Bioresource Technology 128 (2013) 337344

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

Ultrasound assisted extraction of carbohydrates from microalgae as feedstock

for yeast fermentation
Guili Zhao, Xue Chen, Lei Wang, Shixiao Zhou, Huixing Feng, Wei Ning Chen , Raymond Lau
School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore

h i g h l i g h t s

" Carbohydrates from microalgae can be a promising feedstock for yeast culture to produce biofuels.
" Pretreatment of microalgae and carbohydrates extraction from algal cell were performed.
" Four factors of ultrasound assisted extraction were examined by fractional factorial design.
" Rened model was conrmed as a good tting model via analysis of variance (ANOVA).
" Yeast biomass of the group with glucose from microalgae was much higher than that of control group.

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

Article history: Recently, carbohydrates biomass from microalgae is considered as a promising and inexpensive feedstock
Received 16 July 2012 for biofeuls production by microorganism fermentation. The main obstacle of the process is microalgae
Received in revised form 10 October 2012 pretreatment and carbohydrates extraction from algal cell. In this study, comparison of three pretreat-
Accepted 11 October 2012
ment methods was performed and the results showed that ultrasonic assisted extraction (UAE) was very
Available online 22 October 2012
effective. The effects of four parameters (ultrasonic power, extraction time, ow rate and algal cell con-
centration, respectively) on extraction efciency were also investigated. Additionally, in order to identify
Keywords:
signicant factors for glucose yield, combination of these four parameters was examined by using frac-
Microalgae
Ultrasound
tional factorial design (FFD) and the regression model was obtained. Meanwhile, the rened model
Carbohydrates extraction was conrmed as a good tting model via analysis of variance (ANOVA). After extraction, glucose
Yeast growth obtained from microalgae was used as substrate for Rhodosporidium toruloides fermentation and yeast
biomass was much higher than that of control culture.
2012 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae can serve as an excellent feedstock for the biofuels

It is well known that energy has an essential inuence on one
countrys economy and its continued economic growth and devel-
opment. However, due to large consumption of fossil fuels and its
non-renew ability, there exists serious energy crisis in the whole
world and fossil fuels will be exhausted one day. Additional, global
warming is increasingly severe and it is regarded as a consequence
of large emissions of greenhouse gas such as carbon dioxide result-
ing from fossil fuels burning (Lam and Lee, 2012). Therefore, it is
urgent to develop sustainable and clean energy from renewable re-
sources. At present, biofuels produced from microorganisms are
recognized as an attractive alternative of fossil fuels, such as bio-
diesel, bioethanol and biohydrogen (Chisti, 2008; Kito-Borsa
et al., 1998; Wang and Wan, 2009; Levin et al., 2004).
Corresponding authors. Tel.: +65 6316 2870.
E-mail addresses: WNChen@ntu.edu.sg (W.N. Chen), WMLau@ntu.edu.sg (R.
Lau).
production. For example, many paper reported that microalgae
can accumulate lipids in the range 2050% of dry cell weight
(DCW) for biodiesel production (Chisti, 2007; Zhao et al., 2012). Be-
sides, according to the type of feedstock used, biofuels is classied
into two generations that are produced from sugar/starch crops
through conventional technology and lignocellulosic biomass,
respectively (Nigam and Singh, 2011; Chiaramonti, 2007). Bioeth-
anol production from microalgae is considered as the third gener-
ation biofuels, which can overcome the major drawbacks of rst
and second generation biofuels to great extent (John et al., 2011).
More important, some microalgae species like Chlorella, Dunaliella,
Chlamydomonas, Scenedesmus, Spirulina have high carbohydrates
(mainly starch) content (>50% of the DCW) (Ueda et al., 1996).
The carbohydrates from microalgae can be hydrolyzed and con-
verted into glucose which is a very signicant substrate for hetero-
trophic microorganisms (like yeast, bacteria and fungus) to
produce biofuels. For instance, in a study, Chlamydomonas could
contain around 60% carbohydrates (44% of which was starch)

0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.10.038
338 G. Zhao et al. / Bioresource Technology 128 (2013) 337344

which was hydrolyzed and converted into glucose. Then ethanol Table 1
fermentation was conducted by Saccharomyces cerevisiae using Effects of three factors on carbohydrates extraction from Chlorella sp. using
conventional solvent extraction.
hydrolyzate and 235 mg ethanol/g algae were obtained (Choi
et al., 2010). A recent paper reported a novel process that was con- Run Time (min) Temperature Ratio Glucose yield
version carbohydrate in the algae into glucose prior to lipid extrac- (C) (liquid/solid) (ml/ (g/100 g
g) DCW)
tion, which could increase up to 15% lipid accumulation by other
oleaginous microorganism (Trzcinski et al., 2012). Apart from high 1 30 100 30 6.67 0.11
2 60 100 30 6.97 0.17
lipid/carbohydrates content, microalgae can be considered as an 3 90 100 30 8.34 0.18
excellent feedstock because of other advantages, such as fast 4 90 60 30 6.44 0.05
growth, no competition with food crops for land surfaces and high 5 90 80 30 7.50 0.16
CO2 xation efciency. Thus, it will decrease the cost of biofuels 6 90 100 20 8.29 0.15
7 90 100 40 9.06 0.05
production using glucose produced from microalgae via photosyn-
thesis as the substrate for other microorganisms fermentation. In
this study, Chlorella sp. was cultivated using CO2 as carbon source
for carbohydrates accumulation and glucose obtained was used to
Table 2
feed Rhodosporidium toruloides for lipid production.
Effects of three process parameters on carbohydrates extraction from Chlorella sp.
Meanwhile, the main obstacle of carbohydrates/glucose pro- using uidized bed extraction.
duction is that algae rigid cell walls are hard to break, so pretreat-
Run Time (min) Air ow rate Cell concentration Glucose yield
ment or extraction is the most important step so that intercellular
(L/min) (g/L) (g/100 g DCW)
carbohydrates can be released (Choi et al., 2010; Libessart et al.,
1 90 6.12 1.0 3.53 0.31
1995). Previous paper reported carbohydrates could be extracted
2 120 6.12 1.0 3.77 0.28
using many pretreatment methods, such as enzymatic pretreat- 3 150 6.12 1.0 3.93 0.19
ment (Choi et al., 2010; Correiaa and Beiro-da-Costa, 2012), 4 120 4.37 1.0 2.91 0.20
chemical extraction method like alkaline pretreatment (Correiaa 5 120 7.65 1.0 4.56 0.13
and Beiro-da-Costa, 2012; Ray and Lahaye, 1995) and physical 6 120 6.12 0.5 2.16 0.21
7 120 6.12 1.5 4.81 0.23
method, such as hot-water treatment, microwave-assisted
extraction and ultrasonic assisted extraction (Velmurugan and
Muthukumar, 2012; Ying et al., 2011; Donot et al., 2012; Yoshida
et al., 2010). There exist some selection criteria of extraction meth- in the laboratory. The composition of the medium was as follow-
od that the pretreatment is effective for qualitatively and quantita- ing: Glucose 10 g/L, Peptone 5 g/L, Yeast extract 3 g/L, and Malt ex-
tively, and the technology is simply to operate and economical for tract 3 g/L, Agar 2%. In this study, the yeast cells were cultivated in
scale up. For instance, the composition of cell wall is complicated the YM medium under different glucose concentration or in the YM
and unknown to some microalgae species, and some enzymes medium with glucose obtained from microalgae. The yeast culture
are very expensive. Thus, enzymatic pretreatment is not a feasible was carried out using sterilized 50 ml tube (with 15 ml medium)
method. In order to obtain effective extraction method of carbohy- under 25 C and 200 rpm. R. toruloides is oleaginous yeast and con-
drates, comparison of three methods was carried out and effects of tains high lipid content up to 57% (Wu et al., 2011).
several operation parameters on glucose yield for each method
were studied in this paper. The results showed that ultrasonic 2.2. Conventional solvent extraction (CSE) method
assisted extraction was more effective than the other methods.
Then signicant factors of ultrasonic assisted extraction using Conventional solvent extraction was carried out in water bath
FFD were found out in this work. Finally, carbohydrates extracted so that the temperature could be controlled. Algal cells were har-
from microalgae were converted into glucose to feed R. toruloides vested and lyophilized using freeze drier, then about 1.0 g dry mic-
culture for biofuels production. roalgae was mixed with distilled water in 50 ml tube using vortex
mixer. Extraction was performed total 7 runs according to different
conditions, such as extraction time, extraction temperature and ra-
2. Methods tio of liquid to solid. Details were shown in Table 1.

2.1. Strains and culture conditions 2.3. Fluidized bed extraction (FBE) method

The strain of Chlorella sp. (American Type Culture Collection,
ATCC 14854) was provided by Dr. Raymond Lau and the algal cells
were cultivated using modied R-medium. The modied medium
increased the specic growth rate and biomass (Chen et al.,
2011) and the lower sulfur and nitrogen adding could improve
starch accumulation in microalgae cells (Brnyikov et al., 2011).
The composition of modied medium was: sodium citrate 0.5 g/
L, sodium acetate 1.804 g/L, MgSO47H2O 0.1 g/L, NH4NO3 0.3 g/L,
KH2PO4 0.2 g/L, K2HPO43H2O 0.393 g/L, FeCl36H2O 0.01 g/L,
CaCl22H2O 0.053 g/L, H3BO4 1.0 mg/L, MnSO4H2O 0.303 mg/L,
ZnSO47H2O 1.0 mg/L, Na2MoO42H2O 0.2 mg/L, CuSO45H2O
0.0625 mg/L, CoCl26H2O 0.2 mg/L. The Chlorella sp. was cultured
in 5 L air lift photo-bioreactor with mixture gas (air and CO2)
contained 2% (v/v) CO2 under 25 C and 100 lmol/m2s. The total
ow rate was set at 0.26 vvm.
The strain of R. toruloides (ATCC 10788) was obtained from
American Type Culture Collection and stored using YM medium
Fluidized bed extraction was carried out in a uidized reactor
with high ow rate of air. The Chlorella sp. culture was harvested
and washed with distilled water, then diluted to certain cell con-
centration using distilled water. The obtained algal culture and
about 10 ml glass beads were added into uidized reactor with
air aeration. Extraction was conducted referring to the conditions
shown in Table 2.
2.4. Ultrasonic assisted extraction (UAE) method
An ultrasonic processor (UIP 1000hd ultrasonic mixer, Hiescher)
was used to extract carbohydrates from microalgae in ultrasonic
pretreatment. The algal cells were harvested and washed with dis-
tilled water, and diluted to certain biomass concentration. After
that, 500 ml obtained algal culture was used for every run in
extraction process. The microalgae culture could ow in a circle
in ultrasound equipment but ultrasonic wave appeared only in
 G. Zhao et al. / Bioresource Technology 128 (2013) 337344
Table 3
Comparison of cell breakage rate under different ultrasonic power and extraction time.
Run 1 2
Different power: (time: 40 min; ow rate: 1.52 L/min; cell concentration: 0.3 g/L)
Power (w) 600 800
Cell breakage rate (%) 77.5 2.2 89.7 1.7
Different time: (power: 800 w; ow rate: 1.52 L/min; cell concentration: 0.3 g/L)
Time (min) 20 40
Cell breakage rate (%) 73.9 1.2 89.7 1.7
Each data is expressed the mean SD.
ultrasound chamber, so this equipment could work continuously.
In addition, cool water could help to reduce heat and control the
temperature. In order to study the effect of conditions on carbohy-
drates extraction, experiments were done at different ultrasonic
power (600, 800 and 1000 w), different working time (20, 40, 60,
80 and 100 min, respectively), different ow rate (0.92, 1.52 and
2.1 L/min) and different cell concentration (0.3, 1.0 and 3.0 g/L).
2.5. Carbohydrates conversion, determination of glucose concentration
and analysis of carbohydrates after hydrolysis
After extraction, the algae solution was centrifuged at 8000 rpm
for 5 min. The supernatant and sediment part were obtained for
next step. The chlorophyll in supernatant could be removed by
centrifuge after heating. After that, 1.5 ml supernatant in 2 ml tube
was evaporated to about 0.2 ml using a rotary evaporator at 55 C
under vacuum. The resulting solution was mixed with 0.8 ml dehy-
drated ethanol (keep ethanol nal concentration at 80%) under 4 C
overnight. Then the solution was centrifuged at 8000 rpm for
10 min, and washed several times with dehydrated ethanol (Zhong
and Wang, 2010). The precipitate extract was crude carbohydrates
from microalgae. The supernatant was evaporated again using the
rotary evaporator at 55 C under vacuum to collect remain carbo-
hydrates. Then 1 ml 30% perchloric acid was added into the tube
reacting for 45 min at 30 C so that carbohydrates could be hydro-
lyzed and converted into glucose. For sediment part, sediment was
dried using freeze-drier, and about 6 mg dry sediment powder was
mixed with 1.5 ml 80% (v/v) ethanol to extract chlorophyll at 70 C
for 15 min. Then supernatant was discarded after centrifuge and
this extraction process was repeated two times. After that, the
resulting sediment was hydrolyzed using 1.5 ml perchloric acid
(30%) stirring for 15 min at 30 C, and the extract was collected
after centrifuge. The procedure was repeated two times, and the
extract was prepared for glucose detection.
The glucose concentration was determined using anthrone-
sulfuric acid method (Brnyikov et al., 2011). The crude glucose
extracts from supernatant and sediment part were diluted with
30% perchloric acid to certain concentration. Thereafter, 0.3 ml
extract was mixed and reacted with 1.5 ml anthrone solution [2 g
anthrone in 100 g 98% (v/v) sulfuric acid] in cooled water. The mix-
ture in glass tube was placed into 100 C water bath for 10 min.
After cooled to room temperature, absorbance of the mixture
was detected at 620 nm using UV/Visible spectrophotometer
(BioSpec-mini, Shimadzu, USA). The standard curve was obtained
using different glucose concentration at 0, 10, 20, 30, 40 60, 80
and 100 lg/ml, respectively. The glucose concentration was
expressed as following regression equation: y = 0.0048x  0.0047
(R2 = 0.9969, P < 0.05), where y (lg/ml) is absorbance of glucose
solution at 620 nm, x is the glucose concentration. Finally, for
one sample, the total glucose yield (supernatant and sediment)
was obtained for per 100 g dry cell weight.
Crude carbohydrate obtained from microalgae was hydrolyzed
in a tube using 1% H2SO4 solution for 120 min at 80 C. After that,
the monosaccharide composition was analyzed by a Shimadzu
QP2010Plus GCMS system (Shimadzu, Japan).
339
3 4 5
1000
97.9 0.3
60 80 100
94.9 1.1 98.4 0.5 99.0 0.5
2.6. Calculation of algal cell breakage rate
The Chlorella sp. culture before and after pretreatment were di-
luted with distilled water to appropriate concentration and the al-
gal cells were counted using blood counts plate under microscope.
Algal cell breakage rate was calculated as following formula:
Cell breakage rate
the total cells before treatment  cells left after treatment
 100%
the total cells before treatment
2.7. Fractional factorial designs
Fractional factorial designs are experimental designs consisting
of a fraction runs of a full factorial design, and less experimental
runs can help to nd out important variables using FFD. Referring
to a study (Montgomery, 2005), some factors which affected carbo-
hydrates yield signicantly were identied with the aid of 24-1 FFD.
Thus, in this paper, 24-1 FFD was designed to obtain signicant ef-
fects on ultrasonic assisted extraction, and eleven experimental
runs in low (coded 1) and high (coded +1) levels with triplicate
center points were carried out. These factors were ultrasonic
power, working time, ow rate and cell concentration and the de-
tails of design were based on preliminary experiments (shown in
Table 4). The following regression equation was obtained by the
least-square method (LSM) using statistical software JMP 10 (SAS
institute):
X
k X
k
Y i b0 bi X i b1i X 1 X i
i1 i1
where yi is the glucose yield obtained from microalgae; xi is the
independent variables; and bs are the regression coefcients. After
that, ANOVA was performed to conrm the model t and validation.
2.8. Preparation of yeast medium
For yeast medium preparation, carbohydrates extracted from
microalgae were collected and hydrolyzed by 1% (w/w) H2SO4 for
90 min at 80 C, and the ratio of carbohydrate extracts to 1%
H2SO4 solution is about 1:2 (v/v). After carbohydrates conversion,
the hydralyzate solution was added into yeast YM medium (no glu-
cose) to feed yeast fermentation. Before that, crude glucose solu-
tion from microalgae was mixed with distilled water and other
YM chemicals. Due to addition a small amount of 1% H2SO4 for
hydrolysis, then pH was adjusted to 56 using NaOH solution. It
will not obviously affect the subsequent cultivation of yeast.
2.9. Determination of yeast biomass and analysis composition of lipid
from yeast
Optical density measurement was used to determine biomass
concentration of R. toruloides at 600 nm using an UV/Visible spec-
trophotometer (BioSpec-mini, Shimadzu, USA). Every sample was
340 G. Zhao et al. / Bioresource Technology 128 (2013) 337344

Table 4
Independent variables of the FFD and the yield of glucose obtained from microalgae.

Run Coded variables Real variables Glucose yield (g/100 g DCW)

x1 x2 x3 x4 X1 X2 X3 X4
1 0 0 0 0 900 60 1.22 0.65 30.55 1.36
2 0 0 0 0 900 60 1.22 0.65 33.91 1.78
3 0 0 0 0 900 60 1.22 0.65 31.67 2.01
4 +1 +1 1 1 1000 80 0.92 0.3 37.35 2.28
5 +1 1 +1 1 1000 40 1.52 0.3 36.85 1.35
6 +1 +1 +1 +1 1000 80 1.52 1.0 36.21 1.45
7 1 1 1 1 800 40 0.92 0.3 22.87 2.45
8 1 1 +1 +1 800 40 1.52 1.0 19.24 1.99
9 +1 1 1 +1 1000 40 0.92 1.0 31.14 2.74
10 1 +1 +1 1 800 80 1.52 0.3 36.94 2.46
11 1 +1 1 +1 800 80 0.92 1.0 27.44 1.65

X1, X2, X3 and X4 represent ultrasonic power (w), extraction time (min), ow rate (L/min) and cell concentration (g/L), respectively.

diluted with distilled water to make sure an absorbance within the
range 0.11.0 if optical density was more over 1.0.
After fermentation, 3 ml certain concentration of yeast biomass
was collected by centrifuge and washed twice using distilled
water. Then 3 ml dehydrated methanol was added into yeast
sludge. After mixing completely, the yeast cells were broken using
ultrasound, and 2 ml distilled water and 1 ml chloroform were
added into mixture, respectively. After 1 h of lipid extraction in
the shaker, mixture was centrifuged again and the lower phase
solution was transferred into a 2 ml tube for rotary evaporation
to dryness. Next, 0.5 ml BF3CH3OH (14%, v/v) was added into
the tube and reacted for 20 min at 90 C. After that, 0.05 ml satu-
rated NaCl solution and 0.3 ml hexane were added into the tube,
respectively, and then mixed and centrifuged. The upper phase
samples with fatty acid were analyzed using GCMS. The detection
conditions were as follows: DB-5 fused-silica capillary column
(Agilent J&W Scientic, USA), 30 mm (length)  250 lm (inner
diameter)  0.25 lm (thickness); the solvent cutoff time was set
at 3.5 min; the injection and ion source temperatures were 280
and 200 C, respectively; the oven temperature was kept at
100 C for 4 min and raised to 320 C (remaining for 1.56 min) by
the rate of 4 C/min; carrier gas was helium which was controlled
at the ow rate of 1.1 ml/min.
3. Results and discussion
3.1. CSE and FBE of carbohydrates from Chlorella sp.
CSE is the most common and classical method for carbohy-
drates extraction. At the same time, the process of CSE is simple
and easy to control. The effect of different operational conditions
on glucose yield obtained from Chlorella sp. was investigated in this
work (Table 1). The results showed that glucose yield was about
6.59.0 g/100 g DCW of Chlorella, and different condition had cer-
tain inuence on carbohydrates extraction and glucose yield. Spe-
cically, long extraction time and high temperature could improve
the efciency of carbohydrates extraction, which was due to the
fact that algal cells could be broken more effectively under these
conditions. Meanwhile, maximum glucose yield was obtained at
the highest ratio of liquid to solid (40 ml/g). As a consequence,
the optimal condition of CSE was obtained at 90 min, 100 C and
40 ml/g.
There is few paper related to uidized bed extraction of carbo-
hydrates. In fact, FBE was designed and carried out by the author
according to equipment in the laboratory. With the help of large
amount of glass beads and high ow rate of air, the friction
between algal cells and glass beads could be increased, which
leaded to serious damage to Chlorella sp. cell wall. Finally, carbohy-
drates could be released to exterior solvent. Table 2 indicated the
results of carbohydrates extraction efciency under different
extraction time, air ow rate and cell concentration of microalgae.
As shown in Table 2, it was found that glucose yield of FBE was low,
and maximum glucose yield reached only 4.81 0.23 g/100 g DCW.
However, air ow rate and cell concentration affected carbohy-
drates extraction and glucose yield signicantly. More specic,
high air ow rate enhanced glucose yield from microalgae, and
the largest glucose yield was obtained at maximum cell concentra-
tion of 1.5 g/L. The two phenomena illustrated that friction be-
tween algal cells and glass beads was very important for
extraction efciency. Therefore, FBE method could be improved
and modied by adding more glass beads and raising air ow rate.
3.2. Effects of operation parameters of UAE on carbohydrates
extraction
Effects of four factors (including ultrasonic power, extraction
time, ow rate and algal cell concentration) of UAE on glucose yield
from microalgae were shown in Fig. 1. For ultrasonic power stud-
ied in the rst experiment (Fig. 1a), other extraction conditions
were xed as following: extraction time of 40 min, ow rate of
1.52 L/min, and cell concentration of 0.3 g/L. Obviously, ultrasonic
power had a positive impact on extraction efciency. There was a
signicant increase in total glucose yield with the enhancement of
ultrasonic power, and maximum yield (36.85 1.35 g/100 g DCW)
was gained at the highest power provided by ultrasonic equipment
(1000 w). Meanwhile, stronger ultrasonic power boosted the car-
bohydrates extraction efciency in both supernatant and sediment
part. Thus, ultrasonic power was an extremely important factor for
carbohydrates extraction from microalgae. The results obtained
were very similar to a previous report (Liu et al., 2010). As a result,
in this one-factor experiment, 1000 w was the optimal sonic
power.
Fig. 1b described the effect of extraction time on yield of glucose
obtained from Chlorella sp. cell at the ultrasonic power of 800 w,
ow rate of 1.52 L/min and cell concentration of 0.3 g/L. As shown
in Fig. 1b, the total glucose yield obtained increased rapidly with
the extension of extraction time. However, the total glucose yield
was stable after 80 min, and especially, glucose yield in sediment
had a remarkable increase before 80 min extraction but decreased
rapidly after 80 min. The phenomena maybe illustrated that almost
all carbohydrates were extracted from algal cell. Consequently, the
optimum extraction time was 80 min with the maximum glucose
yield reaching around 36.94 2.46 g/100 g DCW. The results indi-
cated that extraction time played an essential role in carbohy-
drates extraction using UAE.
Effects of ow rate on carbohydrates extraction was showed in
Fig. 1c, and the condition was xed at ultrasonic power of 800 w,
extraction time of 40 min and cell concentration of 0.3 g/L. Section
 G. Zhao et al. / Bioresource Technology 128 (2013) 337344 341

a 40
Supernatant
b 45
Supernatant
35
Sediment 40 Sediment
Total Total

Glucose yield (g/100g DCW)

Glucose yield (g/100g DCW) 35
30

30
25
25
20
20
15
15

10
10

5 5

0 0
600 800 1000 20 40 60 80 100
Power (w) Time (min)

c 35
Supernatant
d 45
Supernatant
Sediment 40 Sediment
30 Total
Total
35
Glucose yield (g/100g DCW)
Glucose yield (g/100g DCW)

25
30

20 25

20
15

15
10
10

5
5

0 0
0.92 1.52 2.1 0.3 1.0 3.0

Flow rate (L/min) Cell concentration (g/L)

Fig. 1. Effect of four process parameters on glucose yield from microalgae using ultrasound assisted extraction: (a) ultrasonic power, (b) extraction time, (c) ow rate and (d)
cell concentration. Each data indicates the mean SD.

2.4 mentioned that the microalgae culture could ow a circle in
ultrasound machine, and the cell would pass the ultrasound cham-
ber one time for every circle. Therefore, the ow rate of microalgae
culture would have an impact on extraction efciency. As shown in
Fig. 1c, maximum total glucose yield was obtained at the moderate
ow rate of 1.52 L/min, and there was slight difference at lower
ow rate of 0.92 L/min for carbohydrates extraction. However, glu-
cose yield decreased signicantly from 25.00 1.65 g/100 g DCW
to 19.81 1.81 g/100 g DCW when the ow rate increased from
1.52 to 2.10 L/min. The above results demonstrated that high ow
rate had a negative effect on total glucose yield obtained from
microalgae.
The effects of algal cell concentration on carbohydrates extrac-
tion were studied and the results were shown in Fig. 1d. The
extraction conditions were xed as following: ultrasonic power
of 800 w, extraction time of 80 min and ow rate of 1.52 L/min.
Fig. 1d showed that cell concentration also affected extraction ef-
ciency signicantly. Total glucose yield declined obviously as the
cell concentration increased from 0.3 to 3.0 g/L. However, total glu-
cose yield was still very high (31.35 3.15 g/100 g DCW) at the cell
concentration of 1.0 g/L.
According to previous discussion, the highest amount of glucose
(about 36.94 2.46 g/100 g DCW) was obtained after 80 min pre-
treatment and glucose yield would be stable with time extension
(Fig. 1b). Meanwhile, it can be seen from Fig. 1a and c that the
maximum glucose yields were also around 36 g/100 g DCW. Be-
sides, as shown in Fig. 1b, glucose from supernatant part increased
as time extended after 80 min while that decreased from sediment
counterpart, and as a result, the total glucose yield was the same at
the two points. These above phenomena indicated that almost car-
bohydrates were extracted from both supernatant and sediment
under certain optimal condition. However, under other pretreat-
ment conditions, the sum of glucose from supernatant and sedi-
ment was much lower than 36 g/100 g DCW. It might because
that microalgae cell could not be broken completely but certain
damage on cell wall, so glucose could not be extracted thoroughly
from microalgae cell or sediment.
A similar study reported (Ying et al., 2011) that UAE had larger
yield of polysaccharides with less extraction time compared to CSE,
and the maximum polysaccharides yield was obtained using UAE
method between three pretreatment methods (they were CSE,
microwave-assisted extraction and UAE, respectively). Meanwhile,
in that study, the sample concentration (ratio of liquid to solid) had
a very slight effect on polysaccharides yield using CSE method. The
result of this work was similar and showed that carbohydrates
yields were quite low at any ratio of liquid to solid under CSE. Thus,
compared with CSE and FBE, it was found that UAE was an effective
method for carbohydrates extraction from Chlorella. Generally,
the total glucose yield was over 20 g/100 g DCW which was
much more than that of CSE and FBE. In addition, at high algal
342
concentration, high extraction efciency could also be achieved by
extending extraction time and increasing extraction power since
the extraction time and power affected extraction efciency signif-
icantly. Therefore, high algal concentration could be appropriate
for large scale pretreatment. Furthermore, referring to Section
2.4, ultrasonic equipment in this study was much different from
previous batch ultrasound machine. The microalgae culture was
forced to ow in a circle by a pump and ultrasonic wave was pro-
duced in ultrasound chamber, so microalgae sample could be pre-
treated continuously. Thus, UAE could be used in a larger scale and
might be improved and tried in industrial application.
3.3. Determination of cell breakage rate after ultrasound assisted
extraction
In order to interpret the phenomena and results obtained in
Section 3.2, the cell breakage rate of Chlorella sp. using UAE under
different conditions was calculated in this study. Table 3 showed
the cell breakage rate of microalgae under different ultrasonic
power and extraction time. More specic, the results indicated that
the change trend of total glucose yield were consistent with the
change of cell breakage rate, and total glucose yield increased with
cell breakage rate increasing when sonic power and extraction
time was raised respectively. Cell breakage rate was affected sig-
nicantly by power and extraction time. In addition, cell breakage
rate reached nearly 100% at 1000 w and 80 min respectively, so al-
most all carbohydrates were extracted from microalgae under
these conditions (nearly maximum total glucose yield). These re-
vealed that strong ultrasonic power and long extraction time en-
hanced disruption of cell wall so that they could heighten the
total yield of glucose from microalgae.
The cell breakage rate of different ow rate and cell concentra-
tion along with time was shown in Fig. 2. Referring to above anal-
ysis, as shown in Fig. 2, the trend of total glucose yield was the
same as the change of cell breakage rate with ow rate and cell
concentration. At 40 min data points, cell breakage rate of 1.52 L/
min was the highest value so that optimum extraction efciency
was gained under this condition, and cell breakage rate of 2.10 L/
min was very low resulting to less glucose yield (Fig. 2a). In fact,
it could be seen from Fig. 2a that the trend was maintained before
60 min extraction. That maybe due to the fact that algal cell passed
ultrasonic chamber very quickly every time under high ow rate of
2.10 L/min, so there was no enough time to break cell wall for one
time. However, after 80 min of extraction time, the damage differ-
ence of cell wall was very slight due to experiencing long time
110
a 0.92 L/min
100
1.52 L/min
90 2.10 L/min
80
Cell breakage rate (%)
70
60
50
40
30
20
10
0
0 20 40
Time (min)
Fig. 2. Cell breakage rate of different ow rate (a) and cell concentration (b). Each point is expressed as the mean SD.
G. Zhao et al. / Bioresource Technology 128 (2013) 337344
accumulation. It could be found directly that the effect of cell con-
centration on extraction efciency was more signicant than that
of ow rate from Fig. 2. However, there might exist different extent
of damage on algae cell and cell wall even at the similar cell break-
age rate, especially after long time pretreatment, which due to the
fact that only intact cells and regular shape cells were counted un-
der microscope and naked eye identication. Thus, there would be
error in the relationship between cell breakage rate and damage
extent. As a result, after long time pretreatment, glucose yield
might be different while cell breakage rate was very close, but
the trend that glucose increased as the cell breakage rate increased
was obviously shown.
3.4. Fractional factorial design of ultrasound assisted extraction
Four independent variables combined were examined and the
effects of ultrasonic assisted extraction on carbohydrates were per-
formed by using FFD. The conditions and results of FFD were
shown in Table 4. The data described the responses of the design
for the yield of glucose from microalgae. Based on the analysis of
the data, the Eq. (1) of the regression model was obtained as
follows:
Y 31:29 4:38x1 3:48x2 1:31x3  2:50x4  2:09x1 x2
 0:16x1 x3 0:79x1 x4 1
From the above equation, the positive coefcients of ultrasonic
power, extraction time and ow rate illustrated that these factors
had a positive effect on the yield of glucose extracted from microal-
gae, while cell concentration had a negative impact on glucose yield
with a negative coefcient (2.50).
ANOVA of the regression model was summarized in Table 5. For
model validation, the model P-value obtained from ANOVA should
not exceed 0.05 and the coefcient of determination (R2) value
should be greater than 0.9 (Benita, 1996). The P-value of model
(0.0179) was less than 0.05, so it was signicant at the probability
level of 5%, which suggested that the model t the data. Mean-
while, the correlation coefcient (R2) value of was 0.977 and this
indicated that the sum of square of the model accounted for
97.7% of total sum of square and the model could explain 97.7%
variability of the response variables. Besides, the P-value of lack
of t test was more than 0.05, which indicated that there was no
lack of t in the model at 95% condence level. As a result, the pre-
diction model could be accepted.
110
b 0.3 g/L
100
1.0 g/L
90 3.0 g/L
80
Cell breakage rate (%)
70
60
50
40
30
20
10
0
-10
60 80 100 0 20 40 60 80 100
Time (min)
 G. Zhao et al. / Bioresource Technology 128 (2013) 337344
Table 5
Analysis of variance for regression models of FFD.
Degree of freedom Sum of square
Regression model of FFD
Model 7 354.060
Residual 3 8.206
Lack of t 1 2.352
Pure error 2 5.853
Total 10 362.266
R2 0.977
Screening regression model of FFD
Model 4 335.295
Residual 6 26.971
Lack of t 4 21.118
Pure error 2 5.854
Total 10 362.266
R2 0.925
*
Represents signicant at the probability level of 5%.
Fig. 3. The standardized effect of variables on glucose yield for Eq. (1) showed in Pareto chart. X axis indicated the t ratio of the variables. The bars passed the blue vertical line
illustrating values achieving statistical signicance (a = 0.05). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of
this article.)
In order to nd out the important and signicant variables for
glucose yield in Eq. (1), Pareto chart was constructed in Fig. 3.
The chart showed the effect of the independent variables and their
interactions on glucose yield. The t-ratio in the chart was com-
pared to a critical t value which was shown as the value of the blue
vertical line in the Pareto chart. As shown in Fig. 3, the independent
variables whos chart length (t-ratio) passed the blue vertical line
(tcritical at P > 0.05) were signicant factors on the yield of glucose
from microalgae (ultrasonic power, extraction time, cell concentra-
tion and interaction of power and time, respectively). Thus, in or-
der to simplify the process parameters, the rened model was
obtained as following Eq. (2) by retaining signicant factors:
Y 31:29 4:38x1 3:48x2  2:50x4  2:09x1 x2
Table 5 described the ANOVA for the rened model. It could be
found that the P-value of the rened model was signicant enough
(0.0016) with no signicance in lack of t (0.387). And the coef-
cient of determination value (R2 = 0.925) also indicated good tting
for the model.
3.5. Composition analysis of carbohydrates after hydrolysis
Carbohydrates extracted from microalgae were hydrolyzed by
diluted sulfuric acid and the monosaccharide composition was
analyzed using GCMS. The results showed that there were mainly
three monosaccharides in the carbohydrates hydrolyzate, includ-
ing glucose, galactose and arabinose. The content proportion of
these three sugars was also obtained using a mixed standard
solution via GCMS detection. The results showed that the most
predominant monosacchride in the total sugars was glucose
(around 80%) which was the main carbon source for R. toruloides
fermentation. The above results were very similar to the previous
research (Choi et al., 2010) which reported that glucose obtained
343
Mean of square F-value P-value
50.580 18.491 0.0179*
2.735
2.352 0.804 0.4646
2.927
83.824 18.647 0.0016*
4.495
5.279 1.804 0.387
2.297
from microalgae was considered as feedstock for yeast
fermentation.
3.6. Yeast fermentation using glucose obtained from Chlorella sp.
After extraction, the carbohydrates obtained from Chlorella sp.
were collected and then hydrolyzed by acid. Finally, carbohydrates
were converted to glucose which was added into YM medium pre-
paring for yeast fermentation. Meanwhile, glucose concentration
was 2.4 g/L in normal YM medium group (with chemical glucose)
and the group with glucose from microalgae, respectively; and
the glucose-free culture was the control group. The results were
2 shown in Fig. 4 for 36 h fermentation of R. toruloides. It was found
that yeast could grow well in the culture with glucose extracted
from Chlorella. Obviously, yeast maximum biomass of the group
with glucose from microalgae was much higher than that of the
control culture, and was no much difference from the biomass in
the normal medium. The results demonstrated that carbohydrates
extracted from Chlorella sp. could be considered as a promising and
inexpensive feedstock for R. toruloides growth.
Furthermore, other microorganism could use carbohydrates
biomass from microalgae as substrate to produce biofuels. A study
reported that 0.77 g/L starch accumulated in Chlamydomonas rein-
hardtii was feedstock for biohydrogen production by anaerobic fer-
mentation with Clostridium butyricum (Kim et al., 2006). Another
paper reported that 3.83 g/L bioethanol yield was obtained from
10 g/L lipid-extracted microalgae biomass via Saccharomyces
bayanus fermentation (Harun et al., 2010). Additionally, carbohy-
drates-rich Chlorella vulgaris (with 57% carbohydrates content)
was used as an excellent feedstock as biohydrogen production by
C. butyricum fermentation without any additional organic carbon
sources, and maximum hydrogen yield of 1.15 mol/mol reducing
sugar was achieved in a recent study (Liu et al., 2012). The future
344 G. Zhao et al. / Bioresource Technology 128 (2013) 337344
22
20
18
16
Cell concentration (OD600)
14
12
10
8 NY, pp. 209251.
6 Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26,
4 Choi, S.P., Nguyen, M.T., Sim, S.J., 2010. Enzymatic pretreatment of Chlamydomonas
Glucose from microalgae (2.4g/L)
2 Glucose (2.4g/L)
Glucose-free
0
-5 0 5 10 15 20 25 30 35
Time (h)
Fig. 4. Comparison of the biomass of Rhodosporidium toruloides in different
medium, culture without glucose (h), normal culture (d) and culture with glucose
from microalgae (j), respectively. Each data point represents the mean SD.
work of this study will be focus on biofuels production from R. tor-
uloides using carbohydrates obtained from microalgae. Moreover,
yeast biomass yield of the groups with glucose will be boosted
by increasing glucose concentration in the medium.
3.7. Composition analysis of lipid from yeast fermentation
After fermentation, lipid was extracted from yeast cells cultured
using glucose obtained from microalgae, and composition of the li-
pid was analyzed by GCMS. The results showed that the lipid from
R. toruloides mainly consisted of components of 16 and 18 carbons,
and they were C16:0 (36.9% of total lipid), C18:0 (47.7%), C18:1
(8.1%) and C18:2 (7.3%), respectively. The results indicated that
the lipid composition was quite similar to plants oil which was a
conventional raw material for biodiesel production. Therefore,
the lipid from R. toruloides also could be used for biofuels produc-
tion and carbohydrates from microalgae could be regarded as a
promising feedstock for yeast fermentation.
4. Conclusions
For carbohydrates extraction from microalgae, comparison of
three pretreatment methods was carried out in this study. The re-
sults showed that UAE was an effective method, and the maximum
glucose yield among FFD runs could reach 36.94 2.46 g/100 g
DCW under the extraction condition at ultrasonic power of
800 w, extraction time of 80 min, ow rate of 1.52 L/min and cell
concentration of 0.3 g/L. The combination of the four variables
was examined and regression model was obtained by using FFD.
Finally, R. toruloides could grow well utilizing the glucose extracted
from microalgae as feedstock.
Acknowledgements
The authors acknowledge funding support from the National
Research Foundation of Singapore and ultrasound equipment sup-
port from Wintershine Asia Pte Ltd.
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