JOHN WILEY & SONS, LTD.

, THE ATRIUM, SOUTHERN GATE, CHICHESTER P019 8SQ, UK

***PROOF OF YOUR ARTICLE ATTACHED, PLEASE READ CAREFULLY***
After receipt of your corrections your article will be published initially within the online version of the journal.
PLEASE AIM TO RETURN YOUR CORRECTIONS WITHIN 48 HOURS OF RECEIPT OF YOUR PROOF, THIS
WILL ENSURE THAT THERE ARE NO UNNECESSARY DELAYS IN THE PUBLICATION OF YOUR ARTICLE
READ PROOFS CAREFULLY
ONCE PUBLISHED ONLINE OR IN PRINT IT IS NOT POSSIBLE TO MAKE ANY FURTHER
CORRECTIONS TO YOUR ARTICLE
This will be your only chance to correct your proof
Please note that the volume and page numbers shown on the proofs are for position only
ANSWER ALL QUERIES ON PROOFS (Queries are attached as the last page of your proof.)
List all corrections and send back via e-mail to the production contact as detailed in the
covering e-mail, or mark all corrections directly on the proofs and send the scanned copy via
e-mail. Please do not send corrections by fax or post
CHECK FIGURES AND TABLES CAREFULLY
Check size, numbering, and orientation of figures
All images in the PDF are downsampled (reduced to lower resolution and file size) to facilitate
Internet delivery. These images will appear at higher resolution and sharpness in the printed
article
Review figure legends to ensure that they are complete
Check all tables. Review layout, title, and footnotes
COMPLETE COPYRIGHT TRANSFER AGREEMENT (CTA) if you have not already signed one
Please send a scanned signed copy with your proofs by e-mail. Your article cannot be
published unless we have received the signed CTA
OFFPRINTS
Free access to the final PDF offprint or your article will be available via Author Services only.
Please therefore sign up for Author Services if you would like to access your article PDF
offprint and enjoy the many other benefits the service offers.
Additional reprint and journal issue purchases
Should you wish to purchase additional copies of your article, please click on the link and follow
the instructions provided: http://offprint.cosprinters.com/cos/bw/
Corresponding authors are invited to inform their co-authors of the reprint options available.
Please note that regardless of the form in which they are acquired, reprints should not be
resold, nor further disseminated in electronic form, nor deployed in part or in whole in any
marketing, promotional or educational contexts without authorization from Wiley. Permissions
requests should be directed to mailto: permissionsuk@wiley.com
For information about Pay-Per-View and Article Select click on the following link:
http://olabout.wiley.com/WileyCDA/Section/id-404512.html
1
Research Article
Received: 16 January 2011 Revised: 12 April 2011 Accepted: 12 April 2011 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jctb.2652
Microalgae biodiesel via in situ methanolysis
R.M. Carvalho J unior,
a
J.V.C. Vargas,
a
L.P. Ramos,
b
C.E.B. Marino
a
TS1
and J.C.L. Torrens
c
Abstract
BACKGROUND: Microalgae have recently been considered a potential feedstock for biodiesel production, since they do not
compete with agricultural land, unlike oil crops. However, the production processes must be energetically and economically
viable. Therefore, an in situ methanolysis process is proposed for biodiesel production directly from microalgae biomass, to
avoid the need for the separation and extraction steps.
RESULTS: Biodiesel was obtained using methanol as the methylation reactant for the transesterication reaction and
hydrochloric acid as the catalyst precursor, at 80

C for 2 h of reaction. A mass return of 23.07 2.76% (m/m) was obtained.

Spectrometry in the infraredregion showedthat the product hadequivalent bands of axial deformationof C O, COandCH,
i.e. an ester. Tests showed the chromatographic prole of fatty acids in the sample. A process energetic efciency value of 1.17
was obtained for microalgae derived biodiesel, which is higher than fromsoybean and sunower, reportedly 1.06 and 1.12.
CONCLUSIONS: Industrial sustainability results fromlowenergetic, economic and environmental losses. The microalgae in situ
methanolysis process showed greater fuel available energy than energy consumption, therefore is energetically sustainable.
Economic and environmental issues should still be addressed.
c 2011 Society of Chemical Industry
Keywords: biodiesel; microalgae; in situ methanolysis
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
NOTATION
A
EI
peak area corresponding to methyl heptade-
canoate
C gas chromatography mass spectrometer peak
percentage, Equation (2)
C
EI
concentration, in milligrams per milliliter, of the
methyl heptadecanoate solution
CN cetane number of the biodiesel, Equation (5)
CN
ME
cetane number of individual methyl esters
CFPP cold lter plugging point, Equation (6)
DU degree of unsaturation, Equation (3)
E
biogas
energy content of biogas
E
oil
energy content of algal oil
E energy (kJ)
h
v
requiredspecicenthalpychangetovaporizewater
produced during fuel combustion (kJ kg
1
)
HHV higher heating value (kJ mol
1
), Equation (10)
I current (A)
IV Iodine value [g
Iodine
(100 g)
1
sample
], Equation (8)
LCSF long chain satured factor, Equation (4)
LHV lower heating value (kJ mol
1
), Equation (12)
m mass (g)
M molecular weight (g mol
1
)
n number of mols
N total number of compounds
P
oil
surface productivity of the oil (kg ha
1
year
1
)
P
biomass
surface productivity of the dead microalgae
(kg ha
1
year
1
)
P
biogas
surface productivity of the biogas (m
3
ha
1
year
1
)
q
h
sensible specic enthalpy change to heat water up
to the saturation point (kJ kg
1
)
q
v
water specic enthalpy of vaporization (kJ kg
1
)
SV saponication value (mg
KOH
g
1
sample
), Equation (9)
t reaction time (h)
U internal energy (kJ)
V voltage (V)
V
EI
volume, inmilliliters, of themethyl heptadecanoate
solution
X
ME
weight percentage of each methyl ester
Y
B
mass yield of monoesters (%)
Y
E
total annual energy yield (GJ ha
1
year
1
)
Y
n
net energy yield (GJ ha
1
year
1
)
Greek letters
t duration of process stage (s)

Correspondence to: J.V.C. Vargas, Setor de Tecnologia, Programa de P os

Graduac ao em Engenharia em Ci encia dos Materiais, Universidade Federal
do Paran a, CP 19011, Curitiba, PR, 81531-990, Brazil.
E-mail: jvargas@demec.ufpr.br
a Setor de Tecnologia, Programa de P os Graduac ao em Engenharia em Ci encia
dos Materiais, Universidade Federal do Paran a, CP 19011, Curitiba, PR, 81531-
990, Brazil
b Departamento de Qumica, Universidade Federal do Paran a, CP 19011,
Curitiba, PR, 81531-990, Brazil
c N ucleo de Pesquisa e Desenvolvimento de Energia Auto Sustent avel,
Universidade Federal do Paran a, CP 19011, Curitiba, PR, 81531-990, Brazil
J ChemTechnol Biotechnol 2011; 86: 0 www.soci.org c 2011 Society of Chemical Industry
2
www.soci.org RM Carvalho J unioret al.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
energetic efciency, Equation (7)

A total peak area from the methyl ester in C

14
to that
in C
24 : 1
Subscripts
B biodiesel monoesters obtained by the reaction in
the in situ methanolysis
DBB dry microalgae biomass to biodiesel
H
2
O water
s sample
T total consumption during the reaction steps
INTRODUCTION
According to the Intergovernmental Panel on Climate Change
(IPCC) data, the CO
2
concentration in the atmosphere has
increased from 280 ppm to 379 ppm.
1
To ease the buildup
of these gases, photosynthetic organisms can play a major
role in carbon dynamics. The photosynthesis performed by
chlorophyllous organisms converts solar energy into chemical
energy, with subsequent formation of organic matter (biomass)
and release of oxygen through the metabolism of water and
carbon dioxide removed from the medium
2
, as follows:
6CO
2(g)
+6H
2
O
(l)
+light energy C
6
H
12
O
6(l)
+6O
2(g)
(1)
It is recognized by the scientic community that forestry
projects are able to mitigate the greenhouse effect by xating
the carbon in plant biomass
3
as shown in Table 1, which is
followed by other benets such as rehabilitation of degraded
areas and biodiversity conservation. However, when tilling the soil
for planting, large amounts of CO
2
are releasedtothe atmosphere,
which might cancel the benecial effect of CO
2
absorption and
its incorporation into the biomass of plants throughout their life.
4
Thus, theinvestigationof other photosynthetic organisms withthe
capacity toabsorbCO
2
fromtheatmospheremay helptominimize
the effects of reforestation, with regard to carbon sequestration.
Microalgae are one of these organisms.
Microalgae are mainly responsible for the absorption of
atmospheric CO
2
in the oceans.
5
As Fig. 1 illustrates, microalgae
Table 1. Forest carbon sequestration
3
Species
Productivity
carbon (ton ha
1
year
1
)
CO
2
equivalent
(ton ha
1
year
1
)
Swietenia macrophylla King 5.60 20.55
Hymenaea courbaril L. 5.60 20.55
Anadenanthera colubrina 8.60 29.36
Cedrela ssilis Vell. 5.60 20.55
Pinus taeda 10.34 37.95
Havea sp. 5.17 18.97
Acacia mangium 10.55 38.72
Apuleia leiocarpa 12 44.04
Inga sp. 8.00 29.36
have the capacity to absorb up to 15 times more CO
2
than
the rainforests. A portion of the CO
2
absorbed by microalgae is
transferred to the ocean oor in a process known as a biological
pump.
6
Thus, microalgae cultivation for carbon sequestration
could help to reduce the buildup of greenhouse gases in the
atmosphere.
In addition to the potential biotechnological applications,
719
studies show that biofuels obtained from microalgae can be used
as an alternative to fossil fuels.
2024
The main advantages of
microalgae with respect to higher plants are greater biomass
production per area, possibility of using industrial waste in
cultivation, greater capacity, and easier CO
2
bioxation to
synthesize lipids. Thus microalgae with 30% lipids by dry weight
can produce 58 700 L ha
1
of oil in raceway ponds, and is
considereda potential source for biodiesel production, withbetter
productivity than conventional crops such as corn (172 L ha
1
),
soybeans (446 L ha
1
), canola (1,190 L ha
1
) and palm (5,950 L
ha
1
)
20
.
Microalgae biomass is considered one of the most promising
feedstocks for biodiesel, with the potential to meet the goal of
replacing diesel oil. Chisti
20
reported that the use of microalgae
for biodiesel production of 50%biodiesel for transportation in the
C
O
2

(
t
o
n

h
a
-
1

y
e
a
r
-
1
)
Species
Figure 1. Comparison of carbon sequestration by microalgae forests.
3
wileyonlinelibrary.com/jctb c 2011 Society of Chemical Industry J ChemTechnol Biotechnol 2011; 86: 0
3
Microalgae biodiesel via in situ methanolysis www.soci.org
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
USA would require the use of 2.5% of the agricultural territory of
the country, or about 10 times less than the land required for palm
oil production for the same use.
The production of biodiesel from microalgae oil has been
reported in the literature using the conventional route,
21,22
which involves the extraction of lipids from the microalgae
biomass, followed by its conversion into biodiesel and glycerol.
Thus experiments were conducted with three types of solvent
for extracting lipids from microalgae (l-butanol, ethanol, and
2-propanol) The most efcient solvent for extraction was l-
butanol, followed by 2-propanol and then ethanol. Regarding
the yield of methyl esters of fatty acids, these were obtained
by transesterication using 0.6 mol L
1
of hydrochloric acid
in methanol for 1 h at 70

C, reaching 68% conversion of

monoalkylated ester.
21
Aiming to increase biofuel production, an integrated method
was studied to produce biodiesel from microalgae oil using
Chlorella protothecoides, with the lipids extracted by n-hexane
as in a soxhlet extractor. The best yield of methyl esters derived
from fatty acid were obtained with 56 : 1 molar ratio (alcohol : oil),
temperature 30

C and reaction time 4 h.

22
Other authors have also achieved success with such heteroge-
neous production systems. Umdu et al.
23
assessed the effect of
MgOand CaOsupported on Al
2
O
3
as a catalyst for the transesteri-
cation of oil frommicroalgae, obtaining 97.5%conversion, using
a molar ratio of 30 : 1 (alcohol : oil) at 50

C and 4 h reaction, using

2% catalyst (relative to the mass of oil) CaO supported on Al
2
O
3
.
More recently an in situ reaction for biodiesel production from
microalgae was studied. According to Ehimen et al.
24
the best
results were found when 60 mL methanol was used with 15 g
of microalgae biomass, at a temperature of 60

C, reaching 92%
conversion with 1 h reaction time and constant stirring.
Several studies have demonstrated at laboratory scale the
possibility of producing biodiesel from microalgae. However, the
industrial engineering aspects, equipment and processes are yet
to be properly addressed. Scaling up of microalgae biodiesel
production results in thermodynamic losses that drastically affect
the plant energy balance. From the operational point of view,
economic or environmental losses are detrimental to the entire
production chain.
In this context, the present work focuses on the production
of biodiesel from microalgae by in situ methanolysis, in order
to evaluate the process energetic efciency at laboratory scale,
but with the objective of assessing the potential process
thermodynamic losses in future industrial scale installations.
MATERIALS ANDMETHODS
The experiments used the microalgae Nannochloropsis oculata,
which was cultivated by the Group of Integrated Aquaculture
and Environmental Studies at the Federal University of Paran a
(UFPR GIA). The microalgae were cultivated in an autotrophic
culture with a Guillard F/2 medium,
25
and natural photoperiod.
After growth was stopped, the microalgae were occulated and
dried using the spray drying method.
Production of fatty acid methyl esters (FAME)
The synthesis of microalgae fatty acid monoesters was initially
carried out with 2 g of microalgae biomass in a Soxhlet apparatus,
using 204 mL of a methanol : HCl : chloroform reactant solvent
in a 10 : 1:1 volumetric ratio. The reaction was carried out at
Monoesters
Reaction co-products
Microalgae
Figure 2. Products stratication after the centrifugation stage of in situ
C
o
l
o
r
F
i
g
u
r
e
-
O
n
l
i
n
e
o
n
l
y
methanolysis.
80

C for 2 h under constant magnetic stirring. Once the reaction

procedure was completed, the system was cooled down to room
temperature and 2 mL of water (HPLC grade) were added to
the reaction media, to retain polar substances and to help the
separation of phases ester-gliceride, along with 200 mL of a 4 : 1
(v/v) hexane : chloroform mixture in order to keep the esters
in organic phase. The addition of such solvents is because cell
breaking is expected before lipid extraction,
26
i.e. the contact with
nonpolar solvents could weaken the association between lipids
and cell structure. Although the use of these chemicals might
limit interest in in situ methanolysis, an investigation of possible
signicant gains in the overall energy balance of the procedure
compared with conventional methanolysis could compensate for
this.
After thorough mixing, the resulting material was centrifuged
at 14 000 rpm for 10 min and the organic phase transferred
to a round bottom ask. The resulting microalgae fatty esters
were recovered after evaporation of the extraction solvents.
Reaction co-products and the residual microalgal biomass were
discarded as shown in Fig. 2. In general, microalgae co-products
are composed of proteins, pigments (primarily chlorophyll) and
carbohydrates.
27
Such compounds were not characterized in the
residual microalgae biomass because this was not part of the
present research.
A parametric study of the reaction conditions was also carried
out using a 2
2
central composite design in which the two main
reaction variables were involved, time and methanol : HCl ratio.
The maximum and minimum levels (1; +1) for time were 2 and
4 h, andfor themethanol : HCl ratio, theselevels were2 : 1and20 : 1
(v/v), respectively. To check the experimental error and perform
the analysis of variance (ANOVA), a center point (3 h and a 6 : 1
methanol : HCl volumetric ratio) was carriedout inthree replicates.
The experimental data were treatedcomputationally toobtainthe
response surface and the primary and secondary effects of the
selected reaction variables, having the alkyl monoesters mass
yield (Y
B
) as the response factor.
Chemical characterization of the product
Fourier transforminfrared spectroscopy (FTIR)
FTIR analyses were performed with an FTIR spectrometer man-
ufactured by Bomem. The spectra were obtained in the range
J ChemTechnol Biotechnol 2011; 86: 0 c 2011 Society of Chemical Industry wileyonlinelibrary.com/jctb
4
www.soci.org RM Carvalho J unioret al.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
4000400 cm
1
with resolution of 4 cm
1
and 64 scans per
spectrum. For the analysis, approximately 20 mL of the sam-
ple, supported by chips of 100 mg KBr, were used. These analyses
dened the main structural characteristics of the samples.
Technique for gas chromatography/mass spectrometry (GC-MS)
A gas chromatograph Shimadzu model 17A GC with split/splitless
injector, equipped with a capillary column lled with Carbowax
PolyethyleneGlycol 30 m(length) 0.25 mm(internal diameter)
0.25 m(lmthickness), was employed. Theinjector operatedin
split mode with a rate of 50 mL min
1
, with block temperature of
250

C. Theovenwas programmedtowork intheisothermal mode

at 210

C. The temperature of the detector (mass spectrometric)

was 250

C and carrier gas He was maintained at a constant

ow of 1 mL min
1
. The identication process followed after the
separation by high resolution gas chromatography using a mass
spectrometer Shimadzu model GCMS-QP 5050, where the mass
spectrum was compared with data from the internal library of
the National Institute of Standards and Technology NIST, which
were available in the equipment data bank. This made possible
the assessment of the fatty prole of the sample obtained in
the synthesis. The quantication of the sample followed DIN EN
14 013, using the internal standard methyl heptadecanoate and
the following equation to identify the corresponding percentage
of each peak:
C =

A
EI
A
EI

C
EI
V
EI
m
s
10
3
100% (2)
Properties of the fuel monoesters obtained
Biodiesel fuel properties depend on the fuels fatty acid ester
prole, which is determined by the composition of the parent oil
and the techniques employed for extraction and production of
the oil. Thus, analyzing the composition of the esters obtained
in the in situ transesterication it is possible to assess some of
the properties needed in order to characterize it as biodiesel
(accordingtocurrent norms). Recent studies showthat theamount
of saturated, monounsaturated and polyunsaturated esters that a
biodiesel presents may be used in order to estimate its properties.
In the search for a simple methodology to predict the cetane
number (CN) and cold lter plugging point (CFPP) of different
biodiesel fuels, Ramos et al.
28
used two empirical parameters.
The rst, the degree of unsaturation (DU), takes into account
the amount of monounsaturated and polyunsaturated fatty acid
esters, while the second, the long chain saturated factor (LCSF),
takes into account the composition of saturated fatty acid esters,
putting more weight on the long chain esters. Both parameters,
DU and LCSF, are dened as follows:
DU = (monounsaturated, wt %) +2(polyunsaturated, wt%)
(3)
LCSF = 0.1 C16(wt%) +0.5 C18(wt%) +C20(wt%)
+1.5 C22(wt%) +2 C24(wt%) (4)
The cetane number (CN) is a widely used parameter for diesel
and biodiesel quality, once it is related to the ignition delay,
combustion quality and pollutant emissions.
29
The experimental
procedure required to determine the cetane number is expensive
and impractical, which motivated the development of various
prediction methods, e.g. Freedman and Bagby
30
correlated the
CN of methyl esters with different physical properties. The greater
potential of the prediction methods, however, is to correlate the
CN directly to the composition of the parent oil. For that, the
following correlation, formulated by Clements,
31
was used in this
work for the estimation of the biodiesel cetane number as a
weighed average of all methyl ester cetane numbers present in
the fuel, as follows:
CN =
N

i=1
(X
ME
CN
ME
)
i
(5)
where N is the total number of compounds found in the biodiesel.
Based on the linear regression of experimentally obtained data
for CFPP (R
2
= 0.996), Ramos et al.
28
proposed the following
equation:
CFPP = 3.1417 LCSF 16.477 (6)
Calculation of energetic efciency
The thermodynamic (or energetic) efciency of the process is
dened as the ratio between the energetic content of the
generated product and the energy consumed in the biodiesel
production processes. Through the calculation of such a quantity,
it is possible to quantify the efciency of obtaining biodiesel from
dry microalgae biomass by in situ methanolysis, as follows:

DBB
=
n
B
LHV
E
T
(7)
The efciency calculation starts with estimation of the iodine
value, saponication number, higher and lower heating values.
Based on the fatty ester prole obtained, it is possible to estimate
the iodine value, according to the AOCS method Cd 1c-85
32
as
follows:
IV = (%C16 : 1 0.9976) +(%C18 : 1 0.8986)
+(%C18 : 2 1.810) +(%C22 : 1 0.7497) (8)
Using data from the average molecular weight obtained with
the gas cromatograph coupled with mass spectrometer (GCMS)
analysis it is possible to calculate the saponication number by
the method AOCS Cd 3a-94,
33
as follows:
SV =
3 56.1 1000
[(M
s
3) +92.09] (3 18)
(9)
Next, the higher (HHV)
34
and lower heating (LHV) values are
determined as follows:
HHV =

618 000
SV
0.08 IV 430

4.18 (10)
In order to determine the LHV it is necessary to take into
account the mass of water produced in the combustion reaction.
Bydenition, LHV is theHHVminus therequiredenergytovaporize
the mass of water produced, which is determined through the
combustion chemical equation.
Therefore, the required energy to vaporize the mass of water
is the sum of the required sensible specic enthalpy change to
heat the water produced to its saturation temperature at the
wileyonlinelibrary.com/jctb c 2011 Society of Chemical Industry J ChemTechnol Biotechnol 2011; 86: 0
5
Microalgae biodiesel via in situ methanolysis www.soci.org
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
pressure at which the reaction occurs, and the specic enthalpy of
vaporization at such conditions, which is calculated as follows:
h
v
= q
h
+q
v
(11)
LHV = HHV h
v
m
H
2
O
10
3
(12)
where q
h
is the required sensible specic enthalpy change to
increase water temperature up to the saturation point and q
v
the
water specic enthalpy of vaporization at the reaction conditions.
The global energetic consumption is the sum of all the process
energy requirements, i.e. electrical energy and solvent internal
energies, and is given by
E
T
=
3

i=1
E
i
+
3

i=1
U
i
(13)
where
3

i=1
E
i
is the sum of the electrical energy consumed, and
3

i=1
U
i
the sum of the internal energies of the solvents consumed
in the three process stages (i = 1 in situ methanolysis; 2 rotary
evaporation, and 3 centrifugation).
Since the process of obtaining biodiesel monoesters was
conducted in a closed system, i.e. recovering the solvents used
using negligible energy compared with E
1
, E
2
, and E
3
, the total
consumed energy consumed in the process is given by
E
T

=
3

i=1
E
i
(14)
The electrical energy consumed in each process stage is
calculated by
E
i
= V
i
I
i
t
i
10
3
(15)
where V
i
, I
i
and t
i
are the voltage, current and duration of each
process stage (i = 1 in situ methanolysis; 2 rotary evaporation,
and 3 centrifugation).
RESULTS
Fourier transforminfrared spectroscopy (FTIR)
In order to characterize the material obtained, tests were
conducted using FTIR spectroscopy. The results were consistent
with the chemical properties of esters since analysis of the FTIR
spectrum shown in Fig. 3 identied the characteristic bands of
the links that are part of the organizational structure of fatty
monoesters
35
through the transmittance (vertical axis), clearly
characterizing the axial strain of ester C O (1741 cm
1
) and CO
(11681243 cm
1
), in addition to axial and angular deformation
of CHin the regions of 2921, 1461, 1377 and 719 cm
1
, which are
the measured wavenumbers (horizontal axis). All samples showed
similar FTIR proles.
Chromatography
Becausequanticationof thechromatographictest wasperformed
with internal standardization, it was necessary to use a mass spec-
trometer toinvestigate the peaks relatedtothe samples, aimingto
nd their composition. After obtaining the chromatogram reveal-
ing the presence of 10 compounds, which are shown in Table 2,
each peak was compared with data fromthe NIST library, available
in the chromatograph data bank, which led to the identication
of nine methyl monoesters, and only one substance was not iden-
tied. Based on these results, which conrmed the presence of
alkyl monoesters, and based on the fatty prole, it was possible to
estimate some parameters of interest, such as molecular weight,
iodine values, saponication numbers, higher and lower heating
value and energy efciency, and predict two important fuel prop-
erties, the cetane number and cold lter plugging point. Figure 4
4000 3500 3000 2500 2000 1500 1000 500
50
100
150
200
250
300
350
400
450
500
550
600
650
700
T
r
a
n
s
m
i
t
t
a
n
c
e

(
%
)
Wavenumber (cm
-1
)
Figure 3. Monoesters infrared spectrum.
J ChemTechnol Biotechnol 2011; 86: 0 c 2011 Society of Chemical Industry wileyonlinelibrary.com/jctb
6
www.soci.org RM Carvalho J unioret al.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
Table 2. Resulting fatty acid prole of the sample obtained from
in situ methanolysis
Fatty Acid
Molecular
weight
(g mol
1
)
Percentage
of each peak
1- Methyl laurate 200.30 0.58
2- Methyl Miristate 228.37 6.76
3- Unidentied 0.24
4- Methyl palmitate 256.42 28.18
5- Methyl palmitoleate 254.41 22.61
6- Methyl stearate 284.48 1.03
7- Methyl oleate 282.46 6.19
8- Methyl linoleate 280.45 2.71
9- Methyl Behenate 340.58 4.05
10- Methyl Erucicolate 338.57 27.65
Average molecular weight (M
B
) 274.00 g mol
1
100.00
F
a
t
t
y

a
c
i
d

p
r
o
f
i
l
e

(
%
)
(obtained in this work)
Figure 4. Fatty acid composition of some algae and Nannochloropsis
C
o
l
o
r
F
i
g
u
r
e
-
O
n
l
i
n
e
o
n
l
y
oculata

.
shows a comparison of the fatty acids composition of N. oculata
obtained in this work with literature data
36
for other species.
Mass yield of the biodiesel (Y
B
) synthesis
Inorder toquantify the mass yieldof biodiesel (Y
B
), 11 experiments
were performed, resulting in an average yield of 23.07 2.76%
(m/m), with 0.4614 g monoesters obtained from 2 g of microalgal
biomass. Such product in situ transesterication values were
considered satisfactory since the reaction was processed in a
single step without the need for extraction and purication of
the oil.
Thein situmethanolysis of microalgaebiomass was investigated
througha2
2
central compositedesigninwhichtheresponsefactor
was the alkyl monoesters mass yield (Y
B
) and the selected reaction
variables were the reactiontime (h) andthe MeOH: HCl volumetric
ratio. Thestatistical signicanceof theeffects of eachexperimental
variable and of the possible interactions among them was also
evaluated as shown in Fig. 5. According to the Pareto chart, the
variableMeOH: HCl hadapositiveandsignicant effect onthealkyl
monoesters mass yield(Y
B
), whichwas estimatedfromtheStudent-
t distribution. This means that an increase of the ratio MeOH: HCl
could lead to higher monoesters mass yield. The reaction time
and the interaction between reaction time and ratio MeOH: HCl
were not signicant at the 95% condence level of the Student-t
Figure 5. Pareto chart of effects of reaction parameters on the biodiesel
C
o
l
o
r
F
i
g
u
r
e
-
O
n
l
i
n
e
o
n
l
y
mass yield of in situ methanolysis.
Y
B

(
%
)
MeOH:HCl
t (h)
< 30%
< 20%
< 10%
Figure 6. Effect of the ratio MeOH: HCl and reaction time on the resulting
C
o
l
o
r
F
i
g
u
r
e
-
O
n
l
i
n
e
o
n
l
y
biodiesel mass yield (Y
B
) of in situ methanolysis.
distribution. Therefore, the analysis leads to the conclusion that
the most important variable in the process is the ratio MeOH: HCl.
The software Statistica 8.0
37
was applied to produce these
results. ANOVA was performed with the experimental data and
a quadratic model was built to generate the response surface
shown in Fig. 6. In this case, the ANOVA results demonstrated
that both the response surface and estimated effects were valid
because the quadratic model was able to predict 97.37% of the
experimental data. It should be noted that the response surface
of Fig. 6 was not able to show the optimal condition for the in situ
transesterication of microalgae biomass. However, the intention
was not to optimize the system but only to investigate the main
effects of the reaction variables on the response factor.
Figure 6 is a plot of experimental data showing the combined
effects of volume ratio MeOH: HCl and reaction time on the mass
yield of monoesters (Y
B
). Figure 6 shows a tted surface for the
wileyonlinelibrary.com/jctb c 2011 Society of Chemical Industry J ChemTechnol Biotechnol 2011; 86: 0
7
Microalgae biodiesel via in situ methanolysis www.soci.org
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
Y
B
= 30 %
20 %
10 %
M
e
O
H
:
H
C
l
t (h)
Figure 7. Contour isovalues of the resulting biodiesel mass yield (Y
B
) with
C
o
l
o
r
F
i
g
u
r
e
-
O
n
l
i
n
e
o
n
l
y
respect to the ratio MeOH: HCl and reaction time of in situ methanolysis.
experimentally obtained mass yield of monoesters. As the ratio
MeOH: HCl increases the mass yield of the process increases. It
is also observed that for all values of the ratio MeOH: HCl there
seems to be an optimal reaction time (between 3 and 4 h) so that
the mass yield of monoesters is maximized. This behavior is also
observed in Fig. 7, which shows the contour plot isovalues of the
mass yield of monoesters. Therefore, the results of Figs 6 and 7
demonstrate that the ratio MeOH: HCl has the most important
effect on the resulting mass yield of monoesters. This could
be expected since MeOH is one of the reactants in the in situ
methanolysis.
Properties of the fuel monoesters obtained
Using Equation (5), with the CN
ME
values reported in the
literature
3,30,3842
as showninTable 3, andonthe biodiesel methyl
esters composition, it is possible to estimate the cetane number
of the fuel obtained.
Giventhe highamount of saturatedesters foundinthe samples,
a high CN value was expected, estimated at 68.2. Therefore,
regarding the cetane number, the fuel complies with both ASTM
D 675108 and UNE EN-14 214 : 2003 norms, which state that
the diesel cetane number must be greater than 47 and 51,
respectively.
The high amount of saturated esters also affects the cold-ow
properties of a biodiesel, inducing poor performances at low
temperatures. The biodiesel obtained has a long chain saturated
factor of 9.4, calculated using Equation (4), which is much higher
than those obtained with most common feedstocks such as
soybean and palm(3.4 and 7.7
28
), i.e. larger amounts of long chain
saturated esters are present in the microalgae derived biodiesel.
The length of the esters chain is related to the fuels melting
point, and the saturated esters have melting points above room
temperature. Therefore, at low temperatures, such esters tend to
crystallize, and might interrupt the ow of fuel, causing engine
malfunction due to problems in the fuel distribution system, and
increased pollutant emissions.
Using Equation (6), it was possible to estimate the CFPP of the
microalgae biodiesel obtained by in situ transesterication in this
study at 13.7

C. Such an elevated result indicates that this fuel

would not perform properly at low temperatures, and would be
viable only in hot climates with elevated temperatures during
the entire year. Some techniques for reducing the CFPP exist,
such as winterizing, but its economic viability still remains to be
claried.
The microalgae fatty acid prole depends greatly on the
species and cultivation conditions. Thus, optimized cultivation
conditions could lower the CFPP, improve the cold ow fuel
performance, and allow the fuel to be used in low temperature
conditions.
Calculation of energetic efciency
First, the iodine value is calculated using Equation (8), so that
IV = 53.75 g
Iodine
(100 g)
1
sample
. Next, the saponication value is
calculated with Equation (9) resulting in SV = 195.68 mg
KOH
g
1
s
.
Next, the higher (HHV) and lower heating (LHV) values are
determined according to the previously described methodology.
The rst result is HHV = 11 385.99 kJ mol
1
. The combustion
chemical equation was assumed to be similar to the combustion
of methyl oleate which has a molecular weight of 280 g mol
1
,
since the monoesters obtained had an average molecular weight
of 274 g mol
1
calculated as shown in Table 2, which is given by:
C
19
H
36
O
2(l)
+27O
2(g)
19CO
2(g)
+18H
2
O
(l)
(16)
so that the mass of water produced per mol of fuel is
m
H
2
O
(g) = M
H
2
O
(g mol
1
) n
H
2
O
(mol) = 324 g (17)
Therefore, the required energy to vaporize the mass of water
is the sum of the sensible specic enthalpy change to heat the
water to its saturation temperature at atmospheric pressure (1
bar), i.e. 100

C, and the specic enthalpy of vaporization at such

conditions, which was calculated as follows:
h
v
= 4.18 (100 20) +2257.2 = 2591.6 kJ kg
1
(18)
LHV = HHV h
v
m
H
2
O
10
3
= 10 546.31 kJ mol
1
(19)
where the ambient temperature was considered to be 20

C, the
specic heat of water 4.18 kcal kg
1
, and the water enthalpy of
vaporization 2257.2 kJ kg
1
.
The global energetic consumption to process 2 g of dry
microalgae biomass by the in situ methanolysis was calculated
according to Equations (13) to (15). For that, V
i
, I
i
and t
i
, i.e. the
voltage, current and duration of each process stage (i = 1 in situ
methanolysis; 2 rotary evaporation, and 3 centrifugation) were
carefully measured in the laboratory experiments. The result was
E
T
= 12.96 kJ.
Table 3. Fatty acid prole of the sample from in situ methanolysis, and corresponding cetane numbers given in the literature
Me 12 : 0 Me 14 : 0 Me 16 : 0 Me 18 : 0 Me 22 : 0 Me 16 : 1 Me 18 : 1 Me 22 : 1 Me 18 : 2
61.4
36
66.2
39
74.5
39
86.9
39
100
28
513
40
58.9
39
76
3
38.2
40
J ChemTechnol Biotechnol 2011; 86: 0 c 2011 Society of Chemical Industry wileyonlinelibrary.com/jctb
8
www.soci.org RM Carvalho J unioret al.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
Finally, theenergeticefciencyof theprocess toobtainbiodiesel
through in situ methanolysis was then calculated as follows:

DBB
=
n
B
LHV
E
T
=
0.4614
274
10 546.31
12.96
= 1.37 (20)
Based on the values obtained, it was observed that the energy
output of the system, i.e. the energy released by the product is
greater than the energy consumed in the process (energy input).
Therefore, it is possible to conclude that the process as described
is sustainable in terms of the energy balance, especially when the
value obtained is compared with biodiesel derived from soybean
and sunower reported as 1.06 and 1.12, respectively,
43
or when
compared with ethanol, which was reported to be 1.1.
44
However,
Equation (20) does not consider the energy spent on spray drying
the algae biomass, which is included in the calculation in the
next section. Due to the lack of information in the technical
literature on microalgae derived biodiesel conventional methods,
in situmethanolysis usingmicroalgaewas comparedwithsoybean
and sunower derived biodiesel obtained through conventional
methanolysis.
Estimates for net energy yield considering the production
of biogas fromresidual microalgae biomass
The prospect of large-scale production of microalgae for appli-
cations in biofuels is motivated by the high productivity that
such microorganisms can reportedly achieve. In contrast, a large
amount of biomass is generated after the biodiesel production
process. From the perspective of large-scale cultivation, the pos-
sibility of obtaining energy from the residual biomass and the
fertilizers used for cultivation should be considered, thus leading
to a more realistic total energetic efciency.
Anaerobic digestion is a fundamental process that has the
potential to contribute to the solution of the problem of waste
fromthe microalgae biodiesel production process and, in this way,
to reduce energy costs.
45
The total energy balance of the microalgae derived biodiesel
process could be even more positive than ethanol when consider-
ing the co-generation of energy through biogas production from
the residual microalgae biomass, as reported by Chisti.
45
Thus,
applying Chistis methodology
45
to the values found in this study,
the total annual energy yield can be estimated from the potential
total energy generation from microalgae as follows:
Y
E
= E
oil
P
oil
+E
biogas
P
biogas
(21)
In order to estimate P
oil
, the photobioreactor yield is considered
equal to 0.048 kg m
2
day
1
,
20
and a mass efciency equal to 0.9
(90%). Additionally, X
oil
is dened as the fraction of oil contained
in the biomass, which in this case is assumed to be the percentage
of biodiesel produced in this work as reported previously in the
text, plus 10%of glycerin fromthe total contained in the structure
of triglycerides,
46,47
so that X
oil
= 0.33. Therefore P
oil
is calculated
as follows:
P
biomass
= 0.048 kg m
2
day
1
10
4
m
2
ha
1
365 day year
1
0.9 = 157 680 kg ha
1
year
1
(22)
P
oil
= X
oil
P
biomass
= 0.33 157 680
= 52 034.4 kg ha
1
year
1
(23)
Next, in order to estimate P
biogas
, which is the biogas yield per
area, it is assumed that the biogas yield is Y
biogas
= 0.5 m
3
kg
1
,
40
.
Thus,
P
biogas
= (1 X
oil
) P
biomass
Y
biogas
= (1 0.33) 157 680 kg ha
1
year
1
0.5 m
3
kg
1
= 52 822.8 kg ha
1
year
1
(24)
Applyingthecalculatedvalues toEquation (21), thetotal energy
produced per year results as follows:
Y
E
= E
oil
P
oil
+E
biogas
P
biogas
Y
E
= 37.9 10
3
GJ kg
1
52 034.4 kg ha
1
year
1
+25 10
3
GJ m
3
52 822.8 m
3
ha
1
year
1
= 3292.67 GJ ha
1
year
1
(25)
where the E
oil
and E
biogas
values are taken as reported by Chisti.
43
Using the calculated values, it is possible to estimate the net
energy yield (Y
n
) contained in the microalgae biomass as follows:
Y
n
= Y
E

1
1

DBB

= 3292.67

1
1
1.37

= 889.26 GJ ha
1
year
1
(26)
However, Equation (26) does not account for the energy
consumed in drying the microalgae. In the project that suported
the present work,
48
microalgae were dried by spray drying.
Therefore, considering that this work used 2 g of microalgae for
the in situ methanolysis, and assuming a 33%water content in the
ltered microalgae biomass, i.e. 1 g of water, 2.257 kJ (enthalpy
of vaporization of 1 g of water at 1 bar) to dry the microalgae
biomass. Therefore, incorporating that value in calculating the
energetic efciency, the result is

DBB
=
0.4614
274
10 546.31
12.96 +2.257
= 1.17 (27)
Recalculating the net energy yield as described previously with
Equation (26), the result is Y
n
= 478.42 GJ ha
1
year
1
. Based on
this result, the actual energy balance of the microalgae derived
biodiesel was 2.9 times higher than the energy balance of sugar
cane, considering ethanol and bagasse as energy products, which
was reported as equal to 163.9 GJ ha
1
year
1
.
45,49
CONCLUSION
An in situ methanolysis methodology for biodiesel production
frommicroalgae has been investigated experimentally and tested
using the species Nannochloropsis oculata. In the proposed
methodology, there is no need for biomass separation and
oil extraction before the transesterication reaction. Therefore,
energy consumption is reduced compared with classical biodiesel
production processes. The biodiesel mass yield was characterized
through spectrometry in the infrared region demonstrating
that alkyl monoesters were indeed obtained, i.e. biodiesel. In
order to assess the biofuel energy yield and performance, the
thermophysical properties were estimated, including the cetane
number, cold lter plugging point and lower heating value. The
thermodynamic efciencyof theprocess was calculated, andit was
demonstrated that through in situ methanolysis the microalgae
derived biodiesel energy balance was approximately three times
wileyonlinelibrary.com/jctb c 2011 Society of Chemical Industry J ChemTechnol Biotechnol 2011; 86: 0
9
Microalgae biodiesel via in situ methanolysis www.soci.org
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
higher than the energy balance of sugar cane, considering ethanol
and bagasse as energy products, and more efcient than soybean
and sunower derived biodiesel.
The key conclusion of this study is that the in situ methanolysis
methodology for biodiesel production from microalgae improves
the state-of-the-art by the possible elimination of well known
major bottlenecks in microalgae derived biodiesel processes, such
as drying, separation and oil extraction. In fact, the production
of 17% more energy in the form of biodiesel than the energy
consumed in the process stages shows that there is potential for
the future implementation of industrial scale biodiesel production
frommicroalgae, since the process was shown to be energetically
sustainable. Therefore, a lowenergy consumption methodology is
made available with potential for microalgae derived biodiesel
industrial production from any microalgae species. However,
future work needs to be conducted in order to assess the
economical andenvironmental feasibility of the process, including
the effects of the energy consumed in cultivation and biomass
collection.
ACKNOWLEDGEMENTS
The authors acknowledge with gratitude the support of the Brazil-
ian National Council of Scientic and Technological Development,
CNPq.
REFERENCES
1 Christ R, IPCC: CLIMATE CHANGE 2007: The 4th assessment
report to the intergovernamental panel on climate change.
http://www.ipcc.ch [accessed 2 February 2010].
2 Couto LC, Couto L, Watzlawick LF and C amara D, Vias de valorizac ao
energ etica da biomassa. Biomassa & Energia 1:7192 (2004).
3 Conceic ao PS, Morais MM, Biajoli D, Resende LB and Martins
MA, Quantication of carbon sequestration by microalgae and
eucalyptus, in 4th International Congress of Bioenergy, Proceedings
of Bioenergy, Curitiba, PR, Brazil, 1821 (2009).
4 Ara ujo MSM, Relat orio de an alise de Desenvolvimento Limpo MDL,
ANEEL. http://www.ivig.coppe.ufrj.br/doc/r1parte2d.pdf [accessed
20 January 2010].
5 Borges L, Faria BM, Odebrecht C and Abreu PC, Potencial de absorc ao
de carbono por esp ecies de microalgas usadas na aq uicultura:
primeiros passos para o desenvolvimento de um mecanismo de
desenvolvimento limpo. Atl antica 29:3546 (2007).
6 Lalli C and Parsons T, Biological Oceanography: an Introduction.
Butterworth & Heinermann, Oxford (1993).
7 Derner RB, Ohse S, Villela M, Carvalho SM and Fett R, Microalgas,
produtos e aplicac oes. Ci encia Rural 36:19591967 (2006).
8 Richmond A, Handbook of Microalgal Culture: Biotechnology and
Applied Phycology. Blackwell Science, Iowa (2004).
9 Gomez PI and Gonzalez MA, Genetic variation among seven strains
of Dunaliella salina (Chlorophyta) with industrial potential, based
on RAPD bandling patterns and on nuclear ITS rDNA sequences.
Aquaculture 233:149162 (2004).
10 Lorenz RT and Cysewski GR, Commercial potential for Haematococcus
microalgae as a natural source of astaxanthin. Trends Biotechnol
18:160167 (2000).
11 Cordero B, Otero A, Patti no M, Arredondo BO and Fabregas J,
Astaxanthin production from the green alga Haematococcus
pluvialis with different stress conditions. Biotechnol Lett 2:213218
(1996).
12 Borowitzka MA, Commercial production of microalgae: ponds, tanks,
tubes and fermenters. J Biotechnol 70:313321 (1999).
13 Tripathi U, Sarada R, Rao SR and Ravishankar A, Production of
astaxanthin in Haematococcus pluvialis cultured in various media.
Bioresource Technol 68:197199 (1999).
14 Guerin M, Huntley ME and Olaizola M, Haematococcus astaxanthin:
applications for human health and nutrition. Trends Biotechnol
21:210216 (2003).
15 Bennedsen M, Wang X, Will en R, Wadstr om T and Andersen LP,
Treatment of H. pylori infected mice with antioxidant astaxanthin
reduces gastric inammation, bacterial load and modulates
cytokine release by splenocytes. Immunol Lett 70:185189 (1999).
16 Tanaka T, Makita H, Ohnishi M, Mori H, Satoh K and Hara
A, Chemoprevention of rat oral carcinogenesis by naturally
occurring xanthophylls, astaxanthin and canthaxanthin. Cancer
Res 55:40594064 (1995).
17 Chew BP, Soon PJ, Mun WW and Wong TS. A comparison of the
anticancer activities of dietary b-carotene, canthaxanthin and
astaxanthin in mice in vivo. Anticancer Res 19:18491854 (1999).
18 OConnor I and OBrien N, Modulation of UVA light-induced oxidative
stress by beta-carotene, lutein and astaxanthin in cultured
broblasts. J Dermatol Sci 16:226230 (1998).
19 Murillo E, Efecto hipercolesterol emico de la cantaxantina y La
astaxantina en ratas. Archivos Latinoamericanos de Nutrici on
42:409413 (1992).
20 Chisti Y, Biodiesel frommicroalgae. Biotechnol Adv 25:294306(2007).
21 Nagle N and Lemke P, Production of methyl ester fuels from
microalgae. Appl BiochemBiotechnol 24/25:355361 (1990).
22 Miao XandWu Q, Biodiesel productionfromheterotrophic microalgal
oil. Bioresource Technol 97:841846 (2006).
23 Umdu ES, Tuncer MandSeker E, Transestericationof Nannochloropsis
oculata microalgas lipid to biodiesel on Al
2
O
3
supported CaO and
MgO catalysts. Bioresource Technol 14 (2009).
AQ1
24 Ehimen EA, Sun ZF and Carrington CG, Variables affecting the in situ
transesterication of microalgae lipids. Fuel 18 (2009).
AQ2
25 Guillard RL, Cultureof phytoplanktonfor feedingmarineinvertebrates.
CultureMarine Invertebrate Animal 2960 (1975).
AQ3
26 Smedes F and Askland TK, Revisiting the development of the Bligh
and Dyer total lipid determination method. Marine Pollut Bull
38:193201 (1999).
27 Becker EW, Microalgae: Biotechnology and Microbiology. Cambridge
University Press, New York (1995).
28 Ramos MJ, Fernandez CM, Casas A, Rodrguez L andP erez A, Inuence
of fatty acid composition of raw materials on biodiesel properties.
Bioresource Technol 100:261268 (2009).
29 Graboski MS and McCormick RL, Combustion of fat and vegetable oil
derived fuels in diesel engines. Energy Combustion Sci 24:125164
(1998).
30 Freedman B and Bagby MO, Predicting the cetane number of n-
alcohols and methyl esters from their physical properties. J Am Oil
ChemSoc 67:565571 (1990).
31 Clements LD, Blending rules for formulating biodiesel fuel. Liquid and
industrial products from renewable resources, in Proceedings of the
3
rd
Liquid Fuel Conference, Nashville, 4453 (1996).
32 AOCS International, Ofcial methods of analysis. Calculated Iodine
Value: Cd 1c-85 (1997).
33 AOCS International, Ofcial methods of analysis. Calculated Sapon-
ication Value: Cd 3a-94 (1997).
34 Krisnangkura K, Estimation of heat of combustion of triglycerides and
fatty acid methyl esters. J AmOil ChemSoc 68:5658 (1991).
35 Silverstein, RM, Bassler GCandMorrill TC, Identicac aoespectrom etrica
de compostos org anicos, 7
a
edic ao, 65101; 141173 (2006).
36 Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, et al,
Microalgal triacylglycerols as feedstocks for biofuel production:
perspectives and advances. Plant J 54:621639 (2008).
37 Statistica8.0, StatSoft, Tulsa, Oklahoma, USA (2007).
38 Hili AM and Feinberg DA, Fuel from microalgae lipid products. Solar
Energy Research Institute, Portland (1984).
39 Knothe G, Analyzing biodiesel: standards and other methods. J AmOil
ChemSoc 83:823833 (2006).
40 Knothe G, Dependence of biodiesel fuel properties on the structure of
fatty acid alkyl esters. Fuel Process Technol 86:10211026 (2005).
41 Knothe G, Matheus AC and Ryan III TW, Cetane numbers of branched
and straight-chain fatty esters determined in an ignition quality
tester. Fuel 82:971975 (2003).
42 Knothe G, Dunn RO and Bagby MO, Biodiesel: the use of veg-
etable oils and their derivatives as alternative diesel fuels. Oil
Chemical Research, US Department of Agriculture. http://www.
biodieselgear.com/documentation/VegetableOilsAsAlternative
DieselFuels.pdf [accessed on 2 February 2010].
43 Soares LHB, Ara ujo ES, Alves BJR, Boddey RM and Urquiaga S,
Eci encia energ etica comparada das culturas do girassol e soja,
com aptid ao para a produc ao de biodiesel no Brasil. Serop edica:
Embrapa. Relat orio t ecnico (2008).
J ChemTechnol Biotechnol 2011; 86: 0 c 2011 Society of Chemical Industry wileyonlinelibrary.com/jctb
1
0
www.soci.org RM Carvalho J unioret al.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
44 Salla DA, Furlaneto FPB, Cabello C and Kanthack RAD, Avaliac ao
energ etica da produc ao de etanol utilizando como mat eria-prima
a cana-de-ac ucar. Ci encia Rural 39:25162520 (2009).
45 Chisti Y, Biodiesel frommicroalgaebeats bioethanol. Trends Biotechnol
26:126131 (2008).
46 AOCS International, Ofcial methods of analysis. Total, Free and
Combined Glycerol Iodometric Periodic Acid Method: Ca 1456
(1997).
47 Cordeiro CS, Sntese e caracterizac ao de biodiesel etlico para
utilizac aoemmotores dociclo-diesel. Dissertac aoMestrado, Centro
Federal de Educac ao Tecnol ogica do Paran a, Curitiba, PR, Brasil
(2003).
48 SatyanarayanaKG, MarianoABandVargasJVC, Areviewonmicroalgae,
a versatile source for sustainable energy and materials. Int J Energy
Res 34:121 (2010).
49 Macedo IC, Seabra JEA and Silva JEAR, Green house gases emissions
in the production and use of ethanol from sugarcane in Brazil: the
2005/2006 averages and a prediction for 2020. Biomass Bioenergy
32:582595 (2009).
wileyonlinelibrary.com/jctb c 2011 Society of Chemical Industry J ChemTechnol Biotechnol 2011; 86: 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
QUERIES TOBE ANSWEREDBY AUTHOR
IMPORTANT NOTE: Please mark your corrections and answers to these queries directly onto the proof at the relevant place. Do
NOT mark your corrections on this query sheet.
Queries fromthe Copyeditor:
AQ1 Volume number??
AQ2 Volume number??
AQ3 Volume number??
Queries fromthe Typesetter:
TS1 Journal style is that the rst forename of all authors is provided in full. Please supply this information.
WILEY AUTHOR DISCOUNT CLUB
We would like to show our appreciation to you, a highly valued contributor to Wileys
publications, by offering a unique 25% discount off the published price of any of our
books*.
All you need to do is apply for the Wiley Author Discount Card by completing the
attached form and returning it to us at the following address:
The Database Group (Author Club)
John Wiley & Sons Ltd
The Atrium
Southern Gate
Chichester
PO19 8SQ
UK
Alternatively, you can register online at www.wileyeurope.com/go/authordiscount
Please pass on details of this offer to any co-authors or fellow contributors.
After registering you will receive your Wiley Author Discount Card with a special promotion
code, which you will need to quote whenever you order books direct from us.
The quickest way to order your books from us is via our European website at:
http://www.wileyeurope.com
Key benefits to using the site and ordering online include:
Real-time SECURE on-line ordering
Easy catalogue browsing
Dedicated Author resource centre
Opportunity to sign up for subject-orientated e-mail alerts
Alternatively, you can order direct through Customer Services at:
cs-books@wiley.co.uk, or call +44 (0)1243 843294, fax +44 (0)1243 843303
So take advantage of this great offer and return your completed form today.
Yours sincerely,
Verity Leaver
Group Marketing Manager
author@wiley.co.uk
*TERMS AND CONDITIONS
This offer is exclusive to Wiley Authors, Editors, Contributors and Editorial Board Members in acquiring books for their personal use.
There must be no resale through any channel. The offer is subject to stock availability and cannot be applied retrospectively. This
entitlement cannot be used in conjunction with any other special offer. Wiley reserves the right to amend the terms of the offer at any
time.
To enjoy your 25% discount, tell us your areas of interest and you will receive relevant catalogues or leaflets
from which to select your books. Please indicate your specific subject areas below.
Accounting
Public
Corporate
[ ]
[ ]
[ ]
Architecture
Business/Management
[ ]
[ ]
Chemistry
Analytical
Industrial/Safety
Organic
Inorganic
Polymer
Spectroscopy
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
Computer Science
Database/Data Warehouse
Internet Business
Networking
Programming/Software
Development
Object Technology
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
Encyclopedia/Reference
Business/Finance
Life Sciences
Medical Sciences
Physical Sciences
Technology
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
Engineering
Civil
Communications Technology
Electronic
Environmental
Industrial
Mechanical
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
Earth & Environmental Science
Hospitality
[ ]
[ ]
Finance/Investing
Economics
Institutional
Personal Finance
[ ]
[ ]
[ ]
[ ]
Genetics
Bioinformatics/
Computational Biology
Proteomics
Genomics
Gene Mapping
Clinical Genetics
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
Life Science
Landscape Architecture
Mathematics
Statistics
Manufacturing
Materials Science
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
Medical Science
Cardiovascular
Diabetes
Endocrinology
Imaging
Obstetrics/Gynaecology
Oncology
Pharmacology
Psychiatry
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
Psychology
Clinical
Forensic
Social & Personality
Health & Sport
Cognitive
Organizational
Developmental & Special Ed
Child Welfare
Self-Help
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
[ ]
Non-Profit [ ] Physics/Physical Science [ ]
Please complete the next page /
REGISTRATION FORM
For Wiley Author Club Discount Card
I confirm that I am (*delete where not applicable):
a Wiley Book Author/Editor/Contributor* of the following book(s):
ISBN:
ISBN:
a Wiley Journal Editor/Contributor/Editorial Board Member* of the following journal(s):
SIGNATURE: Date:
PLEASE COMPLETE THE FOLLOWING DETAILS IN BLOCK CAPITALS:
TITLE: (e.g. Mr, Mrs, Dr) FULL NAME: .
JOB TITLE (or Occupation): ..
DEPARTMENT: ..
COMPANY/INSTITUTION:
ADDRESS:

TOWN/CITY:
COUNTY/STATE: .
COUNTRY: .
POSTCODE/ZIP CODE:
DAYTIME TEL:
FAX:
E-MAIL:
YOUR PERSONAL DATA
We, John Wiley & Sons Ltd, will use the information you have provided to fulfil your request. In addition, we would like to:
1. Use your information to keep you informed by post of titles and offers of interest to you and available from us or other
Wiley Group companies worldwide, and may supply your details to members of the Wiley Group for this purpose.
[ ] Please tick the box if you do NOT wish to receive this information
2. Share your information with other carefully selected companies so that they may contact you by post with details of
titles and offers that may be of interest to you.
[ ] Please tick the box if you do NOT wish to receive this information.
E-MAIL ALERTING SERVICE
We also offer an alerting service to our author base via e-mail, with regular special offers and competitions. If you DO wish to
receive these, please opt in by ticking the box [ ].
If, at any time, you wish to stop receiving information, please contact the Database Group (databasegroup@wiley.co.uk) at John Wiley & Sons Ltd,
The Atrium, Southern Gate, Chichester, PO19 8SQ, UK.
TERMS & CONDITIONS
This offer is exclusive to Wiley Authors, Editors, Contributors and Editorial Board Members in acquiring books for their personal use. There should
be no resale through any channel. The offer is subject to stock availability and may not be applied retrospectively. This entitlement cannot be used
in conjunction with any other special offer. Wiley reserves the right to vary the terms of the offer at any time.
PLEASE RETURN THIS FORM TO:
Database Group (Author Club), John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, PO19 8SQ, UK author@wiley.co.uk
Fax: +44 (0)1243 770154