no correlation can be established between the viscosities of the products
and the operational parameters of the reaction. All the samples
were composed of 9193% FAMEs. Mass spectrometry revealed
the presence of methyl esters of low carbon chain carboxylic acids
and branched fatty acids, fatty acids ethyl esters (FAEE), some propyl esters and long chain alcohols which prove the advanced degradation level of the AFR. Glycerol and mono- di- and triglycerides
were detected in some samples at very low levels. Beside of these
components the presence of sulfur and chlorine imbedded in some
molecules were detected in few tested samples. Other physical and
chemical properties are listed in Table 4.The viscosity was slightly
higher than the maximum limit of EN14214 for some samples.
Although, based on the standards, the FAME content of biodiesel
must be higher than 96.5%, the obtained values of between 91%
and 93% might be sufcient as unidentied FAMEs and other alcohol fatty acid esters were present.
Since the ash point is very sensitive to the presence of volatile
compounds in the products, small compositional differences can
explain the difference between sample ash points.
From the composition of the samples of biofuel it can be concluded that the transesterication reaction was successfully
achieved after 5 h of reaction but that the esterication process

Table 2
Experimental acid values of the products at the design points.
Duration [h]

Catalyst [%]

1.8

3.6

5.4

3
1.8

3.6

5.4

5
1.8

3.6

5.4

Products acid value [mgKOH/gbiofuel]

Methanol [%]
30
40
50

18.5
15.7
9.1

15.3
11.9
7.5

13.7
10.3
5.5

12.1
8.7
4.6

8.4
4.9
2.8

5.0
3.3
1.7

8.2
5.1
2.6

5.0
2.6
1.4

2.5
1.6
1.3

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S. Awad et al. / Bioresource Technology 129 (2013) 315320

Fig. 4. Viscosities of product obtained after the rst acid-catalyzed transesterication carried out for 5 h.

Table 4
Properties of biodiesel compared with European norm (EN14214).
EN14214

Products after 5 h

Property

Units

min

max

min

max

Density at 15 C
Kinematic viscosity at 40 C
Flash point
Fatty Acid Methyl Esters
Water content
Sulfur content
Acid value
Methanol
Monoglyceride content
Diglyceride content
Triglyceride content
Free glycerine content
Total glycerine content

g/l
mm2/s
C
% m/m
mg/kg
mg/kg
mgKOH/g
% m/m
% m/m
% m/m
% m/m
% m/m
% m/m

860
3.50
101
96.50

900
5.00

500
10
0.50
0.20
0.8
0.2
0.2
0.02
0.25

870
4.7
128
>93


1.3







870
5.7
140


8.2







not detected.

Fig. 2. (a) Interactions between reaction time and methanol amount; (b) interactions between methanol and catalyst amounts.

16

5.4% 30%

3.4. Effect of fat degradation on the reaction

5.4% 40%

14

5.4% 50%

AV [mgKOH/gbiodiesel]

species of these types of fatty acids were found in samples produced using 1.8% of catalyst and 30% of methanol; this number
was reduced to two when using 5.4% of catalyst and 50% of methanol, namely: undecanoic acid, 2-methyl and 19,19-dimethyl eicossa-8, 11-dienoic acid.

12
10
8
6
4
2
0
0.5

1.5

2.5

3.5

4.5

Reaction time [h]

Fig. 3. Evolution of acid value of product obtained after the rst acid-catalyzed
transesterication at high levels of operating parameters.

A comparison between the results of the present work and

those obtained in other studies is presented in Table 5. The acidcatalyzed esterication of hydrolyzed soap stock used by Haas
(2005), resulted in the highest yield with respect to the amounts
of catalyst and alcohol used in the reaction.
The AFR required lower amounts of reagents, a lower temperature and shorter duration in order to reach yields equivalent to
those obtained with other raw materials. This outcome can be explained by the high degradation level of the fat that resulted in a
lower viscosity (Fig. 1). The degradation also caused the formation
of mono, di-glycerides and glycerol and converted some of the
long-chain fatty acids into shorter chains that could react easier
with methanol in the presence of acid.
3.5. Second step of reaction

was not completed. The esterication process is sensitive to the

formation of water during the reaction and also to the presence
of branched and long-chain carboxylic acids (Bankole, 2011). Seven

In order to reduce the acid value of the product obtained after

the rst acid-catalyzed transesterication to meet with European
norms, conversion of FFA into ester is required. Since the analysis

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S. Awad et al. / Bioresource Technology 129 (2013) 315320

Table 5
Comparison between different studies on the acid catalyzed transesterication of different raw materials.
Reference

Raw material

H2SO4

Methanol

Duration

Temp.(C)

Final FFA (%)

Yield (%)

Acid catalyzed pre-treatment

Tiwari et al. (2007)
Haas (2005)
Dias et al. (2009)

Jatropha 14% FFA

Soap stock 95% FFA
AF 7.3% FFA

2.6%
1.9%
2%

28%
6.7%
22%

88 min
14 h
5h

60
65
65

1
10
5

89
65

2.3%
4%
3.5%
8.720%

22%
74%
112%
180%

4h
10 h
69 h
6h

60
95

80 (P = 1.8 bar)

10
>90
99
97

1.8%
5.4%

30%
50%

5
5

60
60

4.1
0.65

>95
>95

Acid catalyzed Transesterication

Dos Santos et al. (2008)
Palm oil
Wang et al. (2006)
Cooking oil
Freedman et al. (1984)
Cooking oil
Zhang et al. (2003)
Cooking oil
Present work
AFR 30% FFA
AFR 30% FFA

Table 6
Optimization of the second step of reaction.
First step

Second step

Catalyst[%]

Methanol[%]

Time[h]

Catalyst[%]

Methanol[%]

Time[h]

AV[mgKOH/gbiodiesel]

1.8
1.8
1.8
1.8
3.6
3.6
3.6
3.6
5.4
5.4
5.4
5.4

30
30
30
30
30
30
30
30
30
30
30
30

5
5
5
5
5
5
5
5
5
5
5
5

0.9
1.8
1.8
0.9
0.9
1.8
1.8
0.9
0.9
1.8
1.8
0.9

5
5
10
10
5
5
10
10
5
5
10
10

1
1
1
1
1
1
1
1
1
1
1
1

3
1.8
1
2.5
1.7
0.9
0.3
1.2
1
0.7
0.2
0.2

of the regression in Section 3.3 showed a limitation caused by

water formation during reaction, water removal was necessary
prior to the second transesterication step.
This second step is usually accomplished as an alkali-catalyzed
process due to its speed of conversion; however, since the level of
esterication was already close to the required standard after the
rst step, the use of an acid catalyst was acceptable. Although
the reaction would be expected to be slow, saponication and mass
losses could be avoided. A reaction with 3.6% catalyst and 30%
methanol for 5 h for the rst step and a reaction involving 1.8% catalyst and10% methanol 1 h resulted in a nal product with acid value of 0.3 mgKOH/gbiodiesel (Table 6). The second acid-catalyzed
process was only used to transform FFAs to FAMEs, and since the
amount of FFA remaining after the rst step was less than 4%, its
conversion to FAME did not affect the physical or chemical properties of the nal product. Thus the physical and chemical properties
of the nal product were identical to those presented in Table 4.
The yield of the reaction was calculated from the chromatographic
analysis to be 98%.

4. Conclusions
Biodiesel was produced from animal fat residues collected from
fat traps. The reaction was conducted using a two-step acid catalyzed process. A response surface methodology approach showed
that the time of reaction was the most important parameter. The
effect of methanol was slightly more important than that of the
catalyst. The characteristics of the biodiesel obtained from rst
step conformed to European norms except its acid value, which
needed to be lowered in a second reaction. Chromatographic analysis did not reveal the presence of glycerides which proves the to-

tal transformation of fat into FAMEs. Degradation of lipids in AFRs

appears to have facilitated the transesterication reaction in comparison to that of animal fat and vegetable oils.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2012.
11.086.
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