Review

Marine macroalgae: an untapped

resource for producing fuels and
chemicals
Na Wei1, Josh Quarterman2, and Yong-Su Jin1,2
1
2

Institute for Genomic Biology, University of Illinois at UrbanaChampaign, Urbana, IL 61801, USA
Department of Food Science and Human Nutrition, University of Illinois at UrbanaChampaign, Urbana, IL 61801, USA

As world energy demand continues to rise and fossil fuel

resources are depleted, marine macroalgae (i.e., seaweed) is receiving increasing attention as an attractive
renewable source for producing fuels and chemicals.
Marine plant biomass has many advantages over terrestrial plant biomass as a feedstock. Recent breakthroughs
in converting diverse carbohydrates from seaweed biomass into liquid biofuels (e.g., bioethanol) through metabolic engineering have demonstrated potential for
seaweed biomass as a promising, although relatively
unexplored, source for biofuels. This review focuses
on up-to-date progress in fermentation of sugars from
seaweed biomass using either natural or engineered
microbial cells, and also provides a comprehensive overview of seaweed properties, cultivation and harvesting
methods, and major steps in the bioconversion of seaweed biomass to biofuels.
Biofuels to meet increasing energy demands and
societal needs
Since the time of the Industrial Revolution, the ever-increasing demand for energy has been met primarily by
fossil fuel resources such as coal, oil, and natural gas. In
2009, the U.S. Energy Information Administration estimated that 86% of the 483 quadrillion BTU (British thermal unit) of total primary energy consumption in the world
was derived from these fossil fuel derivatives [1]. However,
as rapid growth of low- and medium-income economies and
moderate increases in world population are expected to
increase consumption of gas, coal, and oil by 26% over the
next 20 years [2], there is growing concern about meeting
the vast energy demands of the future with limited, nonrenewable resources. In addition, there is a heightened
awareness of environmental concerns and economic challenges associated with overutilization of fossil fuel
resources, including greenhouse gas emissions, global
warming, receding of glaciers, rising sea levels, loss of
biodiversity [3], increasing crude oil prices [4], and energy
insecurity. All of these factors have emphasized the need
for alternative, sustainable, efficient, cost-effective, and
cleaner-burning energy sources to meet present and future
demands [5].
Corresponding author: Jin, Y-S. (ysjin@illinois.edu)
Keywords: macroalgae; biofuel; metabolic engineering.

70

Biofuels provide an excellent alternative to traditional

fossil fuel-derived energy sources, as they can be produced
from abundant supplies of renewable biomass. During
growth of the biomass feedstocks for biofuel production,
the plants utilize sunlight and carbon dioxide in the atmosphere for synthesis of organic molecules, particularly
carbohydrates and lipids. Subsequently, these biomolecules can be used for conversion to various fuels and
chemicals through chemical or biochemical catalysis. In
general, the overall life-cycle analysis for biofuels showed
an improvement over traditional fossil fuel resources as
the greenhouse gas emitted during combustion is mitigated by the inorganic carbon fixed from atmospheric carbon
dioxide during growth of the biomass feedstock [6]. Also,
biofuels such as ethanol are less toxic, are biodegradable,
and generate fewer pollutants than petroleum fuels [7].
The utilization of renewable sources for production of
biofuels shows definite progress toward limiting greenhouse gas levels, improving air quality, achieving energy
independence and security, and finding renewable energy
resources that are geographically more distributed than
fossil fuels [5]. Although there are numerous efforts on
developing core technologies (production, harvest, storage,
depolymerization, and bio/chemical conversion) for producing biofuels from terrestrial plant biomass, production of
biofuels from marine plant biomass received less attention.
As such, this review focuses on using macroalgae (seaweed)
as a potential feedstock for the production of liquid biofuels
(especially bioethanol) and examines up-to-date efforts and
progress in demonstrating fermentation of sugars from
seaweed biomass using either natural microorganisms or
engineered microbial cells.
Benefits of producing biofuels from seaweeds
Challenges in biofuel production from terrestrial
biomass
Currently, the two most abundant and feasible biofuels for
large-scale production are ethanol from corn or sugarcane
and biodiesel from oil crops such as soy or oil palm [8].
These food crops are attractive and widely used as raw
materials for biofuel synthesis because of well-established
farming practices and simple, cheap processes for release
of starches, sugars, or oils. However, the status quo of using
food crops for biofuel production is only a partial solution
and cannot satisfy the full demand for renewable fuels. For

0167-7799/$ see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2012.10.009 Trends in Biotechnology, February 2013, Vol. 31, No. 2

Review
example, in order to achieve the 2020 federal mandate for
renewable fuel in the United States with corn ethanol,
approximately 100% of the domestic corn crop currently
available would be required [9]. In recent years, the increased demand on food crops for fuel applications has
resulted in concern about food scarcity, higher prices of
food commodities, and pollution of agricultural land [7].
The revised Renewable Fuel Standard (RFS2) thus caps
corn ethanol at 15 billion gal/year (see http://www.epa.gov/
otaq/fuels/renewablefuels/regulations.htm), which necessitates the use of alternative renewable feedstocks. One
plausible alternative that has been a focus of extensive
research is the use of terrestrial non-food lignocellulosic
biomass such as agricultural residues, wood waste, or
energy grasses as raw materials for biofuel production.
These biomass sources are advantageous because of low
cost, minimal land use change, and avoidance of the competition between food and fuel. However, current chemical
and biological technologies have yet to overcome the significant obstacles to releasing sugars from recalcitrant
lignocellulose and efficiently converting hexoses and pentoses to target fuel molecules with high yields and productivities [9].
Marine algae as an alternative source
In the context of the previously mentioned challenges,
marine algae (including macroalgae and microalgae) are
an attractive renewable source for biofuel production with
many advantages over biomass from food or cellulosic
materials. Algae include a wide variety of photosynthetic
organisms living in many diverse environments and present in all existing ecosystems on Earth [5]. Under normal
conditions, autotrophic algae use sunlight and fix inorganic
carbon from the atmosphere for assimilation in the form of
carbohydrates and lipids, which can be exploited for biofuel
production [7]. The marine algae have many advantages
for renewable energy applications. First, marine algae
have relatively high photon conversion efficiency and
can therefore rapidly synthesize biomass through assimilating abundant resources in nature such as sunlight,
carbon dioxide, and inorganic nutrients [10]. Therefore,
production yields of algae per unit area are significantly
higher than those for terrestrial biomass [11,12]. Also, as
marine algae have a higher rate of carbon dioxide fixation
compared to terrestrial biomass, they may have greater
potential for carbon dioxide remediation [12,13]. Second,
marine algae lack hemicellulose and lignin, which are
essential for structural support in most terrestrial plants
[7], and thus can be depolymerized relatively easily as
compared to lignocellulosic biomass [14]. Finally, marine
algae do not require arable land and can be grown in a
variety of marine environments including fresh water, salt
water, or municipal waste water. Algaes ability to grow in
salt water or waste water is critical for sustainable biofuel
production to avoid competition with food crops that require fresh water and cultivable land [7].
Seaweed cultivation and biofuel production
Seaweed production
Currently, the macroalgae industry is primarily focused on
food products for human consumption, which account for

Trends in Biotechnology February 2013, Vol. 31, No. 2

8390% of the global value of seaweed. Algal hydrocolloids

extracted from macroalgae, such as alginate, agar, and
carrageenan, account for most of the remaining value
[15,16]. The vast majority of the macroalgae for these
products is produced by aquaculture, which included some
3.1 million dry metric tons of annual global production in
2006 as compared to only 22 000 dry metric tons by
harvesting of wild algae stocks. The top ten countries
harvesting wild stocks are more geographically distributed
throughout the world, but the production from aquaculture
is focused on Asia with China accounting for 72% of global
annual production [15]. The five generaLaminaria,
Undaria, Porphyra, Euchema, and Gracilariarepresent
76% of the total tonnage of macroalgae production by
aquaculture [15].
Pretreatment and hydrolysis of seaweed biomass
After cultivation and harvesting of seaweed (Box 1), the
biomass must be pretreated for most biofuel applications.
The first step of pretreatment is to remove foreign objects
and debris such as stones, sand, snails, or other litter that
may be caught in the biomass either manually or by
washing [17]. In many cases, chopping or milling is then
required to increase the surface area/volume ratio and
improve the efficiency of hydrolysis [15]. Finally, the
biomass should be dewatered to 2030% to increase shelf
life and reduce transportation costs in situations where it
must be stored for long periods or transported over long
distances before further processing [15,17]. After pretreatment, hydrolysis of the seaweed biomass is necessary to
release the sugars locked up in the structural polysaccharides for the subsequent fermentation step. Seaweed composition and sugars released by hydrolysis are
summarized (see Table I in Box 2). The basic options for
hydrolysis of seaweed include dilute acid hydrolysis and/
or enzymatic hydrolysis. One study found that seaweed
carbohydrates from all three classes of macroalgae
(brown, red, and green) can be effectively hydrolyzed to
monosaccharides by dilute H2SO4 treatment at high temperature [18]. The four key factors for optimal saccharification during sulfuric acid hydrolysis were identified as (i)
reaction temperature, (ii) reaction time, (iii) acid concentration, and (iv) seaweed concentration. Another study
reported that sodium chlorite pretreatment of Ceylon
moss (Gelidium amansii) can greatly improve the efficiency of enzymatic hydrolysis with cellulase, xylanase, and bglucosidase and thus increase glucose yield from 5% in
non-pretreated samples to 70% in treated samples [19]. A
third study demonstrated the effectiveness of combining
acid hydrolysis and enzymatic hydrolysis for saccharification of seaweed, which resulted in a maximum sugar yield
of 0.566 g/g and 0.376 g/g for G. amansii and Laminaria
japonica, respectively [20].
Liquid biofuels from seaweeds
Liquid biofuels such as ethanol and butanol are produced
from algae by bioconversion of sugars from the plant
biomass using microbial cells such as yeast or bacteria.
Being rich in carbohydrates, marine macroalgae (seaweed)
may be used to obtain fermentable sugars for producing
liquid biofuels (Boxes 2 and 3). The overall process of
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Trends in Biotechnology February 2013, Vol. 31, No. 2

Box 1. Seaweed cultivation and harvesting

Cultivation methods
In general, seaweed can either be cultivated vegetatively or by a
separate reproductive cycle [42]. For vegetative cultivation, small
algal pieces are grown in a suitable aquatic environment with
appropriate temperature, light, salt content, nutrients, and water
movement. When the seaweed has reached a mature stage, it is
harvested by either leaving a small piece that will grow again or
removing the whole plant and cutting small pieces as seed for further
cultivation [42]. Although this strategy is quite simple and costeffective, some of the brown macroalgae such as Laminaria cannot be
cultivated by the vegetative method but rather require a reproductive
cycle with alternation of generations. Cultivation by a separate
reproductive cycle is much more expensive as seed production and
raising of young seedlings must be conducted in land-based facilities
under carefully controlled conditions [42].
There are at least three options for macroalgal farming sites:
offshore farms, nearshore coastal farms, and land-based ponds [15].
Offshore farming of L. hyperborea has been tested in the North Sea
with success, but the cost of such a technologically advanced system
is still very high [16,43]. Nearshore farms are currently in use for
culturing macroalgae in some countries such as China and Japan.
However, government regulations and environmental concerns about
aquaculture in coastal waters have prevented this option in the United
States and Europe [15]. Land-based pond systems for cultivation of
macroalgae have many advantages over farming on open marine
water such as ease of nutrient applications and avoidance of bad
weather, disease, and predation. Also, land-based systems can be
integrated with the aquaculture of other species such as fish to
provide waste materials as a cheap supply of nutrients for the
macroalgae. Despite these advantages, the application of open pond
systems for providing biomass to a biofuels marketplace would
require reduction in pond construction costs and technology for
considerable scale-up from current practice [15].
Harvesting
Manual harvesting of seaweed has been used for centuries and is still
common for some species grown on the shore in the intertidal zone.
The recent demand for larger quantities of seaweed for hydrocolloid

biofuel production from seaweed is illustrated (see Figure I

in Box 1). Generally, the seaweed biomass can be generated either by cultivation and harvesting or by collecting wild
drift seaweed. Then, seaweed is processed by dewatering
for the benefit of storage and transportation and to remove
any impurities for the downstream bioconversion process.
The processed biomass can be used for oil production,
liquid renewable fuel (e.g., ethanol or butanol) production
by fermentation, and/or methane production through anaerobic digestion (which is the most established process
among these options). For bioethanol production, the preprocessed seaweed is sent for hydrolysis to release sugars,
which then can be converted to ethanol or other advanced
biofuels by microbial fermentation. Instead of covering all
the potential fuel production options, this review will
specifically focus on the bioconversion of macroalgae (seaweed) for production of liquid biofuels (especially bioethanol). Recent advances in metabolic engineering have
permitted the overproduction of certain target compounds
by engineering of cells and their metabolic pathways. New
approaches to biology are being shaped by the genomics
revolution with unprecedented ability to transfer genes,
modulate gene expression, and engineer proteins [21]. In
the future, we believe that more and more concepts from
these fields will be used to overcome obstacles for economic
biofuel production from seaweed.
72

extraction has led to the development of mechanical harvesting

systems [17]. The type of mechanized harvesting that is used depends
on the type of macroalgae and the method of cultivation. For example,
Macrocystis and other attached seaweed that tend to stand upright may
be well suited for mowing with rotating blades. Floating seaweeds (e.g.,
Sargassum) or low- growing attached forms (e.g., Gracilaria) may
require other forms of harvesting such as suction or dredging with a
cutter. Some species of Laminaria are grown on rings and must be
harvested by transporting the growth structure with attached seaweed
to shore [15]. Improved mechanization for rapid and efficient harvesting
of seaweed will be another important prerequisite to supply ample
feedstock for biofuel production (Figure I).

[(Box_1)TD$FIG]

Seaweed
culvaon
Harvest or
collecon
Wild
seaweed

Anaerobic
digeson

Feedstock
processing
(cleaning, dewatering,
crushing, slurrying, etc)

Solid
waste

Bio-gas

Wastewater
and debris

Pretreatment and
hydrolysis/
saccharicaon

Fermentaon
Value-added
byproducts
Disllaon and
dehydraon

Ethanol
TRENDS in Biotechnology

Figure I. Major steps for bioethanol and/or biogas production from seaweed
biomass.

Production of biofuels from seaweed using

unengineered microbes
Hydrolysis of seaweeds converts the storage carbohydrate
into simple fermentable sugars, which can be easily used to
produce ethanol by some natural microorganisms
(Table 1). As an example, Saccharomyces cerevisiae, the
most widely used yeast for ethanol fermentation, was
presented in previous studies for bioethanol production
from seaweed biomass [20,22]. One study used the enzyme
laminarinase to hydrolyze laminarin in Saccharina latissima to glucose, which then can be fermented easily to
ethanol by S. cerevisiae [22]. Seaweed slurry pretreatments were performed on stirring blocks at pH 6, 23 8C
for 30 min, and after fermentation by S. cerevisiae produced 0.45% v/v ethanol within 40 h [22], which is less than
40% of the theoretical yield. Another study [20] reported
that 7.09.8 g/l ethanol was produced from 50 g/l sugar in
dilute-acid pretreated biomass of brown algae L. japonica
by simultaneous saccharification and fermentation with S.
cerevisiae. The ethanol yields were relatively low because
S. cerevisiae could only consume glucose in the hydrolysates but not mannitol, which was determined to be 81% of
the total sugars in the study.
Mannitol cannot be easily fermented, and only a
few organisms can utilize it. Mannitol needs to be converted to fructose-6P before being further metabolized.

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Trends in Biotechnology February 2013, Vol. 31, No. 2

Box 2. Distribution, diversity, and composition of seaweeds

Based on the presence or lack of phytopigments other than
chlorophyll, marine macroalgae can be classified into three major
classes: brown algae (Phaeophyceae), red algae (Rhodophyceae), and
green algae (Chlorophyceae) [44].
Brown macroalgae
Brown macroalgae include almost 1800 species of multicellular
algae with a characteristic olive-green to dark brown color derived
from an abundance of fucoxanthin, a yellow-brown pigment that
masks the green color of chlorophyll. This group includes the
largest and most complex of the macroalgae, the kelp (Laminaria),
which may reach lengths of 100 m and grow as much as 50 cm/day
(see http://dtc.pima.edu/nschmidt/gallery/algae/algae_index.html).
Kelp are found at depths below the low tide level in temperate and
polar regions and are farmed extensively in Asia as food products,
especially in China, Japan, and South Korea [22]. The composition
of brown macroalgae such as Laminaria includes up to 55% dry
weight of the carbohydrates laminarin and mannitol [22] (for
structures, see Figure I in Box 3). Laminarin can be easily
hydrolyzed by laminarase (endo-1,3(4)-b-glucanase) to release
glucose monomers [22]. Mannitol is a sugar alcohol that can be
readily converted to fructose by mannitol dehydrogenase for
bioconversion into ethanol [25]. Brown macroalgae also contain
alginate and cellulose (see Figure I in Box 3), which are two
structural polysaccharides abundant in the cell wall for mechanical
strength to prevent ripping during currents and tidal fluctuations
(see http://www.cornishseaweedresources.org/index.htm). Although
the sugars from laminarin and mannitol can be easily extracted from
milled seaweed for bioconversion, the full potential of biofuel
production from brown macroalgae has not yet been realized because

industrial microbes are not able to metabolize the alginate component

[14,26].
Red macroalgae
Red macroalgae include almost 6000 species of algae having a
characteristic red or pink color from the pigments phycocyanin and
phycoerythrin, which allow growth in relatively deep waters (see http://
www.cornishseaweedresources.org/index.htm). Red algae are found in
the intertidal and subtidal zones of the sea at depths up to 40 m or
occasionally as deep as 250 m (see http://www.seaweed.ie/index.html).
The composition of red macroalgae varies from species to species but
generally consists of cellulose, glucan, and galactan. The cell wall of red
seaweed is constructed with cellulose and two kinds of long-chain
structural polysaccharides that are valued for their gel-forming abilities
agar and carrageenan (see Figure I in Box 3). Agar can be readily
hydrolyzed to release the galactose subunits. Carageenans can be
classified as lambda (l), kappa (k), or iota (i) based on their gel-forming
ability and are used for thickening foods such as yogurt, ice cream, and
pudding [44].
Green macroalgae
Green macroalgae include an estimated 1500 species, of which only
about 15% are marine and the remainder live in freshwater or
terrestrial environments. Because of its need for more light for
photosynthesis, green seaweeds live mostly in the shallowest waters,
including the intertidal pools that fill and drain with the tides. They are
common in bays or estuaries where salt water and fresh water mix
together (see http://nature.ca/explore/di-ef/isap_ts_e.cfm). In most
cases, the composition of green macroalgae includes starch (Box 3)
for food reserves with cellulose (Box 3) and pectin as the main
structural polysaccharide in the cell wall [45].

Table I. Seaweed composition and sugars released by hydrolysis (% w/w dry biomass) for a variety of species
Seaweed

Class

Gelidium amansii
Gelidium amansii
Gelidium amansii
Laminaria japonica
Laminaria japonica
Sargassum fulvellum
Ulva lactuca
Ulva pertusa

Red

Carbohydrate
composition
Agar, Carrageenan,
Cellulose

Brown

Laminarin, Mannitol,
Alginate, Fucoidan,
Cellulose

Green

Starch, Cellulose

Total
carbohydrates (%)
75.2
77.2
83.6
51.9
59.5
39.6
54.3
65.2

Three possible pathways include (i) mannitol ! fructose

! fructose-6P, (ii) mannitol ! mannitol-1P ! fructose-6P
[23], and (iii) mannitol ! mannose ! mannose-6P !
fructose-6P. All of these pathways include redox reactions
[24], which reduce NAD(P)+ to NAD(P)H and may lead to
excess NAD(P)H accumulation under anaerobic conditions. Zymobacter palmae was found to be capable of
growing in a synthetic mannitol medium under oxygenlimited conditions and producing ethanol with a yield of
0.38 g/g mannitol [25]. Also, fermentation of mannitol
from Laminaria hyperborea hydrolysate extract by Z.
palmae was demonstrated, but laminarin was not utilized
probably because of the lack of b-(1!3)-glucanase [25].
Another study found that the yeast Pichia angophorae was
able to ferment both mannitol and laminarin simultaneously and produced ethanol from brown seaweed
extracts [26]. The maximum ethanol yield was 0.43 g
ethanol/g substrate at pH 4.5 and 5.9 mmol O2/l/h.
Another microorganism Brettanomyces custersii, was
suggested to be more suitable for galactose fermentation

Lipid
(%)
0.6
1.1
0.9
1.8
1.5
1.4
6.2
2.6

Protein
(%)
18.5
13.1
12.2
14.8
8.1
13.0
20.6
7.0

Ash
(%)
5.7
8.6
3.3
31.5
30.9
46.0
18.9
25.2

Sugars released
by hydrolysis (%)
34.6
56.6
67.5
37.6
34
9.6
19.4
59.6

Sugar
composition
Glucose,
Galactose
Glucose,
Mannitol
Glucose

Refs
[46]
[20]
[18]
[20]
[18]
[20]
[20]
[18]

than S. cerevisiae in the presence of mixed sugars [27].

A mutant B. custersii strain KCTC 18154P was reported to
produce ethanol from hydrolysates of red seaweed G.
amansii. Dilute sulfuric acid hydrolysis was applied to
G. amansii biomass, resulting in a hydrolysate solution
containing galactose as the major sugar and glucose as the
minor sugar, and some sugar degradation products such as
5-hydroxymethyl-2-furaldehyde (5-HMF) and organic
acids. Fermentation by B. custersii produced 11.8 g/l ethanol from 90 g/l sugar in a batch reactor (YEtOH = 0.13 g/g),
and 27.6 g/l ethanol from 72.2 g/l sugar in a continuous
reactor (YEtOH = 0.38 g/g) [27].
In addition to ethanol, n-butanol was produced using
seaweed hydrolysates. Clostridium beijerinckii and Clostridium saccharoperbutylacetonicum, which are known as
butanol-producing bacteria, were used to ferment sulfuric
acid-treated hydrolysates of Ulva lactuca, a species of
green macroalgae. The hydrolysate had a sugar composition of 27% glucose, 57% arabinose, and 16% xylose. The
butanol concentration reached about 4 g butanol/l out of
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Trends in Biotechnology February 2013, Vol. 31, No. 2

Box 3. Polysaccharides abundant in seaweed biomass

Information on major polysaccharides present in seaweed biomass is
introduced below.
 Laminarin is the main storage polysaccharide isolated from brown
macroalgae, consisting mostly of linear b-1,3-linked glucose units
with small amounts of b-1,6-linkages (Figure I).
 Alginate is a linear polymer consisting of 1,4-linked b-D-mannuronic
acid (M) and a-L-guluronic acid (G) in varying sequences [44]
(Figure I).
 Cellulose is the structural component of the primary cell wall of
green plants and many forms of algae, and it is a polysaccharide
consisting of a linear chain of several hundred to more than 10 000
b-1,4-linked D-glucose units.
 Starch is a storage polysaccharide found in green macroalgae that
may be a linear or branched molecule, consisting of D-glucose units
joined by a-1,4 and a-1,6 linkages.
 Fucoidan is a heterogeneous polysaccharide in brown macroalgae,
consisting primarily of 1,2-linked a-L-fucose-4-sulfate units with
very small amounts of D-xylose, D-galactose, D-mannose, and uronic
acid.
 Carageenan is a sulfated polysaccharide molecule consisting of
alternate units of b-D-galactose and a-D-galactose (Figure I) [44].
 Agar is a linear polymer of altering 3-linked b-D-galactopyranosyl
and 4-linked 3,6-anhydro-a-L-galactopyranosyl subunits and is
commonly used as a gelling compound in food products and in
culture mediums for research or industry [44].

15.2 g sugar/l in the hydrolysate, and the pilot study

recovered 0.29 g butanol/g sugar [28].
Metabolic engineering for producing biofuels from red
seaweed
Although there are natural microorganisms able to utilize
seaweed sugar extracts, economically feasible biofuel production from seaweed requires more efficient conversion of
mixed sugars in seaweed hydrolysates by robust strains.
Thus, metabolic engineering has been applied to improve
fermentation of sugars that can be recovered from seaweeds. Galactose is a major sugar compound in the hydrolysate of red marine algae, composing up to 23% in the
hydrolysate from red seaweed Ceylon moss [29]. Therefore,
research effort has been made to enhance galactose fermentation to ethanol by engineering S. cerevisiae. The wild
type yeast S. cerevisiae is capable of galactose fermentation, but there are two major issues that limit the ethanol
yield and productivity. First, ethanol production rate and
yield from galactose are considerably lower than from

[(Figure_I)TD$G]
Cellulose

Laminarin

1,4

1,6
1,3

Starch

Alginate

1,6

1,4

1,4

Fucoidan

Agar

1,2
1,4

S
Carrageenan

Key:

1,3

= Glucose

= Mannuronic
acid

= Galactose

= Guluronic
acid

= 3,6-anhydrogalactose

= Fucose

= Sulfate

1,4

1,3

= -linkage
= -linkage

TRENDS in Biotechnology

Figure I. Structural information about polysaccharides abundant in seaweed

biomass.

glucose [3032]. Second, the presence of glucose represses

utilization of galactose because of stringent transcriptional
repression of GAL genes coding for enzymes for galactose
metabolism [3336]. These two issues lead to a diauxic
consumption of glucose and galactose in red seaweed
hydrolysates, which significantly reduces overall ethanol
productivity [33,35]. Therefore, metabolic engineering
strategies to achieve more efficient galactose fermentation
not only focused on improving galactose metabolism itself
but also on cofermentation of galactose and other carbohydrate from red seaweed [37].
Galactose fermentation by S. cerevisiae can be improved
by engineering of gene regulatory network controlling
expression levels of galactose metabolic enzymes or target
metabolic genes. Orthogonal approaches have been applied
in recent studies. First, improved galactose utilization was
achieved by overexpressing a positive regulator GAL4 and
deleting three negative regulators (GAL6, GAL80, and
MIG1) of the galactose assimilating pathway in yeast
[30]. This approach increased specific galactose uptake

Table 1. Microorganisms that convert seaweed biomass to ethanol/butanol

Microorganisms
Natural strains
Saccharomyces cerevisiae
Zymobacter palmae
Pichia angophorae
Brettanomyces custersii
Clostridium beijerinckii, Clostridium
saccharoperbutylacetonicum
Engineered strains
Saccharomyces cerevisiae (engineered
for improved galactose fermentation)
Escherichia coli (engineered for alginate
metabolism)
Escherichia coli KO11
74

Seaweed source

Major sugars utilized

Refs

Saccharina latissima
Laminaria japonica
Laminaria hyperborea
Laminaria hyperborea
Gelidium amansii
Ulva lactuca

Glucose
Glucose
Mannitol
Mannitol, laminarin
Galactose, glucose
Glucose, arabinose, xylose

[22]
[20]
[26]
[25]
[27]
[28]

Red seaweed
(e.g., Ceylon moss)
Saccharina japonica

Galactose, or simultaneous cofermentation

of galactose and cellobiose
Glucose, mannitol and alginate

[13,30,32,37]

Laminaria japonica

Glucose, mannitol

[20]

[14]

Metabolic engineering for producing biofuels from

brown seaweed
Major sugars from brown seaweeds include glucan (laminarin or cellulose), mannitol, and alginates. However,
natural microorganisms as introduced in the earlier section cannot utilize these various sugars concurrently, because they lack the ability to use alginate, and thus ethanol
production from brown seaweed can hardly reach its maximum level [15]. An ethanogenic Escherichia coli strain
KO11was developed by integrating Zymomonas mobilis
ethanol production genes into the pflB gene and was able to
ferment a mixed sugar solution containing glucose, galactose, xylose, L-arabinose, and mannitol with an ethanol
yield of 0.350.37 g/g total sugar [20]. The strain was also
used to convert glucose and mannitol to ethanol in simultaneous saccharification and fermentation of HCl acidtreated brown algae L. japonica, which resulted in an
ethanol yield of 0.4 g/g of sugars [20].
A recent breakthrough in the efficient use of brown
seaweed biomass arose from an engineered E. coli platform
capable of alginate degradation and metabolism. Here,
ethanol was produced through cofermentation of glucose,
mannitol, and alginate from brown seaweed [14]. Fermentation of alginate to ethanol requires surplus reducing
equivalents (NADH or NADPH), which can serve as a
counterbalance to the reducing equivalents generated
from oxidative mannitol metabolism, and by coupling the

(a)

Glactan (60%)

Pretreatment
and hydrolysis

Galactose
GAL2

Cellulose (20%)

Engineered
S. cerevisiae
GAL1

Cellobiose

GAL7

CDT1
(NCU00801)
-glucosidase
(NCU00130)

GAL5

Glucose

Glycolysis

Ethanol

(c)

[Ethanol]

[Cellobiose & Galactose]

(b)

[Ethanol]

rate by 19% (to 3.57 mmol gal/g cell h) and enhanced

specific ethanol production rate by 153% (to 2.71 mmol
ethanol/g cell h). Second, overexpression of a key metabolic
gene PGM2 coding for phosphoglucomutase increased galactose uptake rate by 70% compared to the parental strain
[32]. For another example, overexpression of a truncated
TUP1 coding for a transcription repressor was shown to
remarkably increase galactose consumption rate and ethanol productivity [13]. In addition, the lag time between
glucose and galactose fermentation was significantly shortened by overexpression of the truncated TUP. These
approaches have led to progress in improving galactose
fermentation by S. cerevisiae. However, efficient fermentation of mixed sugars in hydrolysates from seaweed remains
a challenge, because glucose repression on galactose fermentation delays the utilization of the latter.
To circumvent glucose repression, a cellobiose (a dimer
of glucose) utilization pathway was expressed in S. cerevisiae by expressing genes coding for cellodextrin transporter (cdt-1) and intracellular b-glucosidase (gh1-1) from
Neurospora crassa [37]. The strategy used in the study is
illustrated in Figure 1. Simultaneous cofermentation of
cellobiose and galactose was achieved because glucose was
generated from cellobiose by b-glucosidase intracellularly
and did not repress galactose metabolism (Figure 1c). By
this approach, ethanol productivity during cofermentation
of cellobiose (40 g/l) and galactose (40 g/l) increased significantly (0.72 g ethanol/l h) as compared to individual sugar
fermentation (0.37 g ethanol/l h for cellobiose, 0.61 g ethanol/l h for galactose) or two-stage sequential fermentation
of glucose and galactose (0.58 g ethanol/l h). This also
reduces the enzyme cost because there is no need for
addition of b-glucosidase.

Trends in Biotechnology February 2013, Vol. 31, No. 2

[(Figure_1)TD$IG]

[Glucose & Galactose]

Review

Time

Time
(d)

Pretreatment
and hydrolysis

Alginate
Glucose

NAD+

NADP+ NADPH
6-phosphogluconolactone
Glucose-6P
NADH

Mannitol

Fructose-6P

EDP

Glycolysis

NADH

Introcellular
Oligoalginate

6-P-gluconate

Fructose-1,6-biP
DihydroxyGlyceraldehyde-3P
acetone-P
NAD+

Oligoalginate

2-keto-3-deoxy6-P-Gluconate

DEH NADH
NAD+
2-keto-3-deoxy
-gluconate

Engineered pathway for

alginate metabolism

P-enol-pyruvate

TCA

Pyruvate

Acetaldehyde
Ethanol
NADH NAD+

Acetyl-CoA

TRENDS in Biotechnology

Figure 1. (a) Metabolic engineering strategies for the simultaneous fermentation of

hexose and pentose sugars derived from red seaweed hydrolysates. (b) Glucose inhibits
the uptake and the metabolism of galactose, which results in a two stage fermentation
of the mixture. (c) Heterologous expressions of a cellobiose transporter and intracellular
b-glucosidase enable engineered Saccharomyces cerevisiae to simultaneously
coferment glucose and galactose, resulting in efficient ethanol production. (d)
Metabolic pathway for the simultaneous fermentation of sugars derived from brown
seaweed hydrolysates by the engineered Escherichia coli platform reported in [14].
Cofactors involved redox reactions is illustrated. Alginate catabolism pathway and
mannitol catabolism complement each other in terms of cofactor usage, enabling
ethanol fermentation from the mixed sugars. EDP, EntnerDoudoroff pathway; TCA,
tricarboxylic acid cycle; DEH, 4-deoxy-L-erythro-5-hexoseulose uronic acid.

redox-complementary pathways ethanol fermentation

from all sugars in brown algae was realized in this study
[14] (Figure 1d). A secretable alginate lysase (Aly) was
75

Review
engineered in E. coli-enabled depolymerization of alginate
into oligomers without thermal and chemical pretreatment
or enzymatic saccharification. Then, a 36-kbp DNA fragment responsible for alginate degradation, transport, and
metabolism from Vibria splendidus was integrated into the
genome of the engineered E. coli. The pathway was improved by expression of auxiliary genes for alginate degradation, and also ethanol production phenotype was
enhanced by heterologous expression of a homoethanol
pathway. The constructed E. coli platform fermented dry
milled brown macroalgae Saccharina japonica directly for
ethanol production and reached a yield of 0.41 g ethanol/g
total sugars including alginate, mannitol, and glucan, which
is more than 80% of the maximum theoretical yield.
Byproduct utilization for producing value-added
products
As production of biofuels mainly uses the carbohydrate
fraction of seaweed biomass, utilization of other components such as plant protein, alginates (if not used), and
phenolic compounds need to be considered to enhance
economic value of seaweed fuel production process. Moreover, fermentation of hydrolysates from seaweed biomass
produces not only ethanol, but also many byproducts, such
as glycerol, organic acids (e.g., acetate, succinate), biomass
protein, and other minor products. Biofuel industry using
seaweed will be more economical when the byproducts are
put into good use, just like the petroleum industry where
many products besides gasoline are profitable. For example, the fermentation byproduct glycerol has various applications in manufacturing of food, pharmaceutical and
personal care products, and other value-added chemicals.
Organic acids are high-demand chemical feedstock for
producing deicing salts, food additives, and so on. Moreover, because seaweed biomass does not contain lignin,
residuals after fermentation can be used as animal feel or
feed supplement. Furthermore, using macroalgae biomass

Trends in Biotechnology February 2013, Vol. 31, No. 2

residue from saccharification and fermentation to produce

methane via anaerobic digestion is possible [29,38], and
this strategy will have obvious benefits in that both products are valuable for two categories of energy supply
bioethanol as a transport fuel and methane for electricity
generation.
Challenges and potential environmental impacts
Although seaweed has several favorable traits as a renewable feedstock, challenges and potential impacts on ecosystems need to be considered. First, polymers in seaweed
biomass are generally made up of mixed sugars, and some
are not found in terrestrial biomass (Box 3). Thus, although
depolymerization of seaweed polysaccharides is relatively
easy, efficient conversion of the unusual sugars to biofuels is
challenging, and ongoing efforts are being made to develop
strategies for this problem. Second, the application of seaweed biofuels will require increasing seaweed farming (expansion of farming area or increased intensity of current
seaweed farming area), and thus special attention should be
paid to the potential impacts on marine and coastal environments. Potential impacts include alteration of natural
habitats, change of hydrology (e.g., sedimentation and water
movement), nutrient depletion, decrease of biodiversity
(e.g., removal of mangrove or sea grass), degradation of
water quality, and disturbance of coral reefs [39]. Meanwhile, environmental impacts of seaweed farming may be
minor in some cases [40] and may even have beneficial
effects on increasing populations of fish and invertebrates
in the area [41]. A balance must be attained between seaweed biofuel production and its environmental cost.
Concluding remarks and future perspectives
Advanced metabolic engineering approaches based on systems and synthetic biology offer new opportunities to
improve bioconversion of sugars derived from plant biomass to biofuels and value added chemicals. Marine plant

Box 4. Current status and efforts in seaweed biofuel development

Compared to terrestrial biomass, marine macroalgae has received
less attention so far; however, with the urgent challenges in the
pursuit of cost-effective renewable fuels as well as concerns over
terrestrial biofuel crops, increasing effort and investment have been
put in developing seaweed biofuels.
 In Europe, the UK and Irish joint project Sustainable Fuels from
Marine Biomass (the BioMara Project; see http://www.biomara.org/) aims to demonstrate the feasibility and viability of
producing third generation biofuels from marine biomass. The
seaweed cultivation and harvest process has been successfully
established in Scotland, and the project is investigating ethanol
production and/or methane generation from seaweed. Statoil and
Bio-Architecture Lab (BAL) also aim to commercialize the production of ethanol and co-products from seaweed in Norway and in
Europe.
 In the United States, the DOEs Advanced Research Projects
Agency-Energy (ARPA-E) is supporting the development of process
to convert seaweed biomass into isobutanol (see http://arpa-e.
energy.gov/ProgramsProjects/OtherProjects/BiomassEnergy/
MacroAlgaeButanol.aspx), which is undertaken by DuPont and BAL
under Technology Investment Agreement. The program focuses on
improving seaweed aquaculture, converting seaweed biomass to
fermentable sugars, isobutanol production from sugars, and
economic and environmental optimization of the production
process. Furthermore, BAL is leading a project supported by the
76

Chilean Economic Development Corporation to develop renewable

fuels from seaweed in Chile.
 In Asia, a project lead by the Mitsubishi Research Institute in Japan
plans to start demonstration of ethanol production with waste
seaweed in 2012, to develop cultivation technologies by 2016, and
set up a production process by 2020. South Korea National Energy
Ministry has started a 10-year project with the aim of producing
nearly 400 million gallons a year of ethanol by 2020. The Philippine
government also has invested more than $US5 million in building
up an ethanol plant with seaweed bioethanol technology from
South Korea.
 There are several biotechnology companies that are in the process
of commercializing bioethanol production from seaweed biomass,
including Butamax, Seaweed Energy Solutions, Green Gold Algae,
and Seaweed Sciences, Inc. Because seaweed cultivation is well
established in some Asian countries and has been grown at
commercial scale for food products for decades, collaborative
effort that can bring together the advanced knowledge in converting seaweed biomass to biofuels and the established large-scale
cultivation and harvesting technologies will be beneficial in
promoting seaweed biofuel development. For example, Novozymes is collaborating with Indias Sea6 Energy to develop a
seaweed bioethanol process, with the former focusing on the
development of bioconversion process and the latter sharing its
knowledge of offshore seaweed cultivation.

Review
biomass such as seaweed has several advantages over
terrestrial plant biomass and can be a promising feedstock
for biofuel production because of its wide geographic
distribution. Efforts are now underway to use seaweed
biomass for liquid biofuel production (Box 4). As illustrated
in this review, metabolic engineering is evidently essential
to the development of technologies to convert seaweed
biomass to various biofuels, because complex and diverse
carbohydrate composition of seaweed requires fermenting
microorganisms to be able to metabolize mixed sugars.
Although bioethanol production from seaweed has been
reported in several research studies, large-scale production at low cost still faces numerous challenges. In order to
realize converting algae biomass into biofuels and valueadded chemicals, technologies allowing economically feasible design of each step, including seaweed cultivation,
harvesting and transporting, pretreatment and hydrolysis,
fermentation with high yields and productivity, and costeffective utilization of byproducts and biomass residues,
are needed. At the same time, potential impacts of largescale cultivation of seaweeds on marine ecosystems need to
be carefully assessed.
Acknowledgment
This work was supported by funding from the Energy Biosciences
Institute.

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