Professional Documents
Culture Documents
Regular Article
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
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
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
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
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
Chlorophyta
Dinophyta
Cryptophyta
Euglenophyta
Rhodophyta
Leucosin/lipid
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.
4.1. 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.
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.
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
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. 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
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.
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
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