Bioresource Technology 194 (2015) 179186

Contents lists available at ScienceDirect

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

Biodiesel production from wet microalgae feedstock using

sequential wet extraction/transesterication and direct
transesterication processes
Ching-Lung Chen a, Chien-Chang Huang b, Kao-Chia Ho a, Ping-Xuan Hsiao a, Meng-Shan Wu a,
Jo-Shu Chang a,c,
a
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
b
Department of Cosmetic Science, Providence University, Taichung, Taiwan
c
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan

h i g h l i g h t s

 Over 97% biodiesel conversion was obtained using wet microalgae as feedstock.
 Wet extraction was more efcient after microwave and methanol pretreatments.
 Chlorophyll removal was achieved during the process of biodiesel production.
 Direct transesterication with algae cake was operated at a low temperature (45 C).
 Nearly 100% biodiesel conversion was achieved with the direct transesterication.

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

Article history: Although producing biodiesel from microalgae seems promising, there is still a lack of technology for the
Received 13 May 2015 quick and cost-effective conversion of biodiesel from wet microalgae. This study was aimed to develop a
Received in revised form 2 July 2015 novel microalgal biodiesel producing method, consisting of an open system of microwave disruption, par-
Accepted 3 July 2015
tial dewatering (via combination of methanol treatment and low-speed centrifugation), oil extraction,
Available online 10 July 2015
and transesterication without the pre-removal of the co-solvent, using Chlamydomonas sp. JSC4 with
68.7 wt% water content as the feedstock. Direct transesterication with the disrupted wet microalgae
Keywords:
was also conducted. The biomass content of the wet microalgae increased to 56.6 and 60.5 wt%, respec-
Wet microalgae
Biodiesel
tively, after microwave disruption and partial dewatering. About 96.2% oil recovery was achieved under
Microwave disruption the conditions of: extraction temperature, 45 C; hexane/methanol ratio, 3:1; extraction time, 80 min.
Oil extraction Transesterication of the extracted oil reached 97.2% conversion within 15 min at 45 C and 6:1 sol-
Direct transesterication vent/methanol ratio with simultaneous Chlorophyll removal during the process. Nearly 100% biodiesel
conversion was also obtained while conducting direct transesterication of the disrupted oil-bearing
microalgal biomass.
2015 Elsevier Ltd. All rights reserved.

1. Introduction production has attracted considerable international scrutiny and

Biodiesel has many advantages, such as high ash point, high
lubricity, and high biodegradability, and can be used in conven-
tional diesel engines without any modication, making it one of
the most promising alternatives to fossil fuels (Zabeti et al.,
2009). However, the use of plant-based oil feedstock for biodiesel
Corresponding author at: Department of Chemical Engineering, National Cheng
Kung University, Tainan, Taiwan. Tel.: +886 275 7575x62651; fax: +886 234 4496.
E-mail address: changjs@mail.ncku.edu.tw (J.-S. Chang).
criticism (Williams and Laurens, 2010). Therefore, increasing
attention is being paid to using microalgae as an alternative feed-
stock for biodiesel production, because cultivation of microalgae
does not require arable land and has the advantages of a high
growth rate, short maturity, high biomass productivity, and low
environmental impact (Goncalves et al., 2013). However, develop-
ing the technology for the conversion of microalgae-based oil to
biodiesel is more challenging when compared with plant oil (such
as palm oil), due mainly to the high water content of microalgae
and the difculty in extracting the oil from the microalgal biomass,

http://dx.doi.org/10.1016/j.biortech.2015.07.021
0960-8524/ 2015 Elsevier Ltd. All rights reserved.
180 C.-L. Chen et al. / Bioresource Technology 194 (2015) 179186

leading to the higher production cost of microalgae-based biodie-

sel (Milledge and Heaven, 2013). There is thus an urgent need to,
develop a more economically feasible method for producing bio-
diesel from microalgae.
The common process for obtaining oil from microalgae consists
of microalgae harvesting, biomass drying and oil extraction
(Cooney et al., 2009), of which the drying step consumes a large
amount of energy. Therefore, recent studies have focused on using
wet microalgae as the raw materials to produce biodiesel
(Olmstead et al., 2013; Tran et al., 2013). In addition, some of the
downstream technologies still suffer the drawbacks of high energy
consumption and low treatment capacity (as seen, for example,
with centrifugation and supercritical uid extraction), limiting
the commercialization of these methods. This study thus presents
a new process for producing microalgae-based biodiesel from the
wet biomass of microalgae, consisting of microwave disruption,
partial water removal, wet oil extraction, transesterication, and
direct transesterication, skipping the wet extraction step.
Moreover, a homogeneous base catalyst (i.e., NaOH) and heteroge-
neous base catalyst (i.e., Sr2SiO4) (Chen et al., 2012) were used to
convert microalgae oil to biodiesel, and their catalytic effects on
transesterication were compared. The aim of this work was to
develop a low cost and effective method for biodiesel production
from wet microalgal biomass, with the capacity to be scaled up
for commercial applications.

2. Methods

2.1. Materials

The microalgae strain used in this study was Chlamydomonas sp.

JSC4, which was provided by the Center for Bioscience and
Biotechnology, National Cheng Kung University, Tainan, Taiwan
Fig. 1. The owchart of biodiesel production from wet microalgae.
and was shown to possess high lipid content (Ho et al., 2015;
Nakanishi et al., 2014). The culture conditions were based on the
optimal conditions reported by Nakanishi et al. (2014). After culti-
through the pretreatment process consisting of microwave disrup-
vation, the microalgae biomass was collected with a centrifuge at
tion and concentration via methanol-driven occulation, which
22,400g, and the resulting microalgae slurry had a dry biomass
consisted of occulation with methanol and dehydration with a
content of 31.3% and an oil content of 26.3% per dry weight of
spin dryer. The resulting microalgae cake (with a solid content of
biomass.
ca. 60 wt%) was then used to produce biodiesel. Two processes
Hexane (ACS) and sodium hydroxide (NaOH, ACS) were
were conducted for the production of biodiesel from the wet
obtained from Macron Fine Chemicals (Pennsylvania, USA).
microalgae cake, as follows (Fig. 1). Process 1: wet oil extraction
Strontium nitrate (Sr(NO3)2, 98%) and tetraethyl orthosilicate
was rst conducted and the extracted microalgal oil was subjected
(TEOS, 99.9%) were obtained from Showa Co. (Tokyo, Japan).
to transesterication using a homogeneous base catalyst (i.e.,
Ammonia hydroxide (25% NH3 basis) was purchased from
NaOH) or heterogeneous base catalyst (i.e., Sr2SiO4). Process 2:
Sigma-Aldrich (Missouri, USA). The methanol (>99%) was pur-
the wet microalgae cake was directly used to carry out transester-
chased from Uni Ward (Miaoli, Taiwan).
ication without the oil extraction step.

2.2. Preparation of the solid base catalyst 2.3.1. Pretreatment

The solid base catalyst was prepared using a modied method
as reported by Chen et al. (2012). In summary, 48.5 g strontium
nitrate and 25.1 mL TEOS were dissolved in 100 mL ammonia
hydroxide and 200 mL methanol, respectively. The two solutions
were then mixed with each other, and the resulting mixture was
heated in an oil bath to generate the solid precursor. Finally, by cal-
cining the precursor at 1100 C, the solid catalyst was obtained and
was analyzed with X-ray diffraction (XRD; Rigaku Ultima IV). The
catalyst was identied as Sr2SiO4 by comparing the XRD pattern
with the JCPDS le 39-1256 (Chen et al., 2012).
2.3. Procedures of biodiesel production from wet microalgae
The owchart of biodiesel production from wet microalgae car-
ried out in this study is shown in Fig. 1. Wet microalgae rst went
One hundred grams of microalgae sludge was placed into a
500 mL serum bottle, and then 100 mL methanol was added to
the bottle and mixed for 20 minutes at 400 rpm to increase the u-
idity of the sludge. The microalgae-methanol mixture was then
input into an open microwave cell disruption system, consisting
of a microwave oven (Samsung MW630WA), an open reactor, a
cooling system (Fig. 2) with underwent heating at 350 W for
10 min to achieve cell wall disruption. After that, another 200 mL
methanol was added to the bottle and the mixture was stirred at
100 rpm for 20 min. The nal mixture was poured into a commer-
cial ltration bag, and the bag was centrifuged with a spin dryer to
yield the microalgae cake.
2.3.2. Wet oil extraction
The 2 g microalgae cake was placed into a 100 mL serum bottle,
and then 4 mL methanol and various amounts of hexane (i.e., 8, 12,
 C.-L. Chen et al. / Bioresource Technology 194 (2015) 179186
Fig. 2. Schematic description of the microwave cell disruption system.
16, 20, and 24 mL) were added to the bottle. The wet extraction of
microalgal oil was carried out at an agitation rate of 600 rpm,
extraction temperatures of 25, 35, 45, 55, and 65 C, and extraction
times of 20, 40, 60, 80, 100, and 120 min to identify the suitable
conditions for microalgae oil recovery. After each of the
above-mentioned tests was complete, the mixture was separated
into the oil phase, water phase, and microalgae residue by centrifu-
gation at 6000 rpm for 3 min. The hexane solution containing
microalgae oil was then collected and stored at 4 C.
2.3.3. Transesterication
Following the best conditions determined in the earlier experi-
ments, three batches of 100 g microalgae biomass pastes were pre-
treated and then subjected to wet oil extraction to obtain adequate
amounts of microalgae oil (dissolved in hexane) for the transester-
ication experiments. Twelve milliliters of oil-containing hexane
solution (microalgae oil content = 0.0253 g/mL) and the desired
amount of methanol containing 0.5% (w/v) NaOH were poured into
a 100 mL serum bottle, and the volume ratios of hexane solution to
methanol were 2:1, 4:1, 6:1, 8:1, and 10:1. In addition, the reaction
temperatures of 25, 35, 45, 55, and 65 C and reaction times of 5,
10, 15, 20, 25, and 30 min were used to determine the best condi-
tions for biodiesel production. The transesterication reaction was
performed at a stirring speed of 600 rpm under the conditions
mentioned above.
In addition to using NaOH as the catalyst for transesterication
of the extracted microalgae oil, a solid base catalyst (Sr2SiO4), pre-
pared as described in Section 2.2, was also used for transesterica-
tion of microalgae oil. The volume ratio of oil-containing hexane
solution to methanol, reaction temperature, extraction time, and
stirring speed were 12 mL:2 mL, 45 C, 15 min, and 600 rpm,
respectively. Moreover, the catalyst doses examined were 2%, 4%,
6%, 8%, and 10% (w/v) based on methanol.
As for the direct transesterication experiments, another 100 g
of microalgae biomass paste was occulated by mixing with
methanol to yield microalgae cake. Several 100 mL serum bottles
loaded with 2 g cake and 12 mL hexane were then prepared.
After that, a 4 mL methanol solution with 0.25% (w/v) NaOH, as
181
well as 4, 8, and 12 mL methanol solutions with 0.5% (w/v)
NaOH, were added into the bottles to perform direct transesteri-
cation at 45 C and 600 rpm for 15 min.
After the above-mentioned transesterication steps were com-
plete, all samples were centrifuged at 6000 rpm for 3 min, and then
the hexane phase containing produced biodiesel was separated
and preserved at 4 C for analysis.
2.4. Product analysis
The composition of the FAMEs dissolved in hexane solution was
analyzed with a gas chromatograph (Shimadzu GC-14B) equipped
with a ame ionization detector and a capillary column, Agilent
DB-17ht. Nitrogen was used as the carrier gas. The oven tempera-
ture was initially held at 150 C for 2 min, heated at a rate of
10 C/min until reaching 250 C, and then held at 250 C for
5 min. The temperatures of the injector and detector were both
set at 50 C. The oil recovery was calculated according to Eq. (1):
FAMEhexane  Vhexane
Oil recovery % 1
OilMA  W SDW
where, FAMEhexane is the concentration of FAMEs in hexane, which
was transformed by the transesterication reaction at a hexane
solution/methanol (containing 0.5 wt% NaOH) ratio of 2:1 at 65 C
and 600 rpm for 30 min. Vhexane is the hexane volume after the
wet oil extraction process. OilMA is the oil content in microalgae
on the dry biomass basis (26.3%). WSDW is the dry weight of the
sample used to conduct the wet oil extraction process, and was cal-
culated based on Eq. (2):
W SDW W sample  C cake 2
where Wsample is the weight of the microalgae cake sample used for
the wet oil extraction process. Ccake is the solid content of the wet
microalgae cake.
Fig. 3. The effects of the extraction time1, extraction temperature2, and hexane/
methanol ratio3 on the recovery of microalgae oil. 1 The experiments were
performed under the following conditions: cake weight, 2 g; amount of methanol,
4 mL; extraction temperature, 25 C; hexane-to-methanol ratio, 2:1; mixing speed,
600 rpm. 2 The experiments were performed under the following conditions: cake
weight, 2 g; amount of methanol, 4 mL; hexane-to-methanol ratio, 2:1; mixing
speed, 600 rpm; extraction time, 80 min. 3 The experiments were performed under
the following conditions: cake weight, 2 g; amount of methanol, 4 mL; extraction
temperature, 45 C; mixing speed, 600 rpm; extraction time, 80 min.
182 C.-L. Chen et al. / Bioresource Technology 194 (2015) 179186

In addition, the transesterication efciency was calculated for the direct transesterication process, which was also calculated
based on Eq. (3): using Eq. (2).

Transesterification efficiency %
3. Results and discussion
FAMENaOH-T or FAMESr2 SiO4 -T  V 00hexane
 100% 3
FAMEhexane  V 0hexane 3.1. Pretreatment of microalgal biomass and wet oil extraction

where FAMENaOH-T and FAMESr2 SiO4 -T are the concentrations of
FAMEs in the hexane solution produced by NaOH-catalytic transes-
terication and Sr2SiO4-catalytic transesterication, respectively,
following the procedures mentioned in Section 2.3.3. V00 hexane is
the hexane volume after the transesterication reaction. V0 hexane is
the hexane volume of the sample used for the transesterication
test. Moreover, the biodiesel production after the wet oil extraction
process and the transesterication process was evaluated by Eq. (4):
Biodiesel production %
Oilrecovery %  Transesterification efficiency %
The biodiesel production by direct transesterication was cal-
culated using Eq. (5):
FAMEDT  V DT
Biodiesel conversion %
OilMA  W DT
where FAMEDT and VDT are the FAMEs concentration in hexane and
the volume of the hexane solution after the direct transesterica-
tion process, respectively. WDT is the dry weight of the sample used
Fig. 3 shows the effects of extraction time, extraction tempera-
ture and the hexane-to-methanol ratio on oil recovery by the wet
oil extraction process. The results indicate that the oil recovery
increased rapidly with an increase in the extraction time, extrac-
tion temperature, and the hexane-to-methanol ratio in the early
stage of the experiments. The increase in oil recovery then slowed
down as these parameters continued to rise, and nally reached a
maximum oil recovery level. The trends shown in Fig. 3 are quite
similar to those reported in a recent study (Dai et al., 2014). At
room temperature (25 C), the oil recovery increased along with
4 the extraction time, and reached a plateau starting at 80 min,
and thus this was selected as the suitable extraction time. As for
the extraction temperature, although the highest oil recovery
occurred when the temperature was increased to 65 C, when con-
sidering the safety of an open extraction system, a lower tempera-
5 ture of 45 C was selected for oil extraction. In addition, an increase
in the hexane/methanol ratio also led to an increase in oil recovery,
which reached the highest level of 96.2% when the hex-
ane/methanol ratio was 3:1 (12 mL:4 mL), and thus this was
selected as the suitable condition. In summary, the selected

Fig. 4. The effects of the reaction temperature1, hexane solution-to-methanol ratio2, and reaction time3 on the transesterication of the extracted microalgae oil dissolved in
hexane. 1 The experiments were performed under the following conditions: amount of oil-hexane solution, 12 mL; hexane solution-to-methanol ratio, 2:1; mixing speed,
600 rpm; reaction time, 20 min. 2 The experiments were performed under the following conditions: amount of oil-hexane solution, 12 mL; temperature, 45 C; mixing speed,
600 rpm; reaction time, 20 min. 3 The experiments were performed under the following conditions: amount of oil-hexane solution, 12 mL; hexane solution-to-methanol ratio,
6:1; temperature, 45 C; mixing speed, 600 rpm.
 C.-L. Chen et al. / Bioresource Technology 194 (2015) 179186
conditions for the wet oil extraction from the microalgae cake were
an extraction time of 80 min, extraction temperature of 45 C, and
hexane-to-methanol ratio of 3:1.
The success of a wet oil extraction process can be attributed not
only to the use of suitable extraction conditions, but also to the
pretreatment process. Previous studies indicated that microwave
radiation is an excellent tool for disrupting the cells of microalgae
(De Souza Silva et al., 2014; Guldhe et al., 2014; Prabakaran and
Ravindran, 2011), although most of the related studied reported
using a high microwave power, low microalgae concentration,
and small amount of the sample, thus making them infeasible for
commercial applications. In this study, however, the
microwave-assisted cell disruption was performed at a low power
(only 350 W), as well as a relatively high microalgae concentration
(31.3 wt%) and sample loading (100 g), and these conditions have
the potential for large-scale operations. Moreover, after cell disrup-
tion was conducted, fragments of microalgal biomass formed that
were very difcult to separate from the liquid phase. This caused
severe problems in performing the oil extraction from wet microal-
gae, since the oil-containing microalgae were located in dilute
solutions containing microalgae fragments that were difcult to
concentrate. To cope with this problem, the present study tried
to utilize the properties of the cell wall-associated polysaccharides
of microalgae to induce occulation and precipitation of the
microalgal biomass by treating it with a sufcient amount of
methanol (Guo et al., 2013; Xu et al., 2014). After the microwave
disruption and methanol occulation steps, the concentrated
microalgae biomass with a solid content of 56.660.5% could be
easily and effectively collected using a commercial ltration bag
and a spin dryer, thus signicantly reducing the volume of the
microalgae feedstock for the subsequent oil extraction.
3.2. Transesterication
Approximately 920 mL of hexane solution containing microal-
gae oil at a concentration of 0.0253 g oil/mL was prepared from
three batches of 100 g wet microalgal biomass after going through
the pretreatment and wet oil extraction processes with 94.3% oil
recovery efciency. This oil-containing hexane solution was used
as the feedstock to determine the optimal conditions of the trans-
esterication process. As shown in Fig. 4, the transesterication
efciency rapidly increased when the temperature was raised from
25 C to 35 C, but then remained about the same even when the
temperature was further increased from 35 C to 65 C. The trans-
esterication efciency was higher than 96% when the reaction
was conducted at temperatures greater than 45 C, which was thus
considered the most suitable reaction temperature for transesteri-
cation. The transesterication efciency fell from 98.4 1.0% to
87.3 3.4% as the hexane-to-methanol ratio was increased from
2:1 to 10:1 (Fig. 4). The optimal ratio was set at, 6:1, because the
transesterication efciency at the ratio of 8:1 was below 95%
(92.1 2.5%). The transesterication efciency increased sharply
to 86.1 3.7% with a reaction time of 5 min, and then rose more
gradually to over 95% (97.2 12%) as the reaction time was
extended to more than 15 min, which was thus considered the
suitable reaction time for transesterication.
Although it was found in this study that the effects of the reac-
tion temperature, oil-to-methanol ratio, and reaction time on the
efciency of the transesterication of microalgal oil were similar
to those reported in related studies (Dang et al., 2013; Zu et al.,
2010), the efciency obtained in this work was higher than that
in the related literature when the reaction was conducted at a
low temperature (e.g., 35 C). It is thought that the presence of hex-
ane as a co-solvent played a major role in improving the transes-
terication efciency. Similar results were also observed when
using hexane as the co-solvent (Chen et al., 2012), and the reasons
183
for the observation could be that using acetone as the co-solvent
can reduce the time and temperature required for the reaction
(Thanh et al., 2013). In addition, Tang et al. (2013) also indicated
that the presence of co-solvents could avoid the occurrence of
saponication reaction during biodiesel production. This may
explain why the saponication did not take place when using the
processes proposed in this study.
Moreover, chlorophyll, which is an undesirable component of
microalgae biodiesel, is usually present in the transesterication
products if the conventional extraction method is used (Soh and
Zimmerman, 2011). Chlorophyll can be removed by using complex
processes, such as one consisting of hydrolysis, centrifugation, pre-
cipitation, and heat hexane washing (Sathish and Sims, 2012).
However, a recent study showed that a small amount of chloro-
phyll was still present in FAMEs when the transesterication was
conducted with a catalyst loading of greater than
0.2 g-NaOH/g-oil, a methanol-to-oil weight ratio of greater than
15.2, and a mixing speed of 3000 rpm for 3 h (Afy et al., 2010).
In contrast, Fig. 5 shows that the Chlorophyll content was easily
removed when the transesterication reaction of the microalgal
oil in hexane was conducted at 45 C for 15 min with a
hexane-to-methanol ratio of 6:1. This condition is identical to a
methanol to microalgae oil weight ratio of 5.22:1 and a catalyst
loading of 0.033 g-NaOH/g-microalgae oil (see the TSBP set in
Fig. 5).
The solid base catalyst, Sr2SiO4, was also used for the transester-
ication of the extracted microalgal oil. Fig. 6 shows that the trans-
esterication efciency increased along with the Sr2SiO4 dose. This
efciency was more than 95% and chlorophyll was almost totally
removed when the dose was more than 0.12 g per 12 mL of
oil-containing hexane solution, which is equal to a catalyst loading
of 0.40 g-Sr2SiO4/g-microalgae oil. This catalyst dose is 12 times
Fig. 5. Comparison of the biodiesel yield between the direct transesterication
process and the two step (oil-extraction and transesterication) process of biodiesel
conversion. TSBP: biodiesel production using a two-step (oil-extraction and
transesterication) process. The transesterication was performed under the
following conditions: amount of oil-hexane solution, 12 mL; hexane solution-to-
methanol (with 0.5 wt% NaOH) ratio, 6:1; temperature, 45 C; mixing speed,
600 rpm; reaction time, 15 min. DT01: biodiesel production using a direct trans-
esterication process was performed under the following conditions: weight of
microalgae cake, 2 g; amount of methanol, 12 mL; temperature, 45 C; hexane-to-
methanol (with 0.25 wt% NaOH) ratio, 3:1; mixing speed, 600 rpm, reaction time,
15 min. DT02: biodiesel production using a direct transesterication process
performed under the following conditions: weight of microalgae cake, 2 g; amount
of methanol, 12 mL; temperature, 45 C; hexane-to-methanol (with 0.5 wt%
NaOH) ratio, 3:1; mixing speed, 600 rpm; reaction time, 15 min. DT03: biodiesel
production using a direct transesterication process performed under the following
conditions: weight of microalgae cake of 2 g, the amount of methanol, 12 mL, the
temperature of 45 C, hexane-to-methanol (with 0.5 wt% NaOH) ratio of 3:2, and
the mixing speed of 600 rpm; reaction time, 15 min. DT04: biodiesel production
using a direct transesterication process performed under the following conditions:
weight of microalgae cake, 2 g; amount of methanol, 12 mL; temperature, 45 C;
hexane-to-methanol (with 0.5 wt% NaOH) ratio, 3:3; mixing speed, 600 rpm;
reaction time, 15 min.
184 C.-L. Chen et al. / Bioresource Technology 194 (2015) 179186

in the dose of base catalysts that were mixed with methanol at a

xed catalyst to methanol ratio. First, chlorophyll may dissolved
in methanol and then moved to the methanol (polar) phase. This
will cause a decrease in the chlorophyll content in the biodiesel
produced (mainly located in the hexane phase). Second, the base
catalyst may easily react with chlorophyll causing its decomposi-
tion as mentioned in the literature (Han et al., 2013).

3.3. Direct transesterication

Fig. 5 also shows the results of the direct transesterication

Fig. 6. The effects of the Sr2SiO4 dose on the transesterication efciency of the
extracted microalgae oil in hexane. The transesterication was performed under the
following condition: amount of oil-hexane solution, 12 mL; hexane solution-to-
methanol ratio, 6:1; temperature, 45 C; mixing speed, 600 rpm; reaction time,
15 min.
higher than the dose of the homogeneous catalyst (0.01 g
NaOH/12 mL) used. Moreover, after the transesterication process,
the color of Sr2SiO4 became green, which might result from the
adsorption of chlorophyll present in the microalgal oil extracts
on the catalyst surface. This makes it very difcult to reuse the
solid catalyst Sr2SiO4, thus negating the major advantage of using
the heterogeneous catalyst exist (Lam et al., 2010). These results
indicate that the solid base catalyst, Sr2SiO4, seems to be less effec-
tive, when compared with NaOH, and unsuitable for use to catalyze
the transesterication of the extracted microalgae oil under the
current conditions.
There are two possible reasons that may cause the decrease in
chlorophyll content during the transesterication with an increase
experiments using NaOH as the catalyst. At the same catalyst load-
ing of 0.01 g/12 mL oil-hexane solution, the biodiesel conversion
by direct transesterication (DT01) was quite low (only 2.5%),
and the color of the oil-hexane solution was dark green. When
the dose in the direct transesterication increased to
0.04 g/12 mL oil-hexane solution (DT03), which is four times the
dose used for TSBP, the biodiesel conversion suddenly increased
to nearly 100%. A further increase in NaOH loading (DT04) also
resulted in nearly 100% biodiesel conversion, but it would not be
economically viable to use such an excessive amount of NaOH in
practice.
Most of the in situ/direct transesterication studies for the con-
version of microalgae to biodiesel use dried microalgal biomass as
the feedstock (Table 1), and there are relatively fewer cases in
which wet microalgal biomass is applied (Table 2). Moreover, a
high solvent-to-microalgae biomass ratio (Carvalho Jnior et al.,
2011; Velasquez-Orta et al., 2012), high catalyst loading (Haas
and Wagner, 2011; Velasquez-Orta et al., 2012) or high reaction
temperature (e.g., >90 C) (Cao et al., 2013; Dong et al., 2013;
Kumara et al., 2014; Tsigie et al., 2012) are required to achieve a
high biodiesel yield. In addition, some studies (Cao et al., 2013;
Kumara et al., 2014) indicate that the water content has signicant,
negative effects on the one-step direct transesterication process.
Compared with the related studies, the combined pretreatment
and direct transesterication process developed in this work can

Table 1
Comparison of the conditions and performances in the related studies for the production of biodiesel from dried microalgae using an in situ/direct transesterication process.

Microalgae and oil Sample type and amount Process (catalyst loading; MeOH/sample ratio; temperature; mixing FAME yield Reference
content speed; time)
Chlorella sorokiniana Lyophilized biomass: 1 g Acid-catalyzed esterication process 94.87 0.86% Dong et al.
UTEX 1602 12.83% Amberlyst-15, 30 wt%; 2 mL/g; 90 C; 120 rpm; 60 min (2013)
Base-catalyzed transesterication process
KOH, 0.3 wt%; 4 mL/g; 90 C; 10 min
Chlorella vulgaris Dried biomass: 7 g Acid-catalyzed esterication (AE) AE: 96.9 6.3% Velasquez-Orta
26.9 0.4% H2SO4/lipid molar ratio = 0.35:1; MeOH/oil molar ration = 600:1; et al. (2012)
60 C; 380 rpm; 20 h
Base-catalyzed transesterication (BT) BT: 77.6 2.3%
NaOH/lipid molar ratio = 0.15:1; MeOH/oil molar ration = 600:1;
60 C; 380 rpm; 75 min
Chlorella pyrenoidosa Dried biomass: 1 g 0.5 M H2SO4 in MeOH; hexane/MeOH/sample = 6 mL/4 mL/1 g; 95% Li et al. (2011)
56.3% 90 C; 2 h
Nannochloropsis Dried biomass: 1 g H2SO4/oil = 0.8:1 (N. oculata) or 0.35:1 (Chlorella sp.); MeOH/lipid N. oculata: 73 5% Velasquez-Orta
oculata 26.8 4.9% molar ratio = 600:1; 60 C; 450 rpm Chlorella sp.: 92 2% et al. (2013)
Chlorella sp.
11.3 0.4%
Nannochloropsis Spray-dried biomass: 2 g HCl in 204 mL reaction solvent, MeOH/HCl/CHCl3 volumetric 23.07 2.76% (base on Carvalho Jnior
oculata ratio = 10:1:1; solvent/sample = 204 mL:2 g; 80 C; rpm; 2 h dried biomass weight) et al. (2011)
Scenedesmus sp. Lyophilized biomass: acid- AE: 5% H2SO4 (v/v) in MeOH; 15:1 (v/w); 70 C; 500 rpm; 10 h AE: 48.41 0.21% Kim et al.
catalytic esterication (2014)
(AE): 15 g; base-catalytic BT: 0.5 wt% NaOH; 10:1 (v/w); 60 C; 500 rpm; 2 h BT: 55.07 2.18%
trans-esterication (BT): 5 g
Commercial Washed and oven dried H2SO4, 23.5 mmol:2.5 g; 14 mL:2.5 g; 65 C; 150 rpm; 2 h 98 2% Haas and
microalgae biomass: 2.5 g Wagner (2011)
20.9 0.4%
 C.-L. Chen et al. / Bioresource Technology 194 (2015) 179186 185

Table 2
Comparison of the conditions and performances in the related studies for the production of biodiesel from wet microalgae using an in situ/direct transesterication process.

Microalgae and oil Sample type and amount
content
Chlorella pyrenoidosa 47% Lyophilized biomass: 1 g; 100 mg
Chlorella vulgaris ESP-31 Wet microalgae (water content:
1463% 8691%): 0.51 g
Chlorella vulgaris Wet biomass: 5 g (1 g dried
biomass + 4 mL DI water)
Chlorella sp. FC2 IITG Wet biomass (WB): 100 mg
(lyophilized biomass: 30 mg);
lyophilized biomass (LB): 20 mg
Chlorella vulgaris ESP-31 Microalgae cake: 2 g (solid content:
26.3% 56.660.5%)
Process (catalyst loading; MeOH/sample FAME yield References
ratio; temperature; mixing speed; time)
1st process 1st process Cao et al. (2013)
0.5 M H2SO4 in MeOH; hexane/MeOH/DI 90 C: 91.4% (water
water/sample = 6 mL/4 mL/09 mL/1 g; 90, content = 0) to 10.3%
120, and 150 C; 120 min (90%)
120 C: 100% (030%) to
24.8% (90%)
150 C: 100%
Process with the optimal conditions (best Best process: 92.5%
process)
0.5 M H2SO4 in MeOH; hexane/MeOH/ /
sample = 8 mL/4 mL/1 g (water content: 90%);
120 C; 180 min
202818,950 U/g, xed lipase; hexane/ 91.1995.74% Tran et al. (2013)
methanol/oil = 4.1558.12/4.1517.57/1
(w/w/w); 4045 C, 500600 rpm; 48 h
No catalyst (subcritical condition); 4 mL/1 g; 0.29 g/g (base on dried Tsigie et al. (2012)
175 C, 22 bar; 4 h biomass weight)
One step (M2) One step (M2) Kumara et al. (2014)
0.5 N NaOH in MeOH; 1 mL/100 mg or Both: <10 wt% (base on
1 mL/20 mg; 90 C; 150 rpm; 40 min dried biomass weight)
Two step (M7) Two step (M7)
1st step WB: about 10 wt%
0.5 N NaOH in MeOH; 1 mL/100 mg or LB: about 40 wt% (the
1 mL/20 mg; 90 C; 150 rpm; 20 min most one) (base on dried
2nd step biomass weight)
5% (v/v) H2SO4 in MeOH; 1 mL/100 mg or
1 mL/20 mg; 90 C; 150 rpm; 20 min
0.5 wt% NaOH in MeOH; hexane/MeOH/ 101 2.7% This study
sample = 12 mL/8 mL/2 g; 45 C; 600 rpm;
15 min

overcome the above-mentioned problems. Although the required References

catalyst loading in the direct transesterication process could be
higher than that used in the two-step process (e.g., wet oil extrac-
tion followed by transesterication), the high biodiesel yield and
simple operating procedures still encourage the use of the pro-
posed direct transesterication process for efcient production of
microalgae-based biodiesel.
4. Conclusions
This work demonstrated feasible processes for producing bio-
diesel from wet biomass of Chlamydomonas sp. JSC4 following
the microwave disruption, wet oil extraction, transesterication,
and direct transesterication steps. The pretreatment process
transformed wet microalgae into more concentrated microalgae
cake, allowing large scale operation of microalgae oil extraction.
The transesterication of the extracted microalgae oil in hexane
was very efcient at a low reaction temperature of 45 C with
simultaneous removal of the unwanted chlorophyll content.
Furthermore, direct transesterication of the pretreated microal-
gae cake for biodiesel production was successful and showed high
potential for practical applications.
Acknowledgements
This work was nancially supported by the Ministry of Science
and Technology, Taiwan, in the framework of the project MOST
104-3113-E-006-003 and MOST 103-2221-E-006-190-MY3. This
work is also partially supported by the Top University Grants for
National Cheng Kung University issued by Taiwans Ministry of
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