Applied Energy 87 (2010) 38–46

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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
School of Chemical Engineering and Technology, China University of Mining and Technology, China
b
School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China
c
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 a b s t r a c t

Article history: Biodiesel has received much attention in recent years. Although numerous reports are available on the
Received 27 April 2009 production of biodiesel from vegetable oils of terraneous oil-plants, such as soybean, sunflower and palm
Received in revised form 9 June 2009 oils, the production of biodiesel from microalgae is a newly emerging field. Microalgal biotechnology
Accepted 10 June 2009
appears to possess high potential for biodiesel production because a significant increase in lipid content
Available online 8 July 2009
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
Keywords:
the various modes of cultivation for the production of oil-rich microalgal biomass, as well as the subse-
Microalgae
Biodiesel
quent downstream processing for biodiesel production. The advances and prospects of using microalgal
Lipids biotechnology for biodiesel production are discussed.
Fatty acids Ó 2009 Elsevier Ltd. All rights reserved.
Production

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2. Microalgal biotechnology for lipids production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.1. Biosynthesis of lipids/fatty acids in microalgae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.1.1. The formation of acetyl coenzyme A (acetyl-coA) in cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.1.2. The elongation and desaturation of carbon chain of fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.1.3. The biosynthesis of triglycerides in microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.2. Phototrophic cultivation of microalgae for lipids production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.3. Heterotrophic cultivation of microalgae for lipids production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.4. Genetic engineering for lipids production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3. The extraction of oils from microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.1. Pyrolysis technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.2. Liquefaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4. Transesterification technologies in the production of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

1. Introduction lacking conventional fuel resources. In the 1930s and 1940s, veg-
etable oils has been used as diesel fuels in the emergency situ-
The production of biodiesel has recently received much atten- ation. With the rapid development of the modern industry, the
tion worldwide. Because of the world energy crisis [1], many demand for energy has been greatly increased in recent years,
countries have started to take a series of measures to resolve and therefore alternative energy sources are being explored.
this problem [2]. Finding alternative energy resources is a press- Thus, the term ‘‘biodiesel” has appeared very frequently in many
ing mission for many countries, especially for those countries recent reports [3].
The world total biodiesel production was estimated to be
* Corresponding author.
around 1.8 billion liters in 2003 [4]. Although there was no
E-mail address: huangghclara@yahoo.com.cn (G. Huang). increase in biodiesel production between 1996 and 1998, a sharp

0306-2619/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2009.06.016
 G. Huang et al. / Applied Energy 87 (2010) 38–46
increase in biodiesel production was observed in the past several
years. It is speculated that the production of biodiesel will be fur-
ther 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 re-
lease 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 com-
bustion could be reduced, and the decrease in carbon monoxide
is owing to the relatively high oxygen content in biodiesel. More-
over, biodiesel contains no aromatic compounds and other chemi-
cal 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 significant economic
potential because as a non-renewable fuel that fossil fuel prices
will increase inescapability further in the future [8]. Finally, biodie-
sel is better than diesel fuel in terms of flash point and biodegrad-
ability [9].
Conventional biodiesel mainly comes from soybean and vege-
table oils [10], palm oil [11], sunflower 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 14–18 carbons. The struc-
tural 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 bio-
diesel 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 prob-
lem of using these low-cost oils and fats is that they often con-
tain large amounts of free fatty acids (FFA) which is difficult to
convert to biodiesel through transesterification [16]. Raw materi-
als that contain large proportions of fatty acid triglycerides are
preferred. For example, plant oil is found to contain more fatty
Table 1
Comparison of types of sources for the oils production.
Type of organism Advantages
Microalgal oils (1) Fatty acid constitutions similar to common vegetable oils
(2) Under certain condition it may be as high as 85% of the dry weight
(3) Short-time growth cycle
(4) Composition is relative single in microalgae
Bacteria oils (1) Fast growth rate
Oleaginous yeasts (1) Resources are abundant in the nature
and mildews (2) High oil content in some species
(3) Short-time growth cycle
(4) Strong capability of growth in different cultivation on conditions
(5) Conversion and utilization of scrap fiber 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
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 mid-
1980s. 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, seek-
ing for lipid-rich biological materials to produce biodiesel effec-
tively has attracted much renewed interest. Oleaginous
microorganisms are favorably considered for their short growth
cycles, high lipid contents and ease of being modified by biotech-
nological means (see Table 1). Some microalgae appear to be suit-
able 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 efficiency, higher biomass production and higher
growth rate compared to other energy crops [21–23]. Moreover,
according to biodiesel standard published by the American Society
for Testing Materials (ASTM), biodiesel from microalgal oil is sim-
ilar in properties to the standard biodiesel, and is also more stable
according to their flash point values (Table 2).
2. Microalgal biotechnology for lipids production
Microalgae have high potentials in biodiesel production com-
pared 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. Sec-
ond, microalgae grow extremely rapidly and many algal species are
rich in oils. For instance, heterotrophic growth of Chlorella prototh-
ecoides 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 20–50% are common in microalgae
[20]. The whole technical process in the production of biodiesel
Disadvantages
(1) Most algal lipids have lower fuel value than diesel fuel
(2) The cost of cultivation is higher compared to common crop oils
currently
(1) Most of bacteria can not yield lipids but complicated lipoid
(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
(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) 38–46

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
1
Density (kg L ) 0.864 0.838 0.84–0.90
Viscosity (mm2 s 1, cSt at 40 °C) 5.2 1.9–4.1 3.5–5.0
Flash point (°C) 115 75 Min 100
Solidifying point (°C) 12 50 to 10 –
Cold filter plugging point (°C) 11 3.0 (max 6.7) Summer max 0
Winter max < 15
Acid value (mg KOH g 1) 0.374 Max 0.5 Max 0.5
Heating value (MJ kg 1) 41 40–45 –
H/C ratio 1.81 1.81 –

from microalgae has been well investigated in recent years. ter can be formed after several reaction steps. The formation of
Third, the entire production process ranging from the cultivation short carbon chain fatty acids is similar in the cells of advanced
of high-lipid microalgae to the production of biodiesel from the plants, animals, fungi, bacteria, and algae. For example, in the cell
microalgal oils has also been explored. In the laboratory conditions, of green algae, the reaction routes of primer such as palmitoleic
the ideal oil content could reach 56–60% of total dry biomass by acid, oleic acid, linoleic acid, linolenic acid in fatty acid synthesis
genetic engineering or heterotrophic culture techniques. These are similar to that in plant cells and yeast cells [27]. The desatura-
technological advances suggest that the industrial production of tion of carbon chain of fatty acid occurs from C18 and further elon-
biodiesel from microalgal oils may be feasible in the near future. gation of carbon chain takes place to produce long-chain fatty acids
which are unusual in normal plant oils (Fig. 2). Long-chain fatty
2.1. Biosynthesis of lipids/fatty acids in microalgae acids (C20–C22) often exist in microalgae and the content varies
from species to species [28]. Normally, short-chain fatty acids
It is known that both inorganic carbon (CO2) and organic carbon (C14–C18) which are the main components of biodiesel are majority
sources (glucose, acetate, etc.) can be utilized by microalgae for lip- of fatty acids in Chlorella sp., but high content of long-chain fatty
ids production. The components and contents of lipids in microal- acid and hydrocarbons exist in some specific species of microalgae.
gal cells vary from species to species. The lipid classes basically are So, It is vital to choose proper microalgae species as materials of
divided into neutral lipids (e.g., triglycerides, cholesterol) and polar biodiesel production.
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 O
of carbon chain of fatty acids; and (c) the biosynthesis of triglycer- COO-
CH2C-SCoA
ides in microalgae. CH2
C O Econd
2.1.1. The formation of acetyl coenzyme A (acetyl-coA) in cytoplasm O NADP+
SCoA ACP-SH
The metabolism flux route on the utilization of carbon dioxide RC-S-ACP
and glucose for the formation of acetyl-coA in microalgae is de- O NADPH(H+)
scribed by Yang et al. [25]. It is concluded that glyceraldehyde COO - O
RC-S-Econd
phosphate (GAP) is a key intermediate both for the two metabo- HSCoA CH2 RCH=CHC-S-ACP
lism systems. The formation of acetyl-coA in photosynthetic reac-
C O
tions, including the light reactions, Calvin cycle and synthesis, is CO2
located in chloroplast. GAP is withdrawn from Calvin cycle and ex- S ACP OH O
ported to cytoplasm for consumption. After the export of GAP from O O
RCH-CH2C-S-ACP
chloroplast to cytoplasm, the flow of carbon is directed to the syn- RC-CH2C-S-ACP
HS-Econd
thesis 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] re- NADPH (H+) NADP+
ported that glucose was easy to be stored as starch without prior
conversion to GAP and then uptake by the chloroplast which sug- Acetyl-CoA-ACP acyl transferase
gested starch is the main storage formation for carbon source in
Chlorella sp. Therefore, one part of the exogenous glucose was di- malonyl CoA-ACP acyl transferase
rectly converted to starch, and the remainder was oxidized
through glycolytic pathway. β-ketoacyl-ACP Condensing enzyme)

2.1.2. The elongation and desaturation of carbon chain of fatty acids β-ketoacyl-ACP reductase
The elongation of carbon chain of fatty acids is mainly depen-
dent on the reaction of two enzyme systems including acetyl-coA
β-hydroxyacyl-ACP dehydrase
carboxylic enzyme (ACCE) and fatty acid synthase (FAS) in most
organisms. In the process of synthesis of fatty acids (Fig. 1), acet-
yl-coA is the primer. The process of carbon chain elongation needs enoyl-ACP reductase
the cooperation with malonyl-coA, the substrate on which enzyme Fig. 1. Reaction process of the FFA biological synthesis system (Shen and Wang
act are acetyl-ACP and malonyl-ACP. The C16–C18 fatty acid thioes- [62]).
 G. Huang et al. / Applied Energy 87 (2010) 38–46 41

C18:0 acetyl-coA to form Lysophosphatidic acid and later combines

9 desaturase 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
C18:1-9
phosphatidate phosphatase to form diglyceride which is then
12 desaturase
combined with the third acetyl-coA to complete the biosynthesis
of triglycerides. The last reaction step is catalyzed by glyceryl
C18:2-9,12
diester transacylase.
15 desaturase
2.2. Phototrophic cultivation of microalgae for lipids production
C18:3-9,12,15
6 desaturase Microalgae can transform carbon dioxide from the air and light
C18:4-6,9,12,15 energy through photosynthesis to various forms of chemical ener-
gies such as polysaccharides, proteins, lipids and hydrocarbons.
Fatty acid elongase Compared to higher plants, microalgae have a number of advanta-
C18:4-8,11,14,17 ges including higher photosynthetic efficiency and growth rate
[20]. In phototrophic culture, usually microalgae can be grown in
5 desaturase
two systems such as open ponds and enclosed photobioreactors.
C20:4-8,11,14,17 Enclosed photobioreactor system is more suitable for some micro-
algae which are readily contaminated by other microbes, except for
elongation
some special microalgae which can survive well in extreme envi-
C22:5-7,10,13,16,19 ronments such as high pH (e.g., Spirulina) and high salinity (e.g.,
Dunaliella) or can grow very rapidly (e.g., Chlorella) in the open
4 desaturase pond.
C22:6-4, 7,10,13,16,19 Because of better environmental control, enclosed photobiore-
actor system has been suggested for the production of high-value
Fig. 2. The elongation and desaturation of carbon chain of fatty acids (modified long-chain fatty acids (e.g., DHA, EPA). Nevertheless, due to the
from Guschina and Harwood [63]). high cost in terms of operation and capital investment and the
small scale due to the complexity of bioreactor design compared
2.1.3. The biosynthesis of triglycerides in microalgae to open pond system, it might not be economical to produce bio-
Like other higher plant and animal, microalgae are able to diesel on a large scale by enclosed photobioreactors.
biosynthesize triglycerides to store substance and energy. Gener- Open pond system is perhaps more suitable for cultivating mic-
ally, L-a-phosphoglycerol and acetyl-coA are two major primers roalgae for biodiesel because of its relatively cheap operating cost
in the biosynthesis of triglycerides. The L-a-phosphoglycerol compared to the enclosed photobioreactors. The basic require-
mainly derives from phosphodihydroxyacetone which is the ments for microalgal phototrophic growth should include carbon
product of the glycolysis process. The reaction steps are shown dioxide, other macro- and micro-nutrients, as well as light. Carbon
in Fig. 3. One of the hydroxyl in L-a-phosphoglycerol reacts with 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) 38–46
Water Nutrients
Open pond
Waster CO2
Fig. 4. Open pond photosynthesis system (OPSS).
amounts of waste gases (mainly CO2) daily. Typical coal-fired
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-fired 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]. Be-
sides, the wastewater may contain abundant nutrients (e.g., inor-
ganic 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 flue gas [30].
2.3. Heterotrophic cultivation of microalgae for lipids production
Although microalgae can utilize light efficiently, 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, het-
erotrophic 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 be-
cause of higher cell density obtained in heterotrophic culture of
microalgae [34]. In heterotrophic culture, both cell growth and
biosynthesis of products are significantly influenced 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 heterotrophi-
cally. 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-
Algae oil biodiesel
recovery system production
Motorized paddle
Waste water
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 hydroly-
sate (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
beneficial 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 re-
duced the cost of production, which is important for the biodiesel
production by microalgae in terms of economical significance.
Nitrogen is also an essential macronutrient in lipids produc-
tion. 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 simul-
taneously. Industrial wastewater rich in nitrogen also can be con-
sidered 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 microal-
gae growing under nitrogen limitation show enhanced lipid
content. In the late 1940s, it was noted that nitrogen starvation
is most influential on lipid storage and lipid fractions, and as a re-
sult of nitrogen starvation, the lipid content as high as 70–85% of
dry weight was reported [39]. It was also reported that Prophyri-
dium cruentum might double its total lipid content (mainly
neutral lipids) under nitrogen starvation [39]. Nevertheless, nitro-
gen 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 (28.4–38.4%) 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) 38–46
It is also noticeable that the overall rates of oil production might
be lower in the case of nutrient deficiency due to the overall low-
er biomass achieved. Therefore factors other than nitrogen should
be considered altogether.
Initial carbon to nitrogen (C/N) ratio in the medium has signif-
icant 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 ef-
fect of C/N ratio and aeration on the fatty acid composition of het-
erotrophic 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 con-
tent at low temperature might be explained by the fact that the al-
gae need to produce more PUFAs to maintain cell membrane
fluidity. 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 bio-
synthesis of PUFAs [42]. However, the effect of temperature on cell
growth and PUFAs production may not be always the same as men-
tioned above [31]. Therefore, a specific 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 dif-
ferent cultivation modes greatly affect the lipid accumulation in
microalgae. Heterotrophically grown microalgae usually accumu-
late 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 pro-
duction of fatty acids and lipids should be useful for further inves-
tigation 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 gen-
ome 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 efficiently ex-
pressed 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. Na-
tional Renewable Energy laboratory in the USA (NREL) has estab-
lished 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 5–20% 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 re-
search has focused on choosing a proper molecular carrier, making
ACC gene full expression in bacteria, yeast, and plant. Furthermore,
the modified ACC gene is being introduced into microalgae to ob-
43
tain more efficient expression. The utilization of engineered micro-
algae for the production of biodiesel has important economic and
environmental benefits. Its superiorities include high yield of mic-
roalgae; 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 inves-
tigated for fuel production of internal-combustion engines by
transesterification of fatty acids [48], industrial biodiesel produc-
tion from microalgal oils is still not well developed. At least, high
content of microalgal oils is required for this method to realize
economic benefits. Since it is difficult 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 mic-
roalgae to produce liquid fuel was first 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 defi-
ciency and high temperature. Pyrolysis previously was first 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 (450–550 °C) and
high heating rate (103–104 °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 15–20% 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 rea-
sons: (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 re-
quires less energy compared to the slow prolysis process. Fast
pyrolysis tests of microalgae were performed in the fluid 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) flow
rate of 0.4 m3 h 1 and a vapor residence time of 2–3 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 experi-
ments have demonstrated that fast pyrolysis (Table 3) is an effi-
cient method to produce useful fuels and gases from microalgae.
As mentioned above, not only the crude fat, but also other chem-
ical 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) 38–46

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 Fast pyrolysis 773 – 52.0 Peng et al. [53]
C. protothecoides Fast pyrolysis 773 600 °C/s 18 Miao et al. [52]
Microcystis aeruginosa Fast pyrolysis 773 600 °C/s 24 Miao et al. [52]
Heterotrophic C. protothecoides Fast pyrolysis 723 600 °C/s 57.9 Miao et al. [41]
C. protothecoides Fast pyrolysis 773 10 K/s 53.3 Demirbas et al. [66]

3.2. Liquefaction
High content of water often exists in microalgae after harvest-
ing 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 hydro-
gen 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 57–64% 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 thermochem-
ical liquefaction. The study suggested that microalgae consume
low amounts of nutrients and accumulate high caloric materials,
and nutrient resources which are produced without energy wast-
ing processes encourage the recovery of oil from microalgae and
CO2 mitigation.
4. Transesterification 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 transesterification of microalgal oils will
greatly reduce the original viscosity and increase the fluidity.
Few reports on the production of biodiesel from microalgal oils
(Table 4) may be applied in transesterification [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 tri-
glycerides to methyl esters are too slow [57]. Alkali catalysts have
higher reaction rate and conversion than acid catalysts for the
transesterification of triglyceride. Alkali-catalyzed transesterifica-
tion is about 4000 times faster than the acid catalyzed reaction
[58]. So, alkali-catalyzed transesterification 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 re-
sults in the loss of alkali catalysts in the process of reaction. There-
fore, 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 first convert FFAs to methyl eaters (Fig. 6) in order
to reduce the contents of FFAs, and the low FFAs pretreated oil is
transesterified 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
O O
+-
HO-C-R + KOH K O-C-R + H2O
Fatty Acid Potassium Hydroxide Potassium soap Water
Fig. 5. Transesterification by alkali catalyst.

are available [20]. Nevertheless the technologies of the biodiesel

production for vegetable oils can be applied to the biodiesel pro-
duction of microalgal oils because of the similar physical and
O O
chemical properties. In the process of transesterification, alcohols H2SO4
are key substrates in transesterification. The commonly used alco- HO-C-R + CH3OH CH3-O-C-R + H2O
hols are methanol, ethanol, propanol, butanol, and amyl alcohol Fatty Acid Methanol Methyl ester Water
but methanol is applied more widely because of its low-cost and
physical advantages. Alkali, acid, or enzyme catalyzed processes Fig. 6. Transesterification by acid catalyst.

Table 4
Application of transesterification technologies.

Type of Advantages Disadvantages

transesterification
Chemical catalysis (a) Reaction condition can be well controlled (a) Reaction temperature is relative high and the process is complex
(b) Large scale production (b) The later disposal process is complex
(c) The cost of the production process is cheap (c) The process need much energy
(d) The methanol produced in the process can (d) Need a installation for methanol recycle
be recycled
(e) High conversion of the production (e) The waste water pollutes the environment
Enzymatic catalysis (a) Moderate reaction condition (a) Limitation of enzyme in the conversion of short chain of fatty acids
(b) The small amount of methanol required in (b) Chemicals exist in the process of production are poisonous to enzyme
the reaction
(c) Have no pollution to natural environment
Supercritical fluid (a) Easy to be controlled (a) High temperature and high pressure in the reaction condition leads to high cost of
techniques (b) It is safe and fast production and wastes energy
(c) Friendly to environment
 G. Huang et al. / Applied Energy 87 (2010) 38–46
to meet the ASTM fuel specification [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; cata-
lysts, 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
transesterification 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 transesterified 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
transesterified technologies for biodiesel [61].
5. Conclusion
Since oil crisis in the mid 1970s, finding 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 pres-
ent, the high cost of oleaginous materials is the main problem hin-
dering commercial production of biodiesel. Therefore, finding
cheaper oleaginous materials and improving transesterification
technologies are the key to producing biodiesel successfully. To
date, large scale commercial production of biodiesel from microal-
gal oils has not been reported. The low lipid content and low bio-
mass 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 pyroly-
sis may lead to a high yield of bio-oils on a large scale. Research in
genetic engineering coupled with advanced cultivation and down-
stream technologies will benefit the future development of micro-
algae for biodiesel production.
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