CHINESE JOURNAL OF BIOTECHNOLOGY

Volume 24, Issue 3, March 2008

Online English edition of the Chinese language journal

Cite this article as: Chin J Biotech, 2008, 24(3), 341348. REVIEW PAPER

Exploitation of Oil-bearing Microalgae for Biodiesel

Donghui Song1,2, Jingjuan Fu2, and Dingji Shi2,3

1 Tianjin Key Laboratory of Marine Resources and Chemistry, Tianjin University of Science & Technology, Tianjin 300457, China
2 Laboratory of Cyanobacterial and Algal Biotechnology, College of Marine Science & Engineering, Tianjin University of Science &
Technology, Tianjin 300457, China
3 Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

Abstract: As a renewable energy source to replace conventional fossil fuel, biodiesel fuel has been becoming increasingly
necessary for the global fuel market. Biodiesel derived from oil crops cannot realistically satisfy even more fraction of the raw
material existing costs and soil competitive demand for its growth. Microalgae appear to be the advantage of costs depending on its
capability of higher photosynthetic efficiency, larger biomass and faster growth rate compared to that of oil crops. Oil content of
many microalgae is usually 80% of its dry weight. Genetic microalgae with oil-bearing productivity by genetic manipulations are
capable of making microalgal biodiesel economically competitive with petrodiesel through large-scale production of genetic
microalgal biomass. As demonstrated here, the current application of biodiesel at home and aboard are introduced, and the low cost
advantage of microalgae as the raw material for biodiesel is analyzed. In addition, the role of key enzymes in microalgal fatty acid
biosynthesis and problems in manipulation of lipid production in microalgae via genetic engineering, especially proposals and
measures for exploitation and utilization of oil-bearing transgenic microalgae for biodiesel production are also discussed in this
review.

Keywords: genetic engineering; microalgae; oil-bearing plant; biodiesel

Introduction benefits. Reviewers intend to introduce current utilization of

In a world of rapidly changing economic requirements
for power production, oil has been internationally an important
power need for helping economic and power development.
China is the second largest economic complex of power
needs and consumption across the world. Oil shortage has
been a real dominant influencing factor on Chinese
economic and societys development; so it is an important
strategic plan to form some new sustainable power sources
to replace conventional fossil fuel. Biomass powers,
especially biodiesel fuel, have been recently becoming a
major contribution to international renewable power needs,
while providing substantial environmental and economic
biodiesel in domestic and international biomass fuel market,
further to analyze the feasibility and progress in manipulation
of lipid production in microalgae via genetic engineering.
At last, the reviewers offer some proposals for reducing the
producing cost and promoting wide adoption of microalgal
biodiesel. It is possible that these will be useful documents,
both when planning biodiesel use and as an ongoing
resource.
1 Biodiesel offers many advantages
Biodiesel is a fossil diesel replacement fuel that is
manufactured from vegetable oils, recycled cooking greases

Received: July 23, 2007; Accepted: September 5, 2007

Supported by: the National Science Funds of China (No. 30670493), Tianjin Key Projects of Science and Technology (No. 04318271), Tianjin Science and
Technology Developmental Funds of Universities and Colleges (No. 20060724) and Exceptional Talented Persons Initial Funds of TUST (No. 20060411)
Corresponding author: Donghui Song. Tel: +86-22-60601101; Fax: +86-22-60600358; E-mail: dhsong@tust.edu.cn

Copyright 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights
reserved.
 Donghui Song et al. / Chinese Journal of Biotechnology, 2008, 24(3): 341348

or waste oils, animal fats, or microalgal lipids. Because Ethanol fuel made from fermentable starchy and
plants (including microalgae) produce oils from sunlight lignocelluloses plants can also reduce tailpipe emissions,
and air, and can do so year after year on cropland, these oils including air toxics. However, the heating value of ethanol
are renewable. Animal fats are produced when the animal fuel is only 2/3 of gasoline and more less than petrodiesel.
consumes plant oils and other fats, and they too are The combustibility of ethanol fuel may descend due to its
renewable. Used cooking oils are mostly made from any blend ratio with water[1].Because biodiesel has its fine
vegetable oils and animal fats, and also are both recycled characteristic advantage over other biomass fuel, many
and renewable. The biodiesel is made by chemically governments worldwide successively developed the
combining any natural oil or fat with an alcohol such as research programs and the industrial production of biodiesel
methanol or ethanol. These chemicals are called long chain raw material. The aim is to displace fossil fuel which
mono alkyl esters, or also referred to as long chain fatty acid reserves reduces and seriously pollutes the environment day
methyl esters. by day.
Biodiesel offers more advantages than petroleum-based
diesel and ethanol fuel: (1) It is energy efficient. The biggest 2 Development of material for biodiesel
benefit to using biodiesel is that it is easy. Biodiesel has
physical and chemical properties similar to petrodiesel (Tab. 2.1 Raw material for overseas biodiesel
1) and can in some cases be used in existing diesel According to the current technique of the preparation of
applications with little or no modification to the engine or biodiesel from plant lipids and animal grease, the cost of
fueling system. (2) Biodiesel is renewable. It may be raw material accounts for the total production cost
continually obtained from plants, animals and microalgae by 5085%[2]. Therefore, the cost of material is the dominant
cultivation and breeding year after year. (3) Biodiesel can factor in the price formation of biodiesel. At present different
play a role in reducing emissions of many air pollutants, material is selected to prepare biodiesel according to their
especially those targeted in urban areas. native planting composition characteristics in main biodiesel
These include emissions of particulate matter, carbon producing countries in the world. In Europe Union and
monoxide, hydrocarbons, sulfur oxides, nitrogen oxides and United States, the preparation of biodiesel is made from
air toxics. Biodiesel has significantly lower sulfur than rapeseed oil, soybean oil and mustard seed oil. As a
todays diesel fuel, while providing a significant increase in renewable energy nation of big power in the world, Brazils
lubricity. Biodiesel also contains 11% oxygen by weight, as biodiesel is mainly produced by using castor bean oil. Yet
well as a slightly higher cetane number, which provides for the Japanese make use of waste fried oil and cattle grease to
more complete combustion and a reduction in most emissions. produce biodiesel. In Southeast Asia countries, such as
South Korea, India and Malaysia, palm tree oil as the
important material is used to develop biodiesel fuel.
Tab. 1 Selected properties of typical No. 2 diesel and Biodiesel
2.2 Raw material for Chinese biodiesel
fuel in the United States in 2006
China is a country with a large population, while its per
Fuel property Diesel Biodiesel capita amount of arable land area lag is far behind the worlds
Lower heating value (Btu/gal) ~129,050 ~118,170 average level. The government has to guarantee first to
Kinematic viscosity (40C) 1.34.1 4.06.0 supply enough food and edible oils to meet daily needs of the
Specific gravity (kg/L, 60C) 0.85 0.88 people. Statistics have indicated, in 2003, that China
Density (lb/gal, 15C) 7.079 7.328 imported rape and rapeseed oil reached 2.50 million tons to
Carbon (wt %) 87 77 meet the growing needs of people. In the first 11 months of
2004, China imported a lot of 6.25 million tons edible fat
Hydrogen (wt %) 13 12
and vegetable oil from overseas, of which the soybean oil of
Oxygen, by dif (wt %) 0 11
import was 2.39 million tons, up 56% over the same period
Sulfur, (wt %) 0.05 max 0.0 to 0.0015
last year, and the rapeseed oil of import was 0.32 million
Boiling point (C) 180 to 340 315 to 350
tons, up 118%, and also the palm oil of import was 2.16
Flash point (C) 60 to 80 100 to 170 million tons, up 120%[3]. In the following year of 2005, the
Cloud point (C) 15 to 5 3 to 12 direct import of edible fat and vegetable oil came to 6.53
Pour point (C) 35 to 15 15 to 10 million tons, of which palm oil, soybean oil and rapeseed oil
Cetane number 4055 4865 was 4.33 million tons, 1.69 million tons and 0.51 million
Lubricity SLBOCLE (g) 20005000 >7 000 tons, respectively. These data indicated that most edible oil
Lubricity HFRR (microns) 300600 <300 products need to be imported in China in the next year,
while edible oils demand in 2010 is projected as 13.50
million tons, accounting for 21% of the total amount of food
 Donghui Song et al. / Chinese Journal of Biotechnology, 2008, 24(3): 341348
supplies[4].
In such cases, it is possible to take China a long time to
produce enough edible oils, instead of developing biodiesel
fuel using these deficient edible oils in the future. If Chinese
biodiesel was made mostly from oil crops as material, the vast
farmland would be planted with these plants to meet the
need of biodiesel production. This may be a disaster to China
who has a large population and relatively little land
available for cultivation. At the same time, if the massive
soybean oil and the rapeseed oil were imported to China to
produce biodiesel, the price of biodiesel would be too high
to that of fossil fuels to use widely, and the high-priced
biodiesel has bad competitiveness in the fuel market that it
would need for the massive governmental subsidy. All of the
above cases do not accord with China's national state.
How to develop the industry of Chinas biodiesel in
conformity with the national conditions? In January 2003,
the Chinese Academy of Engineering (CAE) held in Beijing
Symposium on Biodiesel Fuel from Oil-bearing Plants
Material. Prof. Shi Dingji, one of authors of this article,
was invited to make a key report about the development of
biodiesel industry by microalgal biotechnology on this
symposium, and won praises of Chair Xu Kuangdi and other
academicians. This conference has become an important
milestone of the industrial development of material for
biodiesel in China. On November 12, 2006, CAE held also
in Beijing 2006 Strategy Forum on the Development of
Chinese Biomass Energy and defined the following policy
of developing biomass energy: Chinese biomass energy has
mainly focused on the non-grain crops, and the state will
formulate more favorable policies in income tax for the
purpose of promotion and rapid development of biomass
energy industry[5].
China is blessed with natural resources of woody
oil-bearing crops, such as Barbadosnut, Chinese pistache,
Chinese tallow and Tung oil tree in which the oil content of
seeds reach approximately to 20%. Therefore, the state has
focused at present on seeds of wild woody oil-bearing crops,
waste animal fats and plant oils, as material of biodiesel, to
produce biodiesel and glycerin. It is a theoretical possibility
that, in China, a plenty of wild Barbadosnut and Chinese
pistache can be used as material to produce biodiesel with
an annual output of 5 million tons, and the recycling of
waste animal fats and plant oils material may produce the
average yearly output of 2 million tons biodiesel[6]. But
these cannot be put into practice because of material cost
and productions cost. The problem of costs will be
described in the following sections.
The Chinese industrialization progress of biodiesel first
began from a private enterprise. At the end of 1990s, Hainan
Zhenghe Biomass Energy Inc. used seeds of Chinese
pistache as raw material to extract and produce biodiesel
fuel. Now the company had its raw material base of near
8000 ha mountainous area for planting Chinese pistache,
and an experimental station of 10 thousand-ton biodiesel-
producing equipment, and two biodiesel production bases
with an annual output of 50 thousand-ton. In the meantime,
Sichuan Gushan Oil and Grease Chemical Co., Fujian
Zhuoyue New Energy Development Co. Ltd and Liaoning
Fushun Yangtze Biodiesel Technology Research Institute
also developed a series of production technology of
biodiesel with independent intellectual property rights.
However, because of relatively small productions capacity
and low output of biodiesel products, the cost of these
companies biodiesel-producing from woody oil-bearing
crops always stay at a high level which resulted in the
difficulty with popularity of biodiesel and more difficult to
compete with the fossil diesel of low-cost and low price.
3 Advantage of microalgae as a new source of
biodiesel
Biodiesel fuel and fossil fuel essentially originated from
the photosynthesis products of green plants. Any of a group
of organic compound that includes sugars, starches,
celluloses and lipids is produced by photosynthetic plants.
Heterotrophic organism, including most microorganism and
all animals, depend on plants photosynthesis to supply their
food and energy. All lipids formation of live-forms is also
converted from organic compound of plant photosynthesis
products. In principle, it is possible that any kind of fatty
acid obtained from plant, animal and microbe are sources of
biodiesel. So the plants or microbe possessing of high lipids
content are potentially the best source of biodiesel.
3.1 Cost of existing oilseed plants as material for
biodiesel
Green plants with high oil content are the herb
oil-bearing crops, such as soybean and rapeseeds, of which
the seeds harvesting, storage and processing are easy and
reliable to operate. Nevertheless, large amounts of soybean
oil and rapeseeds oil were recently imported to meet the
daily needs of people in China, and the state is not possible
to produce biodiesel by importing oilseeds. Other inedible
oil plants like castor-oil plant and peanuts may also become
the source of biodiesel, but growing them will be bound to
compete with crops and cotton for farmlands, water and
fertilization, and so on. So it is unrealistic to produce
biodiesel by use of the herb oil-bearing crops.
Woody oil-bearing crops, such as Barbadosnut and
Chinese pistache can be grown on the barren hills and
wastelands which do not contend with crops for farmland.
Growth of these plants has also much effect on greening
environment and improvement of the citys ecological
environment. But a harvest festival of these woody oilseed
plants with high storage cost is always about once a year.
Once mass-produced biodiesel are based on the woody
 Donghui Song et al. / Chinese Journal of Biotechnology, 2008, 24(3): 341348
oilseed plants, the growing season of oilseed plants are the
worst bottleneck and impediment to the industrial development
of biodiesel.
3.2 Benefits of microalgae as material for biodiesel
Microalgae are one of the oldest living organisms. They
are unicellular species which exist individually, or in chains
or groups. Depending on the species, their sizes can range
from a few micrometers (m) to a few hundreds of
micrometers. As a group of lower plant, microalgae live
annually in oceans, rivers and lakes. Moreover, they have
been long known to be essential components of coral reefs.
Microalgae comprise a vast group of photosynthetic,
heterotrophic organisms which have an extraordinary
potential for cultivation as energy crops. Cyanobacteria of
microalgae, so-called blue algae or blue greens, are
gram-negative photoautotrophic prokaryotes which can be
cultivated without nutrition of carbon and nitrogen. The
advantages of such cyanobacteria make that they are
generally suitable for a low investment, very cost-effective
and easy to manage.
Microalgae can be cultivated under aqueous
conditions ranging from freshwater to situations of
extreme salinity and are able to produce a wide range of
commercially interesting byproducts,, such as fats, oils,
sugars and functional bioactive compounds. Large-scale
algae production can be achieved in open raceway ponds
or photobioreactor systems. Microalgae cell growth time
of duplicating is in 24 h and even in 3.5 h during the
logistic phase of cell multiplication[7].
Microalgae are remarkably efficient biological factories
capable of taking a waste (zero-energy) form of carbon and
converting it into a high density liquid form of energy
(natural oil). Microalgae-based biodiesel has emerged as a
viable resource. Microalgae may contain significant quantities
of lipids (fats and oil) with compositions similar to those of
vegetable oils. The photosynthesis productivities of
hydrocarbons and lipids in some eukaryotic diatom, green
algae and brown algae isolated from ocean and lakes reach
as high as 50 g/m2/d. The lipids of these algal species are
also rich in essential fatty acids, such as C18 and C16 acids
and their C20 derivatives[8]. While it is common to find
levels of 20%80% lipids on a dry basis, on occasion the
quantities of lipids found in microalgae can be
extraordinarily high. For example, in one particular species,
Botryococcus, concentration of hydrocarbons in the dry
matter may exceed 80%, under certain conditions.
Meanwhile, the levels of 15%30% lipids on a dry basis
were found in vegetable oils[9,10].
Chinese mainland coastline stretches for some 18 000 km
with a large swamp wetland and marshes, suitable for large-
scale cyclic cultivation of oil-bearing microalgae. Because
of the advantages of its enormous groups, gigantic biomass
and very cost-effective management, microalgae and its
based biodiesel are becoming a remarkable source of new
biofuel and one of developmental trend in clean energy in
the future.
4 Selection and construction of high lipid
content microalgae
4.1 Physiological and biochemical research of oil-bearing
microalgae
From 1978 to 1996, the United States Department of
Energys Office of Fuel Development funded the Aquatic
Species Program (ASP). The focus of the program was to
develop renewable biofuel from algae. By the end of the
study the researchers had identified around 300 strains of
algae, as varied as the diatoms (genera Amphora, Cymbella,
Nitzschia, etc.) and green algae (genera Chlorella in
particular), which are the most suitable for producing
biodiesel from over 100 000 different species of algae. They
all have high growth rates, oil content, and are capable of
growing in harsh climates. Of which, some algal oil content
was up to 80% with the photosynthesis productivity of 50
g/m2/d[9,10].
The researchers focused their efforts on using physiology
and biochemistry to manipulate the algae to have higher oil
content. For example, they found that although the nitrogen
and silicon deficiency did increase the oil content of the
algae it does not lead to increased oil productivity because it
reduces the growth rates of the algae. During this study,
researchers also attempted to genetically modify certain
algae species so that they would produce more oil content
and also enable them to grow in very harsh environments.
Although the researchers did make significant discoveries
they were unable to demonstrate increased oil production in
the cells[9,10]. The development of medicine and agriculture
during that period indicated that methods based on classical
experience had not promoted the leap-forward development
of science and technology.
4.2 Construction of high oil content genetic modified
microalgae
The photosynthesis productivity of microalgae are more
10-folds amount of that of oil crops under the similar growing
condition; so lipid metabolism in these algae for fatty acid
biosynthesis are more active than others. The pathway of
fatty acid metabolism in microalgae could be regulated by
the use of genetic engineering technology to enhance the
ability of de novo fatty acid biosynthesis; thus the
construction of the high oil content genetic modified
microalgae for biodiesel will be realized.
4.2.1 Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2)
in fatty acid biosynthesis
ACCase is an enzyme involved in many important
metabolic pathways in plant, animal and bacterial cells.
Until now, ACCase is known to play an important role in
 Donghui Song et al. / Chinese Journal of Biotechnology, 2008, 24(3): 341348
Fig. 1 Reaction mechanism of E. coli ACCase
The overall reaction of ACCase proceeds by a two-step mechanism. In
the first half-reaction, biotin is carboxylated by bicarbonate in an
ATP-dependent reaction to form carboxybiotin catalyzed by BC,
whereas in the second half-reaction, the carboxyl group is transferred
from carboxybiotin to acetyl-CoA to form malonyl-CoA catalyzed by
CT
fatty acid synthesis, because the enzyme catalyses the
irreversible carboxylation of acetyl-coenzyme A (acetyl-
CoA) to produce malonyl-CoA through its two catalytic
activities, biotin carboxylase (BC) and carboxyltransferase
(CT) (Fig. 1) [11]. ACCase is a multi-subunit enzyme in most
prokaryotes, whereas it is a large, multi-domain enzyme in
most eukaryotes. The enzyme catalyzes the rate-limiting
reaction for fatty acid synthesis by which acetyl-CoA is
carboxylated to form malonyl-CoA. The activity of ACCase
can be controlled at the transcriptional level, as well as by
small molecule modulators and covalent modification.
Recent studies of ACCase in eukaryotic plant
up-regulating lipid mechanism found that the high oil
content specie of soybean, from the beginning to mid-period
of development, exhibits about a 2-fold increase in ACCase
activity than that of low oil content specie of soybeans[12].
This suggests that soybean seeds in the beginning of
development showing an increase in expression of ACC
gene were related to the total oil content of mature seeds. It
has also been found that Brassica napus napin
storage-protein gene promoters were fused to the
Arabidopsis ACC gene and transformed into B. napus with
the results of plants expressing the ACC gene increasing 10-
to 20-fold higher ACCase activity in mature seeds in which
the total oil content was increased approximately by 5%[13],
suggesting that ACCase overexpression altered seed fatty
acid composition with the largest effect being an increase in
oil content. It has been different from the above ACCase
up-regulation that the reduction of ACCase activity in B.
napus seeds by antisense expression reduces seeds lipid
content[14]. These results indicated that ACCase activity in
developing oilseed crops affected predominantly seed fatty
acid synthesis. In prokaryotes like Escherichia coli, the
same can be said of the alteration of ACCase activity
increasing an effective amount in fatty acid. The effects of
four ACCase subunits of E. coli overexpression resulted in a
6-fold increase in the rate of fatty acid synthesis [11]. All of
the above evidences suggest that overexpression of ACC
gene is able to increase ACCase activity and the total oil
content of mature seeds from transformed plants under
certain conditions.
In manipulation of genetic modified algae for high oil
content, extensive research was conducted on the
manipulation of algae lipid mechanism by the researchers of
the ASP in National Renewable Energy Laboratory (NREL),
which was supported by the United States Department of
Energys Office of Fuel Development during 19911996[8].
During the period, researchers of ASP attempted to use
genetic engineering to manipulate the microalgal
biosynthetic pathways to produce algal strains with
constitutively high lipid levels. They thought that it could be
done by transforming ACC gene to diatom in which some
species had been selected as special high oil content strains
of algae[15]. The enzyme which was found to consist of a
homo-tetramer of 185 kD subunits was purified and cloned
from the diatom Cyclotella cryptica using biochemistry and
genetic engineering[16].
Scientists in NREL during the early 1990s developed
genetic transformation methods for microalgae and
accomplished successfully ACC gene transformation of C.
cryptica strains with demonstrated potential for biodiesel
production in 19931995[8,15,16]. This was the first report of
cloning a full-length ACCase sequence and its genetic
transformation from any photosynthetic organism.
Unfortunately, funding levels for ASP decreased during this
period from the high funding levels. Laboratory experiments
emphasizing biochemistry, molecular biology, and genetic
engineering was closed eventually with no budget in 1996 [8].
Although researchers in other labs proceeded to focus their
efforts on using biochemistry and genetic engineering to
manipulate microalgae to have higher lipid content, little
was known about the molecular biology of oleaginous
microalgae and the genetic regulation of lipid biosynthesis
pathways resulting in no significant discoveries for
increasing oil production in microalgal cells.
Increasing information of microalgal ACCase sequences,
including some model cyanobacteria like Anabaena sp. PCC
7120 and Synechocystis sp. PCC 6803 (Tab. 2), have been
exploited in GenBank Database (http://www.ncbi.nlm.
nih.gov/) in recent years. Studies on cyanobacterial ACCase
for manipulating fatty acid biosynthesis are now ongoing in
the research group and its final goal is to produce algal
strains with constitutively high lipid levels, and studies on
screening transgenic microalgal strains with fused ACC
gene are now in progress (unpublished).
4.2.2 Phosphoenolpyruvate carboxylase (PEPC, EC
4.1.1.31) in fatty acid biosynthesis
PEPC is an enzyme that catalyzes the addition of CO2 to
 Donghui Song et al. / Chinese Journal of Biotechnology, 2008, 24(3): 341348

Tab. 2 The ACC gene of Escherichia coli and partial cyanobacteria (includes deductive homological genes)*
ACC
Microbe species mRNA (nt) Amino acid number Molecular weight (kD) GenBank Access No.
gene
Escherichia coli 960 320 35.3 D83536
Nostoc punctiforme PCC 73102 981 326 35.9 NZ_AAAY02000034
Nostoc sp. PCC 7120 981 326 35.7 NC_003272
accA
Synechococcus elongatus PCC 6301 984 327 35.7 AP008231
Synechococcus PCC 7942 984 327 35.7 U59236
Synechocystis sp. PCC 6803 981 326 36.0 NC_000911
Escherichia coli 471 156 16.7 M80458
Nostoc punctiforme PCC 73102 558 185 19.7 NZ_AAAY02000072
Nostoc sp. PCC 7120 549 182 19.2 NC_003272
accB
Synechococcus elongatus PCC 6301 477 158 16.9 AP008231
Synechococcus PCC 7942 477 158 16.9 U59235
Synechocystis sp. PCC 6803 465 154 16.3 BA000022
Escherichia coli 1350 449 49.3 M80458
Nostoc punctiforme PCC 73102 1344 447 49.3 NZ_AAAY02000007
Nostoc sp. PCC 7120 1344 447 49.1 NC_003272
accC
Synechococcus elongatus PCC 6301 1362 453 49.7 AP008231
Synechococcus PCC 7942 1362 409 44.7 U59234
Synechocystis sp. PCC 6803 1347 448 49.2 NC_000911
Escherichia coli 915 304 33.3 M68934
Nostoc punctiforme PCC 73102 951 316 35.5 NZ_AAAY02000009
Nostoc sp. PCC 7120 951 316 35.5 NC_003272
accD
Synechococcus elongatus PCC 6301 918 305 33.8 AP008231
Synechococcus PCC 7942 918 305 33.8 U59237
Synechocystis sp. PCC 6803 981 326 36.4 NC_000911

*The data cuts off June 13, 2007

phosphoenolpyruvate (PEP) to form the four-carbon
compound oxaloacetate. This reaction is used for carbon
fixation in the so-called C4 plants (plants which initially
form C4 acids), where it plays a key role in photosynthesis.
Besides plants, the enzyme is also found in
photoautotrophic microalgae like cyanobacteria, but not in
animals or fungi[17].
It has been known that the modulation of fatty acids
biosynthesis in oil crop depended on the relative activity of
ACCase and PEPC[18]. ACCase catalyzes the formation of
malonyl-CoA from acetyl-CoA which is derived from
pyruvic acid in animal, plant and bacterial cells. Of which
malonyl-CoA is an essential substrate for de novo fatty acid
synthesis and fatty acid elongation. Pyruvic acid is an
intermediate compound in the metabolism of proteins,
because it is converted into the amino acid alanine and then
input to protein metabolic processes. Actually, the
biosyntheses of either protein or lipid take PEP as the
common substrate. It is converted into oxaloacetic acid by
PEPC and then directly participates in the anabolism of
protein. On the other hand, PEP can be converted into
pyruvate by the pyruvate kinase. Pyruvate is then
transformed to acetyl-CoA by pyruvate dehydrogenase,
yielding malonyl-CoA by the ACCase, finally participated
in the anabolism of fatty acid in this form[18]. Overall, the
relative activity of PEPC and ACCase determines the flow
direction of PEP and the ration of proteins/lipids produced
in oilseeds crops (Fig. 2).
Many evident roles of PEPC in lipids biosynthetic
pathways in oilseed crops have been reported. In the early
1950s, it was found that the contents of proteins and lipids
were inversely correlated to each other in plants, and that
soybean protein content was positively related to the activity
of PEPC at the end of 1980s[19]. It looks as if the oil content
would negatively relate to the activity of the PEPC.
Recently, Chen et al found that antisense expression of B.
napus PEPC gene reduced the activity of PEPC resulting in
PEPC transgenic rapeseeds had 6.4%18% higher oil
content; the highest to 50%, than control seeds[18,20,21].
Antisense expression of soybean PEPC gene increased oil
content in transgenic plant seeds and yielded a new strain of
high oil content transgenic soybean[22]. Therefore, the
 Donghui Song et al. / Chinese Journal of Biotechnology, 2008, 24(3): 341348
antisense expression of PEPC gene for the modulation of
fatty acids biosynthesis in oil crops is an effective method.
In the recent 20 years, the DNA sequence of PEPC in many
microorganisms, including Anabaena sp. PCC 7120[23],
Anacystis nidulans[24], E. coli[25], Microcystis aeruginosa
NIES-843[26], Synechococcus vulcanus[27] and Synechocystis
sp. PCC 6803[28], have been sequenced with the detailed
functional notes (http://bacteria. kazusa.or. jp/cyanobase/).
However, no studies on expression of PEPC for the
modulation of fatty acids biosynthesis have been reported;
also, there were no reports about the role of PEPC in
regulating oil content of microalgae. In the research group,
studies on the antisense expression of PEPC gene for oil
content of microalgae are now in progress with primary
research results of that the reduced activity of PEPC
increased the lipids content in the cells of Synechococcus sp.
PCC 7002 by antisense expression of PEPC gene
[unpublished].
Fig. 2 Possible role of PEPC in biochemical processes of fatty
acid biosynthesis pathway
PEPC catalyzes the fixation of carbon dioxide with PEP to produce
oxaloacetate, which is the main input for a series of reactions known as
the Krebs cycle. PEP is also converted to pyruvate by pyruvate kinase
which reaction is strongly exergonic and irreversible. Pyruvate can be
converted to fatty acids through acetyl-CoA, which is the main
substrate for the formation of malonyl-CoA catalyzed by ACCase.
Malonyl-CoA is afterwards transferred to the acyl carrier protein (ACP)
and to start fatty acid synthesis. While the activity of PEPC is reduced
by antisense inhibition, the concentration of PEP increases to high level
to help to the formation of more pyruvate. Meanwhile, along with
consumption of acetyl-CoA for the formation of malonyl-CoA, the low
level of acetyl-CoA is able to activate the biochemical process of PEP
convert to pyruvate.
5 Problems and possible measures on research
and development for microalgae biodiesel
Research on microalgae genetic engineering is quickly
developing in domestic and overseas in the recent years.
The growth in the number of researchers and of the
technology achievements has all exceeded more than the
other countries in the field of microalgae genetic
engineering since Chinese national programs for high
technology research and development in marine science and
technology (namely Marine 863 Programs) was started in
1998. Many achievements in the expression of exogenous
genes in microalgae made China the leading nation in the
field of microalgae genetic engineering. Based on the
achievements for decades, the research group realizes that,
besides national policies, personnel quality and research
experiments, and so on, there are at least two major
problems in the technological level that need to be solved in
microalgae genetic engineering; one is the growth rate of
microalgae, and the other is the expression efficiency of
exogenous genes in microalgae.
5.1 Existing two major problems in microalgae genetic
engineering
5.1.1 A problem of the growth rate of microalgae with
large-scale culture
The problem was even a major obstacle for selecting
microalgae as the host of alien gene in a long time.
Large-scale high cell density culture of the microalgae is the
only way for increasing the growth rate of microalgae,
reducing the cost of production and realizing the
industrialization of microalgae genetic engineering. At
present, there are two approaches to realize large-scale
culture of the microalgae; one is the outdoor raceway pond
model and the other is the enclosed biophotoreactor model.
The outdoor raceway pond model is always used to
mass-produce commercially special high nutritive value
microalgae like Spirulina and Chlorella. Although the
advantages of the model are a relatively simple structure
and operation with the low management cost, the
application and extension of the model are deeply limited
because of its disadvantages, such as the relatively low cell
density culture of microalgae, easily polluted products,
unsteady process of production and coverage of a vast area.
Different from the outdoor raceway pond model, the
enclosed biophotoreactor model is usually used for
large-scale culture of most genetic engineering microalgae
which are unable to produce in open ponds, or the model is
also suited to those microalgae with high economic value.
The enclosed biophotoreactor model has some obvious
highlights, such as simple operation, easy parameters
control for culture, steady cultural condition and high
quality of products. Under the model, the axenic culture of
purified strains of microalgae are able to be realized all the
year round, and the cell density of microalgae is also greatly
increased to a high level which is close to the level of E. coli
in the growth rate and biomass.
In recent years, the enclosed biophotoreactor has been
used for the commercial mass production with high cell
density for active substances derived from microalgae.
 Donghui Song et al. / Chinese Journal of Biotechnology, 2008, 24(3): 341348
Nevertheless, the relative high cost of microalgal production
made it a necessity to adjust the balance between the most
suitable cultural condition and the lowest cost containment,
including the selection of optimum illumination, improving
the utilization of light energy and selection of the
appropriate equipments with the most amount of mass
culture.
How to find the solution to the problem about the
utilization of light energy for the increase of microalgal
growth rate? It is an alternative solution that, besides the
sunlight and the external light source, the minimum
transmission route and the maximized surface area of
lighting for illumination could be adopted, and the
toughened fiber-glass biophotoreactor installed with a
built-in light source could also be applied to increase the
growth rate of microalgae.
The quantity of CO2 venting should also be considered in
the mass production of microalgae. The problem of the
carbon source could be solved by utilizing CO2 from coastal
power plants. In addition, mixed culture of photoautotrophic
and heterotrophic stage of microalgal cells under the
condition of light or dark is also to be possible for the
purpose of high cell density culture. In recent years, the
method of mixed culture in a 100 liter biophotoreactor with
CO2 venting has been applied to improve the increasing rate
and high cell density culture of microalgae in the group[29].
5.1.2 Expression efficiency of exogenous genes in
microalgae is possibly the focal point at home and abroad.
The status shows that the expression efficiency of
exogenous genes is mostly 0.1%0.9% of soluble proteins
in host cells of microalgae. In the group, the method of
exogenous genes expression in cyanobacteria resulted in a
6-fold increase in the transformation efficiency, which
makes one warrant that the national invention patent has
been authorized[30]. The collaborators with the group have
created 15 transgenic cyanobacteria harboring exogenous
genes that have not yet been reported internationally in the
past 14 years, including six kinds of microalgae harboring
exogenous medical genes, which occupied nearly 50% in all
15 transgenic microalgae harboring medical genes[3037].
There is a need to improve plasmid vectors, promoters,
enhancers, terminators and SD sequences of microalgae for
increasing expression efficiency of exogenous genes in
transgenic algae, and the endeavor resulted in the amount of
transgenic expressed in soluble proteins increasing from
the initial 0.01% to now 3%5% which is the highest
expression efficiency in the world[3037].
5.2 Strategies and proposals on developing the
material for biodiesel
The cost of material at a high level is the main limiting
factor for the development and application of biodiesel
utilizations. In recent years, several researches on the
resources of the material for biodiesel are proceeding with
various plans in China. The development of international
biological technology indicates that China possesses obvious
superiority and many proprietary of intellectual property
rights in genetic engineering of microalgae. Based on such
an essential feature, the following proposals were made for
the development and utilization of oil-bearing transgenic
microalgae.
5.2.1 Combining fundamental researches with applied
researches
Research programs on the manipulation of genetic
modified algae for high oil content should be supported
financially by the National Ministry of Science and
Technology, National Science Funds of China and other
related organizations.
The above fundamental researches should also be
combined with the applied researches which includes the
upstream, the middle state and the downstream of
microalgal genetic engineering. It not only develops the
transgenic technology of microalgae, but also develops new
large-scale bioreactors with the most suitable condition for
the culture of microalgae. Expressions of exogenous genes
in cyanobacteria have relative matured technologies and
develop very quickly, but the technology for gene
transformation in eukaryotic algae is still slower than that of
prokaryote and needs to be strengthened further.
5.2.2 Using mature technology of genetic engineering
Survey programs on resources of oil-bearing microalgae
should be mapped out for a share plan of action for its
exploitation by Ministry of Agriculture, the State Oceanic
Administration, China National Petroleum Corporation,
China Petrochemical Corporation and China National
Offshore Oil Corporation and so on. The program should be
funded by every publicly listed organization. Key scientific
and technological problems in the program should be
tackled by cooperation amongst relative academics, algae
breeding farms and oil refineries.
Expression efficiency of exogenous genes in high oil
content microalgae can be increased greatly by the
application of the existing knowledge and technology of
genetic engineering, such as cloning of genes related to
lipids metabolism, analysis of new regulation sequences and
construction of new transformation system. Total lipids
content in the cells of transgenic microalgae should also be
increased by effective methods, such as the regulation of
C/N ratio in culture medium and extension cell growing
period. The oil-bearing microalgae should be domesticated
using sea water as a culture medium which helps to reduce
effectively the cost of mass production.
5.2.3 Measures for large-scale culture of microalgae
There are likely to be questions about the long-term
supply of purified microalgae on a large scale, the
harvesting of mass production and lipids extraction from
mass production for biodiesel.
 Donghui Song et al. / Chinese Journal of Biotechnology, 2008, 24(3): 341348
(1) The algae breeding farms should strengthen their ties
with oil refineries by cooperation between the two sides. It
is also necessary to take into account the rational way to
coordinate the continuous production of oil-bearing
microalgae with the large-scale refinery's annual production.
(2) The suitability of transgenic microalgae for biodiesel
production and its oil-productivity for biodiesel should be
further improved according to environmental conditions in
the different cultivation areas and to requirements of
biodiesel refinery. Existing coastal swamp wetland and
marshes could be used as the basis of large-scale culture
with sea water medium to cultivate oil-bearing microalgae
for the purpose of resolving the long supply of biodiesel
material. For example, the large-scale bioreactors for
oil-bearing microalgae could be built in some factories in
close proximity to power plants on the coastal beach and
tidal flat areas from which a large volume of exhaust CO2
could be used as the carbon source for large-scale
cultivation and the whole year aseptic pure culture of algae.
(3) The harvesting process in commercial production of
microalgae is a crucial step that can be resolved by combined
techniques of chitosan flocculants and foam fractionation
unit to simplify the process of separation, reduce the cost for
harvesting, and keep oil-bearing microalgae nature[38]. For
instance, as an alternative new technology the integrating the
reaction with methyl-ester-exchange-separation in fluidized
bed bioreactors can be used to extract fat and lipids from
microalgae and then to recycle the byproduct, high quality
glycerol.
(4) In addition, the new patent technology of immobilized
enzymes for preparation of biodiesel under normal
temperature and pressure can eliminate effectively the
negative impact of methanol and glycerin by-product on the
enzymatic activity and stability[39]. Application of new
techniques in the conversion of high value product 1,
3-propanediol from glycerin can significantly improve the
economic benefits of producing microalgal biodiesel.
5.2.4 Establishing a perfect biodiesel sales networks
To promote the utilization of biodiesel in the country, the
national marketing network should be established gradually
in planned steps. It is possible that the biodiesel fuel sales
have first accessed the oil market for automobile relying on
the existing Sino-Pechs and other gas station system on the
basis of the government organizing and coordinating the
relevant departments and organizations in the initial stage of
biodiesel fuel development. Once biodiesel is generally
accepted and popularized in the nation, the specialized
nationwide sales networks for biodiesel would be
established quickly.
REFERENCES
[1] Yuan ZH, Wu CZ, Ma LL, et al. The principle and
technology of biomass utilization. Beijing: Chemical
Industry Press, 2005.
[2] Gerpen JV. Business management for biodiesel producers.
NREL Technical Report, NREL/SR-51036342, 2004.
[3] SINA-China Finance. 2004-2005 National edible fat
market: Review and Outlook. http://finance.sina.com.cn/
futur-emarket/futures roll/20050121/10251312384.shtml.
[4] www.toponey.com. A investigation report on Chinese
edible oil market. http://www.toponey.com/Html/
20069139505-1.html.
[5] www.chinanews.com.cn.http://www.chinanews.com.cn/cj/ews/
2006/11-12/819402.shtml.
[6] Jia HS, Xu YN. World biodiesel utilization and
development strategies in china. J Plant Ecol, 2006, 30:
221230.
[7] Chisti Y. Biodiesel from microalgae. Biotechnol Advan,
2007, 25: 294306.
[8] Sheehan J, Dunahay T, Benemann J, et al. A look back at
the U. S. Department of Energys Aquatic Species
Program:biodiesel from algae. Close-out report.
NREL/TP-580-24190, 1998.
[9] Metting FB. Biodiversity and application of microalgae. J
Ind Microbiol Biotechnol, 1996, 17: 477489.
[10] Spolaore P, Joannis-Cassan C, Duran E, et al. Commercial
applications of microalgae. J Biosci Bioengineer, 2006,
101: 8796.
[11] Davis MS, Solbiati J, Cronan JE. Overproduction of
Acetyl-CoA carboxylase activity increases the rate of fatty
acid biosynthesis in Escherichia coli. J Biol Chem, 2000,
275: 2859328598.
[12] Somers DA, Wyse DL, Gronwald JW, et al. Transgenic
plants expressing maize acetyl CoA carboxylase gene and
method of altering oil content. United States Patent
6222099, 2001.
[13] Ohlrogge JB, Roesler KR, Shorrosh BS. Methods of
increasing oil content of seeds. United States Patent
5925805, 1999.
[14] Sellwood C, Slabas AR, Rawsthorne S. Effects of
manipulating expression of acetyl-CoA carboxylase I in
Brassica napus L. embryos. Biochem Soci Trans, 2000, 28:
598600.
[15] Dunahay TG, Jarvis EE, Roessler PG. Genetic
transformation of the diatoms Cyclotella cryptica and
Navicula saprophila. J Phycol, 1995, 31: 10041012.
[16] Roessler PG, Ohlrogge JB. Cloning and characterization
of the gene that encodes acetyl-coenzyme A carboxylase
in the alga Cyclotella cryptica. J Biol Chem, 1993, 268:
1925419259.
[17] Izui K, Matsumura H, Furumoto T, et al.
Phosphoenolpyruvate carboxylase: A new era of structural
biology. Ann Rev Plant Biol, 2004, 55: 6984
[18] Chen J, Lang C, Huang R, et al. A method for producing
seeds having altered protein/fatty acid composition.
European Patent EP1229120. 2002
[19] Sugimoto T, Tanaka K, Monma M, et al.
Phosphoenolpyruvate carboxylase level in soybean seed
 Donghui Song et al. / Chinese Journal of Biotechnology, 2008, 24(3): 341348
highly correlates to its contents of protein and lipid. Agr
Biol Chemy, 1989, 53: 885887.
[20] Chen JQ, Lang CX, Hu ZH, et al. Antisense PEP gene
regulates t ratio of protein and lipid content in Brassica
napus seeds. Chin J Agr Biotechnol, 1999, 7: 316320.
[21] Chen JQ, Huang YZ, Lang CX, et al. Molecular cloning
and sequencing of the PEP gene from Brassia napus and
the construction of the antisense PEP gene. J Zhejiang
Univ (Agr & Life Sci), 1999, 25: 365367.
[22] Zhao GL, Chen JQ, Yin AP, et al. Transgenic soybean
lines harbouring anti-PEP gene express super-high oil
content. Mol Plant Bre, 2005, 3: 792796.
[23] Luinenburg I, Coleman JR. Identification, characterization
and sequence analysis of the gene encoding
phosphoenolpyruvate carboxylase in Anabaena sp. PCC
7120. J General Microbiol, 1992, 138: 685691.
[24] Katagiri F, Kidaki T, Fujita N, et al. Nucleotide sequence of
the phosphoenolpyruvate carboxylase gene of the
cyanobacterium Anacystis nidulans. Gene, 1985, 38:
265269.
[25] Sabe H, Kondo S, Shmuzev A, et al. Properties of
human.interleukin-2 receptors expressed on non-lymphoid
cells by cDNA transfection. Mol Biol Medi, 1984, 2:
379396.
[26] Kaneko T, Nakajima N, Okamoto S, et al. Complete
genomic structure of the bloom-forming toxic
cyanobacterium Microcystis aeruginosa NIES-843. DNA
Res, 2007, 14: 247256.
[27] Chen L, Omiya T, Hata S, et al. Molecular
characterization of a phosphoenolpyruvate carboxylase
from a thermophilic cyanobacterium, Synechococcus
vulcanus with unusual allosteric properties. Plant Cell
Physiol, 2002, 43: 159169.
[28] Sato S, Shimoda Y, Muraki A, et al. A large-scale
protein-protein interaction analysis in Synechocystis sp.
PCC6803. DNA Research, 2007, 14: 207216
[29] Zhu XY, Li Q, Liu DL, et al. Studies on the culture of the
transgeneic TNF- Anabaena IB02 in 100L photobioreactor.
Xiamen: Proceedings of the Symposium for National Marine
Biotechnology and Marine Pharmaceuticals Academic
Meeting, 2006, 220-222.
[30] Shi DJ, Ran L, Li Y, et al. The efficient expression box
and its vector of filament cyanobacterium. Chinese Patent,
ZL00132268.0.2004-10-13.
[31] Shi DJ, Deng YG, Zhao XG, et al. Cyanobacterial genetic
engineering technology for recombinant pharmaceutical
products. Proceedings of 2004 National Medicine
Bioengineering Seminar of Chinese Society of
Biotechnology. 2004, 54.
[32] Song DH, Liu J, Shi DJ, et al. The expression of
granulocyte colony-stimulating factor (G-CSF) in
Synechococcus sp. PCC 7002. Proceedings of the
Symposium for National Marine Biotechnology and
Marine Pharmaceuticals, 2006, 200203.
[33] Shi DJ, Duan R, Song DH, et al. Cyanobacterial gene
engineering: promising biotechnological application. In:
The 10th International Conference on Applied Phycology,
Qingdao, 2005.
[34] Ren L, Shi DJ, Dai JX, et al. Expression of the mouse
metallothionein-I gene conferring cadmium rersistance in
a transgenic cyanobacterium. FEMS Microbiol Lett, 1998,
158: 127132.
[35] Liu FL, Zhang HB, Shi DJ, et al. Construction of shuttle
vector expressing human tumor necrosis factor- gene and
expression of the gene in Anabaena sp. PCC 7120. Sci in
China (Series C), 1999, 29: 217224.
[36] Luo N, Ning Y, Shi DJ, et al. Cloning and expression of
human pro-urokinase gene in the cyanobacterium
Synechococcus sp. PCC 7002. Acta Bota Sini, 2000, 42:
931935.
[37] Dai W, Shi DJ, Zhang H, et al. Expression of human
epidermal growth factor gene in cyanobacteria. Acta Bota
Sini, 2001, 43: 12601264.
[38] Csordas A, Wang JK. An integrated photobioreactor and
foam fractionation unit for the growth and harvest of
Chaetoceros spp. in open systems. Aquacul Engineer,
2004, 30: 1530.
[39] Deng L, Tan TW, Wang F. Studies of enzymatic synthesis
of biodiesel. Chin J Biotech, 2003, 19: 97101.