Applied Energy 87 (2010) 3846

Contents lists available at ScienceDirect

Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Biodiesel production by microalgal biotechnology

GuanHua Huang a,*, Feng Chen b,c, Dong Wei c, XueWu Zhang c, Gu Chen c
a
b
c

School of Chemical Engineering and Technology, China University of Mining and Technology, China
School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China

a r t i c l e

i n f o

Article history:
Received 27 April 2009
Received in revised form 9 June 2009
Accepted 10 June 2009
Available online 8 July 2009
Keywords:
Microalgae
Biodiesel
Lipids
Fatty acids
Production

a b s t r a c t
Biodiesel has received much attention in recent years. Although numerous reports are available on the
production of biodiesel from vegetable oils of terraneous oil-plants, such as soybean, sunower and palm
oils, the production of biodiesel from microalgae is a newly emerging eld. Microalgal biotechnology
appears to possess high potential for biodiesel production because a signicant increase in lipid content
of microalgae is now possible through heterotrophic cultivation and genetic engineering approaches. This
paper provides an overview of the technologies in the production of biodiesel from microalgae, including
the various modes of cultivation for the production of oil-rich microalgal biomass, as well as the subsequent downstream processing for biodiesel production. The advances and prospects of using microalgal
biotechnology for biodiesel production are discussed.
2009 Elsevier Ltd. All rights reserved.

Contents
1.
2.

3.

4.
5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microalgal biotechnology for lipids production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Biosynthesis of lipids/fatty acids in microalgae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1.
The formation of acetyl coenzyme A (acetyl-coA) in cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
The elongation and desaturation of carbon chain of fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.3.
The biosynthesis of triglycerides in microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Phototrophic cultivation of microalgae for lipids production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Heterotrophic cultivation of microalgae for lipids production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Genetic engineering for lipids production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The extraction of oils from microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Pyrolysis technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Liquefaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transesterification technologies in the production of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
The production of biodiesel has recently received much attention worldwide. Because of the world energy crisis [1], many
countries have started to take a series of measures to resolve
this problem [2]. Finding alternative energy resources is a pressing mission for many countries, especially for those countries
* Corresponding author.
E-mail address: huangghclara@yahoo.com.cn (G. Huang).
0306-2619/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2009.06.016

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lacking conventional fuel resources. In the 1930s and 1940s, vegetable oils has been used as diesel fuels in the emergency situation. With the rapid development of the modern industry, the
demand for energy has been greatly increased in recent years,
and therefore alternative energy sources are being explored.
Thus, the term biodiesel has appeared very frequently in many
recent reports [3].
The world total biodiesel production was estimated to be
around 1.8 billion liters in 2003 [4]. Although there was no
increase in biodiesel production between 1996 and 1998, a sharp

G. Huang et al. / Applied Energy 87 (2010) 3846

increase in biodiesel production was observed in the past several

years. It is speculated that the production of biodiesel will be further tremendously increased because of increasing demand for
fuels and cleaner energy globally.
Biodiesel is made from biomass oils, mostly from vegetable oils.
Biodiesel appears to be an attractive energy resource for several
reasons. First, biodiesel is a renewable resource of energy that
could be sustainably supplied. It is understood that the petroleum
reserves are to be depleted in less than 50 years at the present rate
of consumption [5]. Second, biodiesel appears to have several
favorable environmental properties resulting in no net increased
release of carbon dioxide and very low sulfur content [6,7]. The release of sulfur content and carbon monoxide would be cut down by
30% and 10%, respectively, by using biodiesel as energy source.
Using biodiesel as energy source, the gas generated during combustion could be reduced, and the decrease in carbon monoxide
is owing to the relatively high oxygen content in biodiesel. Moreover, biodiesel contains no aromatic compounds and other chemical substances which are harmful to the environment. Recent
investigation has indicated that the use of biodiesel can decrease
90% of air toxicity and 95% of cancers compared to common diesel
source [64]. Third, biodiesel appears to have signicant economic
potential because as a non-renewable fuel that fossil fuel prices
will increase inescapability further in the future [8]. Finally, biodiesel is better than diesel fuel in terms of ash point and biodegradability [9].
Conventional biodiesel mainly comes from soybean and vegetable oils [10], palm oil [11], sunower oil [6], rapeseed oil [12]
as well as restaurant waste oil [13]. The number of carbon in the
carbon chain of the diesel oil molecular is about 15, which is
similar to that of the plant oil with 1418 carbons. The structural characteristic of biodiesel determines that biodiesel is a
feasible substitute for conventional energy. Nevertheless, the
production cost is generally high for biodiesel. The price of biodiesel is approximately twofold that of the conventional diesel at
present. The production cost of biodiesel consists of two main
components, namely, the cost of raw materials (fats and oil)
and the cost of processing. The cost of raw materials accounts
for 60% to 75% of the total cost of the biodiesel fuel [14]. Though
there might be large amounts of low-cost oil and fats available
such as restaurant waste and animal fats [15], the major problem of using these low-cost oils and fats is that they often contain large amounts of free fatty acids (FFA) which is difcult to
convert to biodiesel through transesterication [16]. Raw materials that contain large proportions of fatty acid triglycerides are
preferred. For example, plant oil is found to contain more fatty

39

acid triglycerides and therefore has been used in the production

of biodiesel for some years [17].
Earlier studies on liquid fuel from microalgae had begun in mid1980s. During the world war II, although some German scientists
attempted to extract lipids from diatom in order to resolve energy
crisis [18], and soon later in the USA, research was conducted by a
group of scientists at the Carnegie Institution of Washington, and
their experiences had been summarized in a book [65] entitled
Algal Culture from Laboratory to Pilot Plant, but the technologies
of making microalgae as fuels had not been fully exploited. The
reasons could be as follows. First, as a source of lipids, microalgae
are less known than plants and animals. Second, the prices for
most plant oils are relatively low and animal fats are even cheaper;
therefore, processes for the microbial oils production have mainly
focused on high-valued products that cannot be produced by
plants, such as omega-3 polyunsaturated fatty acids, especially
EPA and DHA [19].
In order to resolve the worldwide energy shortage crisis, seeking for lipid-rich biological materials to produce biodiesel effectively has attracted much renewed interest. Oleaginous
microorganisms are favorably considered for their short growth
cycles, high lipid contents and ease of being modied by biotechnological means (see Table 1). Some microalgae appear to be suitable group of oleaginous microorganism for lipids production [20].
Microalgae have been suggested as potential candidates for fuel
production because of a number of advantages including higher
photosynthetic efciency, higher biomass production and higher
growth rate compared to other energy crops [2123]. Moreover,
according to biodiesel standard published by the American Society
for Testing Materials (ASTM), biodiesel from microalgal oil is similar in properties to the standard biodiesel, and is also more stable
according to their ash point values (Table 2).
2. Microalgal biotechnology for lipids production
Microalgae have high potentials in biodiesel production compared to other oil crops. First, the cultivation of microalgae dose
not need much land as compared to that of terraneous plants
[20]. Biodiesel produced from microalgae will not compromise
the production of food and other products derived from crops. Second, microalgae grow extremely rapidly and many algal species are
rich in oils. For instance, heterotrophic growth of Chlorella protothecoides can accumulate lipids as high as 55% of the cell dry weight
after 144 h of cultivation with feeding of corn powder hydrolysate
in fermenters [24]. Oil levels of 2050% are common in microalgae
[20]. The whole technical process in the production of biodiesel

Table 1
Comparison of types of sources for the oils production.
Type of organism

Advantages

Disadvantages

Microalgal oils

(1)
(2)
(3)
(4)

(1) Most algal lipids have lower fuel value than diesel fuel
(2) The cost of cultivation is higher compared to common crop oils
currently

Fatty acid constitutions similar to common vegetable oils

Under certain condition it may be as high as 85% of the dry weight
Short-time growth cycle
Composition is relative single in microalgae

Bacteria oils

(1) Fast growth rate

(1) Most of bacteria can not yield lipids but complicated lipoid

Oleaginous yeasts
and mildews

(1) Resources are abundant in the nature

(2) High oil content in some species
(3) Short-time growth cycle

(1) Filtration and cultivation of yeasts and mildews with high-content oils
are required
(2) Process of oils extracted from oleaginous yeasts and mildew is complex
and new technology should be exploited to resolve it
(3) the cost of cultivation is also higher compared to common crop oils
currently

(4) Strong capability of growth in different cultivation on conditions

(5) Conversion and utilization of scrap ber to yield useful oils and the
application for dealing with waste oils in environment
Waste oils

(1) The waste oils is cheap compared to crop oils

(1) Containing a lot of saturated fatty acids which is hard to be converted to

biodiesel by catalyst

40

G. Huang et al. / Applied Energy 87 (2010) 3846

Table 2
Comparison of properties of microalgal oil, conventional diesel fuel, and ASTM biodiesel standard [24].
Properties

Biodiesel from microalgal oil

Diesel fuel

ASTM biodiesel standard

Density (kg L )
Viscosity (mm2 s 1, cSt at 40 C)
Flash point (C)
Solidifying point (C)
Cold lter plugging point (C)

0.864
5.2
115
12
11

0.838
1.94.1
75
50 to 10
3.0 (max

Acid value (mg KOH g 1)

Heating value (MJ kg 1)
H/C ratio

0.374
41
1.81

Max 0.5
4045
1.81

0.840.90
3.55.0
Min 100

Summer max 0
Winter max < 15
Max 0.5

from microalgae has been well investigated in recent years.

Third, the entire production process ranging from the cultivation
of high-lipid microalgae to the production of biodiesel from the
microalgal oils has also been explored. In the laboratory conditions,
the ideal oil content could reach 5660% of total dry biomass by
genetic engineering or heterotrophic culture techniques. These
technological advances suggest that the industrial production of
biodiesel from microalgal oils may be feasible in the near future.
2.1. Biosynthesis of lipids/fatty acids in microalgae
It is known that both inorganic carbon (CO2) and organic carbon
sources (glucose, acetate, etc.) can be utilized by microalgae for lipids production. The components and contents of lipids in microalgal cells vary from species to species. The lipid classes basically are
divided into neutral lipids (e.g., triglycerides, cholesterol) and polar
lipids (e.g., phospholipids, galactolipids). Triglycerides as neutral
lipids are the main materials in the production of biodiesel. The
synthesis routes of triglycerides in microalgae may consist of the
following three steps: (a) the formation of acetyl coenzyme A
(acetyl-coA) in the cytoplasm; (b) the elongation and desaturation
of carbon chain of fatty acids; and (c) the biosynthesis of triglycerides in microalgae.
2.1.1. The formation of acetyl coenzyme A (acetyl-coA) in cytoplasm
The metabolism ux route on the utilization of carbon dioxide
and glucose for the formation of acetyl-coA in microalgae is described by Yang et al. [25]. It is concluded that glyceraldehyde
phosphate (GAP) is a key intermediate both for the two metabolism systems. The formation of acetyl-coA in photosynthetic reactions, including the light reactions, Calvin cycle and synthesis, is
located in chloroplast. GAP is withdrawn from Calvin cycle and exported to cytoplasm for consumption. After the export of GAP from
chloroplast to cytoplasm, the ow of carbon is directed to the synthesis of sugars or oxidation through the glycolytic pathway to
pyruvate. Sugars including sucrose are the major storage products
in the cytoplasm of plant cells. Akazawa and Okamoto [26] reported that glucose was easy to be stored as starch without prior
conversion to GAP and then uptake by the chloroplast which suggested starch is the main storage formation for carbon source in
Chlorella sp. Therefore, one part of the exogenous glucose was directly converted to starch, and the remainder was oxidized
through glycolytic pathway.
2.1.2. The elongation and desaturation of carbon chain of fatty acids
The elongation of carbon chain of fatty acids is mainly dependent on the reaction of two enzyme systems including acetyl-coA
carboxylic enzyme (ACCE) and fatty acid synthase (FAS) in most
organisms. In the process of synthesis of fatty acids (Fig. 1), acetyl-coA is the primer. The process of carbon chain elongation needs
the cooperation with malonyl-coA, the substrate on which enzyme
act are acetyl-ACP and malonyl-ACP. The C16C18 fatty acid thioes-

6.7)

ter can be formed after several reaction steps. The formation of

short carbon chain fatty acids is similar in the cells of advanced
plants, animals, fungi, bacteria, and algae. For example, in the cell
of green algae, the reaction routes of primer such as palmitoleic
acid, oleic acid, linoleic acid, linolenic acid in fatty acid synthesis
are similar to that in plant cells and yeast cells [27]. The desaturation of carbon chain of fatty acid occurs from C18 and further elongation of carbon chain takes place to produce long-chain fatty acids
which are unusual in normal plant oils (Fig. 2). Long-chain fatty
acids (C20C22) often exist in microalgae and the content varies
from species to species [28]. Normally, short-chain fatty acids
(C14C18) which are the main components of biodiesel are majority
of fatty acids in Chlorella sp., but high content of long-chain fatty
acid and hydrocarbons exist in some specic species of microalgae.
So, It is vital to choose proper microalgae species as materials of
biodiesel production.

COO-

CH2C-SCoA

CH2
C O

Econd

SCoA

ACP-SH

NADP+

RC-S-ACP
NADPH(H+)

O
-

COO
HSCoA

CH2

RCH=CHC-S-ACP

C O
S

RC-S-Econd

ACP

CO2
O

HS-Econd

OH

RCH-CH2C-S-ACP

RC-CH2C-S-ACP
NADPH (H+)

NADP+

Acetyl-CoA-ACP acyl transferase

malonyl CoA-ACP acyl transferase
-ketoacyl-ACP Condensing enzyme)
-ketoacyl-ACP reductase
-hydroxyacyl-ACP dehydrase
enoyl-ACP reductase
Fig. 1. Reaction process of the FFA biological synthesis system (Shen and Wang
[62]).

G. Huang et al. / Applied Energy 87 (2010) 3846

C18:0

41

acetyl-coA to form Lysophosphatidic acid and later combines

with another acetyl-coA to form phosphatidic acid. These two
reactions are catalyzed by glycerol phosphate acyl-transferase.
In the following steps, lysophosphatidic acid is hydrolyzed by
phosphatidate phosphatase to form diglyceride which is then
combined with the third acetyl-coA to complete the biosynthesis
of triglycerides. The last reaction step is catalyzed by glyceryl
diester transacylase.

9 desaturase
C18:1-9

12 desaturase
C18:2-9,12

15 desaturase

2.2. Phototrophic cultivation of microalgae for lipids production

C18:3-9,12,15

6 desaturase
C18:4-6,9,12,15

Fatty acid elongase

C18:4-8,11,14,17

5 desaturase
C20:4-8,11,14,17

elongation
C22:5-7,10,13,16,19

4 desaturase
C22:6-4, 7,10,13,16,19
Fig. 2. The elongation and desaturation of carbon chain of fatty acids (modied
from Guschina and Harwood [63]).

2.1.3. The biosynthesis of triglycerides in microalgae

Like other higher plant and animal, microalgae are able to
biosynthesize triglycerides to store substance and energy. Generally, L-a-phosphoglycerol and acetyl-coA are two major primers
in the biosynthesis of triglycerides. The L-a-phosphoglycerol
mainly derives from phosphodihydroxyacetone which is the
product of the glycolysis process. The reaction steps are shown
in Fig. 3. One of the hydroxyl in L-a-phosphoglycerol reacts with

Microalgae can transform carbon dioxide from the air and light
energy through photosynthesis to various forms of chemical energies such as polysaccharides, proteins, lipids and hydrocarbons.
Compared to higher plants, microalgae have a number of advantages including higher photosynthetic efciency and growth rate
[20]. In phototrophic culture, usually microalgae can be grown in
two systems such as open ponds and enclosed photobioreactors.
Enclosed photobioreactor system is more suitable for some microalgae which are readily contaminated by other microbes, except for
some special microalgae which can survive well in extreme environments such as high pH (e.g., Spirulina) and high salinity (e.g.,
Dunaliella) or can grow very rapidly (e.g., Chlorella) in the open
pond.
Because of better environmental control, enclosed photobioreactor system has been suggested for the production of high-value
long-chain fatty acids (e.g., DHA, EPA). Nevertheless, due to the
high cost in terms of operation and capital investment and the
small scale due to the complexity of bioreactor design compared
to open pond system, it might not be economical to produce biodiesel on a large scale by enclosed photobioreactors.
Open pond system is perhaps more suitable for cultivating microalgae for biodiesel because of its relatively cheap operating cost
compared to the enclosed photobioreactors. The basic requirements for microalgal phototrophic growth should include carbon
dioxide, other macro- and micro-nutrients, as well as light. Carbon
source can be obtained from power plants which release large

Fig. 3. The biosynthesis of triglycerides in microalgae.

42

G. Huang et al. / Applied Energy 87 (2010) 3846

Water Nutrients

Algae oil
recovery system

biodiesel
production

Open pond

Motorized paddle

Waster CO2

Waste water

Fig. 4. Open pond photosynthesis system (OPSS).

amounts of waste gases (mainly CO2) daily. Typical coal-red

power plants emit fuel gas from their stacks containing up to
13% CO2. This high concentration of CO2 enhances transfer and
uptake of CO2 in the pond system. The concept of combining a
coal-red power plant with algae cultivation provides a feasible
approach to recycle CO2 from coal combustion into useable liquid
fuel (Fig. 4). When grown in large outdoor ponds, microalgae could
use CO2 from fuel gas directly injected into the culture [29]. Besides, the wastewater may contain abundant nutrients (e.g., inorganic irons) which are necessary for microalgal growth. Chlorella
vulgaris was grown in waste water from a steelmaking plant with
the aim of developing an economically feasible system to remove
ammonia from wastewater and CO2 from ue gas [30].
2.3. Heterotrophic cultivation of microalgae for lipids production
Although microalgae can utilize light efciently, phototrophic
growth of microalgae is often slow because of light limitation at
high cell densities on a large scale [31] or photoinhibition due
to excessive light, especially in sunny days [32]. In view of these
disadvantages associated with photoautotrophic cultivation, heterotrophic growth of microalgae in conventional fermentors
should be favorably considered [33]. Heterotrophic cultivation of
microalgae offers several advantages over phototrophic cultivation
including elimination of light requirement, good control of the
cultivation process, and low-cost for harvesting the biomass because of higher cell density obtained in heterotrophic culture of
microalgae [34]. In heterotrophic culture, both cell growth and
biosynthesis of products are signicantly inuenced by medium
nutrients and environmental factors. Carbon sources are the most
important element for heterotrophic culture of microalgae in the
production of lipids. For example, although the green microalgae
C. protothecoides can grow photoautotrophically or heterotrophically. heterotrophic growth of C. protothecoides using acetate,
glucose, or other organic compounds as carbon source results in
much higher biomass as well as lipid content in cells [35]. More-

over, heterotrophic microalgae might utilize other carbon sources

such as ethanol, glycerol, and fructose depending on the microalgal
species used [36]. Liu et al. [37] compared several carbon sources
and concluded that glucose was preferred. In order to lower the
production cost of microalgal oils as biodiesel, cheaper carbon
sources should be considered. For instance, corn powder hydrolysate (CPH) or molasses instead of glucose may be used as organic
carbon source in heterotrophic culture. It was reported occasionally
CPH was superior to glucose solution, because CPH contained some
benecial components to Chlorella, and as a result, C. protothecoides
produced 55.2% crude lipids in the cells with a cell dry weight
concentration of 15.5 g L 1 [24]. The utilization of corn powder
hydrolysate instead of glucose in heterotrophic culture greatly reduced the cost of production, which is important for the biodiesel
production by microalgae in terms of economical signicance.
Nitrogen is also an essential macronutrient in lipids production. Complex nitrogen source might be superior to simple
nitrogen source in heterotrophic culture of microalgae, because
it might provide amino acids, vitamins and growth factors simultaneously. Industrial wastewater rich in nitrogen also can be considered for the cultivation of microalgae. Monosodium glutamate
waste after diluted was well treated as a cheap fermentation
medium for Rhodotorula glutinis to biosynthesize lipids as the
raw material for the production of biodiesel [38]. Many microalgae growing under nitrogen limitation show enhanced lipid
content. In the late 1940s, it was noted that nitrogen starvation
is most inuential on lipid storage and lipid fractions, and as a result of nitrogen starvation, the lipid content as high as 7085% of
dry weight was reported [39]. It was also reported that Prophyridium cruentum might double its total lipid content (mainly
neutral lipids) under nitrogen starvation [39]. Nevertheless, nitrogen starvation might not always result in an increase in total lipid
content in microalgae but a change in lipid composition. Zhila
et al. [40] reported that the alga Botryococcus braunii contained
high content (28.438.4%) of oleic acid under nitrogen limitation,
but the content of total lipids and triacylglycerols did not change.

G. Huang et al. / Applied Energy 87 (2010) 3846

It is also noticeable that the overall rates of oil production might

be lower in the case of nutrient deciency due to the overall lower biomass achieved. Therefore factors other than nitrogen should
be considered altogether.
Initial carbon to nitrogen (C/N) ratio in the medium has significant impact on the biosynthesis of lipids in microalgae. With the
addition of glucose as organic carbon source to the medium and
the tremendous decrease of nitrogen source in the medium, a
crude lipid content up to 55.2% was achieved in heterotrophic C.
protothecoides, which was about 3.4-fold that in photoautotrophic
C. protothecoides [41]. Chen and Johns [34] also investigated the effect of C/N ratio and aeration on the fatty acid composition of heterotrophic Chlorella sorokiniana. When C/N ratio of approximately
20, cell lipid content was at a minimum and increased at both
higher and lower C/N values.
Environmental factors also cannot be neglected in the growth of
microalgae and the formation of fatty acids. The high PUFAs content at low temperature might be explained by the fact that the algae need to produce more PUFAs to maintain cell membrane
uidity. Another reason might be that low temperature could lead
to high level of intracellular molecular oxygen and hence improves
the activities of the desaturase and elongase involved in the biosynthesis of PUFAs [42]. However, the effect of temperature on cell
growth and PUFAs production may not be always the same as mentioned above [31]. Therefore, a specic and careful study of the
individual microalgae is required. Salinity, pH, and dissolved O2
are also important factors affecting the heterotrophic cultivation
of microalgae [43,34]. Besides, it has been demonstrated that different cultivation modes greatly affect the lipid accumulation in
microalgae. Heterotrophically grown microalgae usually accumulate more lipids than those cultivated photoautotrophically [44].
Some phototrophic microorganisms could also be grown on
cheap organic substrates heterotrophically [33]. Evidences have
been shown that the dark metabolism of photosynthetic plants
and microalgae is similar to that of non-photosynthetic organisms
such as yeasts. Recently, an obligate photoautotrophic microalga
Phaeodactylum tricornutum was grown heterotrophically when a
single gene (Glut 1) that encoded the glucose transporter protein
(Glut 1) was introduced into this alga [45]. Such studies on the production of fatty acids and lipids should be useful for further investigation of microalgae as bio-fuels by heterotrophic cultivation.
2.4. Genetic engineering for lipids production
Genetic engineering of microalgae is an upstream technology in
microalgal biotechnology. In 1960s, cyanobacteria were chosen as
ideal material for academic research by scientists. Since the genome of Anabaena PCC7120 was successfully cloned, the number
of cloned functional genes in cyanobacteria has increased to over
130 [46]. Acc1 is a kind of restriction enzyme which was cloned
from oceanic diatom Cylclotella cryptica had been efciently expressed in C. cryptica for the production of bio-fuel [47]. A new
method for biodiesel production from microalgal oils has been
developed by the application of genetic engineering recently. National Renewable Energy laboratory in the USA (NREL) has established engineered microalgae which belong mostly to diatom
species. The lipid content of the engineered microalgae increased
to above 60% in laboratory conditions and above 40% in outdoors
cultivation, whereas the lipids content in microalgae is 520% in
common natural conditions. The improvement of lipid content in
engineered microalgal cells is mainly due to the high expression
of acetyl-coA carboxylase gene, which plays an important role in
the control of the level of lipid accumulation. At present, the research has focused on choosing a proper molecular carrier, making
ACC gene full expression in bacteria, yeast, and plant. Furthermore,
the modied ACC gene is being introduced into microalgae to ob-

43

tain more efcient expression. The utilization of engineered microalgae for the production of biodiesel has important economic and
environmental benets. Its superiorities include high yield of microalgae; saving agricultural resources by using seawater as natural
medium; the cellular content of lipids in microalgae is several
times higher than that in terrestrial plants.

3. The extraction of oils from microalgae

3.1. Pyrolysis technologies
Although oils extracted from microalgal cells have been investigated for fuel production of internal-combustion engines by
transesterication of fatty acids [48], industrial biodiesel production from microalgal oils is still not well developed. At least, high
content of microalgal oils is required for this method to realize
economic benets. Since it is difcult to obtain microalgae with
high fatty acid content conventionally, it has been considered
that only crude fat (lipid) is used for conversion into substitutes
for traditional fuels [49]. Recent research on the production of
energy from renewable sources such as bio-oils production by
pyrolysis of biomass has received much interest. Pyrolyzing microalgae to produce liquid fuel was rst put forward in Germany
in 1986. It was reported that the method of catalytic pyrolysis
could yield gasoline with high content of aromatic hydrocarbon
and octane number [21]. Pyrolysis is a phenomenon related to
decomposition of biomass under the condition of oxygen deciency and high temperature. Pyrolysis previously was rst used
for the production of bio-oils or bio-gases from lignocellulose.
However, such a technology may be more suitable for microalgae
because of the lower temperature required for pyrolysis and the
higher-quality oils obtained [50]. Moreover, the cost of pyrolysis
of lignocellulose is relatively higher than that of microalgae.
Compared to lignocellulose, microalgae contain high content of
cellular lipids, resolvable polysaccharides and proteins, which
are easier to be pyrolyzed to bio-oils and bio-gases. Compared
to slow pyrolysis [22,23], fast pyrolysis is a new technology,
which produces bio-fuel in the absence of air at atmospheric
pressure, with a relatively low temperature (450550 C) and
high heating rate (103104 C s 1) as well as short gas residence
time to crack into short chain molecules and be cooled to liquid
rapidly [50]. The main products of slow pyrolysis are char and
char-oils with a 1520% yield, whereas, the products of fast
pyrolysis are oils and gases with a yield of approximately 70%
[51]. Fast pyrolysis has proved to be a promising way to produce
bio-oils compared to slow pyrolysis [41] for the following reasons: (1) less bio-oils were produced from slow pyrolysis; (2)
the viscous bio-oils from slow pyrolysis is not suitable for liquid
fuels; and (3) the fast pyrolysis process is time saving and requires less energy compared to the slow prolysis process. Fast
pyrolysis tests of microalgae were performed in the uid bed
reactor [52]. It was reported that the experiment was completed
500 C with a heating rate of 600 C s 1, a sweep gas (N2) ow
rate of 0.4 m3 h 1 and a vapor residence time of 23 s. The fast
pyrolysis of C. protothecoides and Microcystis aeruginosa yielded
18% and 24% of liquid products, respectively. Compared to the
slow pyrolysis from microalgae in autoclave, a great amount of
high quality bio-oils can be directly produced from continuously
processing microalgae feeds at a rate of 4 g min 1. Many experiments have demonstrated that fast pyrolysis (Table 3) is an efcient method to produce useful fuels and gases from microalgae.
As mentioned above, not only the crude fat, but also other chemical components such as protein and water-soluble carbohydrate,
can be converted easily into fuel oil or gas by thermochemical
techniques [53].

44

G. Huang et al. / Applied Energy 87 (2010) 3846

Table 3
The application of fast pyrolysis for some microalgal species.
Samples

Type of pyrolysis

Optimal pyrolysis temperature (K)

Heating rate

Oil yield (% biomass dry weight)

References

C. protothecoides
C. protothecoides
Microcystis aeruginosa
Heterotrophic C. protothecoides
C. protothecoides

Fast
Fast
Fast
Fast
Fast

773
773
773
723
773

600 C/s
600 C/s
600 C/s
10 K/s

52.0
18
24
57.9
53.3

Peng et al. [53]

Miao et al. [52]
Miao et al. [52]
Miao et al. [41]
Demirbas et al. [66]

pyrolysis
pyrolysis
pyrolysis
pyrolysis
pyrolysis

3.2. Liquefaction
High content of water often exists in microalgae after harvesting which requires a great deal of energy to remove moisture in
the algal cells in the period of pretreatment. Liquefaction has been
developed to produce bio-fuel directly without the need of drying
microalgae [22,23]. Moreover, wet microalgae can provide hydrogen for hydrogenolysis. It was reported that Dunaliella tortiolecta
cells with 78.4% water content converts to oils directly. The yield
of oils reached 37% of the total organic matters [23]. Dote et al.
[22] reported that B. braunii produced liquid oils at 5764% of
dry weight under the conditions of a N2 pressure of 10 MPa at
300 C in warm water and catalyzed by NaCO3. Sawayama et al.
[54] investigated the energy balance and CO2 mitigating effect of
a liquid fuel production process from B. braunii using thermochemical liquefaction. The study suggested that microalgae consume
low amounts of nutrients and accumulate high caloric materials,
and nutrient resources which are produced without energy wasting processes encourage the recovery of oil from microalgae and
CO2 mitigation.

(Table 4) may be applied in transesterication [56]. The use of acid

catalyst has found to be useful for the conversion of high free fatty
acid feedstocks to esters but the reaction rates for converting triglycerides to methyl esters are too slow [57]. Alkali catalysts have
higher reaction rate and conversion than acid catalysts for the
transesterication of triglyceride. Alkali-catalyzed transesterication is about 4000 times faster than the acid catalyzed reaction
[58]. So, alkali-catalyzed transesterication is most frequently
used commercially. The free fatty acid (FFA), however, may react
with the alkali catalyst to form soap and water (Fig. 5) which results in the loss of alkali catalysts in the process of reaction. Therefore, additional catalysts must be added to compensate for the
catalyst loss to soap. When the FFA level is above 5%, the soap will
inhibit separation of the methyl esters and glycerol and causes
emulsion formation during the water washing. Therefore, it is
necessary to rst convert FFAs to methyl eaters (Fig. 6) in order
to reduce the contents of FFAs, and the low FFAs pretreated oil is
transesteried with an alkali catalyst to convert triglycerides to
methyl esters. In contrast, enzymes exhibit good tolerance to
the FFA level of the feedstock, but the enzymes are expensive
and may not be able to provide the degree of reaction required

4. Transesterication technologies in the production of

biodiesel
The viscosities of vegetable oils and microalgal oils are usually
higher than that of diesel oils [55]. Hence, they cannot be applied
to engines directly. The transesterication of microalgal oils will
greatly reduce the original viscosity and increase the uidity.
Few reports on the production of biodiesel from microalgal oils
are available [20]. Nevertheless the technologies of the biodiesel
production for vegetable oils can be applied to the biodiesel production of microalgal oils because of the similar physical and
chemical properties. In the process of transesterication, alcohols
are key substrates in transesterication. The commonly used alcohols are methanol, ethanol, propanol, butanol, and amyl alcohol
but methanol is applied more widely because of its low-cost and
physical advantages. Alkali, acid, or enzyme catalyzed processes

HO-C-R

Fatty Acid

+-

K O-C-R + H2O

KOH

Potassium Hydroxide

Potassium soap

Water

Fig. 5. Transesterication by alkali catalyst.

HO-C-R +
Fatty Acid

CH3OH
Methanol

H2SO4

CH3-O-C-R + H2O
Methyl ester

Water

Fig. 6. Transesterication by acid catalyst.

Table 4
Application of transesterication technologies.
Type of
transesterication

Advantages

Disadvantages

Chemical catalysis

(a) Reaction condition can be well controlled

(b) Large scale production
(c) The cost of the production process is cheap
(d) The methanol produced in the process can
be recycled
(e) High conversion of the production

(a) Reaction temperature is relative high and the process is complex

(b) The later disposal process is complex
(c) The process need much energy
(d) Need a installation for methanol recycle

Enzymatic catalysis

(a) Moderate reaction condition

(b) The small amount of methanol required in
the reaction
(c) Have no pollution to natural environment

(a) Limitation of enzyme in the conversion of short chain of fatty acids

(b) Chemicals exist in the process of production are poisonous to enzyme

Supercritical uid
techniques

(a) Easy to be controlled

(b) It is safe and fast
(c) Friendly to environment

(a) High temperature and high pressure in the reaction condition leads to high cost of
production and wastes energy

(e) The waste water pollutes the environment

G. Huang et al. / Applied Energy 87 (2010) 3846

to meet the ASTM fuel specication [59]. Immobilization of the

enzyme and multiple enzymes may provide more choices in the
future [60].
Besides, the effects of molar ratio of glycerides to alcohol; catalysts, reaction temperature and time, the contents of FFAs and
water in oils and fats have been reported in some recent reports
[9]. It was reported that biodiesel could be produced by acidic
transesterication with 56:1 M ratio of methanol to microalgal
oil at temperature at 30 C and 100% catalyst quantity (based on
oil weight) was achieved [44]. The most abundant composition of
microalgal oil transesteried with methanol is C19H36O2, which is
suggested to accord with the standard of biodiesel [24]. In recent
years, there have been many reports about the applications of
transesteried technologies for biodiesel [61].
5. Conclusion
Since oil crisis in the mid 1970s, nding new energy resources
to replace petroleum has been a hot topic worldwide. Because of
the many advantages over the conventional energy resources, the
production of biodiesel has attracted much attention in recent
years. There have been numerous publications on the production
of biodiesel made from vegetable oils and other oil-plants. At present, the high cost of oleaginous materials is the main problem hindering commercial production of biodiesel. Therefore, nding
cheaper oleaginous materials and improving transesterication
technologies are the key to producing biodiesel successfully. To
date, large scale commercial production of biodiesel from microalgal oils has not been reported. The low lipid content and low biomass that can be achieved leading to the high cost of biodiesel
from microalgal oils are the bottleneck for industrial production.
Heterotrophic cultivation of lipid-rich microalgae with fast pyrolysis may lead to a high yield of bio-oils on a large scale. Research in
genetic engineering coupled with advanced cultivation and downstream technologies will benet the future development of microalgae for biodiesel production.

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