Biochemical Engineering Journal 78 (2013) 110

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular Article

Microalgae-based carbohydrates for biofuel production

Chun-Yen Chen a , Xin-Qing Zhao b , Hong-Wei Yen c , Shih-Hsin Ho d , Chieh-Lun Cheng d ,
Duu-Jong Lee e , Feng-Wu Bai b, , Jo-Shu Chang a,d,f,
a

University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan
School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China
Department of Chemical and Materials Engineering, Tunghai University, Taichung, Taiwan
d
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
e
Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan
f
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan
b
c

a r t i c l e

i n f o

Article history:
Received 23 November 2012
Received in revised form 4 March 2013
Accepted 8 March 2013
Available online 15 March 2013
Keywords:
Microalgae
Biogas
Growth kinetics
Integrated Processing
Biofuels
Biorenery

a b s t r a c t
Microalgae are considered as the most promising renewable feedstock for biofuel production and bioreneries, due to their advantages of fast growth, efcient carbon dioxide xation, not competing for arable
lands and potable water, and potentially accumulating high amounts of lipids and carbohydrates. Since
carbohydrates in microalgae biomass are mainly cellulose in the cell wall and starch in the plastids
without lignin and low hemicelluloses contents, they can be readily converted into fermentable sugars.
However, to date there are very few studies focusing on the use of microalgae-based carbohydrates for
biofuel production, which requires more understanding and knowledge to support the technical feasibility of this next-generation feedstock. This review article elucidates comprehensive information on the
characteristics and metabolism of main fermentable microalgal carbohydrates (e.g., starch and cellulose),
as well as the key factors and challenges that should be addressed during production and saccharication of microalgal carbohydrates. Furthermore, developments on the utilization of microalgae-based
feedstock in producing liquid and gaseous biofuels are summarized. The objective of this article is to
provide useful knowledge and information with regard to biochemistry, bioprocess engineering, and
commercial applications to assist in the viable technology development of for biofuels generation from
microalgae-based carbohydrates.
2013 Elsevier B.V. All rights reserved.

1. Introduction
The fast growth of the global population and the rise of developing countries, such as China and India, have led to a rapid increase
in demand for energy [1]. Currently, about 90% of energy needs
come from coal, natural gas and petroleum, and sustainable energy
supplies need to be developed due to the dwindling reserve of
these fossil fuel resources [2,3]. With current consumption trends,
world oil reserves may run out by 2050 [1,4]. Moreover, the
problems of environmental pollution and climate change are also
mainly attributed to the over-consumption of fossil fuels [5]. Therefore, a number of countries have expressed increased interest in

Corresponding author at: National Cheng Kung University; Department of

Chemical Engineering; Tainan 701; Taiwan. Tel.: +886 6 2757575x62651;
fax: +886 6 2357146.
Co-corresponding author at: School of Life Science and Biotechnology, Dalian
University of Technology, Dalian 116024, China. Tel.: +86 411 8470 6308;
fax: +86 411 8470 6329.
E-mail addresses: fwbai@dlut.edu.cn (F.-W. Bai), changjs@mail.ncku.edu.tw
(J.-S. Chang).
1369-703X/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bej.2013.03.006

developing alternative energy sources that are renewable, economically competitive and environmentally friendly [6].
Biomass can be converted to energy by biological or thermochemical methods. Biological conversion includes fermentation of
degradable components to produce energy carriers like bioethanol,
biobutanol, biohydrogen and biogas, or extraction of oils for
biodiesel production. Thermo-chemical conversion includes direct
combustion for heat and electricity, as well as indirect processes
like pyrolysis and gasication [7]. The most widely used biofuel is
bioethanol, which is produced from sugar-based (sugar beets, sugarcane) and starch-based (corn, wheat, barley, etc.) feedstocks [8],
while technology leading to conversion of lignocellulosic materials (bagasse, corn stover, rice straw, switchgrass, and so on) into
ethanol is under development worldwide.
The choice of biomass feedstock depends on social, environmental, economic and industrial factors, such as availability and
cost of raw materials. However, the challenges associated with
most of the current feedstock lies in the need for arable lands and
freshwater for the cultivation of plants, and thus possible competition with food production, seasonal and geographical variations
in productivity, as well as the need for herbicides [9]. Although

C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 110

lignocellulosic biomass is cheap and plentiful to utilize, the cost

of converting it into ethanol is still relatively high. Moreover,
the lignin component is very difcult to ferment or degrade biologically [10], and thus a more sustainable feedstock should be
developed to overcome these barriers. Microalgae have been recognized as an alternative, a so-called third generation feedstock that
does not compete for arable land or portable water, and marine
microalgae species growing in seawater can also reduce freshwater consumption. In addition, the microalgae-based carbohydrates
have low lignin content (high fermentable sugars), and their saccharication is much easier, thus being a more promising and
sustainable biomass source for bioethanol production [1,11]. In
this review, we provide up-to-date and comprehensive information on the metabolism, characteristics, production technologies,
and applications of microalgae-based fermentable carbohydrates.
In particular, recent advances in the production and utilization of
carbohydrate-rich microalgae feedstock for biofuels are covered to
promote the idea of using microalgae as a practical feedstock.
2. Evolution of feedstock for biofuels production
As bulk commodities, the cost of biofuels mainly comes from
feedstock consumption. For example, the cost of feedstock consumption is as high as 6070% for fuel ethanol produced from
starched-based feedstocks [8]. Therefore, the availability of lowcost feedstock is always the biggest concern for biofuels production
if it is to successfully meet the global demand for energy. Due to
the issues of cost, land/freshwater requirement, food competition,
and environmental concerns, the feedstock for biofuels production
has been switched from sugar or starch-based crops (rst generation feedstock) to lignocellulosic materials (second generation
feedstock), and then to microalgae (third generation feedstock).
The advantages and limitations of these feedstocks are addressed
below.
2.1. Conventional feedstocks and their limitations
Biofuels produced from sugar, grain and oleaginous crops are
known as rst generation biofuels. Currently, ethanol is the main
biofuel, which is primarily produced from sugar- and starch-based
feedstocks that are easy to ferment, and thus have high biofuel
conversion efciency. However, sugarcane, the major sugar-based
feedstock, requires tropical or temperate climates, with Brazil the
only successful example of sugarcane ethanol use [12]. Starchbased feedstocks are mostly grains, and thus are not sustainable
for biofuels production, taking into account the global population
situation and increased demand for grains as the food or animal
feed. For example, fuel ethanol production in the United States
increased from 1.6 to 13.2 billion gallons from 2000 to 2010, consuming one third of the corn harvest of US and causing a signicant
increase in global grain prices [13]. A similar situation also applies
with biodiesel, another major biofuel which is produced from animal fats or vegetable oils through transesterication with alcohols
catalyzed by either enzymes or chemical catalysts [14]. Much like
sugarcane, palm trees, which have the highest oil yield among
oleaginous plants, are limited to tropical regions such as Southeast Asia, while other vegetable oils from soybeans, peanuts and
rapeseeds are edible ones. Therefore, rst generation feedstock has
signicant limitations and raises issues with regard to food versus
fuel. Therefore, alternative feedstocks are needed if biofuels are to
be more widely used.
2.2. Lignocellulosic biomass and challenges
Lignocellulosic biomass, such as agricultural and forest residues,
is not food-related and abundantly available at low cost without

competition for arable land. It was estimated that the United States
alone produces about 1.3 billion tons of lignocellulosic biomass
annually, which could be converted into 60 billion gallons of
ethanol with negligible impact on the food supply and signicant
environmental benets [15]. Therefore, lignocellulosic biomass has
been widely acknowledged as an ideal feedstock for the sustainable
production of biofuels. However, due to its recalcitrance to degradation, lignocellulosic biomass still cannot be converted to biofuels
in an efcient and economical way [16]. The major components
of lignocellulosic biomass are cellulose, hemicelluloses and lignin,
which interact and entangle to form the lignincarbohydrate complex [17]. This unique crystalline structure prevents cellulases from
binding onto cellulose surfaces to liberate sugars for biofuels production, and pretreatment is thus needed to deconstruct it, which
is energy-intensive and consumes signicant amounts of chemicals
[18]. On the other hand, cellulases, the enzymes responsible for catalyzing cellulose hydrolysis, are more expensive than amylases and
glucoamylases, and the cellulase dosage required for effective cellulose saccharication is usually very high [19]. Furthermore, there
are still problems arising from inefcient fermentation of pentose from the hemicellulose content in lignocellulosic feedstock,
although intensive research and development to engineer various
microorganisms has been carried out for decades [20]. In addition,
the lignin part is very difcult to ferment, thereby decreasing the
overall biomass to biofuels yield, and also leading to higher waste
treatment costs

2.3. Carbohydrate-based microalgae as innovative feedstock for

biofuels production
Although lignocellulosic biomass is much cheaper for biofuels
production than sugar- and starch-based feedstock, a recent outlook report predicted that large-scale production of the second
generation biofuels will take another ve to ten years for development [21]; needless to say the ongoing controversy over land
use, even for marginal land used for the cultivation of herbaceous
energy crops. However, aquatic microalgae that can grow with
high photosynthetic efciency could be a solution to this problem
[22]. Microalgae are photosynthetic organisms with relatively simple requirements for growth when compared to other sources of
biomass. Through the process of photosynthesis, the algae convert
CO2 , water and light into biomass. Some microalgae may contain
a large amount of carbohydrates in the cell wall (mainly in the
form of cellulose and soluble polysaccharides) [23] and plastids
(mainly in the form of starch) [24], which can potentially be used as
carbon sources for fermentation [11]. Moreover, some microalgae
species are also known to accumulate lipids under stress conditions, and thus could also be used as the raw material for biodiesel
production [2,25].
Using microalgal feedstock has several advantages over traditional feedstock. These include: (1) the high growth rate and
high area (or volumetric) productivity; (2) the absence of lignin
and low hemicellulose content, and thus only minor pretreatment
being needed; (3) efcient CO2 capture via photosynthesis, thereby
mitigating greenhouse gas emissions; (4) being able to grow on
salt or wastewater streams (saline/coastal water, brackish seawater, domestic/municipal/industrial wastewater), thereby reducing
freshwater use; (5) being able to grow in areas unsuitable for agricultural purposes (e.g. desert and seashore lands), and thus there
is no competition with arable land for food production; (6) having a very short harvesting cycle (110 days) compared with other
feedstocks (which are harvested once or twice a year), thus providing enough supplies to meet ethanol production demand; and (7)
compatibility with integrated production of fuels and co-products
within bioreneries. These advantages thus allow microalgae to be

C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 110

preferentially selected as a clean, efcient, and sustainable feedstock for bioethanol production [1].

Table 2
Carbohydrate or starch content of green microalgal species.
Microalgal species

Carbohydrate or starch
content (%)

References

Chlorella vulgaris IAM C-534

C. vulgaris CCAP 211/11B
C. vulgaris P12
C. vulgaris
Chlamydomonas reinhardtii UTEX 90
C. reinhardtii IAM C-238
Chlorococum sp.
Chlorococcum sp. TISTR8583
S. acutiformis TISTR 8495
S. obliquus CNW-N
Tetraselmis sp.CS-362

37.0 (starch)
55.0
41.0 (starch)
55.0 (starch)
60.0
55.0 (starch)
32.5
26.0 (starch)
16.4 (starch)
51.8
26.0

[67]
[31]
[86]
[87]
[67]
[32]
[88]
[58]
[89]
[11]
[90]

3. Microalgae-based carbohydrates
Carbohydrates are the major products derived from photosynthesis and the carbon xation metabolism (i.e., the Calvin cycle)
[4]. These carbohydrates are either accumulated in the plastids as
reserve materials (e.g., starch), or become the main component
of cell walls (e.g., cellulose, pectin, and sulfated polysaccharides). However, the composition and metabolism of carbohydrates
(mainly starch and cellulose) in microalgae may differ signicantly
from species to species [24,26]. It is thus of great importance to
select microalgae with high carbohydrate productivity as well as
suitable sugar composition for biofuels or chemical production.
For instance, microalgae that contain glucose-based carbohydrates
are the most feasible feedstock for bioethanol production. However, although increasing attention has been paid to the potential
of using macroalgae (e.g., seaweed) as a sugar source [27], the major
sugars from brown macroalgae are glucan, mannitol, and alginate. The former two are relatively easily assimilated by available
microbial ethanol producers, with a yield of 0.080.12 g ethanol
per g dry biomass weight. However, the inability of industrial
microbes to metabolize alginate is a major hurdle to achieving the
full potential of ethanol production from macroalgal biomass [27].
In contrast, the carbohydrates from microalgae are very suitable
for bioethanol production, although more economic cell harvesting
technology should be developed to make microalgae-based ethanol
commercially viable. The carbohydrate composition and the general carbohydrate metabolism of microalgae are described in the
following sections.
3.1. Carbohydrate composition of microalgae

Table 1
Composition of microalgal cell wall and storage products.
Division

Cell wall

Storage products

Cyanophyta

Lipopolysaccharides,
peptidoglycan
Cellulose, hemicellulose
Absence or contain few cellulose
Periplast
Absence
Agar, carrageenan, cellulose,
calcium carbonate
Naked or covered by scales or
with large quantities of silica

Cyanophycean
starch
Starch/lipid
Starch
Starch
Paramylum/lipid
Floridean starch

Heterokontophyta

starch content of different microalgae species. As indicated in

Table 2, Chlorella sp. has the highest carbohydrate content. In
particular, Chlorella vulgaris can accumulate a large amount of carbohydrates, up to 3755% of its dry biomass. For example, after 14
days cultivation in a low-nitrogen medium, the biomass concentration and carbohydrate content of C. vulgaris CCAP 211/11B can
reach 0.52 g (dry cell weight, DCW)/L and 55%, respectively [31].
Chlamydomonas reinhardtii and Scenedesmus obliquus also has the
potential to serve as a biofuels feedstock due to its high carbohydrate content of 4560%. In a batch culture of C. reinhardtii UTEX
90, the biomass concentration and starch content were found to be
1.45 g DCW/L and 53%, respectively, after 3-day cultivation [32].
Moreover, the biomass concentration and carbohydrate content
of S. obliquus could reach 4.96 g DCW/L and 51.8%, respectively,
under 3-day nitrogen starvation [11]. Therefore, species of Chlorella,
Scenedesmus, and Chlamydomonas are appropriate carbohydratebased feedstock candidates for bioethanol production.
3.2. Carbohydrate metabolism of microalgae

The cell walls of microalgae primarily consist of an inner cell wall

layer and an outer cell wall layer, and can be grouped into three
types, namely those (1) with a trilaminar outer layer, (2) with a
thin outer monolayer and (3) without an outer layer [28]. The composition of the outer cell wall varies from species to species, but
usually contains specic polysaccharides, such as pectin, agar, and
alginate [28]. The inner cell wall layer of microalgae is mainly composed of cellulose and other materials (such as hemicellulose and
glycoprotein) [28]. Table 1 shows the compositions of the cell walls
and the storage products in different microalgal species. For some
microalgae, the glucose polymers produced via cellulose/starch are
the predominant component in the cell walls and stored products of
microalgae [29]. Microalgae are considered a promising feedstock
for bioethanol production because they have cellulose-based cell
walls, with accumulated starch as the main carbohydrate source.
Both starch and most cell wall polysaccharides can be converted
into fermentable sugars for subsequent bioethanol production via
microbial fermentation [30]. Table 2 shows the carbohydrate or

Chlorophyta
Dinophyta
Cryptophyta
Euglenophyta
Rhodophyta

Leucosin/lipid

The accumulation of carbohydrates in microalgae is due to CO2

xation during the photosynthetic process. Photosynthesis is a
biological process utilizing ATP/NADPH to x and convert CO2 captured from the air to produce glucose and other sugars through a
metabolic pathway known as the Calvin cycle [33].
The metabolic pathways of energy-rich molecules (e.g., carbohydrate and lipid) are closely linked. Some studies demonstrated
that there was a competition between lipid and starch synthesis
because the major precursor for triacylglycerols (TAG) synthesis
is glycerol-3-phosphate (G3P), which is produced via catabolism
of glucose (glycolysis) [11,24]. Thus, to enhance biofuels production from microalgae-based carbohydrates, it is vital to understand
and manipulate the related metabolisms to achieve higher microalgal carbohydrate accumulation via strategies like increasing glucan
storage and decreasing starch degradation [34]. The starch forms
around a crystallizing nucleus and is present as an amorphous
starch grain. When a chloroplast gathers enough starch, it may
become an amyloplast. However, the detailed changes in enzymatic activity and metabolic ux of carbohydrate biosynthesis of
microalgae are poorly understood. The manipulation of the carbohydrate metabolisms of microalgae by genetic engineering has also
been proposed [34]. With the development of genetic engineering
of microalgae, and a better understanding of the biochemistry of
microalgae carbohydrate metabolisms, superior strains for carbohydrate accumulation could be developed.
Except the starch in plastids, microalgal extracellular coverings
(e.g., cell wall) are another carbohydrate-rich part which could be
transformed to biofuel [1]. However, the compositions of microalgal extracellular coverings are diverse by species [23]. Among them,
cellulose is one of the main fermentable carbohydrates in most of
green algae [34]. Cellulose synthesis is a complicated process that

C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 110

Fig. 1. Carbohydrate metabolism of green algae. Redrawn on the basis of the reference [33].

includes many enzymatic reactions. The starting substrate for cellulose synthesis is UDP-glucose, which is formed from the reaction
of UDP and fructose catalyzed by sucrose synthase (Fig. 1) [35].
Despite the understanding of main carbohydrate metabolism
in microalgae, in-depth knowledge on its regulation is still lacking. To meet the challenges on economic biofuels production from
microalgae, it is important to integrate updated information of
genomic sequences, transcriptomes, proteomes, and metabolomes
data at systems level. Although systems study of microalgae on carbohydrate metabolisms is currently in its infant stage, omics studies
on microalgae have made signicant progress [24]. Such a strategy
will open a door for efcient carbohydrate metabolic regulation and
genetic engineering of microalgae for biofuels production.

4. Environmental factors affecting microalgae

carbohydrate production
To enhance the economic feasibility of using algal carbohydrates for biofuels production, the productivity is the key parameter
that needs to be improved. Unfortunately, the accumulation of
microalgal carbohydrates usually occurs when the microalgal cells
face environmental stress (typically nutrients limitation), which
often results in poor cell growth (or lower biomass productivity). Therefore, how to enhance the carbohydrate content without
compromising the cell growth rate is crucial to the success of carbohydrate production from microalgae. The carbohydrate content
of microalgae could be enhanced by the use of various cultivation
strategies, such as irradiance [36], nitrogen depletion [37], temperature variation [38], pH shift [39], and CO2 supplement [40].

The effects of these approaches are discussed in more detail in the

following sections.

4.1. Irradiance
Providing appropriate irradiance is essential for the autotrophic
growth of microalgae, since illumination can offer light energy
that is further stored in the form of carbohydrates or lipids in
the microalgal biomass. The efciency of light energy supply thus
becomes one of the major limiting factors for outdoor or large-scale
microalgae cultivation. Therefore, the conguration of microalgae
cultivation systems (such as photobioreactors) should be designed
to provide uniform and sufcient irradiance to the microalgal cells.
The key design factors associated with the light supply would
be the operation depth and agitation. The former strongly affects
the light penetration and availability, while the latter is vital for
enhancing the light distribution and uniformity. In addition, the
light intensity was also found to affect the carbohydrate accumulation in microalgae. Previous studies demonstrate that an
increase in the light intensity in the range of 30400 mol m2 s1
could slightly increase the accumulation of carbohydrates [41].
In a recent report, a signicant increase in starch content from
8.5% (dry weight basis) to 40% was observed when the mean
light intensity was increased from 215 to 330 mol m2 s1 [42].
However, in other cases [41], there was no obvious positive correlation between light intensity and carbohydrate accumulation. This
suggests that the accumulation of microalgal carbohydrates not
only depends on light intensity, but also on other environmental
parameters.

C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 110

4.2. Nitrogen depletion

Nitrogen is an essential nutritional component for the growth
of microalgae, because the formation of proteins, amino acids,
chloroplast, enzymes, coenzymes, and so on is associated with
nitrogen assimilation [43]. A variety of nitrogen sources (such as
nitrate, nitrite, ammonia and urea) can be utilized by microalgae,
while different nitrogen sources may inuence their biochemical
composition. More importantly, when under nitrogen-depletion
conditions many microalgal strains could transform proteins or
peptides to lipids or carbohydrates as energy reserve components
[44]. Illman et al. [31] reported that a 55% carbohydrate content was
achieved when cultivating C. vulgaris on a low-nitrogen-containing
medium. DSouza and Kelly [37] also demonstrated that the cultivation of Tetraselmis suecica under nitrogen starvation with CO2
feeding could dramatically enhance the cellular carbohydrate content from 10 to 57%. There are many other examples in the literature
showing that limitation of nitrogen availability seems to be the
most effective way of triggering the accumulation of carbohydrates in microalgae [4]. However, some studies indicated that
there was a competition between lipid and carbohydrate synthesis under stress environments (e.g., nitrogen-decient) because the
metabolic pathways associated with synthesis and degradation of
energy-rich compounds (e.g., lipids and carbohydrates) are closely
linked [11,24,45]. Precisely, starch biosynthesis of microalgae can
directly proceed away from lipid synthesis. On the contrary, degradation of starch provides the metabolites for producing acetyl-CoA,
which is the precursor of fatty acid synthesis [24,46]. Thus, in some
cases, decreasing starch degradation by genetic modication is necessary to block the synthetic pathway of lipids [34].
4.3. Temperature
The effect of temperature on the accumulation of microalgal carbohydrates are still inconclusive [38]. Some researchers observed
no signicant differences in biochemical composition under the
stress of temperature variations in some microalgal species [47],
whereas some researchers pointed out that temperature is potentially able to change the biochemical composition of microalgae.
Therefore, the effect of temperature on carbohydrate accumulation in microalgae seems to be highly dependent on the microalgal
strains used.
4.4. pH
pH is an important environmental condition for the metabolism
of microorganisms, affecting not only cell growth rate but also
biochemical composition [39]. In general, the adequate pH for carbohydrate accumulation differs based on the type of microalgal
species used. Khalil et al. [39] indicated that the pH value significantly affects the accumulation of total carbohydrates in both D.
bardawil and C. ellipsoidea and their maximum carbohydrate accumulation was reached at pH 7.5 and 9.0, respectively. Furthermore,

Taraldsvik and Myklestad. [48] reported that the extracellular carbohydrate production of a marine diatom Skeletonema costatum
was dramatically increased from 2.1 to 17.7% when it was grown
at pH 9.4.

4.5. CO2 supplementation

The addition of CO2 is required for the autotrophic growth
of microalgae, and is considered to be positively related to the
efciency of photosynthesis, with the synthesis of carbohydrates
as the end product. A sufcient supply of CO2 is thus one of
the key factors inuencing the accumulation of carbohydrate in
microalgae. Some studies found that carbohydrate accumulation in
microalgae is improved by increasing the percentage of CO2 in the
inlet gas [49,50]. For example, Xia and Gao [49] pointed out that
increasing dissolved CO2 concentration from 3 to 186 mol/L in
the cultivation of C. pyrenoidosa and C. reinhardtii could elevate the
carbohydrate content from 9.30 to 21.0% and 3.19 to 7.40% (w/w),
respectively. However, many researchers believe that increasing
the CO2 concentration not only provides more carbon source for
photosynthesis to promote microalgae growth, but also induces the
synthesis of relevant proteins, which may inuence the cell physiology. In some microalgal species, increases in the CO2 concentration
result in an increase in the protein content, but a decrease or no
obvious change in the carbohydrate content [51]. However, under
nitrogen starvation conditions and with an adequate supply of CO2
and light energy, the protein content in microalgae can be consumed as a nitrogen source, and the carbohydrate content may
increase signicantly during this process. Therefore, suitable addition of CO2 is a key step to improving the autotrophic growth of
microalgal cells (i.e., improving biomass productivity and protein
content), although it may not directly enhance carbohydrate accumulation in microalgae, unless appropriate stress conditions are
employed.

4.6. Comparison of the carbohydrate production performance of

different microalgae
The carbohydrate production ability of microalgae varies from
species to species. However, as mentioned earlier, the production
of microalgal carbohydrates can be signicantly improved when
appropriate cultivation conditions are applied. Table 3 summarizes
the performance of the biomass and carbohydrates production of
various microalgae species according to recent reports in the literature, showing that the microalgal carbohydrate productivity
varies in a wide range from 0.021 to 0.687 g/L/d (Table 3). Our
recent work showed that C. vulgaris FSP-E can achieve the highest carbohydrates productivity of 0.687 g/L/d due to a high biomass
productivity of 1.363 g/L/d [52]. Information concerning the composition of carbohydrates produced by microalgae is also crucial for
future applications of microalgae feedstock. However, this information is currently very limited, as only two Chlorella strains

Table 3
Comparison of biomass productivity and carbohydrate productivity of microalgae strains reported in the literature.
Microalgae strains

Operation
mode

Cultivation
time (days)

Biomass
production (g/L)

Biomass
productivity (g/L/d)

Carbohydrate
productivity (g/L/d)

References

C. vulgaris (CCAP 211/11B)

C. vulgaris (P12)
C. reinhardtii UTEX 90
C. reinhardtii
Tetraselmis subcordiformis
S. obliquus CNW-N
C. vulgaris
C. vulgaris FSP-E

Batch
Batch
Fed-batch
Batch
Semi-batch
Batch
Batch
Batch

14
N.D.
4
3
N.D.
5
6
5.25

0.52
N.D.
2.40
1.45
N.D.
4.03
1.70
7.30

0.037
0.485
0.507
0.484
N.D.
0.821
0.254
1.363

0.021
0.199 (starch)
0.304
0.257
0.255 (starch)
0.383
0.112
0.687

[31]
[86]
[61]
[32]
[91]
[11]
[92]
[52]

C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 110

(C. vulgaris P12 and C. reinhardtii UTEX 90) have been shown to
produce 41% and 35% starch, respectively (Table 3).
5. Saccharication of the microalgae-based carbohydrates
Saccharication is usually the rate limiting step in biofuels production using lignocellulosic materials or microalgal biomass that
contains a cellulose source. It is known that cellulose and starch are
the two major carbohydrate components in microalgal biomass.
Therefore, to enhance utilization of microalgae-based carbohydrates, it is important to identify efcient methods for the cleavage
of -1,4-glycosidic linkages between the hydroglucose subunits in
cellulose molecules, and the cleavage of -1,4-glycosidic linkages in
starch. While the process of saccharication of microalgae is similar
to that of lignocellulosic materials, the lack of lignin present in the
microalgal biomass simplies the pre-treatment process [1]. Various methods have been applied to produce sugars from microalgae,
such as mineral acids, alkaline, enzymes or hot compressed water
[53], and can be categorized into two major groups, namely enzymatic saccharication and chemical saccharication. The details of
those methods are described in the following subsections.
5.1. Enzymatic saccharication
Enzymatic saccharication processes, involving the use of cellulases, amylases and glucoamylases, are widely used to hydrolyze
microalgae to obtain sugars. For microalgae-based cellulose, which
is mainly located in the inner cell wall, lignin is absent and the hemicellulose content is also very low. Therefore, the lignin-degradation
enzymes (e.g., laccase and lignin peroxidase) and xylanase may not
be necessary in the enzymatic saccharication process. In addition, harsh pretreatment, such as acidic or alkaline pretreatment,
or steam explosion, is also not needed, making it easier and cheaper
to saccharify microalgae-based cellulose when compared with
lignocellulosic materials. To hydrolyze microalgae-based cellulose, endo--1,4-d-glucanase attacks the amorphous cellulose and
cleaves cellulose into small fragments. The exo--1,4-d-glucanase
further hydrolyzes the small fragments into simple sugars, such as
cellobiose and cellodextrin. Finally the cello-oligosaccharides are
degraded to glucose by -glucosidase [54]. To hydrolyze starch
inside the microalgal cells, amylase (endo-amylase) rst attacks
the internal -1,4-glycosidic bond of starch to produce dextrin
and glucoamylase further hydrolyzes dextrin into glucose and
oligosaccharides, such as maltose [55]. Enzymatic hydrolysis has
several advantages over chemical hydrolysis (such as acid or alkaline hydrolysis), including lower equipment costs, as hydrolysis is
conducted at mild conditions, and higher glucose yields without
sugar-degradation products or toxic by-products that may affect
follow-up biofuels fermentation [56]. Choi et al. [57] explored the
effects of two different commercial enzymes (including amylase
from B. licheniformis and glucoamylases from Aspergillus niger) on
the bioethanol conversion efciency of Chlamydomonas reinhardtii
biomass with a carbohydrate content of about 59.7% dry weight
base. The results showed that when algal biomass was hydrolyzed
at pH 4.5 and 55 C for 30 min, better sugar conversion of 0.57 g
sugar/g algal biomass was obtained and bioethanol production was
efcient by the separate hydrolysis and fermentation (SHF) process
[57]. In the other example, Chlorococum humicola was hydrolyzed
by enzymes from Trichoderma reesei, obtaining a saccharication
yield of 64.2% (w/w) under the conditions of 40 C, pH 4.8, and a
microalgal biomass concentration of 10 g/L [58].
5.2. Chemical saccharication
The chemical saccharication process is characterized by its
fast reaction, but usually requires violent reaction conditions

(higher temperature, pressure, and addition of acid and alkali),

resulting in the production of the inhibitors such as furfural and
5-hydroxymethylfurfural, which potentially repress fermentative
biofuels production and also require costly downstream treatment of the waste [59]. Maintaining suitable reaction conditions
(including temperature, moisture content, residence time and reaction agent concentration) is essential to reduce the production
of inhibitors and improve operational efciency [60]. Due to the
short hydrolysis time required, acid/alkaline hydrolysis has been
successfully applied to sugar production from microalgae. In particular, since the microalgae-based cellulose is not associated with
lignin and is thus easier to hydrolyze when compared with lignocelluloses, chemical hydrolysis under relatively mild conditions is
of great interest (i.e., low acid or alkali concentration, low reaction temperature, and so on). Nguyen et al. [61] reported that by
using an extremely low acid concentration (3.0 wt.% sulfuric acid)
a maximum glucose yield of 95.0% can be achieved from hydrolysis of algal biomass at 100 C for 30 min. Nguyen et al. [62] also
demonstrated that a nearly 100% sugar yield of algal starch from C.
reinhardtii was obtained while it was treated by 1.5% HCl. On the
other hand, Harun et al. [1] used alkaline treatment (0.75% NaOH,
120 C, 30 min) to produce sugar from C. infusionum with the highest glucose yield of 0.35 g sugar/g algal biomass. It has also been
shown that bioethanol production from the sugars resulting from
the chemical hydrolysis of microalgal biomass is quite successful
[1]. These results demonstrate the advantages of using microalgaebased carbohydrates for biofuels production, as the conversion of
assimilable sugars from microalgal biomass can be achieved more
efciently via much simpler and cheaper chemical saccharication
processes. In other words, the expensive and energy intensive pretreatment and enzymatic reaction may not be necessary during the
saccharication of microalgal biomass. However, the sugar yield by
acid or alkali hydrolysis may still be lower than that from enzymatic
saccharication [57].

6. Producing biofuels from microalgae-based

carbohydrates
Microalgae possess higher lipids or carbohydrates productivity, making them one of the most promising feedstock to produce
biofuels [63]. The production of liquid and gaseous biofuels from
microalgae biomass (mainly based on carbohydrates) is addressed,
taking ethanol, butanol, hydrogen and methane as examples.

6.1. Liquid biofuels

Two liquid biofuels (i.e., bioethanol and biobutanol) produced from microalgae-based carbohydrates are discussed here.
Carbohydrates-based microalgae can be suitable feedstock for
bioethanol production [64]. Although bioethanol fermentation
generates a large amount of by-product CO2 , this disadvantage
can be overcome when bioethanol fermentation is coupled with
the cultivation of carbohydrate-rich microalgae [4]. The CO2 produced from bioethanol fermentation process can be fully recovered
to grow microalgae for carbohydrates accumulation. The resulting
carbohydrate-rich microalgal biomass is then used as feedstock for
bioethanol production through the fermentation process. This coupling process can efciently achieve the goal of CO2 mitigation and
re-utilization. Moreover, the carbohydrate productivity of microalgae is usually higher than that of lipids, since the accumulation
of the latter requires intensive stress, while carbohydrate production is readily achieved by photosynthesis through the Calvin cycle
[34]. This is one of the advantages of producing bioethanol instead
of biodiesel from microalgae feedstock.

C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 110

Fig. 2. A conceptual process of ethanol production coupled with the cultivation of starch-based microalgae. Starch-based microalgal biomass is supplemented with the
primary feedstocks such as corn and cassava chips, which are subjected to liquication and pre-saccharication. Fermentation is then performed by the simultaneous
saccharication and fermentation (SSF) process. Ethanol is distilled from the fermentation broth and dehydrated as the nal product. Meanwhile, CO2 produced during
ethanol fermentation and the thin distillage with solid residues removed can be used for microalgae culture.

The main component in the carbohydrates of microalgal cells

is starch, and some species such as Chlorella, Dunaliella, Chlamydomonas and Scenedesmus have been reported to accumulate more
than 50% starch based on their dry cell weight [65]. Although
different hydrolysis technologies have been studied for microalgal biomass [57], enzymatic hydrolysis is still the most promising
method, and has been widely used in bioethanol fermentation
from starch-based feedstocks. Microalgal biomass can be used as a
supplement to replace part of the primary feedstocks, taking advantage of existing technologies and facilities. A conceptual process
is thus illustrated in Fig. 2. In addition to being used as supplementary feedstock, the starch accumulated within microalgal cells
can be converted directly into bioethanol under dark and anaerobic conditions, although bioethanol production rate and yield
are much lower [66], making this approach more scientically
signicant in elucidating the fundamental metabolism of photosynthetic microalgal cells under dark conditions, rather than for
use in practical applications for bioethanol production. [67] carried out bioethanol fermentation utilizing C. vulgaris biomass (37%
starch content) as feedstock, and obtained 65% bioethanol conversion when compared with the theoretically maximum bioethanol
conversion from starch. Choi et al. [57] demonstrated that the
bioethanol yield of C. reinhardtii UTEX 90 hydrolyzed by commercial hydrolytic enzymes was 0.235 g bioethanol/g microalgal
biomass with a separate hydrolysis and fermentation (SHF) process.
Chemical pretreatment (such as acid and alkaline) is an alternative way to hydrolyze the carbohydrates of microalgal biomass
into fermentable sugars for bioethanol production. Nguyen et al.
[61] reported that when 5% (w/v dry weight basis) of algal biomass
was pretreated with 3% sulfuric acid at 110 C for 30 min, 28.5 g/L
of glucose was released, representing a glucose yield of 95%. The
subsequent bioethanol yield (g bioethanol/g algae) from algae
biomass through the SHF process was 29.2%. The alkaline pretreatment method has also been shown to be promising option
for bioethanol production. Using 0.75% (w/v) of NaOH to pretreat microalgal biomass at 120 C for 30 min can obtain the

maximum bioethanol yield of 0.26 g bioethanol/g algae [58].

Although chemical saccharication usually gives a higher sugar
production rate, the sugar-containing hydrolysates derived from
microalgal biomass may need to be detoxied to avoid the
inhibitory effect on bioethanol fermentation by the byproducts
(such as furfural and 5-hydroxymethylfurfural) generated during
the pretreatment/hydrolysis.
Biobutanol can also be produced from carbohydrate-based
microalgae as an alternative fuel. Butanol contains more energy
and is less corrosive and water soluble [68], making this compound well suited for use with the existing storage and distribution
infrastructure of petroleum-based transportation fuels. Butanol
also has several advantages over ethanol as a fuel, due to its higher
energy content and lower volatility, being less hygroscopic, and
mixing better with gasoline in any proportion. Biobutanol and
other higher alcohols produced from biomass feedstocks are thus
known as advanced biofuels, and are expected to eventually replace
bioethanol [69].
Butanol is now mainly produced by chemical synthesis using
petroleum as the raw material [70]. Like bioethanol, tremendous
research efforts have also been focused on biobutanol production from conventional carbon sources. However, compared with
bioethanol, biobutanol fermentation is much less efcient and less
productive, with a lower product titer and yield. This is mainly due
to the severe inhibition of biobutanol to host cells [71] and the
unique metabolic pathways of the biobutanol-producing species
(i.e., Clostridium spp.) that produce a signicant amount of byproducts, in particular acetone, bioethanol and various organic
acids [72]. Moreover, the theoretical maximum biobutanol yield
is 1 mol/mol glucose (or 0.41 g/g glucose) if the fermentation can
be controlled to produce biobutanol with CO2 and H2 as the only
byproducts, which is lower than that for bioethanol (i.e., 2 mol/mol
glucose or ca. 0.5 g/g glucose).
Biobutanol production from starch-based microalgae feedstock
could be a simpler process than that of bioethanol production,
since Clostridium spp. are saccharolytic. As a result, the steps

C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 110

for starch liquefaction by amylase and saccharication of dextrins by glucoamylase required by bioethanol fermentation with
Saccharomyces are no longer needed for biobutanol production
with Clostridium spp. However, there is limited research progress
on biobutanol production from carbohydrates-rich microalgae,
whereas butanol produced from macroalgae has been reported
[73]. Efremenko et al. [74] examined the efciency of ABE (acetonebutanol-ethanol) fermentation from various microalgal biomasses
by fermentation with poly(vinyl alcohol)-immobilized C. acetobutylicum cells. The highest biobutanol yields were obtained
when using thermolysis pretreated Arthrospira platensis and Nannochloropsis sp. Ellis et al. [75] investigated the feasibility of using
microalgae biomass cultivated with wastewater as feedstock for
ABE fermentation with Clostridium saccharoperbutylacetonicum N14. They found that proper pretreatment and enzymatic hydrolysis
signicantly improved the ABE yield, while adding 1% glucose also
led to a 1.6 fold increase in total ABE production. The highest total
ABE production yield and productivity obtained was 0.311 g/g and
0.102 g/L/h, respectively.
In addition to the conversion of starch components in microalgae for biobutanol production, the cellulose content in microalgae
can also be converted to biobutanol after appropriate hydrolysis processes. It is known that lignocellulosic materials, such as
corn ber, wheat straw [76] and wheat bran [77], can be converted to biobutanol with Clostridium spp. [77]. However, there
have been no reports describing biobutanol production by using
cellulose originating from microalgae (mainly located in the inner
cell walls). Nevertheless, converting microalgal biomass that contains both starch and cellulose into biobutanol via fermentation
with Clostridium spp. is likely to be growing trend in biobutanol
production [78].
6.2. Gaseous biofuels
In addition to liquid biofuels production from microalgae-based
carbohydrates, gaseous biofuels, such as methane and biohydrogen,
could also be produced through the aerobic/anaerobic fermentation process using microalgal biomass as the carbon source. The
methane formation through the anaerobic digestion of organic
waste or wastewater has been well utilized, especially for the
degradation of complex materials. Hence, microalgae biomass
could also be a suitable substrate for methane fermentation. Biohydrogen can also be produced directly through the metabolic
network of microalgae, but the biohydrogen production efciency
is quite low [79]. In contrast, production of biohydrogen from
anaerobic fermentation using microalgae-based carbohydrates is
another attractive route, which could signicant reduce the production cost of biohydrogen.
When the cultivation of microalgae is associated with
bioethanol or biobutanol production from grain-based feedstocks,
microalgal residues can be processed together with grain residues
as animals feed. Otherwise, the microalgal residues can be digested
for methane production to recover the remaining energy through
an anaerobic digestion process. In general, methane production
facilities can be established to the treat organic wastes produced
in fermentation plants, such as bioethanol production from cassava chips. The same facilities can also use microalgal residues for
methane production. Anaerobic digestion of algal biomass can produce biogas with a high methane content (over 60%) and low sulfur
concentration to avoid corrosion problems in the power generator
[80]. However, the low C/N ratio of microalgal biomass may make it
unsuitable for methane production. Therefore, it was proposed that
microalgal residues and cellulosic materials (such as agriculture
wastes) can be co-digested to balance the C/N ratio in the optimum
range of 20:1-25:1 [81]. The co-digestion of algal biomass (mainly
Spirulina) and waste papers can greatly enhance the biomethane

production rate. Moreover, the co-digestion of microalgal biomass

with other organic materials as well as physical pretreatment on
algal biomass was also applied to improve the digestibility of algae.
In addition to methane production from algal biomass, biohydrogen is another alternative biogas produced through the
digestion of algal cells. Biohydrogen has emerged as one of the
most promising new energy carriers, because it is cleaner and
more efcient, particularly when it is used in fuel cells to directly
generate electricity [82]. Kawaguchi et al. [83] reported that the
starch-containing green alga Dunaliella tertiolecta and C. reinhardtii
were used successfully in biohydrogen fermentation, achieving biohydrogen yields (based on starch) of 61% and 52%, respectively.
Nguyen et al. [62] used enzymatic degradation of starch with thermostable -amylase to enhance the biohydrogen production of
green alga biomass (Thermotoga neapolitana), obtaining a biohydrogen yield of 2.5 mol H2 /mol glucose through the SHF process [62].
Signicant amounts of biohydrogen were produced from lipidextracted microalgal biomass residues (LMBRs) using conventional
anaerobic activated sludge fermentation technology, generating a
new concept for use in bioreneries. Yang et al. [84] showed that
the highest biohydrogen yield of 45.54 mL/g-VS was achieved from
LMBRs that underwent thermo-alkaline pretreatment at 100 C,
with this being approximately three-fold higher than the yield
from untreated LMBRs. In addition, there has recently been growing
research attention focused on biogas production from microalgae
[85]. However, it is worth noting that the anaerobic digestion process cannot be economically competitive unless it is targeted to
treat the microalgal residues remaining after the production of high
grade biofuels, such as bioethanol and biobutanol, or unless it is
integrated into a microalgae biorenery [6].
7. Conclusions
Microalgae strains can accumulate over 50% carbohydrates
intracellularly (in terms of starch and cellulose) under appropriate cultivation conditions. These microalgae-based carbohydrates
have the advantages of easy saccharication and requiring less pretreatment, thereby being highly competitive against lignocellulosic
materials as the feedstock for biofuels production and bioreneries. Understanding of fundamentals underlying the carbohydrate
metabolism of microalgae is a prerequisite for developing more
effective strategies to increase the carbohydrates productivity,
which should be optimized via manipulation of the key operating
factors (such as light supply, nutrients starvation, temperature and
CO2 supplementation). In addition, more economic and effective
saccharication processes should also be developed to enhance the
efciency of biofuel conversion through the microalgae biomass.
To meet the demand of the biofuels market, large scale processes for the cultivation of carbohydrates-rich microalgae should
be developed with appropriate photobioreactor design. Moreover,
economic assessment and life cycle analyses of the microalgaebased biofuels producing system should also be conducted to assess
the commercial feasibility of converting microalgae-based carbohydrates into various biofuel products.
Acknowledgements
The authors gratefully acknowledge the nancial support
provided by the National Basic Research Program of China
(2011CB200905) for research into the self-occulation of microalgal cells, as well as the support from Taiwans Ministry of Economic
Affairs (Grant no. 101-D0204-3) and Taiwans National Science
Council under grant numbers NSC 100-2221-E-006-126, NSC
100-2622-E-011-008-CC2, NSC 101-3113-P-110-002, NSC 1013113-E-006-015, and NSC 101-3113-E-006-016.

C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 110

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