4166

Energy & Fuels 2009, 23, 41664173

Alga-Based Biodiesel Production and Optimization Using Sugar

Cane as the Feedstock
Yun Cheng, Yue Lu, Chunfang Gao, and Qingyu Wu*
Department of Biological Science and Biotechnology, Tsinghua UniVersity,
Beijing 100084, Peoples Republic of China
ReceiVed April 30, 2009. ReVised Manuscript ReceiVed June 4, 2009

The alga Chlorella protothecoides is known to produce oil suitable for biodiesel preparation by heterotrophic
cultivation in media containing glucose as a carbon source. In this study, sugar cane juice was used as alternative
carbon supply for oil production. As a result, the highest oil content of 53.0% by cell dry weight was achieved.
Fermentation in a 5 L bioreactor showed that algae using sugar cane juice hydrolysate (SCH) grow faster than
that using glucose. Conversion ratios of sugar/biomass and sugar/oil using SCH were 15.2 and 8.8% higher
than that using glucose, respectively. Biodiesel prepared from algal oil by transesterification is mainly composed
of 9-octadecenoic acid methyl ester, 9,12-octadecadienoic acid methyl ester, and hexadecenoic acid methyl
ester. Our results suggest that sugar cane is a good feedstock for biodiesel production. Response surface
methodology upon exploring the effect of C/N and concentration of yeast extraction (YE) on the yield of
biomass and oil was performed. The optimal production with the highest output-cost coefficient of 0.061 (
0.004 was achieved when C/N was 26.9 and YE was 0.60 g L-1.

1. Introduction
The recent decade witnessed a surge of interest in algal oil
as a promising supplemental oil source for biodiesel production.
Many specific species of algae can use a carbon source for oil
accumulation under special conditions.1 Not all algal oils are
suitable for producing biodiesel; however, satisfactory oils occur
commonly. Usually triglyceride and fatty acids are the dominating components of algal oil, from which biodiesel can be
prepared by transesterification, yielding monoalkyl esters of fatty
acids and alcohols.2
Algae can grow with CO2 as the carbon source and sunlight
as the energy supply. However, both biomass productivity and
oil content of photoautotrophic cultures are extremely low. The
photoautotrophic technology mainly suffers from a limited
supply of light and lower energy conversion efficiencies.3 In
comparison to photoautotrophic cultivation, heterotrophic fermentation allows algae to accumulate a much higher proportion
of oil within less time and the scale-up is much easier. Thus, it
offers a potential pathway to produce oil for diesel production
in large scale.4 Various strains of algae, including Chlorella
protothecoides,5 Crypthecodinium cohnii,6 and Porphyridium
propureum,7 have been studied to achieve both high biomass
and oil or fatty acids productivity in heterotrophic cultures. In
our previous studies, we have reported a strategy of pilot-scale
* To whom correspondence should be addressed. Telephone/Fax: +8610-62781825. E-mail: qingyu@mail.tsinghua.edu.cn.
(1) Li, Y.; Horsman, M.; Wu, N.; Lan, C. Q.; Dubois-Calero, N.
Biotechnol. Prog. 2008, 24, 815820.
(2) Meng, X.; Yang, J.; Xu, X.; Zhang, L.; Nie, Q.; Xian, M. Renewable
Energy 2009, 34, 15.
(3) Grobbelaar, J. U. J. Appl. Phycol. 2000, 12, 201206.
(4) Eriksen, N. T. Biotechnol. Lett. 2008, 30, 15251536.
(5) Xiong, W.; Li, X. F.; Xiang, J. Y.; Wu, Q. Y. Appl. Microbiol.
Biotechnol. 2008, 78, 2936.
(6) Barclay, W. R.; Meager, K. M.; Abril, J. R. J. Appl. Phycol. 1994,
6, 123129.
(7) Wen, Z. Y.; Chen, F. Biotechnol. AdV. 2003, 21 (4), 273294.

fermentation of heterotrophic microalgae with high biomass

productivity and oil yield. The oil content of microalgae has
reached up to 50% of dry cell weight.8
To further improve the yield of algal oil and minimize the
cost of fermentation, optimizing the medium to achieve a
maximum performance of oil productivity seems to be of critical
importance, because medium composition can significantly
affect the yield and cost of products. The key factors associated
with oil yield are nitrogen content, C/N (ratio of carbon source/
nitrogen source), and carbon source.1,9 Currently, the cost of
producing biodiesel from algal oil is much higher than that of
diesel derived from petroleum, and the cost of carbon source
represents 50% of the cost of medium for algal cultivation.8
Therefore, economic considerations need to explore cheap and
easily available alternate feedstock.
Sugar cane is a highly efficient C4 crop and can store about
1% of the radiation on biomass per year.10 It is estimated that
750 L of raw sugar cane juice and 250 kg of wet bagasse can
be harvested from 1 ton of sugar cane stalks. The juice contains
18.5% sucrose and 1.7% other fermentable sugars, such as
glucose and fructose, indicating a yield of about 150 kg of
sugars/ton of stalks. Recently, sugar cane has received increasing
attention for its application in ethanol production. The most wellknown example is The Brazilian National Alcohol Program
lcool). A total of 50% of sugar cane
(Programa Nacional do A
production in Brazil is used to satisfy alcohol consumption.11
However, sugar cane has never been reported for biodiesel
production.
The major objective of this study was to evaluate the
feasibility of producing biodiesel from algal oil with sugar cane
(8) Li, X. F.; Xu, H.; Wu, Q. Y. Biotechnol. Bioeng. 2007, 98, 764
771.
(9) Yamaberi, K.; Takagi, M.; Yoshida, T. J. Mar. Biotechnol. 1998, 6,
4448.
(10) Lunn, J. E.; Furbank, R. T. New Phytol. 1999, 143, 221237.
(11) Moreira, J. R.; Goldemberg, J. Energy Policy 1999, 27, 229245.

10.1021/ef9003818 CCC: $40.75 2009 American Chemical Society

Published on Web 06/26/2009

Alga-Based Biodiesel Production Using Sugar Cane

as the feedstock. Our results suggest that sugar cane juice

hydrolysate (SCH) was an available and effective carbon source
for algal fermentation and oil production. We have achieved
the optimized medium composition for algal oil production and
have significantly improved the oil yield and efficiency of carbon
source conversion. This may provide a positive pathway for
industrial application of sugar cane in biodiesel production.
2. Materials and Methods

Energy & Fuels, Vol. 23, 2009 4167

media at an equivalent initial cell density. Heterotrophic cultivation
was carried out in 500 mL flasks at 28 ( 1 C with continuous
shaking (220 rpm). Samples were taken at regular intervals to
determine the cell density (g L-1) and reducing sugar concentration
(g L-1). Curves of cell growth and sugar consumption were recorded
by software Origin 7.0 (OriginLab Corporation, Northampton, MA).
2.5. Experimental Design and Optimization Using Response
Surface Methodology (RSM). Upon exploring the effect of C/N
and YE concentration on the production of biomass and oil, C/N
and YE concentration were chosen as independent variables.
Different amounts of SCH and YE were used to set different C/N
and added into basal medium. According to the central composite
design (CCD), C/N at 9, 15, and 21 was set as -1, 0, and +1. The
YE concentration at 0.1, 0.8, and 1.5 g L-1 was set as -1, 0, and
+1. The oil content (%), conversion ratio, and output-cost coefficient (described below) were used as responses. A two-factor
CCD, with three central points, two factorial points, and six axial
points (R ) 1), was employed for the optimization of oil production,
leading to a total of 11 runs (described below; Table 2). The
conditions of inoculation and heterotrophic cultivation of 11 total
runs were as described in section 2.4. Because the triglycerides
usually accumulate to a maximum amount and remain constant
during the stationary phase in nitrogen-depleted culture,1,13 cell
density was monitored. Then, cells were collected when cultures
reached the stationary growth phase.
Equation 1 was calculated with software Design Expert 7.1.3
(Static Made Easy, Minneapolis, MN) to estimate the responses to
the variables. The quadratic model for predicting the optimal point
was expressed as the equation

2.1. Microorganisms, Maintenance, and Inoculum. C. protothecoides was originally provided by Culture Collection of Algae
at University of Texas (Austin, TX). The methods of preparation
of medium, preparation of inoculum, and culture apparatus were
described before.5 Heterotrophic cells in exponential phase were
used to inoculate fresh media.
2.2. Selection of Nitrogen and Carbon Sources. According
to our previous work, among three inorganic nitrogen sources (urea,
potassium nitrate, and ammonium nitrate) and two organic nitrogen
sources (glycine and yeast extract), yeast extract (YE, 1-10 g L-1)
was studies and considered as the most suitable (1-4 g L-1)
nitrogen source to provide high biomass and oil yield.5 Therefore,
YE was selected as the nitrogen supplement in all cultures. The
most common carbon source maintaining heterotrophic growth was
glucose, which greatly contributed to biomass productivity. To study
the feasibility of SCH as the carbon source for biodiesel production,
glucose was used as a control to evaluate the cultivation kinetics
with that of SCH. In our previous observation, C/N at 10 was used
for oil production.5 In this work, a range of C/N covering 9-21
was investigated for further optimization. Nitrogenous compounds
may be present in sugar cane juice (SC). However, the components
and amount is limited and vary between sugar cane species. The
quantity of amino acids decreases during storage.12 Besides, it is
unknown whether nitrogenous compounds in SC can be used by
alga. Therefore, C/N when referred to cultivations with SCH is
considered as an apparent C/N ratio, which represents the ratio of
added carbon source/added YE.
2.3. Preparation of Sucrose Hydrolysate (SH) and SCH. To
obtain a carbon source suitable for microalgal growth, sucrose and
sugar cane juice were enzymatically hydrolyzed. Concentrated sugar
cane juice was commercially available (ZK007, Bai Guo Yuan Juice
and Food Co., Ltd., China, containing about 600-700 g L-1
sucrose). The sugar cane juice or 500 g L-1 sucrose solution was
supplemented with fructofuranosidase (Valisase Ivertase ANL,
15 000 Valley Summer units g-1) and hydrolyzed according to the
instructions provided by the manufacturer. The concentration of
reducing sugar (hydrolysate) was monitored at regular intervals by
the dinitrosalicyclic acid (DNS) method to determine the complete
digestion.
2.4. Media and Cultivation in a Shake Flask. The components
of basal medium for alga growth were as follows: 0.7 g L-1
KH2PO4, 0.3 g L-1 K2HPO4, 0.3 g L-1 MgSO4 7H2O, 3 mg L-1
FeSO4 7H2O, 0.01 mg L-1 vitamin B1, 1 mL L-1 A5 trace mineral
solution. To investigate the cell growth and oil accumulation in
algal cells using sugar cane as the feedstock, an equivalent amount
of glucose, sucrose, SH, SC, and SCH was added separately into
basal medium with 3 g L-1 YE to prepare media containing 20 g
L-1 different carbon source, in which glucose or sucrose powder
was weighted and directly added into media. Through diluting the
SH or SCH prepared in section 2.3, the concentration of reducing
sugar in SH or SCH media was measured by the DNS method and
adjusted to 20 g L-1. On the basis of the concentration of reducing
sugar in SCH, the content of sucrose in SC can be evaluated. SC
media were prepared by diluting the concentrated SC and adjusting
the amount of sucrose to 20 g L-1. All media and the cultivation
apparatus were sterilized with steam at 112 C and 0.12 MPa for
30 min. C. protothecoides in exponential phase was inoculated into

where Y is the predicted response, A and B are independent variables

that represent C/N and YE concentration, respectively, b0 is the
intercept, b1 and b2 are linear effects, b11 and b22 are squared effects,
and b12 is the interaction term. The variables and corresponding
responses were analyzed on the basis of experimental results to
evaluate the significance of quadratic models. The generated models
were also subjected to analysis of variation (ANOVA) using Design
Expert 7.1.3.
2.6. Fermentation Conditions. Heterotrophic fermentation was
completed in a 5 L bioreactor (Minifors, INFORS AG CH-4103,
Bottmingen, Switzerland) containing 3 L of basal medium. A
concentrated solution of YE (100 g L-1) and glucose or SCH
(400-500 g L-1) were fed to keep the concentration of YE and
carbon source at certain levels. KOH solution (0.5 M) was fed to
keep pH at 6.3 ( 0.3. Temperature was controlled at 28 ( 1 C.
The dissolved oxygen concentration was controlled between 20 and
50% air saturation by airflow and agitation speed. The cell density
and consumption of carbon source were determined and recorded
at regular intervals.
2.7. Cell Collection, Oil Extraction, and Biodiesel Preparation. Cells were collected by centrifugation and lyophilized to a
constant weight. The total oil in algal cells was extracted by Soxhlet
extraction. Alga-based biodiesel was prepared by transesterification
of algal oil from bioreactor cultures. The manipulation was
previously reported in details in a previous publication.14
2.8. Monitor of Cell Growth, Reducing Sugar Consumption, and Oil Content. Cell growth was monitored by optical
density measurements at 540 nm using a UV/vis spectrophotometer
(Pharmacia Biotech Ultrospec 2000, Sweden). Algal cells were
harvested and weighed after lyophilization. The concentration of
reducing sugar was analyzed by the DNS method. The oil content
was analyzed by time-domain nuclear magnetic resonance (TDNMR) on a Bruker Minispec MQ20 NMR analyzer (Bruker,

(12) Li, J. B.; Liu, H. X.; Lu, D. J. Sugarcane Canesugar 2003, 4, 3539 (in Chinese).

(13) Turcotte, G.; Kosaric, N. Biotechnol. Lett. 1989, 2 (9), 637642.

(14) Miao, X. L.; Wu, Q. Y. Bioresour. Technol. 2006, 97, 841846.

Y ) b0 + b1A + b2B + b12AB + b11A2 + b22B2

(1)

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Cheng et al.

Figure 1. Comparison of different carbon sources for microalgal heterotrophic cultivation. (a) Cell density (-b-) and reducing sugar (-O-) in
the medium with 20 g L-1 glucose and cell density ( b ) and reducing sugar ( O ) in the medium without glucose (basal medium).
(b) Cell density (-b-) and reducing sugar (-O-) in the medium with 20 g L-1 sucrose hydrolysate (SH) and cell density ( b ) and
reducing sugar ( O ) in the medium with 20 g L-1 sucrose without hydrolysis. (c) Cell density (-b-) and reducing sugar (-O-) in the
medium with 20 g L-1 sugar cane juice hydrolysate (SCH) and cell density ( b ) and reducing sugar ( O ) in the medium with 20 g
L-1 sugar cane juice without hydrolysis.

Rheinstetten, Germany) with a 35 mm absolute probe, at a

resonance frequency of 19.95 MHz.15
2.9. Composition Analysis of Biodiesel. The composition of
biodiesel produced from extracted algal oil was analyzed on gas
chromatograph (GC)-linked mass spectrometry (GC-MS), dualstage quadrupoles GC apparatus (Thermo, Waltham, MA), equipped
with a Varian VF-5 ms column (30 m 0.25 mm inner diameter,
DF ) 0.25 m), and the GC was manipulated with a flow rate of
10 mL min-1.

3. Results
3.1. Cell Growth and Oil Accumulation in C. protothecoides Using Sugar Cane as the Feedstock. Sugar cane juice
is rich in carbohydrate, including sucrose as well as fructose
and glucose. As far as we know, C. protothecoides was shown
to use hexose, such as glucose, fructose, galactose, etc. To
provide the carbon source available to algal assimilation, sugar
cane juice was processed by hydrolysis. Considering this, media
without carbon source supplementation (basal media with YE)
and glucose media were used as a comparison to evaluate the
effects of SCH media on heterotrophic cultivation of C.
protothecoides. In Figure 1a, algal cells do not exhibit a
significant growth in a shake flask. When glucose is supplemented, algal cells grow fast and the glucose is consumed. It is
known that C. protothecoides can also grow with CO2 and light
as energy. However, the cultivation condition in a shake flask
restrains the air and light supply. Accordingly, cultivation in
medium without an organic carbon source has difficulty in
maintaining algal growth (Figure 1a). Similar to the condition
in the medium without a carbon source supply (Figure 1a), algal
cells hardly grow with nonhydrolyzed sucrose or nonhydrolyzed
SC (panels b and c of Figure 1). Whereas, when hydrolyzed
sucrose (i.e., SH) or hydrolyzed SC (i.e., SCH) is added, algal
cells grow fast. This suggests that sucrose and sugar cane juice
could not be used directly by C. protothecoides before hydrolysis
treatment. However, both SH and SCH are available for algal
use. The lack of enzyme-digesting sucrose may account for this
phenomenon. We observed that the reducing sugar in nonhydrolyzed sugar cane juice was very low (Figure 1c), thus not
enough to support algal growth under heterotrophic conditions.
Table 1 showed that the biomass, oil content, and conversion
ratio obtained in SCH media reached comparable levels to those
of glucose media (Table 1). We also noticed that the oil content
(15) Gao, C. F.; Xiong, W.; Zhang, Y. L.; Yuan, W. C.; Wu, Q. Y. J.
Microbiol. Methods 2008, 75, 437440.

Table 1. Effects of Different Carbon Sources on Heterotrophic

Cultivation [Data Are Given as Means ( Standard Deviation
(SD), n ) 3]a
carbon
source
biomass
(20 g L-1) yield (g, DW)c
glucose
SH
SCH

1.43 ( 0.07
1.22 ( 0.05
1.23 ( 0.07

conversion ratio (%)b

consumed
sugar (g)

oil
content (%)

4.64 ( 0.18 46.7 ( 1.1

4.28 ( 0.20 44.4 ( 2.5
3.65 ( 0.15 43.1 ( 2.3

biomass/
sugar

oil/
sugar

30.8
28.5
33.8

14.4
12.6
14.6

a The final volume of media in flasks was 200 mL. b Conversion ratio
of sugar/biomass (%, g of biomass/g of consumed sugar) and conversion
ratio of sugar/oil (%, g of oil/g of consumed sugar). c The initial
inoculum was subtracted from the final biomass yield. The biomass
yield includes the weight of oil.

and the conversion ratios of sugar/biomass and sugar/oil were

not higher than that in our previous publications.5,8 However,
there is still considerable room for improvement on them after
optimization of C/N and YE concentration (see section below).
3.2. Effects of YE Concentration and C/N on Biomass
Production and Oil Accumulation. Because (1) nitrogen
source and concentration are important for heterotrophic growth
in algal cells and (2) SCH is an efficient carbon source for
heterotrophic growth of C. protothecoides, it is necessary to
investigate the effects of YE concentration and C/N on biomass
production and oil accumulation when using SCH as the
feedstock for alga-based biodiesel production. Data of 11 runs
show that a higher YE concentration leads to a higher biomass
yield (run 3 in Table 2) and a higher biomass productivity
(Figure 2a) but lower conversion ratio of sugar/biomass (Figure
2b). Oppositely, a low YE concentration (0.1 g L-1) leads to a
high conversion ratio of sugar/biomass of 66.5%. However, C/N
showed no significant influence on the cell growth and conversion ratio. Although maximum and minimum points did not
shown up on the response curves in Figure 2, the range of this
design has been effective enough to provide information for
analysis. On the other hand, experiments at higher C/N or higher
YE concentrations are not necessary, because the conversion
ratio of the carbon source will be extremely low (Figure 2b).
Low efficiency of use would not be considered in industrial
application.
The YE concentration was shown to significantly affect
the oil production. Both high and low YE concentrations had
a negative influence on oil content (Figure 3a). The highest
oil content of 53.0% was observed in run 4 when C/N was
set at a high level (Table 2). This result is coincidence with
previous publications, which indicated that a high C/N ratio

Alga-Based Biodiesel Production Using Sugar Cane

Energy & Fuels, Vol. 23, 2009 4169

Table 2. Experimental Design and Estimated Data for Response Surface Analysis
central composite design

conversion ratio (%)

runa

Ab

Bc

SCH (g L-1)

biomass
yield (g, DW)

1
2
3
4
5
6
7
8
9
10
11

9
9
21
21
9
21
15
15
15
15
15

0.8
0.1
1.5
0.8
1.5
0.1
0.8
0.8
0.1
0.8
1.5

7.2
0.9
31.5
16.8
13.5
2.1
12.0
12.0
1.5
12.0
22.5

0.875
0.233
2.173
1.435
1.453
0.279
1.200
1.228
0.261
1.184
1.976

output-cost coefficient f

oil content (%)

consumed
sugar (g)

cultivation
time (day)d

biomass/sugar

oil/ sugar

experimental

estimated

42.5
39.0
49.2
53.0
42.7
38.3
49.6
50.2
40.3
49.5
49.2

1.87
0.35
5.91
3.40
3.38
0.45
2.72
2.66
0.47
2.67
5.80

3.00
2.08
5.00
4.42
3.58
2.25
3.75
3.75
2.25
3.75
4.75

46.8
66.5
36.8
42.2
43.0
61.7
44.1
46.1
55.3
44.4
34.1

16.4
07.0
17.0
20.4
16.4
09.0
19.5
20.7
08.3
19.5
15.6

0.0547
0.0335
0.0339
0.0462
0.0458
0.0399
0.0519
0.0552
0.0371
0.0520
0.0329

0.0547
0.0348
0.0308
0.0500
0.0446
0.0392
0.0518
0.0518
0.0365
0.0518
0.0372

a Runs 7, 8, and 10 are central points. b A represents C/N: -1 (9), 0 (15), and +1 (21). c B represents the concentration of YE (g L-1): -1 (0.1), 0
(0.8), and +1 (1.5). d The cell density was monitored, and cells were collected at stationary phase. The final volume of media in flasks is 260 mL.

Figure 2. Response surface plot representing the effects of C/N and YE concentration on (a) biomass production and (b) conversion ratio of
sugar/biomass. (a) Biomass productivity (g day-1, DW) is defined here as the daily biomass production (g) when cells are collected at stationary
phase.

Figure 3. Response surface plot representing the effects of C/N and YE concentration on (a) oil content and (b) conversion ratio of sugar/oil.

in the medium has been considered to be essential for oil

overproduction.16 In addition, nitrogen starvation usually
triggers the oil accumulation and contributes to a greater
extent of triglyceride content during the stationary phase of
growth.13 Figure 3a indicated that the maximum oil content

was obtained when C/N was 19.8 and YE was 1.16 g L-1. It
means that increasing the C/N ratio further over 19.8 did
not cause a proportionally increasing oil content. However,
53% of oil content in dry algal cells is satisfied in biodiesel
production. We also noticed that the highest conversion ratio

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Cheng et al.

Figure 4. Response surface plot representing the effects of C/N and YE concentration on the output-cost coefficient f.

of sugar/biomass was 66.5% in run 2 (Table 2). The highest

conversion ratio of sugar/oil was 20.7% in run 8, which was
almost similar to that (20.4%) in run 4. In the process of oil
production, alga can be considered as a cell factory that
processes the carbon source into oil. The conversion ratio of
sugar/oil (%) is an indicator of how efficient the algae convert
sugars to oil. Accordingly, we suggest that the high conversion ratio of sugar/oil is more important than the high sugar/
biomass in alga-based biodiesel production.
The cost of carbon source contributes to the main cost of
medium and product. Poor efficiency of carbon source
conversion is therefore not desirable. It showed that a high
or low YE concentration had negative influences on oil
content (Figure 3a) and the conversion ratio of sugar/oil
(Figure 3b). Higher C/N seems to slightly increase the
efficiency of sugar conversion. However, when the nitrogen
source was set constant, higher C/N often leads to a higher
concentration of carbon source. The carbon source at high
concentrations, such as 60 g of glucose L-1, had an inhibition
on both cell growth and conversion efficiency.5 For this
reason, a proper combination of the YE concentration and
C/N leading to both a satisfying oil yield and efficiency of
carbon source use should be considered.
3.3. RSM Analysis and the Optimal Production Based
on Feedstock and Cultivation Time. The main cost for oil
production from algae comes from the cultivation time and
carbon source (sugar) consumed. In the other words, the
commercialization of alga-based biodiesel depends upon its oil
yield, consumed feedstock, and production time, which mainly
refer to labors and consumption of electric power. Therefore,
we set an output-cost coefficient f that is in a direct ratio with
the oil yield (g) and an inverse ratio with both time (days) and
consumed sugar (g) to evaluate and optimize the alga-based
biodiesel production: f ) oil yield/time sugar. Using data
from Table 2 related to oil yield, cultivation time, and consumed
sugar, a response surface plot indicating the effect of C/N and
YE concentration on the output-cost coefficient was achieved
(Figure 4). To optimize the oil production, all data related to
oil yield, carbon consumption, and cultivation time in 11 runs
listed in Table 2 were also conducted and analyzed through

Table 3. ANOVA for the Response Surface Quadratic Model

source
model
A
B
AB
A2
B2
residual
lack of fit
pure error
correlation total

sum of squares DF
7.193 10-4
3.279 10-5
8.012 10-7
8.333 10-5
6.709 10-7
5.693 10-4
5.806 10-5
5.118 10-5
6.878 10-6
7.773 10-4

5
1
1
1
1
1
5
3
2
10

mean square

F value p value

1.439 10-4
3.279 10-5
8.012 10-7
8.333 10-5
6.709 10-7
5.693 10-4
1.161 10-5
1.706 10-5
3.439 10-6

12.39
2.82
0.069
7.18
0.058
49.03

0.0076
0.1537
0.8033
0.0439
0.8196
0.0009

4.96

0.1723

RSM. As a result, the quadratic model eq 2 was generated and

suggested as the best fitting model
Y ) 0.052 - 2.338 10-3A + 3.654 10-4B 4.564 10-3AB + 5.146 10-4A2 - 0.015B2

(2)

Please see eq 1 to understand eq 2.

The optimum values of C/N and YE concentration were
calculated by solving eq 2 through Design Expert 7.1.3. The
optimal production with the highest output-cost coefficient is
suggested to be obtained when C/N was 26.9 and YE concentration was 0.60 g L-1. Media prepared according to the
optimum C/N and YE concentration for the highest output-cost
coefficient may optimize the algal oil production by heterotrophic cultivation with sugar cane as the feedstock.
3.4. Justification of the Quadratic Model Generated by
RSM. To evaluate the fitness of the model eq 2 in practical
application, ANOVA was conducted to fit the model using the
experimental data. As the result, the value of 0.0076 showed
that the model was statistically significant at a 5% level of
significance (Table 3). This means that the combined effects of
C/N and YE concentration contribute significantly to the
variation in oil production. No significant lack of fit is desired
and obtained in this design. When R is set as 0.05, the p value
is 0.1723 and larger than R, which demonstrates a model
representing the actual interaction of tested parameters. The p
value is the probability that the variation between conditions
may have occurred by chance; therefore, coefficients with
smaller p values vary more significantly. Thus, the value of

Alga-Based Biodiesel Production Using Sugar Cane

Energy & Fuels, Vol. 23, 2009 4171

Table 4. Estimated and Experimental Values of Responses

under Optimized Conditionsa
responses

estimated

experimentalb

t valuec

oil content (%)

oil/sugar (%)
biomass/sugar (%)
output-cost coefficient f
time (h)

43.90
18.15
56.40
0.051
72

47.91 ( 0.62
18.98 ( 1.12
46.22 ( 1.95
0.061 ( 0.004
75

3.71
0.42
3.01
1.56

a C/N ) 26.9. YE ) 0.60 g L-1. SCH ) C/N YE ) 16.1 g L-1.

Data were shown as means ( SD. c n ) 3. DF ) 2. R ) 0.05.
Two-tailed t ) 4.303.

Table 5. Effects of Different Carbon Sources on Cell Growth

and Oil Production of Fed-Batch Fermentation in a 5 L
Bioreactor (the Harvesting Volume Is 2.5 L)
conversion ratio (%)
carbon
source

consumed
sugar (g)

biomass
yield (g)

oil
yield (g)

biomass/
sugar

oil/
sugar

glucose
SCH

304.0
296.4

107.8
121.3

51.8
54.7

35.5
40.9

17.0
18.5

3.6. Composition of Biodiesel Produced from Algal Oil

by Transesterification. The main fatty acid methyl esters
detected in biodiesel from both SCH and glucose carbon source
include 9-octadecenoic acid methyl ester, 9,12-octadecadienoic
acid methyl ester, and hexadecenoic acid methyl ester; their
corresponding contents from SCH and glucose carbon source
were 52.56 and 53.75% (w/w), 10.33 and 19.48% (w/w), 12.49
and 11.34% (w/w), respectively. Other minor methyl esters were
also detected (data not shown). This result illustrates that the
main components of biodiesel from sugar cane are similar to
that of glucose. High-quality biodiesel from a glucose carbon
source has previously been characterized by a heating value of
41 MJ kg-1, a density of 0.864 kg L-1, and a viscosity of 5.2
10-4 Pa s (at 40 C).14 We expect that biodiesel produced
from SCH will show a similar performance to that from glucose.
This result further confirms the feasibility of using sugar cane
as the feedstock for biodiesel production through heterotrophic
algal fermentation.
4. Discussion

Figure 5. Heterotrophic fermentation in a 5 L bioreactor using (a)

glucose and (b) SCH as the feedstock, respectively: (b) biomass yield
and (O) concentration of reducing sugar. Arrows indicated the points
of feeding.

0.009 showed that B2 was the most significant term among

others (Table 3).
Three independent experiments in optimized media were
conducted to verify the predictions of the model. The estimated
and experimental responses were compared and subjected to a
two-tailed t test (Table 4). The results demonstrate no significant
differences between estimated and experimental values. Thus,
RSM combining an effective design can be applied to the
optimization of medium composition for algal oil production.
3.5. Heterotrophic Fermentation of Algal Cells in a 5
L Bioreactor. Algal heterotrophic fermentation in a bioreactor,
instead of cultivation in a shake flask, is commonly used in
practical production. Therefore, algal fermentations in a 5 L
bioreactor were conducted with 3 g L-1 YE and 30 g L-1
glucose or SCH as the starting conditions. The carbon source
and YE were batch-fed whenever the carbon source was
exhausted. Within 7 days of cultivation, it is interesting to note
that C. protothecoides provided higher biomass productivity and
oil yield with SCH than with glucose (Figure 5). The conversion
ratios of sugar/biomass and sugar/oil with SCH were higher
than those with glucose by 15.2 and 8.8%, respectively
(Table 5).

4.1. Feasibility of SCH as the Carbon Source for Oil

Production. This work, for the first time, illustrates the
feasibility of sugar cane juice served as a fermentable carbon
source for oil production in C. protothecoides. It means that
sugar cane is not only a good feedstock for fuel-ethanol
production but also for biodiesel production. The production
pathway from sugar cane to biodiesel by microalgae fermentation has some advantages in comparison to fuel-ethanol production by yeast fermentation. For example, the heating value of
alga-based biodiesel is much higher than that of yeast-based
fuel-ethanol. Because algal cells are easily separated from
medium and oil is easily extracted by solvent, it takes a lot of
heating power to prepare ethanol by distillation from yeast
fermentation medium.
The present work focusing on reducing the cost of feedstock
and optimizing oil yield to some extent helps to reduce the cost
in algal oil production. In alga-based biodiesel production by
heterotrophic fermentation, a lower price of carbon source means
less cost of oil production and better economic feasibility. On
the basis of the market in China, the price of industrial glucose
was about 330-360 USD/ton and the price of sugar cane stalks
is about 40 USD/ton (October 2008), indicating that the price
of fermentable sugars from sugar cane juice is about 260 USD/
ton, less than that of glucose.
According to the observation in this study, it is interesting to
note that algal cells grow faster and provide a higher yield of
biomass and oil with SCH than that with glucose. Both
conversion ratios of sugar/biomass and sugar/oil with SCH were
higher than that with glucose. It is reasonable that there are
other unknown nutritions in SCH to benefit cell growth and oil
accumulation.
Although the advantages mentioned above, we are clearly
conscious that this is just a beginning for biodiesel production
using microalgae to meet the commercial needs. There are
bottlenecks in this pathway. In comparison to yeast fermentation
for ethanol production, microalgae accumulate oils as intracel-

4172

Energy & Fuels, Vol. 23, 2009

lular products. The oil/carbon source conversion is dependent

upon the biomass/sugar conversion. As a result, the oil cell
content typically ranges from 30 to 60%, leading to a limitation
of oil/carbon source conversion. Larger quantities of ethanol
could be produced with relatively small amounts of microbial
cells for ethanol fermentation. However, in the future, reasonable
improvements can be introduced to the pathway of alga-based
biodiesel production. For example, microalga is genetically
modified, allowing the oil vesicle to be transported to an
extracellular medium. On the other hand, the algal species used
in this study cannot assimilate sucrose directly. Because of the
lack of corresponding enzyme, pretreatment of SC is inevitable
and requires time and labor. Genetic manipulation offering algal
species with the ability of digesting sucrose is welcome in future
studies.
4.2. Influences of C/N and YE Concentration on Algal
Growth and Oil Production. We observed that 53% of oil
content (run 4 in Table 2), 66.5% conversation ratio of sugar/
biomass (run 2 in Table 2) and 20.7% conversation ratio of
sugar/oil (run 8 in Table 2) were achieved at different C/N and
YE concentrations separately. It indicates the critical importance
of C/N and nitrogen concentration for alga growth and oil
accumulation. For this reason, a proper combination of the YE
concentration and C/N leading to both a satisfying oil yield and
efficiency of carbon source use should be optimized. Using
RSM, the optimal oil production with the highest output-cost
coefficient (0.061 ( 0.004) was achieved when C/N was 26.9
and the YE concentration was 0.60 g L-1, from which the
condition contributing to the highest efficiency of less
input-more output can be judged.
Within the range of the design, a high nitrogen supply was
observed to lead to poor efficiency of sugar use (Figure 2b).
When the YE concentration is constant, a higher concentration
of sugar does not lead to a higher efficiency of sugar use.
Because of the conditions of the experiments, cells were
cultivated in a closed system (shake flask) and air supply and
agitation speed were restrained. The moment cell density reaches
the maximum value (stationary phase), a higher carbon supply
does not contribute to continuous growth and oil accumulation.
In our previous observation, the highest cell density in flasks
was shown at 14-15 g L-1. The dynamics of algal cells in flasks
is different from that in a bioreactor. Under the conditions of
the bioreactor, the cell density will be greatly improved. The
air, pH, and agitation can be automatically controlled. The
strategy of feed batch, continuous flow, and semicontinuous flow
feed have been widely applied in high-density fermentation.
Even parts of cultures can be pumped out from the bioreactor,
allowing further growth. As observed in this study, through SCH
feeding and fermentation process controls, the highest cell
density in the 5 L bioreactor has been achieved at 48 g L-1.
4.3. Photoautotrophic Cultivation versus Heterotrophic
Cultivation. At present, microalgal biomass production has been
achieved by at least two main approaches: one is photoautotrophic cultivation by using solar energy and carbon dioxide,
and the other is heterotrophic fermentation using glucose as the
carbon supply.
From the aspect of energy saving and greenhouse effect relief,
the photoautotrophic alternative seems to be more economical
and preferred. Many works have been published about this
strategy. For example, in a recent study, 30 strains of photoautotrophic algae were screened for biomass productivity and
oil content. When photoautotrophic algae were grown in a
nitrogen-deprived outdoor system, the highest lipid productivity
of 0.204 g L-1 day-1 was achieved (with biomass productivity

Cheng et al.

at 0.30 g L-1 day-1 and lipid content over 60%).17 In another

example, Neochloris oleoabundans was reported to give a
remarkable lipid productivity of 0.133 g L-1 day-1 (with biomass
productivity at 0.40 g L-1 day-1 and lipid content at 34%).1
However, considering the following reasons, biofuel via a
heterotrophic pathway should be further studied and considered
as a transitional strategy. First, fast-growing photosynthetic algal
cells tend to produce poor amounts of oil, whereas those
accumulating high oil content show little growth ability.18
Nitrogen starvation has been widely reported to trigger lipid
accumulation but leads to a decrease in protein synthesis,
chlorophyll content, and cell division.19 The results in this study
consist with previous publications. Second, it is important to
notice that light deficiency is common in photosynthetic
cultivation. Thus, enough ATP and reductant for CO2 fixation
can hardly be supplied. The dilemma is that the synthesis of
fatty acid needs a large amount of ATP and reducing power.7
When light is restricted, it is not likely for alga to prefer
synthesizing fatty acids. Therefore, in-depth research on the
mechanism of oil production in heterotrophic alga is desired.
For example, genetic manipulation performed on critical
enzymes for lipid synthesis may also be possible to improve
oil yield. A strategy of phototrophic/heterotrophic tandem
cultivation can be conducted and may exhibit a benefit to both
environmental and economic aspects.
As for this work, the highest lipid productivity in run 3 is
0.82 g L-1 day-1 (Table 2; with biomass productivity at 1.67 g
L-1 day-1 and lipid content at 49.2%). This result is almost 3
times larger than 0.204 g L-1 day-1 and 5 times larger than
0.133 g L-1 day-1 achieved in phototrophic cultivation. Through
comparison, it is obvious that heterotrophic culture has advantages in both high biomass and oil productivity. Accordingly,
the heterotrophic option nevertheless should not be completely
ignored.
4.4. RSM with a Proper Experimental Design Is an
Efficient Tool in Optimization. Classical methods for medium
designing and optimization include biological mimicry, oneat-a-time, full factorial, partial factorial, etc. Some of them are
laborious and time-consuming, because many experiments are
involved.20 In industrial application, more efficient and easier
approaches are necessary. RSM used in this work is one kind
of such an approach, which is able to generate a mathematical
model that accurately describes a given process. Its advantage
is the reduced number of experimental runs required to generate
sufficient information for a statistically acceptable result.21 In
this study, a quadratic model has been successfully generated
from 11 runs and used to give optimum parameters for the
output-cost coefficient in oil production. This may provide useful
information for further research of biodiesel production involving more variables.
4.5. Feasibility Algal Oil as a Biodiesel Oil Source.
Currently, the main oil sources for biodiesel production are from
oil crops, such as castor, sunflower, soybean, rapeseed, cotton
seed, etc. The dominating fatty acid methyl ester is C19H34O2
(52.3%) in soybean biodiesel, C19H36O2 (64.4%) in rapeseed
(16) Sattur, A. P.; Karanth, N. G. Biotechnol. Bioeng. 1989, 34, 868
871.
(17) Rodolfi, L.; Chini, Z. G.; Bassi, N.; Padovani, G.; Biondi, N.;
Bonini, G.; Tredici, M. R. Biotechnol. Bioeng. 2009, 102 (1), 100112.
(18) Hu, Q.; Sommerfeld, M.; Jarvis, E.; Ghirardi, M.; Posewitz, M.;
Seibert, M.; Darzins, A. Plant J. 2008, 54 (4), 621639.
(19) Macduff, J. H.; Humphreys, M. O.; Thomas, H. Ann. Bot. 2002,
89 (1), 1121.
(20) Kennedy, M.; Krouse, D. J. Ind. Microbiol. Biotechnol. 1999, 23,
456475.
(21) Bibhu, P. P.; Mohd, A.; Saleem, J. Res. J. Microbiol. 2007, 2 (3),
201208.

Alga-Based Biodiesel Production Using Sugar Cane

biodiesel, and C19H36O2 (80.4%) in olive biodiesel,22 respectively. The biodiesel prepared from algal oil in this work is
mainly composed of 9-octadecenoic acid methyl ester
(C19H36O2), 9,12-octadecadienoic acid methyl ester (C19H34O2),
and hexadecenoic acid methyl ester (C17H34O2), which are
(22) Srivastava, A.; Ram, P. Renewable Sustainable Energy ReV. 2000,
4 (2), 111133.

Energy & Fuels, Vol. 23, 2009 4173

closely accordant with that of crop oils. The results proved that
microalgal oil can also be used as another potential oil source.
Acknowledgment. This research was supported by NSF Guangdong Joint Project U0633009, National 863 Project 2007AA05Z400,
and China MOST Overseas Project 20070574.
EF9003818