Application of sweet sorghum for biodiesel production by heterotrophic

microalga Chlorella protothecoides

Chunfang Gao, Yan Zhai, Yi Ding, Qingyu Wu
*
School of Life Science, Tsinghua University, Beijing 100084, PR China
a r t i c l e i n f o
Article history:
Received 21 May 2009
Received in revised form 11 September
2009
Accepted 12 September 2009
Available online 8 October 2009
Keywords:
Biodiesel
Chlorella protothecoides
Enzymatic hydrolysis
Microalgae
Sweet sorghum
a b s t r a c t
Microalga Chlorella protothecoides can grow heterotrophically with glucose as the carbon source and
accumulate high proportion of lipids. The microalgal lipids are suitable for biodiesel production. To fur-
ther increase lipid yield and reduce biodiesel cost, sweet sorghum juice was investigated as an alternative
carbon source to glucose in the present study. When the initial reducing sugar concentration was 10 g L
1
in the culture medium, the dry cell yield and lipid content were 5.1 g L
1
and 52.5% using enzymatic
hydrolyzates of sweet sorghum juice as the carbon source after 120 h-culture in asks. The lipid yield
was 35.7% higher than that using glucose. When 3.0 g L
1
yeast extract was added to the medium, the
dry cell yield and lipid productivity was increased to 1.2 g L
1
day
1
and 586.8 mg L
1
day
1
. Biodiesel
produced from the lipid of C. protothecoides through acid catalyzed transesterication was analyzed by
GCMS, and the three most abundant components were oleic acid methyl ester, cetane acid methyl ester
and linoleic acid methyl ester. The results indicate that sweet sorghum juice could effectively enhance
algal lipid production, and its application may reduce the cost of algae-based biodiesel.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
In the past decade, the development of biofuel production is re-
garded as the national strategy to solve energy and environmental
issues in many countries [1,2]. Biofuel accounted for 1% of the
worlds road fuel consumption in 2006 and the rapid growth in
biofuel production is demanded [3]. Finding cheap and efcient re-
sources is an urgent task worldwide. Under this situation, Biodiesel
production using microalgal lipid has received special attention
[4], and some advances were made in screening strains and pro-
duction systems [5,6]. Oil accumulated in most microalgae is
mainly triglyceride that can be applied to form biodiesel and glyc-
erol through transesterication [7]. In our previous studies, hetero-
trophic fermentation system of alga Chlorella protothecoides was
developed [8]. Compared to photoautotrophic culture model, the
advantage of heterotrophic fermentation strategy lies in that it re-
sulted in higher content of lipid in cells and higher biomass con-
centration within shorter time. The lipid content and biomass
concentration of heterotrophic C. protothecoides were up to 55.2%
of dry cell weight and 51.2 g L
1
, respectively [9]. At present, bio-
diesel was commonly produced from the seeds of plant oil, includ-
ing soybean, rapeseed, jatropha and karanja. Whereas, the oil
content of the seeds was mainly below 40% [10]. In this respect,
biodiesel productivity of heterotrophic C. protothecoides greatly
exceeds that of vegetable oils. However, commercial application
of biodiesel production from C. protothecoides is restricted due to
the high cost, which mostly exists in the fermentation substrate.
According to a previous estimate, the cost of glucose accounted
for 80% of the total medium cost [11]. So in order to realize com-
mercialization of biodiesel production from heterotrophic C. prot-
othecoides, low-cost and effective alternative to glucose is
desirable.
Sweet sorghum {Sorghum biocolor (L.) Moench} is a C4 plant
with high biomass productivity. As a high photosynthetic ef-
ciency crop, it does not only produce grain, but also yields large
amounts of sugar-rich stems. The sugar in stems is mainly sucrose
(up to 55% of dry biomass), fructose and glucose [12], which are
ideal for preparing fermentation media [13,14]. Besides these, its
wide adaptability, resistance to semi-arid soils and water-logging
characterize it as one of the most popular crop throughout the
world. Currently, it is planted in 99 countries around the world
of 44 million ha, mainly in poor and semi-arid areas [15]. The
development of sweet sorghum in semi-arid and other marginal
lands would improve agricultural land use. Moreover, sugar yield
of sweet sorghum is attractive. The typical fresh stem yield of
sweet sorghum is more than 45 t/ha per year [16], and the total su-
gar obtained was 5.110.5 t/ha [17]. Since the duration for sweet
sorghum to mature is just 35 months, the sugar yield would be
doubled in some areas where two crops could be harvested per
year. These outlets would help to ease world energy crisis if they
can be converted to energy efciently.
0306-2619/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2009.09.006
* Corresponding author. Tel./fax: +86 10 62781825.
E-mail address: qingyu@mail.tsinghua.edu.cn (Q. Wu).
Applied Energy 87 (2010) 756761
Contents lists available at ScienceDirect
Applied Energy
j our nal homepage: www. el sevi er. com/ l ocat e/ apenergy
Most of the present studies in sweet sorghum utilization focuse
on ethanol production. The common process for ethanol produc-
tion from sweet sorghum is the suspended or immobilized cell fer-
mentation of soluble sugars in the juice using Saccharomyces
cerevisiae or some other species [14,18,19]. Solid state fermenta-
tion method was also reported [20]. However, sweet sorghum
has not been explored so far as a resource for the production of bio-
diesel [21]. As described above, heterotrophic C. protothecoides was
capable of converting sugars to intracellular oil that could be used
to produce biodiesel efciently. Therefore, in the present study, we
investigated the application of sweet sorghum for biodiesel pro-
duction by heterotrophic C. protothecoides. The efciency of bio-
mass and oil production from glucose and sweet sorghum juice
was compared.
2. Materials and methods
2.1. Microorganism and inoculum preparation
C. protothecoides was originally provided by the Culture Collec-
tion of Algae at University of Texas (Austin, TX, USA). The compo-
nents of basal culture medium and the culture apparatus were the
same as described in previous work [9]. For the preparation of
inoculum, 30 g L
1
glucose and 4 g L
1
yeast extract was added to
the basal medium. Cells cultivated in shaking asks at 28 1 C
and 220 rpm for about 4 days were used for inoculation.
2.2. Sweet sorghum
Sweet sorghum cultivar Chuntian No. 2 was cultivated in the
farm of Xiao Tang Shan, Beijing. After harvest, the leaves of the
fresh crop were stripped by hand and the stems were stored at
4 C. Fresh juice was obtained by squeeze of the stems with a roller
mill. The sweet sorghum juice (SJ) was stored at 20 C until use.
2.3. Hydrolysis of sweet sorghum juice
2.3.1. Acid hydrolysis
The SJ (sweet sorghum juice) was adjusted to pH 12 with
H
2
SO
4
, and hydrolyzed at 112 C in autoclave sterilizer, this step
was also used for sterilizing SJ. The hydrolysate was neutralized
with autoclave sterilized BaSO
4
and centrifuged at 7000 rpm for
5 min. Then, the supernatant was collected and the reducing sugar
concentration in this solution was measured. In the preparation of
culture medium, the acid hydrolysate of SJ (ASJ) was added directly
to the sterilized basal culture medium in the clean bench.
2.3.2. Enzymatic hydrolysis
Invertase ANL (Validase), a liquid fungal invertase, was used for
the hydrolysis of sucrose in SJ. After the addition of Invertase ANL,
the mixture was vortexed well, and then maintained at 55 C. After
hydrolysis, the reducing sugar content in the enzymatic hydroly-
sate of SJ (ESJ) was determined.
2.4. Microorganism cultivation in ask
Microorganism cultivations were performed in 500 ml conical
asks, each of which contained 200 ml culture medium. The cul-
ture mediumsupplemented with 10 g L
1
glucose was used as con-
trol to evaluate the effect of SJ in C. protothecoides growth. The nal
concentration of reducing sugar released from SJ was controlled at
10 g L
1
in the culture medium with the corresponding compo-
nents of basal medium and yeast extract (Table 1). In some exper-
iments, fructose or sucrose is applied instead of SJ. All media and
cultivation apparatus were sterilized with autoclave sterilizer at
112 C, 120 kPa for 30 min. C. protothecoides in logarithmic phase
was inoculated into culture medium. Cultivation was carried out
at 28 1 C and 220 rpm.
2.5. Lipid extraction
At the end of the cultivation, biomass was collected by centrifu-
gation of 7000 rpm for 5 min, and then the cell pellets were lyoph-
ilized to constant weight. Afterwards, dried cell pellets were
pulverized and disrupted in a mortar for 30 min. Lipids were ex-
tracted from the disrupted samples by Soxhlet apparatus with
n-hexane as the solvent. After removing n-hexane by a rotary evap-
orator (N-1000, Eyela, Japan), lipids were obtained. In extraction
experiments, lipid could be obtained also directly from wet pellets.
2.6. Biodiesel preparation by transesterication
Biodiesel from microalgal lipid was derived by acid catalyzed
transesterication, the manipulation is described in detail in our
previous publication [22]. The optimized transesterication condi-
tions were applied. The reaction factors were 1:1weight ratio of
catalyst to oil with 56:1 M ratio of methanol to oil at 30 C for 4 h.
2.7. Analysis
2.7.1. Cell growth
Optical density measurement at 540 nm was used to monitor
cell growth with a UV/Visible spectrophotometer (Pharmacia Bio-
tech Ultrospec 2000). Biomass concentration was calculated by a
Table 1
Composition of culture media.
Ingredient Amount
Basal culture
medium
Glucose culture
medium
Fructose culture
medium
Sucrose culture
medium
SJ culture
medium
Glucose + yeast
extract medium
SJ + yeast extract
medium
KH
2
PO
4
(g L
1
) 0.7 0.7 0.7 0.7 0.7 0.7 0.7
K
2
HPO
4
(g L
1
) 0.3 0.3 0.3 0.3 0.3 0.3 0.3
MgSO
4
7H
2
O (g L
1
) 0.3 0.3 0.3 0.3 0.3 0.3 0.3
Glycine (g L
1
) 0.1 0.1 0.1 0.1 0.1 0.1 0.1
FeSO
4
7H
2
O (mg L
1
) 3 3 3 3 3 3 3
Vitamin B
1
(mg L
1
) 0.01 0.01 0.01 0.01 0.01 0.01 0.01
A5 trace mineral
solution (ml L
1
)
1 1 1 1 1 1 1
Yeast extract (g L
1
) 4 13
Glucose (g L
1
) 10 10
Fructose (g L
1
) 10
Sucrose (g L
1
) 10
SJ reducing sugar (g L
1
) 10 10
C. Gao et al. / Applied Energy 87 (2010) 756761 757
regression equation: y = 0.4155x (R
2
= 0.9933, P < 0.05), where y
(g L
1
) is the dry cell weight, x is the absorbance of the suspension
at 540 nm.
2.7.2. Sugar concentrations
Sucrose, glucose and fructose were three main sugars in SJ and
sucrose can be hydrolyzed to glucose and fructose. The content of
total reducing sugar, including glucose and sucrose, was deter-
mined using the 3, 5-dinitrosalicylic acid (DNS) method. Glucose
concentration of SJ was determined enzymatically with a glucose
oxidase-chromogen reagent (Shandong University). Fructose con-
tent was obtained by subtracting the content of glucose from
reducing sugar. For the determination of sucrose content, SJ was
acid-hydrolyzed in 0.36 M HCl for 20 min at 80 C. After neutraliza-
tion with NaOH, glucose concentration was measured enzymati-
cally and sucrose content was calculated by the increase of
glucose before and after hydrolysis.
2.7.3. Intracellular lipid content
After cultivation, cells harvested by centrifugation were lyoph-
ilized to constant weight. Then the lipid content of biomass was
analyzed by time-domain nuclear magnetic resonance [23].
2.7.4. Composition analysis of biodiesel
The composition of biodiesel produced from microalgal lipid
was analyzed by GC-linked mass spectrometry, using a dual-stage
quadrupoles GC apparatus (Thermo, USA) equipped with a Varian
VF-5 ms column (30 m 0.25 mm ID DF = 0.25 lm) and manipu-
lated with a ow rate of 10 ml min
1
.
3. Results
3.1. Cell growth and lipid accumulation of heterotrophic C.
protothecoides with fructose or sucrose as carbon source
SJ was rich in carbohydrate, including sucrose as well as fruc-
tose and glucose. The contents of sucrose, fructose and glucose in
SJ applied in this study were 101.7, 33.1 and 25.0 g L
1
, respec-
tively. Cell growth and lipid accumulation of heterotrophic C. prot-
othecoides with different sugars as carbon source were shown in
Fig. 1. At 120 h, cell density of C. protothecoides was 3.7, 3.9 and
1.2 g L
1
in culture medium with the initial concentration of
10 g L
1
of glucose, fructose or sucrose, respectively. And the corre-
sponding lipid content was 53.3%, 52.7% and 37.7% of cell dry
weight. The biomass density and lipid content of cells cultured
with glucose and fructose showed no signicant differences, but
these values of cells cultured with sucrose were much lower. The
data suggested that sucrose, which accounted for 63.6% of the total
sugar in sweet sorghum juice, can not be utilized by heterotrophic
C. protothecoides without hydrolysis. Thus, in order to convert SJ to
biodiesel through the cultivation of C. protothecoides in high ef-
ciency, sucrose in SJ should be hydrolyzed to glucose and fructose
prior to use.
3.2. Acid and enzymatic hydrolysis of sweet sorghum juice
In order to obtain carbon source available for heterotrophic
microalgal C. protothecoides cultivation, sucrose should be hydro-
lyzed to reducing sugars. The hydrolytic methods applied were
acid and enzymatic hydrolysis.
The experiments of acid hydrolysis were carried out at 112 C.
The reaction pH and time were two main factors which can inu-
ence the hydrolysis of sucrose, so the hydrolysis ratio of sucrose
with reaction pH 13 and reaction time in the range of 20 to
30 min was investigated. As shown in Table 2, we can appreciate
that the optimal condition of acid hydrolysis was at pH 2 and
112 C for 25 min.
The enzymatic hydrolysis curves of sucrose in SJ with different
dose of invertase ANL were shown in Fig. 2. Within 45 min, the
hydrolysis ratio of sucrose in sweet sorghum juice increased with
the increase of invertase ANL dose. But in 60 min, when invertase
ANL dose was 0.5 and 0.6 ml L
1
, the hydrolysis ratio reached the
Fig. 1. Cell yield (white columns) and lipid content (black columns) of heterotro-
phic C. protothecoides with different sugars as carbon source. C. protothecoides was
cultivated for 120 h.
Table 2
Acid hydrolysis of SJ.
pH 1 2 3
Time (min) 20 25 30 20 25 30 20 25 30
Hydrolysis ratio of sucrose (%) 90.6 98.8
a
91.6 99.0 99.0 72.2 81.4 98.9
a
Some sugar was carbonized.
Fig. 2. Enzymatic hydrolysis curves of sucrose in SJ with different dosages of
invertase ANL. Invertase ANL added were as follows: s, 0.3 ml L
1
; d, 0.4 ml L
1
; N,
0.5 ml L
1
; j, 0.6 ml L
1
.
758 C. Gao et al. / Applied Energy 87 (2010) 756761
same value of 98%. So the optimal enzymatic hydrolysis strategy
was as follows: 0.5 ml L
1
Invertase ANL was added to sweet sor-
ghum juice, the mixture was kept at 55 C for 1 h.
3.3. Cultivation of heterotrophic C. protothecoides in asks
The cell cultivation kinetics of different mediums was presented
in Fig. 3. As shown, the dry cell yield reached the maximum value
of 3.7 g L
1
after 120 h culture with the substrate of glucose, while
the maximum value was 3.3 and 5.1 g L
1
with ASJ (acid hydroly-
sate of sweet sorghum juice) and ESJ (enzymatic hydrolysate of
sweet sorghum juice), respectively. Compared with glucose, the
biomass production of ASJ was decreased by 10.8% and that of
ESJ was increased by 37.8%. The results indicated that, during acid
hydrolysis SJ might produce some inhibitory components to C.
protothecoides that inhibit cell growth, while ESJ might contain
some benecial components to C. protothecoides, such as protein,
amino acids or other components. Therefore, the enzymatic hydro-
lysis of SJ was preferable. Lipid content in algal cells was 53.3%
with glucose feeding, and 52.5% with ESJ feeding, which was not
signicantly different. Thus, the lipid yield of C. protothecoides
was 394.4 mg L
1
day
1
on glucose and 535.5 mg L
1
day
1
on
ESJ. The results indicate that not only can sweet sorghum be used
efciently for C. protothecoides cultivation, but also the lipid yield
from sweet sorghum has exceeded that from glucose.
Yeast extract is one of the most suitable nitrogen sources to pro-
mote growth of C. protothecoides [9]. In order to conrm the opti-
mal yeast extract amount in culture medium, a series of
experiments using ESJ supplemented with 13 g L
1
yeast extract
were carried out. As shown in Fig. 4, when the initial yeast extract
was 1, 2 and 3 g L
1
, the biomass achieved 5.30, 5.85, 6.00 g L
1
,
respectively. However, the lipid content was 50.2%, 49.2% and
48.9%. With the increase of yeast extract from 2 to 3 g L
1
, the bio-
mass increased slightly and the lipid content seldom changed. The
highest lipid mass 586.8 mg L
1
day
1
(6.00 g L
1
48.9%/5 day)
was achieved with the yeast extract concentration of 3 g L
1
,
8.5% higher than that before adding yeast extract.
3.4. Composition of biodiesel produced from heterotrophic C.
protothecoides
After converting the heterotrophic C. protothecoides lipid to
methyl ester by acid transesterication, the composition of biodie-
sel obtained was analyzed by GCMS. The gas chromatograph of
biodiesel produced with ESJ is shown in Fig. 5. The fatty acid
methyl esters of biodiesel from different carbon source feedings
were compared in Table 3. As indicated, the three most abundant
components were the same, including oleic acid methyl ester
(C
19
H
36
O
2
), cetane acid methyl ester (C
17
H
34
O
2
) and linoleic acid
methyl ester (C
19
H
34
O
2
).
The properties of biodiesel from heterotrophic C. protothecoides
such as density, viscosity, ash point, cold lter plugging point,
solidifying point and heating value were determined in our previ-
ous work [8]. These properties were comparable to those of diesel
fuel and most of them comply with the limits established by ASTM
related to biodiesel quality.
4. Discussion
Biodiesel is regarded to be one of the most promising alterna-
tives to fossil fuel because it is renewable and environmental-
friendly. Currently, biodiesel is produced mainly from soybeans,
rapeseed, canola oil, palm oil, animal fat and waste cooking oil. It
is important to note that current supply is far less than demand
and the price is high. A country such as the United States produced
491 million gallons biodiesel in 2007 [24], far below the annual
biodiesel demand. It is not practical to increase biodiesel produc-
tion by increasing planting area of oil crops in concern of limited
land.
Another alternative method for biodiesel production is offered
by fermentation of high lipid yield microorganism. Heterotrophic
C. protothecoides has been reported to be a very good candidate
for biodiesel production because of its high lipid content and cell
density. The high cost of the feedstock glucose is the main obstacle
for commercialization. And in the long run, it is not practical to
produce biodiesel from the food-based sugar.
Sweet sorghum is reported as a key crop for sustainable agricul-
tural development in areas that suffer from aridity and saline/alka-
line soils. In China, it is an agriculture policy to develop sweet
sorghum for improving the use of agriculture land. Not only can
the grain be used for food or feed, but also, the fresh stem which
is rich in sugar has high value for applications. Currently, the com-
prehensive use of stem was not realized yet, and there were few
reports about the application of sweet sorghum in biodiesel pro-
duction. So we investigated the application of sweet sorghum for
biodiesel production by heterotrophic C. protothecoides.
The main sugar in sweet sorghum is sucrose and it can not be
used by C. protothecoides directly, so sweet sorghum juice was
Fig. 3. Growth curve of heterotrophic C. protothecoides with glucose, ASJ and ESJ
culture medium in asks. N, ASJ; d, glucose; j, ESJ.
Fig. 4. Cell yields (white columns) and lipid content (black columns) of heterotro-
phic C. protothecoides with different amounts of yeast extract. C. protothecoides was
cultivated for 120 h.
C. Gao et al. / Applied Energy 87 (2010) 756761 759
hydrolyzed prior to use. The hydrolysis ratio of sucrose can achieve
to 98% in 60 min using the enzyme of invertase ANL. Compared
with acid hydrolysates, enzymatic hydrolyzates can be used more
efciently by heterotrophic C. protothecoides, so the enzymatic
hydrolysis method was selected in subsequent experiments. When
the initial reducing sugar was 10 g L
1
, after cultivation of 120 h
the dry cell yield reached 3.7 g L
1
and 5.1 g L
1
with the substrate
of glucose and ESJ, respectively. These data suggested that ESJ
might contain some benecial components to C. protothecoides
and could promote cell growth. Meanwhile, the lipid content in
cells did not change signicantly. The lipid yield was 394.4 mg L
1
day
1
(glucose feeding) and 535.5 mg L
1
day
1
(ESJ feeding). We
can appreciate that the lipid productivity was increased by 35.7%
when ESJ was applied as carbon source. These illustrate that it
was feasible to use ESJ as organic carbon to cultivate C. prototheco-
ides and enhance the yield of algal lipid for biodiesel production.
The addition of yeast extract could further increase the biomass
and lipid yield to 1.2 g L
1
day
1
and 586.8 mg L
1
day
1
, respec-
tively. The lipid obtained from C. protothecoides of sweet sorghum
feeding was converted to biodiesel through acid catalyzed transe-
sterication efciently. The three most abundant components in
biodiesel prepared were oleic acid methyl ester (C
19
H
36
O
2
), linoleic
acid methyl ester (C
19
H
34
O
2
) and cetane acid methyl ester
(C
17
H
34
O
2
). They were the same with that of glucose feeding.
In this research, the lipid yield reached up to 586.8
mg L
1
day
1
. Typically, the lipid productivity by phototrophic
microalgae was signicantly lower at 17204 mg L
1
day
1
[25].
As regard to carbon efciency, there are three advantages in this
heterotrophic process. Firstly, the lipid productivity in heterotro-
phic process is much higher than the phototrophic system. Thus,
to produce the same amount of lipid, the heterotrophic process
need less energy for maintenance, irradiation, mixing and collec-
tion of microalgae. The save of energy, which is commonly gener-
ated by burning fossil fuel, would reduce the emission of CO
2
.
Secondly, sweet sorghum is directly used as a natural carbon
source, so the feedstock of the process is obtained by bio-xation
of atmospheric CO
2
. Thirdly, the conversion ratio of sugar to lipid
is 29.3% in this process, and it is higher compared to using glucose.
The novel strategy enhanced the carbon conversion efciency.
From the global perspective, the heterotrophic fermentation has
special characteristics, and the improvement of technology would
further increase the carbon efciency and biodiesel productivity.
RT: 2.97 - 39.96
5 10 15 20 25 30 35
Time (min)
0
10
20
30
40
50
60
70
80
90
100
R
e
l
a
t
i
v
e

A
b
u
n
d
a
n
c
e
25.98
22.62
26.41
18.49 39.83 3.40 29.90 36.83 8.26 13.72 9.76
NL:
1.10E8
TIC F:
MS
wuqy-
080529
Fig. 5. Gas chromatograph of fatty acid methyl ester in biodiesel produced from algal oil with ESJ as feedstock.
Table 3
Components of biodiesel produced by glucose and SJ.
No. Molecular formula Fatty acid methyl ester Relative content (%)
Glucose SJ
1 C
19
H
36
O
2
9-Octadecenoic acid methyl ester 53.75 66.80
2 C
19
H
34
O
2
9,12-Octadecadienoic acid methyl ester 19.48 15.12
3 C
17
H
34
O
2
Hexadecenoic acid methyl ester 11.34 12.66
4 C
19
H
38
O
2
Octadecanoic acid methyl ester 4.88 ND
a
5 C
18
H
36
O
2
Heptadecanoic acid methyl ester 1.48 0.54
6 C
20
H
38
O
2
10-Nodadecenoic acid methyl ester 0.75 ND
7 C
21
H
42
O
2
Eicosanoic acid methyl ester 0.51 ND
8 C
18
H
34
O
2
Cyclopropaneoctanoic acid 2-hexyl-methyl ester 0.37 ND
9 C
19
H
38
O
2
Heptadecanoic acid 16-methyl-methyl ester 0.24 4.30
10 C
17
H
32
O
2
11-Hexadecenoic acid methyl ester 0.18 ND
11 C
15
H
30
O
2
1-methylethyl ester ND 0.59
a
ND = not detected.
760 C. Gao et al. / Applied Energy 87 (2010) 756761
Based on the data obtained, a biodiesel yield of 1.4 t ha
1
a
1
of
sweet sorghum will be expected, and the yield would be doubled
to 2.8 t ha
1
a
1
in some areas where two crops could be harvested
per year. The biodiesel production of soybean, rapeseed and palm
oil is 0.36, 0.59, 3.68 t ha
1
a
1
, respectively [26]. The biodiesel
productivity of sweet sorghum is close to that of palm oil. Besides
this, the grain produced by sweet sorghum could be used for food
or feed. In light of price, sweet sorghum also has its advantage. The
price of industrial glucose was about 320366 USD per ton (1.00
USD = 6.83 CNY, April, 2009) in China. The price of sweet sorghum
stem was 2329 USD per ton in China, and the average sugar con-
tent was 17.57% [27] by fresh stem. Moreover, compared with glu-
cose, the application of sweet sorghum sugar resulted in 35.7%
increase of the lipid productivity. So in terms of land utilization
and biodiesel productivity, sweet sorghum appears to be a poten-
tial alternative feedstock for biodiesel production.
In conclusion, this work conrmed the application of sweet sor-
ghum and proved that it is feasible to cut down the cost of biodie-
sel from heterotrophic C. protothecoides by using ESJ as an
alternative feedstock instead of glucose. Further studies on pilot
scale and commercial scale fermentation of biodiesel from sweet
sorghum with low-cost could be continued.
Acknowledgements
This study was funded by the NSF Guangdong joint project
U0633009, NSF project 30670476 and 30970224, the National High
Technology Research & Development Program of China (863 Pro-
gram) 2007AA05Z400, MOST overseas cooperation project
20070574.
References
[1] Balat M, Balat H. Recent trends in global production and utilization of bio-
ethanol fuel. Appl Energy 2009;86:227382.
[2] Yan J, Lin T. Biofuels in Asia. Appl Energy 2009;86:S110.
[3] Zhou A, Thomson E. The development of biofuels in Asia. Appl Energy
2009;86:S1120.
[4] Huang GH, Chen F, Wei D, et al. Biodiesel production by microalgal
biotechnology. Appl Energy 2010;87:3846.
[5] Rodol L, Zittelli GC, Bassi N, et al. Microalgae for oil: strain selection,
induction of lipid synthesis and outdoor mass cultivation in a low-cost
photobioreactor. Biotechnol Bioeng 2009;102:10012.
[6] Sheehan J, Dunahay T, Benemann J, et al. A look back at the US Department of
Energys Aquatic species program: biodiesel from algae. NREL; 1998 [TP-580-
24190].
[7] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:294306.
[8] Miao XL, Wu QY. Biodiesel production from heterotrophic microalgal oil.
Bioresource Technol 2006;97:8416.
[9] Xiong W, Li XF, Xiang JY, Wu QY. High-density fermentation of microalga
Chlorella protothecoides in bioreactor for microbio-diesel production. Appl
Microbiol Biotechnol 2008;78:2936.
[10] Sharma YC, Singh B. Development of biodiesel: current scenario. Renew Sust
Energy Rev 2009;13:164651.
[11] Li XF, Xu H, Wu QY. Large-scale biodiesel production from microalga Chlorella
protothecoides through heterotrophic cultivation in bioreactors. Biotechnol
Bioeng 2007;98:76471.
[12] Billa E, Koullas DP, Monties B, Koukios EG. Structure and composition of sweet
sorghum stalk components. Ind Crops Prod 1997;6:297302.
[13] Ntaikou I, Gavala HN, Kornaros M, Lyberatos G. Hydrogen production from
sugars and sweet sorghum biomass using Ruminococcus albus. Int J Hydrogen
Energy 2008;33:115363.
[14] Laopaiboon L, Thanonkeo P, Jaisil P, Laopaiboon P. Ethanol production from
sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces
cerevisiae. World J Microbiol Biotechnol 2007;23:1497501.
[15] Sakellariou-Makrantonaki M, Papalexis D, Nakos N, Kalavrouziotis IK. Effect of
modern irrigation methods on growth and energy production of sweet
sorghum (var. Keller) on a dry year in Central Greece. Agric Water Manage
2007;90:1819.
[16] Gnansounou E, Dauriat A, Wyman CE. Rening sweet sorghum to ethanol and
sugar: economic trade-offs in the context of North China. Biores Technol
2005;96:9851002.
[17] Zhao YL, Dolat A, Steinberger Y, et al. Biomass yield and changes in chemical
composition of sweet sorghum cultivars grown for biofuel. Field Crops Res
2009;111:5564.
[18] Bulawayo B, Bvochora JM, Muzondo MI, Zvauya R. Ethanol production by
fermentation of sweet-stem sorghum juice using various yeast strains. World J
Microbiol Biotechnol 1996;12:35760.
[19] Liu RH, Shen F. Impacts of main factors on bioethanol fermentation from stalk
juice of sweet sorghum by immobilized Saccharomyces cerevisiae (CICC 1308).
Biores Technol 2008;99:84754.
[20] Yu JL, Zhang X, Tan TW. Ethanol production by solid state fermentation of
sweet sorghum using thermotolerant yeast strain. Fuel Process Technol
2008;89:10569.
[21] Agarwal AK. Biofuels (alcohols and biodiesel) applications as fuels for internal
combustion engines. Prog Energy Combust Sci 2007;33:23371.
[22] Wu Q, Yin S, Sheng GY, Fu J. A comparative study of gases generated from
simulant thermal degradation of autotrophic and heterotrophic Chlorella. Prog
Nat Sci 1992;3:43540 [in Chinese].
[23] Gao CF, Xiong W, Zhang YL, et al. Rapid quantitation of lipid in microalgae by
time-domain nuclear magnetic resonance. J Microbiol Methods
2008;75:43740.
[24] Energy Information Administration. Annual energy review; 2007.
<www.eia.doe.gov/emeu/aer/renew.html> [renewable energy Table 10.3].
[25] Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and
other applications: a review. Renew Sust Energy Rev, in press. doi:10.1016/
j.rser.2009.07.020 [available online 3.8.2009].
[26] Yee KF, Tan KT, Abdullah AZ, et al. Life cycle assessment of palm biodiesel:
revealing facts and benets for sustainability. Appl Energy 2009;86:S18996.
[27] Ji GS, Du RH, Hou SL, et al. Study on the sugar content in the stems of sweet
sorghum. Acta Agric Boreali-Sinica 2006;21:813 [in Chinese].
C. Gao et al. / Applied Energy 87 (2010) 756761 761