Bioresource Technology 97 (2006) 841–846

Biodiesel production from heterotrophic microalgal oil

a,b a,*
Xiaoling Miao , Qingyu Wu
a
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China
b
Department of Biological Sciences, Ningde Teachers College, Fujian, Ningde 352100, PR China

Received 6 September 2004; received in revised form 14 February 2005; accepted 6 April 2005
Available online 4 June 2005

Abstract

The present study introduced an integrated method for the production of biodiesel from microalgal oil. Heterotrophic growth of
Chlorella protothecoides resulted in the accumulation of high lipid content (55%) in cells. Large amount of microalgal oil was effi-
ciently extracted from these heterotrophic cells by using n-hexane. Biodiesel comparable to conventional diesel was obtained from
heterotrophic microalgal oil by acidic transesterification. The best process combination was 100% catalyst quantity (based on oil
weight) with 56:1 molar ratio of methanol to oil at temperature of 30 °C, which reduced product specific gravity from an initial value
of 0.912 to a final value of 0.8637 in about 4 h of reaction time. The results suggested that the new process, which combined bioen-
gineering and transesterification, was a feasible and effective method for the production of high quality biodiesel from microalgal oil.
Ó 2005 Elsevier Ltd. All rights reserved.

Keywords: Biodiesel; Transesterification; Microalgal oil; Heterotrophic; Chlorella protothecoides; Acid catalyst

1. Introduction an alternative fuel since 1988 in many European coun-

Fatty acid methyl esters originating from vegetable
oils and animal fats are known as biodiesel. Biodiesel
fuel has received considerable attention in recent years,
as it is a biodegradable, renewable and non-toxic fuel.
It contributes no net carbon dioxide or sulfur to the
atmosphere and emits less gaseous pollutants than nor-
mal diesel (Lang et al., 2001; Antolin et al., 2002; Vice-
nte et al., 2004). There are four primary ways to make
biodiesel, direct use and blending, microemulsions, ther-
mal cracking (pyrolysis) and transesterification (Ma and
Hanna, 1999). The most common way is transesterifica-
tion as the biodiesel from transesterification can be used
directly or as blends with diesel fuel in diesel engine (Pet-
erson et al., 1991; Zhang et al., 2003). Biodiesel, primar-
ily rapeseed methyl ester, has been in commercial use as
*
Corresponding author. Tel./fax: +86 10 62781825.
E-mail address: qingyu@tsinghua.edu.cn (Q. Wu).
tries (Lang et al., 2001). However, in spite of the favour-
able impact that its commercialization could provide,
the economic aspect of biodiesel production prevents
its development and large-scale use, mainly due to the
high feed cost of vegetable oil (Antolin et al., 2002; Lang
et al., 2001). Biodiesel usually costs over US$0.5/l, com-
pared to US$0.35/l for normal diesel (Zhang et al.,
2003). Exploring ways to reduce the high cost of biodie-
sel is of much interest in recent biodiesel research, espe-
cially for those methods concentrating on minimizing
the raw material cost.
Microalgae have been suggested as very good candi-
dates for fuel production because of their advantages
of higher photosynthetic efficiency, higher biomass pro-
duction and faster growth compared to other energy
crops (Milne et al., 1990; Ginzburg, 1993; Dote et al.,
1994; Minowa et al., 1995). Heterotrophic growth of
some microalgae has been used for efficient production
of biomass and some metabolites such as lipid (Shi
et al., 2000, 2002; Wen et al., 2002), which can reduce

0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.04.008
842 X. Miao, Q. Wu / Bioresource Technology 97 (2006) 841–846
the cost of microalgal biomass production and microal-
gal oil production. However, most of the research has
concentrated on biodiesel production from vegetable
oil such as soybean oil, sunflower oil, palm oil and rape-
seed oil (Siler-Marinkovic and Tomasevic, 1998; Lang
et al., 2001; Antolin et al., 2002; Al-Widyan and Al-Shy-
oukh, 2002). There is no information available regard-
ing the production of biodiesel from heterotrophic
microalgal oil. Chlorella protothecoides is a microalgae
that can be photoautotrophically or heterotrophically
grown under different culture conditions. Heterotrophic
growth of C. protothecoides results in high production of
biomass and accumulation of high lipid content in cells.
In the present study, an integrated method for biodiesel
production from heterotrophic microalgal oil by acidic
transesterification was developed. The characteristics
of acidic transesterification of microalgal oil and the
properties of the biodiesel were investigated.
2. Methods
2.1. Microalgae and sample preparation
The strain of C. protothecoides was provided by the
Culture Collection of Algae at the University of Texas
(Austin, Texas, USA). The culture medium and method
were as described before (Wu et al., 1992). The alga was
grown autotrophically and axenically in batch cultures
under 26 °C (±1) with continuous illumination at inten-
sities of 40 lmol m 2 s 1. Aeration was provided by
bubbling air at regular pressure. For the heterotrophic
growth of C. protothecoides, 10 g/l glucose was added
to the basal medium and the glycine was reduced to
0.1 g/l. The details of the culture of heterotrophic cells
were reported in our previous research (Wu et al.,
1994). Cells were harvested by centrifugation and
washed with distilled water and then dried by a freeze
dryer. The main chemical components of autotrophic
and heterotrophic C. protothecoides were measured as
described before (Miao et al., 2004). Lipid compounds
(referred as microalgal oil) was prepared by pulveriza-
tion of heterotrophic cell powder in a mortar and then
extraction with n-hexane.
The saponification (189.3 mg KOH/g) and acid value
(8.97 mg KOH/g) of the microalgal oil were determined
according to the method of Vicente et al. (2004). The
molecular weight of the oil was calculated from saponi-
fication and acid value. The average molecular weight of
the oil was 933.
2.2. Reaction conditions
Preliminary experiments indicated that the basic cata-
lyst (alkali catalyst) was not suitable for the transesterifi-
cation of microalgal oil probably because of the high acid
value of microalgal oil (8.97 mg KOH/g). Therefore, the
acid catalyst (sulfuric acid) was used in the research.
Experiments were designed to determine effections of
catalyst quantity, molar ratio of methanol to oil, time
and the temperature in reaction on the yield and proper-
ties of biodiesel product. According to the methods of
Al-Widyan and Al-Shyoukh (2002) and Siler-Marinko-
vic and Tomasevic (1998), four levels of the catalyst
quantity (25%, 50%, 60% and 100% H2SO4 based on
oil weight), six levels of molar ratio of methanol to oil
(25:1, 30:1, 45:1, 56:1, 70:1 and 84:1) and three different
temperatures (30, 50 and 90 °C) were used in the exper-
iments. Each 9.12 g (about 0.01 mol) of microalgal oil
was used in all experiments.
2.3. Acidic transesterification of microalgal oil
The acid catalyzed transesterification were carried out
in flasks and heated to the reaction temperature on a sha-
ker or constant temperature bath. A standard reaction
mixture consisted of oil, methanol and concentrated sul-
furic acid. The reaction mixture was heated for the speci-
fied period, cooled, and left to separated into two layers
in a separating funnel. The upper oil layer (biodiesel) was
separated, and washed with petroleum ether and then
washed with hot water (50 °C) until the washing were
neutral. The biodiesel product was obtained by evaporat-
ing the ether solution. Biodiesel yield (wt.%) relative to
the weight of microalgal oil was estimated.
In order to investigate the changes of the product spe-
cific gravity with reaction time, aliquots of samples were
taken from the reaction mixture at specified times and
centrifuged to obtain the upper layer. The specific grav-
ity of the upper layer was precisely measured. Lower
values of the specific gravity indicated that more of the
heavy glycerine was removed, more the reaction was
completed (Al-Widyan and Al-Shyoukh, 2002).
2.4. Analysis of biodiesel from microalgal oil
The properties of biodiesel such as density, viscosity,
flash point, cold filter plugging point, solidifying point
and heating value were measured. The elemental compo-
sitions of biodiesel were determined by a CE-440 ele-
mental analyzer.
3. Results and discussion
3.1. Heterotrophic microalgal oil
As shown in Fig. 1, heterotrophic growth of C. prot-
othecoides resulted in not only the disappearance of
chlorophyll in cells (Fig. 1A) but also accumulation of
high lipid content in cells. Lipid content in heterotrophic
cells reached as high as 55.20% (Table 1), which was
 X. Miao, Q. Wu / Bioresource Technology 97 (2006) 841–846 843

However, the high cost of biodiesel due to the high cost

of vegetable oil prevents its large-scale use and develop-
ment. C. protothecoides is a microalgae that can be
photoautotrophically or heterotrophically grown under
different culture conditions. Heterotrophic growth of
C. protothecoides results in high production of biomass
and accumulation of high lipid content in cells. Large
amount of microalgal oil was efficiently extracted from
these heterotrophic cells by using n-hexane. In addition,
heterotrophic growth can be performed in conventional
microbial bioreactors, which will improve the yield of
biomass and reduce the cost of microalgal biomass pro-
duction and microalgal oil production. It is important as
the low cost is desirable for commercial application in
biodiesel production. Developing high lipid content mic-
roalgae or Ôbioengineering microalgaeÕ would be a new
and the promising way for biodiesel production in the
future.

3.2. Effect of temperature and catalyst quantity

The yield and the specific gravity of biodiesel product

Fig. 1. (A) Growth of the cells of C. protothecoides under autotrophic
(left, green) and heterotrophic (right, yellow) culture conditions. (B, C)
under different temperature and catalyst quantity (on oil
Cells of autotrophic and heterotrophic C. protothecoides under basis) are shown in Fig. 2. At different levels of catalyst
differential interference microscopy. (B) Almost no lipid vesicles were
observed in autotrophic C. protothecoides cells. (C) The cells of
25% ‘Catalyst quantity’
heterotrophic C. protothecoides were full of lipid vesicles. (For 50%
100
interpretation of the references in colour in this figure legend, the 60%
100%
reader is referred to the web version of this article.)
Biodiesel yield (wt.%)

80

Table 1 60
Contents of the main chemical components of cells of autotrophic
(AC) and heterotrophic (HC) C. protothecoides 40
Component (%) AC HC
20
Protein 52.64 ± 0.26 10.28 ± 0.10
Lipid 14.57 ± 0.16 55.20 ± 0.28
Carbohydrate 10.62 ± 0.14 15.43 ± 0.17 0
0 20 40 60 80 100
Ash 6.36 ± 0.05 5.93 ± 0.04
A Temperature (oC)
Moisture 5.39 ± 0.04 1.96 ± 0.02
Others 10.42 ± 0.65 11.20 ± 0.61 25% ‘Catalyst quantity’
50%
60%
0.9 100%
0.895
about four times that in autotrophic cells (14.57%)
(Table 1). The heterotrophic cells were full of lipid 0.89
Specific gravity

vesicles, which could be easily observed under differen- 0.885

tial interference microscopy (Fig. 1C). The lipid-soluble 0.88
compounds from the autotrophic cells appeared in a 0.875
blackish green with chlorophyll and carotenoid as the 0.87
major components, whereas the lipid-soluble com-
0.865
pounds from the heterotrophic cells appeared in a state
0.86
of light yellow grease, which were mainly lipid com- 0 20 40 60 80 100
pounds (referred as oil). The fatty acid composition of B Temperature (oC)
the oil mainly composed oleic acid, linoleic acid, cetane
Fig. 2. The influence of catalyst quantity and temperature on the yield
acid by hydrolysis, esterification and gas chromato-
(A) and the specific gravity (B) of biodiesel product. Reaction
graphical analysis (Wu et al., 1992). conditions: 30:1 molar ratio of methanol to oil, 160 rpm, 5 h of
Biodiesel, primarily made from vegetable oil is being reaction time. Temperature: 30 °C, 50 °C and 90 °C. The catalyst
commercialized in many countries (Lang et al., 2001). quantity based on oil weight: 25%, 50%, 60% and 100%.
844 X. Miao, Q. Wu / Bioresource Technology 97 (2006) 841–846

quantity, high yields of biodiesel products were all ob- 100

tained at 50 °C (Fig. 2A), whereas low values of specific
gravity were all obtained at 90 °C (Fig. 2B). At the same 80

Biodiesel yield (wt.%)

temperature, low values of specific gravity were obtained
at levels of 100% catalyst quantity (Fig. 2B). As reported 60
by Al-Widyan and Al-Shyoukh (2002) and Siler-Mar-
inkovic and Tomasevic (1998), high catalyst concentra- 40
tions (2.25 M or 100% H2SO4) could produce biodiesel
with low specific gravity in much short time. In the pres- 20
ent work, the lowest value of specific gravity (0.862) was
obtained at 90 °C in the presence of 100% catalyst quan-
0
tity, whereas the yield was lowest at these levels. This is 25:1 30:1 45:1 56:1 70:1 84:1
probably because that high temperature and high con- A Molar ratio
centration of H2SO4 could burn some of oil, which cause
low yield of biodiesel product. In spite of this, good 0.91
quality of biodiesel could be obtained in the presence
of 100% acid catalyst at a higher temperature. At level 0.90
of 100% catalyst quantity, there were only slight differ-

Specific gravity
ence in the yields of biodiesel and their specific gravity 0.89
at temperature of 30 °C and 50 °C. The biodiesel prod-
uct yields at temperature of 30 °C and 50 °C were 56% 0.88
and 58% respectively (Fig. 2A), and the values of prod-
uct specific gravity were 0.878 and 0.875 respectively 0.87
(Fig. 2B). On economic basis, the process combination
of 100% catalyst quantity with temperature of 30 °C 0.86
would be best. Production cost in this case is lower than 25:1 30:1 45:1 56:1 70:1 84:1
that when H2SO4 was used at high temperature for a B Molar ratio
long time. Therefore, above conditions were applied to
Fig. 3. The yield (A) and the specific gravity (B) of biodiesel product
the following experiments.
with different molar ratio of methanol to oil. Reaction conditions:
30 °C, 160 rpm, 100% catalyst quantity based on oil weight, 7 h of
3.3. Effect of molar ratio of methanol to oil reaction time.

From the results presented in Fig. 2, 100% catalyst

quantity and the temperature of 30 °C were used in
0.92 30:1 ‘Molar ratio’
the experiments of the effects of molar ratio of methanol 45:1
to oil on the yield and the specific gravity of biodiesel 0.91 56:1
product.
Specific gravity

Fig. 3 shows the product yield and specific gravity 0.90

under different molar ratio of methanol to oil after 7 h
0.89
of reaction time. High yields of biodiesel were obtained
at molar ratio levels of 45:1 and 56:1 (Fig. 3A), they ac- 0.88
counted for 68% and 63%, respectively. The values of
the product specific gravity at these two molar ratio lev- 0.87
els were as low as 0.873 and 0.864 respectively (Fig. 3B).
The level of 30:1 also gave the low product specific grav- 0.86
0 1 2 3 4 5 6 7 8
ity (0.878). Therefore, the changes of the product Reaction time (h)
specific gravity with reaction time were further investi-
gated from these three molar ratio levels as shown in Fig. 4. The variation of product specific gravity with reaction time
under different molar ratio of methanol to oil. Reaction conditions:
Fig. 4. The specific gravity of the product varied with 30 °C, 160 rpm, 100% catalyst quantity based on oil weight.
reaction time. It may be noticed that at any given level
of methanol, the product specific gravity decreased with
reaction time in a roughly exponential fashion (Fig. 4). values of product specific gravity (Fig. 4). The presence
The molar ratio of methanol to oil at level of 56:1 re- of excess methanol in the transesterification process is
sulted in the lowest terminal product specific gravity essential for it is responsible for breaking the glycer-
and in the shortest reaction time (4 h). At 45:1 and ine–fatty acid linkages (Al-Widyan and Al-Shyoukh,
30:1 levels, it took about 5–7 h to reach the asymptotic 2002). However, an excess of methanol in large quanti-
 X. Miao, Q. Wu / Bioresource Technology 97 (2006) 841–846
Table 2
Comparison of properties of biodiesel from microalgal oil and diesel fuel and ASTM biodiesel standard
Properties Biodiesel from microalgal oil
Density (kg/l) 0.864
Viscosity (mm2/s, cSt at 40 °C) 5.2
Flash point (°C) 115
Solidifying point (°C) 12
Cold filter plugging point (°C) 11
Acid value (mg KOH/g) 0.374
Heating value (MJ/kg) 41
H/C ratio 1.81
a
The data about diesel fuel were taken from published literature as indicated in the text.
ties slowed down the separation of the two phases pro-
duced as in the case of the levels of 70:1 and 84:1 molar
ratio. Therefore, the best process combination of acidic
transesterification of microalgal oil was 100% catalyst
quantity with 56:1 molar ratio of methanol to oil at tem-
perature of 30 °C. It reduced product specific gravity
from an initial value of 0.912 to a final value of 0.8637
in about 4 h of reaction time.
3.4. Characteristics of biodiesel from microalgal oil
To assess the potential of biodiesel as a substitute of
diesel fuel, the properties of biodiesel such as density,
viscosity, flash point, cold filter plugging point, solidify-
ing point and heating value were determined. A compar-
ison of these properties of diesel fuel (Ma and Hanna,
1999; Lang et al., 2001; Al-Widyan and Al-Shyoukh,
2002; Antolin et al., 2002; Vicente et al., 2004) and bio-
diesel from microalgal oil and ASTM biodiesel standard
is shown in Table 2. Most of these parameters comply
with the limits established by ASTM related to biodiesel
quality (Antolin et al., 2002). The physical and fuel
properties of biodiesel from microalgal oil in general
were comparable to those of diesel fuel. The biodiesel
from microalgal oil showed much lower cold filter plug-
ging point of 11 °C in comparison with the diesel fuel
(Table 2).
The results suggested that the new process, which com-
bined bioengineering and transesterification, could be a
feasible and effective method for the production of high
quality biodiesel from heterotrophic microalgal oil. The
biodiesel from heterotrophic microalgal oil could be a
competitive alternative to conventional diesel fuel.
4. Conclusions
The present study introduced an integrated method
for the production of biodiesel from heterotrophic mic-
roalgal oil. C. protothecoides is a microalgae that can be
photoautotrophically or heterotrophically grown under
different culture conditions. Heterotrophic growth of
C. protothecoides resulted in the accumulation of a large
845
Diesel fuela ASTM biodiesel standard
0.838 0.86–0.9
1.9–4.1 3.5–5.0
75 Min 100
50 to 10 –
3.0 (Max 6.7) Summer max 0; winter max < 15
Max 0.5 Max 0.5
40–45 –
1.81 –
amount of lipid in cells. Lipid content in heterotrophic
cells reached as high as 55.20%. Large amount of micro-
algal oil was efficiently extracted from these heterotro-
phic cells by using n-hexane. Biodiesel comparable to
conventional diesel was obtained from heterotrophic
microalgal oil by acidic transesterification. Good quality
of biodiesel could be obtained in the presence of 100%
acid catalyst (on oil basis) at high temperature. The best
combination of factors was 100% catalyst quantity
(based on oil weight) with 56:1 molar ratio of methanol
to oil at temperature of 30 °C, which reduced product
specific gravity from an initial value of 0.912 to a final
value of 0.8637 in about 4 h of reaction time. Develop-
ing high lipid content microalgae or Ôbioengineering mic-
roalgaeÕ for biofuels production would be becoming a
new and the promising way in biofuels production in
the future.
Acknowledgements
This research was supported by NSFC project
40272054 and NSFC key project of 40332022 to Q.
Wu. It was also supported by National Key research
plan 2004BA411B05 from Chinese Ministry of Science
and Technology.
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