INTERNATIONAL JOURNAL OF ENERGY RESEARCH

Int. J. Energy Res. (2014)

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/er.3164

REVIEW PAPER

An overview of algae bioethanol production

Kexun Li1,*,†, Shun Liu1 and Xianhua Liu2,‡,§
1
The College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China
2
The College of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China

SUMMARY
Because of rapid growth in population and industrialization, worldwide ethanol demand is increasing continuously. The
first-generation and second-generation biofuels are unable to meet the global demand of bioethanol production because
of their primary value of food and feed. Therefore, algae are among the most potentially significant sources of sustainable
biofuels in the future of renewable energy because of the accumulating high starch/cellulose and because they are widely
distributed in nature. The focus of this paper is to review the production and recent advances in research and development
in the algae bioethanol, including pretreatment, hydrolysis, and fermentation of algae biomass. Despite the many
developments made in the recent years, commercialization of algal bioethanol remains challenging chiefly because of
the techno-economic constraints. Technological breakthroughs in all major aspects must be overcome before it can be a
successfully large-scale and commercialized product. Copyright © 2014 John Wiley & Sons, Ltd.

KEY WORDS
algae; pretreatment; saccharification; fermentation; bioethanol

Correspondence
*Kexun Li, Environmental Engineering Program, College of Environmental science and Engineering, Nankai University, 94 Weijin Road,
Tianjin 300071, China.
†
E-mail: [email protected]
‡
Xianhua Liu, School of Environmental Science and Engineering, Tianjin University, 92 Weijin Road, Tianjin 300072, China.
§
E-mail: [email protected]

Received 30 January 2013; Revised 15 October 2013; Accepted 23 December 2013

1. INTRODUCTION considered as one of the most promising replacements of

gasoline to act as a transport fuel (Table I). Although the
With the expansion of the world population and increase of first-generation bioethanol production from food crops
industrial prosperity, global energy consumption also has such as corn, grain, or sugar cane is well established and
increased gradually. The major energy demand is still sup- the industry is growing throughout the world, the use of
plied from conventional fossil fuels such as oil, coal, and these staple food crops as feedstocks is not ideal because
natural gas. In the International Energy Outlook 2011 of the high price of raw materials, which accounts for
(IEO2011) Reference case, world energy consumption in- almost 40–75% of total ethanol production cost [7]. It
creased by 53%, from 505 quadrillion British thermal units has raised doubts about its possible impact on food supply
(Btu) in 2008 to 770 quadrillion Btu in 2035, consumption and security, which is mainly reduced if its residues are
of petroleum and other liquid fuels increases from 85.7 used for bioethanol production, as studied here [8]. In con-
million barrels per day in 2008 to 112.2 million barrels trast, the second-generation bioethanol is derived from lig-
per day in 2035 [1]. In addition, the usage of fossil fuels nocellulosic feedstock. They are a better option for
is now widely accepted as unsustainable because of the ac- addressing the food and energy security and environmental
cumulation of greenhouse gas (GHG) in the environment concerns. However, conversion of lignocelluloses to
that has already exceeded the ‘dangerously high’ threshold bioethanol is more challenging than the first-generation
of 450 ppm CO2-e [2]. Therefore, depletion of fossil fuels, bioethanol process because of the complex structure of
global warming, and increasing price of petroleum-based plant cell walls. Currently, no commercial-scale cellulosic
fuels are gaining great concern and have led to a move ethanol plants are in operation largely because of the high
toward alternative, sustainable, renewable, efficient, and price of production, which is almost twice that of corn
cost-effective energy sources with lesser GHGs emissions. ethanol [9]. In view of the aforementioned issues, algae
Unlike fossil fuels, bioethanol is more environment are gaining wide attention as an alternative renewable
friendly because it is renewable energy source, and it is source of biomass for production of bioethanol, which is

Copyright © 2014 John Wiley & Sons, Ltd.

K. Li, S. Liu and X. Liu An overview of algae bioethanol production

Table I. Properties of bioethanol as a transport fuel.

Fuel property Units Gasoline Pure ethanol References

Oxygen content w/w 2.7% 35 [3]

Octane number 100% 85–94 112.5–114 [3,4]
3
Energy content MJ/dm 31.3 21.2 [3,5]
Reid vapor pressure psi 7.0–15.0 2.3 [4,6]
Higher heating value BTU/gal 125,000 84,600 [4,6]
Lower heating value BTU/gal 112,000–116,000 75,670–76,100 [4,6]

grouped under ‘third-generation bioethanol’ [10,11]. To a greater unicellular [13]. Unlike higher plants, microalgae do not
extent, the major drawbacks of first-generation and second- have roots, stems, and leaves. Microalgae, capable of
generation bioethanols are overcome by the algae bioethanol. performing photosynthesis, are important for life on earth;
The objectives of this paper are to review the published in- they produce approximately half of the atmospheric
vestigations on algae conversion to bioethanol and present the oxygen and use simultaneously the GHG carbon dioxide to
culture of algae, the available and accessible technologies for grow photoautotrophically. The three most important classes
bioethanol production using algae biomass throughout the of microalgae in terms of abundance are the diatoms
entire process, that is, pretreatment, hydrolysis, and fermen- (Bacillariophyceae), the green algae (Chlorophyceae), and
tation stages, challenges encountered, and future perspective. the golden algae (Chrysophyceae) [14].

2.2. Characteristics and advantage of the

2. ADVANTAGES OF ALGAE AS A algae for bioethanol production
SOURCE OF ENERGY PLANTS FOR
BIOETHANOL There are several commercial advantages of algae
bioethanol production that have combined to capture the
2.1. Brief description of the algae interest of researchers and entrepreneurs around the world.
These include the following. (i) Algae bioethanol produc-
Algae are photosynthetic, eukaryotic organisms that do not tion need not compete with food production in either land
develop multicellular sex organs. All algae contain green or water: both marine and freshwater algae may be used.
chlorophyll; however, they are masked by photosynthetic Additionally, they also need not compete with people for
pigments that give them a distinguishing color that is used food [15]. (ii) The content of carbohydrate in the algae cell
to identify key divisions [12]. Based on their morphology is abundant; the carbohydrates such as starches and sugars
and size, algae are typically divided into two major catego- can be fermented to produce bioethanol, as shown in
ries—macroalgae and microalgae. Table II. (iii)Algae have the advantages of having no lignin
Macroalgae, commonly known as seaweed, are usually and low hemicellulose levels, which result in an increased
found in coastal areas in both intertidal and subtidal habi- hydrolysis efficiency and fermentation yields [24,25]; thus
tats and are composed of multiple cells, which organize they can reduce the cost of the bioethanol production. (iv)
to structures resembling roots, stems, and leaves of higher Algae have the ability to take up CO2 from the atmosphere
plants; some species have gas-filled structures to provide and power plants, and with the use of appropriate technol-
buoyancy [13]. They are classified into three broad groups ogy options, algae bioethanols can yield GHG reductions
based on their pigmentation: (i) brown seaweed relative to fossil and other biobased fuels [26,27]. (v) Al-
(Phaeophyceae), which includes the large kelps; (ii) green gae grow rapidly and can be easily grown in various
seaweed (Chlorophyceae) such as sea lettuce; and (iii) aquatic environments such as fresh water, saline water, or
red seaweed (Rhodophyceae), the most diverse group of municipal waste water [28,29]. (vi) They have a high
all [15]. In contrast, microalgae are a large group of micro- photosynthetic efficiency; the average photosynthetic effi-
scopic photosynthetic organisms, many of which are ciency of aquatic biomass is 6–8%, which is much higher

Table II. Carbohydrate content of algae cell (% of dry matter).

Algae Biomass Carbohydrate Reference

Microalgae Chlorococcum infusionum 32 [16]

Chlamydomonas reinhardtii 59.7 [17]
Porphyridium cruentum 40–57 [18]
Macroalgae Undaria pinnatifida (brown seaweed) 48.5 [19]
Sargassum spp. (brown seaweed) 41.81 [20]
Kappaphycus alvarezii (red seaweed) 64 [21]
Gelidium amansii (red seaweed) 67.3 [22]
Ulva lactuca (green seaweed) 54.3 [23]

Int. J. Energy Res. (2014) © 2014 John Wiley & Sons, Ltd.
DOI: 10.1002/er
An overview of algae bioethanol production K. Li, S. Liu and X. Liu

than that of terrestrial biomass (1.8–2.2%) [30]. (vii)The that 1.00 × 106 tonnes of L. japonica was produced per
microalgal cells have a very fast productivity and harvesting year at one site. Buck and Buchholz [35] cultured
cycle (1–10 days) compared with other feedstock (harvest Laminaria saccharina under rough offshore conditions;
once or twice a year) and thus provide enough supplies to the system not only resists rough weather conditions
meet ethanol production demands [2,29]. (2 m s 1 current velocity, 6 m wave height) but also
can be equipped with culture lines that can be collected
offshore or transported to shore facilities for harvesting.
3. SOURCE OF ALGAE Near-shore coastal environments are already used for
macroalgae culture by countries such as China, Japan,
3.1. Nature stock and Chile, which have viable seaweed aquaculture
industries. However, in the USA and Europe, environ-
The distribution of macroalgae is worldwide. They are mental regulations and popular resistance against use
abundant in coastal environments, primarily in near-shore of coastal regions for aquaculture represent barriers that
coastal waters with suitable substrate for attachment [31]. will need to be overcome because of the conflicting
Another primarily natural source is floating seaweeds, uses of coastal zones [31]. So, the near-shore seaweed
and floating seaweeds are considered one of the most aquaculture regulations will be stricter in the future.
important components of natural materials on the sea
surface [32]. In 1995, about 3.6 million tonnes wet
weight were collected globally from natural stocks. This 3.2.2. Culture of microalgae
was about 48% of the total global seaweed biomass The most common production systems employed for
harvested with the balance produced by aquaculture. microalgae cultivation are outdoor open ponds and
In 2010, the production of seaweeds and other aquatic enclosed photobioreactors (PBRs). Open ponds systems
algae reached 19.9 million tonnes, of which nature are the most widespread growth systems, and all very large
produced 0.9 million tonnes (0.5%). Predictably, commercial systems used today are of this type. Major
aquatic seaweed will be the most prominent in the advantages of open ponds are that they are easy to
production of bioethanol. construct and operate, and their costs are minimal [12].
There are at least 30,000 known species of microalgae, Ponds for algae production are built as a closed
and they are widely distributed in marine waters, freshwa- recirculation loop forming a canal with depth of
ter lakes, and others. As their small size makes subsequent ~0.3 m. The stirring and circulation are provided by a
harvesting more complicated, cultivation is the main way turbine that enforces the motion of the suspension.
to generate biomass from microalgae. The turbine operates continuously, preventing sedimen-
tation of algae. Algal biomass is drained behind the
3.2. Artificial culturing turbine, at the end of the recirculation loop [36]. How-
ever, major limitations in open ponds include poor light
Because of the uneven distribution of the algae, algae utilization by the cells, evaporative losses, diffusion of
occurring in nature are very low in density [14]. To a de- CO2 to the atmosphere, and requirement of large amounts
gree, it causes certain difficulties to bioethanol production of water and land and low biomass productivity [37]. Addi-
on the large scale. In order to meet the fuel demand of tionally, they require highly selective environment because
the exploding population and industrialized production, it of inherent threat of contamination and pollution from other
is inevitable that there should be a year-round supply of algae species and protozoa. And they are highly susceptible
algal biomass [11]. to environmental fluctuations such as swings in temperature
Culture systems are very different between macroalgae and pH [38].
(seaweed) and microalgae. Because of their small (μm) The limitations of open pond systems led to the
size, microalgae have to be cultivated in a system designed development of enclosed PBRs for mass cultivation of
for that purpose (placed on land or floating on water), algae. PBRs are a closed system that allows the cultiva-
while macroalgae can be grown directly in the open tion of single-species culture of microalgae. And PBRs
sea [33]. have the following fundamental advantages: a reduced
contamination risk, no CO2 losses, reproducible cultiva-
3.2.1. Culture of macroalgae tion conditions, controllable hydrodynamics and
The common options for sitting macroalgae farms temperature, and flexible technical design [39]. So
include offshore farms and near-shore coastal farms. PBR photosynthetic production process of microalgal
Offshore aquaculture is one of the new frontiers for ma- biomass obtains simultaneously the reduction of input
rine aquaculture production that could supply growing energy and achievement of high photosynthetic produc-
consumption demand. Yokoyama et al. [34] cultured tion [40]. And it is necessary to make petroleum
seaweed (Laminaria japonica) at offshore marine farms. replacement feasible, with a reasonable amount of land.
The marine farm is anchored in position and floats on There are two major types of enclosed PBRs—tubular
the surface of the sea, with a marine farm of 41.2 km2 and plate types. Table III summarizes the characteristics
area, 5120 m wide and 8050 m long. It was estimated of flat-plate and tubular PBRs.

Int. J. Energy Res. (2014) © 2014 John Wiley & Sons, Ltd.
DOI: 10.1002/er
K. Li, S. Liu and X. Liu An overview of algae bioethanol production

Table III. Characteristics of various culture systems for microalgae.

Productivity
1 1
Culture systems Shapes Prospects Limitations (g L day ) References

Flat-plate Horizontal, vertical, Large illumination Scale-up requires many 2.76 (shape, [13,41]
photobioreactors conical, inclined area, suitable for outdoor compartments and support horizontal; ID,
cultures, good light path, materials, temperature, 3 cm; strain,
good biomass hydrodynamic stress Phaeodactylum)
productivities, relatively
cheap, easy to clean up
Tubular Vertical, horizontal, Large illumination surface Gradients of pH, dissolved 4.3 (shape, inclined; [13,41]
photobioreactors inclined area, suitable for outdoor oxygen and CO2 along ID, 1.3 cm; strain,
cultures, fairly good the tubes, fouling, requires Spirulina platensis)
biomass productivities, large land space
relatively cheap

4. STEPS OF BIOETHANOL account for 33% of the total cost [42]. Because of the costs,
PRODUCTION FROM ALGAE pretreatment has a great potential for improvement of effi-
BIOMASS ciency and is being widely discovered. An effective
pretreatment can destroy physical barriers in the cell wall
Like cellulosic ethanol, bioethanol production from algae and improve enzymatic hydrolysis rates, fermentation,
requires four major unit operations including pretreatment, and other process variables [43]. Because algae biomass
hydrolysis, fermentation, and distillation. Schematic has its own characteristics, such as soft organization and
flowsheet for the bioconversion of algae biomass to high moisture content, the pretreatment of the algae is
bioethanol is shown in Figure 1. easier than that of lignocellulosic biomasses. Physical,
In order to produce sugars from the algae biomass, physico-chemical, chemical, and biological processes have
pretreatment is designed to help separate cellulose, been used for pretreatment of algae materials.
hemicellulose, and lignin so that the complex carbohydrate
molecules in the algae cell can be broken down by
enzyme-catalyzed hydrolysis (water addition) into their 4.1.1. Physical pretreatment
constituent simple sugars. Then the fermentable sugars 4.1.1.1. Mechanical comminution. Milling, chipping,
can be fermented into ethanol by ethanol-producing and grinding are used to reduce seaweeds to particle sizes
microorganisms and finally recover and purify the etha- and cellulose crystallinity that are more efficient to
nol to meet fuel specifications. Additionally, some sepa- process. Smaller particles, with higher surface-area-to-
rated solids can be recovered and utilized as a fuel to volume ratios, will have higher reaction efficiency during
provide process heat and electricity at an alcohol produc- fermentation for bioethanol [31]. The power requirement
tion facility. of mechanical comminution of biomass depends on the
final particle size and the biomass characteristics. It was
4.1. Pretreatment of algal biomass proposed that, if the final particle size is held at a range of
3–6 mm, the energy input for comminution can be kept
Biomass pretreatment is one of the most crucial and expen- below 30 kWh per ton of biomass. The energy consump-
sive process steps in the process of converting biomass to tion is higher than the theoretical energy content available
fermentable sugars [16], and pretreatment is estimated to in the biomass in most cases [44].

Figure 1. Simplified depiction of process steps for production of algae bioethanol.

Int. J. Energy Res. (2014) © 2014 John Wiley & Sons, Ltd.
DOI: 10.1002/er
An overview of algae bioethanol production K. Li, S. Liu and X. Liu

4.1.1.2. Irradiation. Irradiation by gamma rays and process ultimately improves the hydrolysis efficiency of
microwaves can increase sugar yield of various lignocellu- algae into glucose for bioethanol production. The major
losic materials. The combination of the radiation and other advantages are no addition of chemicals and no require-
methods such as acid treatment can significantly accelerate ment of corrosion-resistant materials for hydrolysis
the saccharification process for bioethanol production from reactors in this process [53]. Additionally, this process
marine algae materials. The action mode behind the high might have the advantage of generating less
energy irradiation could be one or more changes of hydroxymethylfurfural (HMF) and other furfurals
features of cellulosic biomass, including increase of spe- degraded from xylose due to the acid and harsh
cific surface area and decrease of the degrees of polymeri- pretreatment conditions. Choi et al. [24] investigated on
zation and crystallinity of cellulose [45]. The effect of how to improve hydrolysis yields of macroalgae Ulva
gamma irradiation on saccharification of Undaria biomass pertusa Kjellman by high-temperature liquefaction process
for bioethanol production was investigated by Yoon et al. (HTLP), and they found it under optimal hydrolysis condi-
[46]. They found that cell wall structures of the Undaria tions, which is 15 MPa and 150 °C for 15 min, with water
biomass were damaged seriously, and the concentration as the solvent. The conversion yields of glucose and xylose
of reducing sugar of hydrolysates significantly increased were 9.08% and 21.14%, respectively. Additionally, the
as a result of gamma irradiation. Microwave is also a feasi- HTLP generated only about 40 mg L 1 of HMF, which
ble method, which uses the high heating efficiency to treat was much less than that from other pretreatment. Other
the lignocellulosic biomass. In the method, internal heat is researchers used hydrothermal treatment to produce
generated in the biomass by microwave radiation, resulting bioethanol from microalgae Schizocytrium sp., under
from the vibrations of the polar bonds in the biomass and optimal conditions (solid loading: 25% (w/w); reaction
the surrounding aqueous medium. Thus a hot spot is cre- temperature: 115.5 °C; reaction time: 46.7 min), and the
ated within the inhomogeneous material. This unique experimental oligomeric sugar yield was 16.6 wt.%. Pro-
heating feature results in an explosion effect among the duction of bioethanol using micro-algal-induced glucan and
particles and improves the disruption of recalcitrant struc- Escherichia coli KO11 was tested with simultaneous sac-
tures of lignocelluloses [47]. This method is widely used charification and fermentation (SSF), which resulted in
in vegetable oil extraction [48]. It was seldom applied in 11.8 g ethanol/L, was produced from 25.7 g/L of glucose
the pretreatment of bioethanol. [54]. Okuda et al. [55] used hydrothermal pretreatment
performed at 423 K for Monostroma nitidum Wittrock (green
4.1.1.3. Ultrasonication. Ultrasound is widely used algae) and at 473 K for Solieria pacifica (red algae) for
for breaking cell walls to release intracellular products. 30 min, which yielded extracted water-soluble components
Ultrasound produces a hydrodynamic shear force in aque- containing monosaccharides of 0.51 g g 1 for the green alga
ous phase due to the rapid collapse of microbubbles and 0.62 g g 1 for the red algae.
formed during cavitations. The hydrodynamic shear force
facilitates the disintegration of coarse particles in slurry 4.1.2.2. Steam explosion. Steam explosion is a
into finer particles, thereby significantly increasing the sur- physico-chemical process, which is one of the most com-
face area for enzyme activity [49]. The ultrasonic treatment monly used pretreatments of lignocellulosic biomass. In
could improve the ethanol production from the lignocellu- this method, size-reduced biomass is rapidly heated by
loses through the glucose enhancement and improvement high-pressure steam for a period, and then the pressure is
of enzymatic hydrolysis [50]. Zhou and Ma [51] used suddenly reduced, which makes the materials undergo an
ultrasound to degrade polysaccharide from Porphyra explosive decompression. Steam explosion is typically
yezoensis (PYPS). They found that the ultrasonic degrada- initiated at a temperature of 160–260 °C (corresponding
tion rate of PYPS solution increases with the increase of pressure, 0.69–4.83 MPa) for several seconds to a few
ultrasonic power and reaction temperature, and the minutes before the material is exposed to atmospheric pres-
decrease of the initial pH value of the solution. Some sure [44]. Manzanares et al. [56] explored steam explosion
researchers found that bioenergy (ethanol/hydrogen) pro- pretreatment on biological conversion of forage sorghum
duction after long-term sonication was almost seven times biomass to ethanol. Under optimum conditions, that is,
higher than that after short-term treatment or the control, 220 °C and 7 min, saccharification efficiency attained
and cells were significantly damaged during sonication. 89% of the theoretical, and the recovery of xylose in the
Ultrasonication was an effective pretreatment method for pre-hydrolyzate accounted for 35% of the amount of
enhancing the fermentative bioenergy production from xylose present in raw material. It can enhance the enzymatic
microalgae biomass [52]. hydrolysis of the lignocellulosic biomass largely [57]. Addi-
tion of chemicals such as sulfuric acid or sulfur dioxide in
4.1.2. Physico-chemical pretreatment steam explosion can improve the conversion rate of all poly-
4.1.2.1. Hydrothermal pretreatment. The hydro- saccharides in the lignocellulosic biomass to monomeric
thermal pretreatment methods have been widely applied sugars [58,59], because in the process of steam explosion,
for pretreatment of lignocellulosic materials. Water under it consumes large amounts of energy. Compared with other
high pressure can penetrate into the biomass, hydrate cellu- pretreatment methods, it has a higher price; therefore, it is
lose, and remove hemicellulose and part of lignin. This rarely used in the pretreatment of algae. Viola et al. [60]

Int. J. Energy Res. (2014) © 2014 John Wiley & Sons, Ltd.
DOI: 10.1002/er
K. Li, S. Liu and X. Liu An overview of algae bioethanol production

investigated the effect of the steam explosion treatment on 4.1.3.2. Acid hydrolysis pretreatment. Acid hy-
the seaweed Zostera marina and the algae Ulva armoricana drolysis is probably the most commonly applied method
conversion in ethanol and methane, and they found that the among the chemical pretreatment methods. It is a method
algal leaves of exploded product are amassed because of that can be used either as a pretreatment before enzymatic
increased surface and cohesion due to steam explosion treat- hydrolysis or as the actual method of hydrolyzing biomass
ment. In best conditions, it can improve sugar extractability to the sugars [66]. There are two types of acid hydrolysis:
and cellulose hydrolysis by enzymes. one that uses concentrated acids and another that uses
diluted acids. Concentrated acids such as H2SO4 and HCl
have been used to treat lignocellulosic materials [44,67].
4.1.2.3. Supercritical carbon dioxide. The super-
Concentrated acid hydrolysis takes place at low tempera-
critical CO2-based pretreatment is similar to the steam
tures (40 °C), using concentrated acids (30–72%). This
explosion pretreatment in principle. It has been considered
method provides high sugar yields (nearly 100% of theo-
as a method to the treatment of lignocellulosic biomass,
retical hexose yields) and has the advantage of being
because of several advantages such as availability at
performed at low temperature [68]. However, concentrated
relatively low cost, non-toxicity, nonflammability, easy re-
acid is toxic, corrosive, and hazardous and requires reac-
covery after extraction, and environmental acceptability
tors that need expensive construction materials resistant
[53]. In supercritical carbon dioxide, the glucose yield
to corrosion. Additionally, the concentrated acid must be
was increased when pressure and temperature increased.
recovered and recycled after hydrolysis to render the
And when compared with the untreated, the surface area
process economically feasible [45]. For these reasons, its
of biomass was increased [61].
commercial potential is small, and little work has been
made in this field. On the other hand, dilute acid hydrolysis
4.1.3. Chemical pretreatment has been extensively reviewed and is considered to be one
4.1.3.1. Alkaline pretreatment. Alkali liquor has of the treatment methods with greater potential for wide-
been widely used in the pretreatment of lignocellulosic scale application. It can be used as a pretreatment to
biomass. It involves the use of bases, such as sodium, increase cellulose accessibility to enzymes or as a method
potassium, calcium, and ammonium hydroxide [62]. The for direct saccharification.
mechanism of alkaline hydrolysis is believed to be sapon- Dilute acid hydrolysis has been successfully developed
ification of intermolecular ester bonds crosslinking xylan for pretreatment of algal materials. The most commonly used
hemicelluloses and other components, for example, lignin acid is dilute sulfuric acid (H2SO4). Miranda et al. [69] found
and other hemicelluloses [45]. The major effect of alkaline that among the physical and physicochemical methods
pretreatment is the removal of lignin from the biomass, tested, the best results were obtained with acid hydrolysis
thus improving the reactivity of the remaining polysaccha- by H2SO4 (2 N), at 120 °C for 30 min. Feng et al. [70] inves-
rides. In addition, alkaline pretreatments remove acetyl and tigated the hydrolysis of Enteromorpha using four different
the various uronic acid substitutions on hemicellulose that acids: H2SO4, HCl, H3PO4, and C4H4O4 (maleic acid). In
lower the accessibility of the enzyme to the hemicellulose the same conditions, H2SO4 was the most effective.
and cellulose surface [63]. The algae biomass does not Dilute H2SO4 pretreatment has been widely used
look like terrestrial plants, which have hard organizations, between microalgae and macroalgae. Lee et al. [71] hydro-
and they have no lignin. Therefore, alkali liquor is rarely lyzed microalgae (Chlorella vulgaris and Chlamydomonas
used in pretreating algae biomass. Harun et al. [16] used reinhardtii) with dilute H2SO4 (1–5% on 5% (w/v) dry
NaOH for bioethanol production from the species solid basis (v/v)) and heated them in an autoclave at
Chlorococcum infusionum, which is green algae. They 100–120 °C; the treatment time was 120 min. They found
found that the highest glucose yield was determined to be that the two microalgae species had different optimal con-
350 mg/g, and the maximum bioethanol yield obtained ditions: only 0.075 g glucose/g biomass was extracted from
was 0.26 g ethanol/g algae using 0.75% (w/v) of NaOH C. reinhardtii at 100 °C, 5% sulfuric acid for 120 min.
and 120 °C for 30 min. Eshaq et al. [25] used 50% of the However, 0.12 g glucose/g biomass, 0.033 g xylose/g
Spirogyra powder, and they found that pretreatment with biomass, 0.047 g galactose/g biomass, and 0.007 g fructose/g
alkaline is not required for the algal material particularly biomass were extracted from C. vulgaris at 110 °C, 5%
for Spirogyra, as Spirogyra cell wall is purely cellulosic sulfuric acid for 120 min. Nguyen et al. [72] employed C.
and cell contains simple starch. In fact, alkaline reinhardtii UTEX 90 as the feedstock, with dry cells of
pretreatment will damage the cellulose leading to less yield 5% (w/v); the algal biomass was pretreated with sulfuric
of ethanol when compared with untreated Spirogyra acid (1–5%) under temperatures from 100 to 120 °C, for
biomass. In addition, alkaline pretreatment reduces the 15–120 min. As a result, the glucose release from the bio-
degree of inhibition during fermentation and provides mass was maximum at 58% (w/w) after pretreatment with
lower production cost and safe handling compared with 3% sulfuric acid at 110 °C for 30 min. Harun et al. [73]
other pretreatment methods [64]. Karunakaran and investigated the influence of acid pretreatment on micro-
Gurusamy [65] cooked Rhodopyhtes feedstock with hot algal biomass (Chlorococcum humicola) for bioethanol
water and alkali to extract the polysaccharides, the carra- production; results showed that the highest bioethanol
geenan yield was in the range of between 23% and 36%. concentration obtained was 7.20 g/L, and this was

Int. J. Energy Res. (2014) © 2014 John Wiley & Sons, Ltd.
DOI: 10.1002/er
An overview of algae bioethanol production K. Li, S. Liu and X. Liu

achieved when the pretreatment step was performed with moss (G. amansii) resulted in glucose yield up to 70% with
15 g/L of microalgae at 140 °C using 1% (v/v) of sulfuric contrast of only 5% obtained from non-pretreated samples.
acid for 30 min. An investigation on HCl and H2SO4 The efficiency of enzymatic hydrolysis is significantly
pretreatment of four marine algae, Ulva lactuca, Gelidium improved by sodium chlorite pretreatment.
amansii, L. japonica, and Sargassum fulvellum, was
carried out by Kim et al. [23]; at high temperature and 4.1.4. Biological pretreatment
pressure, more soluble sugars (or carbohydrates) were re- Biological treatment using various types of rot fungi, a
leased with increasing concentrations of HCl or H2SO4. safe and environmentally friendly method, is increasingly
Among the four algae tested, G. amansii produced the being advocated as a process that does not require high en-
highest amounts of soluble sugars (or carbohydrates) on ergy for lignin removal from a lignocellulosic biomass
acid pretreatment, 9.8% (w/w-dry biomass) with HCl [44]. Because algae have low lignin levels, this method
and 11.6% (w/w-dry biomass) with H2SO4. Wang et al. has rarely been used for algae pretreatment. Some re-
[74] found that the homogenates of the invasive searchers saccharified Saccharina japonica by Bacillus
Gracilaria salicornia was hydrolyzed at a level of 2% sp. JS-1, a marine bacterium. The results show that a
sulfuric acid, 120 °C for 30 min. Glucose concentration in reducing sugar concentration of 45.6 ± 6.8 g/L was reached
the resulting hydrolysates contained approximately 4.3 glu- with treatment of the seaweed slurry with Bacillus sp. JS-1,
cose/kg fresh algal biomass. Most researchers concluded that compared with 13.9 ± 0.8 g/L with thermal acid hydrolysis
the effectiveness of acid hydrolysis pretreatment on algae is and the reducing sugar concentrations increased linearly
strongly infected by four parameters: amount of biomass, with time [7]. Matsumoto et al. [80] investigated using
acid concentration, temperature, and reaction time. However, an amylase-producing bacterium, Pseudoalterimonas
among the parameters investigated, temperature may be the undina NKMB 0074, to saccharify green marine microalga
most critical factor during acid pretreatment of algae for NKG 120701; the marine bacteria were inoculated into
bioethanol production [73]. Recently, acid hydrolysis suspensions containing NKG 120701 cells and increas-
pretreatment had some improvement to its pretreatment effi- ingly reduced suspended sugars with incubation time. Ter-
ciency. Park et al. [22] used facile continuous method for di- restrial amylase and glucoamylase were inactive in saline
lute acid hydrolysis of G. amansii, and they found that there suspension. Therefore, marine amylase is necessary in sa-
are many advantages to the continuous hydrolysis process. line conditions for successful saccharification of marine
For example, the low melting point of the agar component microalgae.
in G. amansii facilitates improved raw material fluidity in
the continuous reactor. In addition, the hydrolysate obtained 4.2. Enzymatic hydrolysis
from the continuous process delivered a high sugar and low
inhibitor concentration, thereby leading to both high yield Saccharification is the critical step for bioethanol produc-
and high final ethanol titer in the fermentation process. tion where complex carbohydrates are converted to simple
Under extremely low acid concentration, Lee et al. used monomers. Compared with acid hydrolysis, enzymatic
L. japonica as feedstock for bioethanol, and they found the hydrolysis requires less energy and mild environment
maximum glucan content of 29.09%, which was fourfold conditions (e.g., pH 4.5–5.0 and temperature 40–50 °C) and
higher than that of raw L. japonica obtained after the ex- does not have a corrosion problem [48,68]. Therefore, one
tremely low acid pretreatment under the optimal condition may expect low utility consumption and low toxicity of the
with sulfuric acid of 0.06% at temperature of 170 °C for hydrolyzates as the main advantages of this process [67].
15 min [75]. Cellulose hydrolysis is catalyzed by a class of enzymes
However, the main drawbacks of acid hydrolysis are for- known as celluloses [42]. These enzymes can be produced
mation of many inhibiting by-products and pH neutralization by both bacteria and fungi. Among the various cellulolytic
requirement for downstream processes. Furfural and HMF microbial strains, Trichoderma is one of the most well-stud-
were known to be severe inhibitors of fermentative microor- ied cellulase-producing and hemicellulase-producing fungal
ganisms. Formation of furfural depended on the retention strains [42,47,67]. During enzymatic hydrolysis, cellulose
time and acid concentration [76], and the inhibition was is degraded by the cellulase to reduce sugars that can be
prevented by increasing cell biomass concentration [77]. fermented by yeasts or bacteria to ethanol. The action of
Recently, some researchers found that biotransformation of cellulolytic enzymes occurs through three steps of adsorp-
HMF to less inhibitory compounds by Scheffersomyces tion, biodegradation, and desorption [42].
stipitis KCTC 7228 could enhance overall fermentation Both enzymatic hydrolysis and chemical hydrolysis
yields of seaweed hydrolysates to ethanol [78]. have been reported on the conversion of algae biomass to
sugars. After enzymatic hydrolysis, the concentrations of
4.1.3.3. Sodium chlorite treatment. In an acidic sugars are much higher than those of chemical hydrolysis
medium, chlorite generates ClO2 molecules that transform (Table IV). Additionally, different algae contain different
lignin into soluble compounds without any significant loss compounds, so cellulose enzyme supplemented with other
of carbohydrate content, and this procedure is widely used enzymes can raise the rate of enzymatic hydrolysis. There
as an analytical method for holocellulose determination. are large amounts of starch contained in algae biomass,
Wi et al. [79] found that sodium chlorite-pretreated Ceylon so amylase can greatly conjugate to hydrolyze algae

Int. J. Energy Res. (2014) © 2014 John Wiley & Sons, Ltd.
DOI: 10.1002/er
K. Li, S. Liu and X. Liu An overview of algae bioethanol production

Table IV. Sugar yields vary in the enzymatic hydrolysis and chemical hydrolysis of algal biomass.

Chemicals hydrolysis Enzymatic hydrolysis

Algal source Procedure Sugar yield Enzymes mixture Sugar yield Reference

Ulva pertusa Kjellman 15 MPa and Glucose 9.08% Cellulose and Glucose 61.1% [12]
150 °C for 15 min amyloglucosidase
Gracilaria salicornia 120 °C and 2% 4.3 g glucose/kg Cellulase 13.8 g glucose/kg [64]
H2SO4 for 30 min biomass biomass
Gelidium amansii 0.2 N H2SO4 at Sugar 9.8% Endo-glucanase and Sugar 56.6% [63]
121 °C for 15 min beta-glucanase
Saccharina japonica 40 mM H2SO4 at Reducing sugar 13.5 g/L Industrial enzyme, Reducing sugar [4]
121 °C for 60 min Termamyl 120 L 20.6 ± 1.9 g/L
Dunaliella tertiolecta 0.5 M HCl at Reducing sugar 29.5% AMG 300 L Reducing sugar 42% [87]
121 °C for 15 min

biomass [24]. Some specific enzymes can promote hydro- However, some researchers used the ethanologenic strain
lysis of polysaccharides from seaweed [81]. E. coli KO11 to ferment the invasive marine macroalgae G.
Several factors influence yields of monomer sugars salicornia; ethanol yield of this study was relatively low.
through enzymatic hydrolysis; temperature, pH, substrate The reason may be that the monosugars contained in inva-
concentration, and enzyme loading are the main factors. sive feedstocks were not readily fermented by E. coli
The optimum pH of cellulose enzyme is 4.5–5, so before KO11 [74].
enzymatic hydrolysis, the hydrolysate should be neutral- Recently, some researchers developed surface aeration
ized by acid and alkali. In addition, in the process of enzy- to improve bioethanol production from the hydrolysate
matic hydrolysis, it often tends to generate acidic containing C5 monosaccharides such as xylose. Yeon
substances, which will affect the stability of the system op- et al. [84] designed a surface-aerated repeated-batch fer-
eration. Researchers often join buffer solutions to address mentor culture, in which the aeration was finely controlled
the issues, such as citrate buffer and acetate buffer at 100 mL/min and delivered into the headspace of a 2.5-L
[23,74,79]. fermentor. When the medium was replaced every 48 h,
However, adding to the commercial constraints is the bioethanol was continuously produced for 200 h under re-
fact that costs of enzymes are almost exclusively accounted peated-batch fermentor culture, where the level of
for and reported in terms of dollars per gallon of bioethanol bioethanol production was about 9 g/L. Lee et al. [85] in-
[82]. And the high cost of starch/cellulose depolymerizing vestigated the feasibility and performance of bioethanol
enzymes for pretreatment of algal biomass makes the cost production from the hydrolysate of seaweed Sargassum
of algal bioethanol several folds higher. So we must de- sagamianum in cornsteep liquor-containing medium,
crease the cost of enzymes to ensure commercial algal where yeast Pichia stipitis was used, and the repeated
bioethanol production. batch was carried out in a surface-aerated fermentor. The
optimal medium replacement time during the repeated op-
4.3. Fermentation eration was determined to be 36 h, and the surface aeration
rates were 30 and 100 mL/min. Under these conditions, the
Fermentation using microorganisms will utilize all five of repeated-batch operation was successfully carried out for
the major biomass sugars, glucose, xylose, mannose, ga- six runs (216 h), in which the maximum bioethanol con-
lactose, and arabinose, which are from enzymatic hydroly- centrations reached about 11–12 g/L at each batch opera-
sis of algae biomass. The best known microorganisms for tionally. In addition, the bioethanol yields were 0.43
ethanol production from hexoses are the yeast Saccharo- (about 84% of theoretical value) and 0.44 (about 86% of
myces cerevisiae and the bacterium Zymomonas mobilis theoretical value) when the surface aeration rates were 30
[42]. Sulfahri et al. [83] investigated bioethanol production and 100 mL/min, respectively.
by means of fermentation using Z. mobilis and S. Enzymatic hydrolysis and fermentation process can be
cerevisiae. The results showed that Z. mobilis was able to accomplished using different strategies: separate hydroly-
survive at high concentrations compared with S. cerevisiae. sis and fermentation (SHF), SSF, and simultaneous sac-
Some researchers applied ethanogenic E. coli KO11 to al- charification and cofermentation (SSCF).
gae fermentation. Kim et al. [23] concluded that acid hy-
drolysis of L. japonica biomass followed by simultaneous 4.3.1. Separate enzymatic hydrolysis and
treatment of the lysate with hydrolytic enzymes and fer- fermentation
mentation with ethanogenic E. coli KO11 resulted in an In this process, pretreated algae biomass is hydrolyzed
ethanol yield of 0.4 g ethanol/g of sugars. This yield was to glucose and subsequently fermented to ethanol in sepa-
achievable because E. coli KO11 was able to utilize manni- rate units. The main advantages of this process include the
tol, which is present in high amounts in the hydrolysate. low cost of chemicals, short residence time, and simple

Int. J. Energy Res. (2014) © 2014 John Wiley & Sons, Ltd.
DOI: 10.1002/er
An overview of algae bioethanol production K. Li, S. Liu and X. Liu

equipment system, all of which promote its large-scale Although more than 100 algae-to-fuel companies have
application. Choi et al. [17] found that approximately been set up around the world mostly in the last couple of
235 mg of ethanol was produced from 1.0 g of algal years, not a single commercial facility has been built so
biomass (C. reinhardtii) by SHF method. The major disad- far. So like any other industrial process, the commerciali-
vantage is that the released sugars inhibit cellulases [86]. zation of algae bioethanol is also dependent on the
economics of the process. There are several barriers in
4.3.2. Simultaneous saccharification and the technology of algae bioethanol production, which must
fermentation be overcome before it can be successfully large scale and
Simultaneous saccharification and fermentation is a single commercialized produced.
process that combines the saccharification and fermentation Pretreatment is estimated to account for about 33% of
processes. In this process, (the enzyme) cellulases and micro- the total cost in the production of algae bioethanol, which
organisms are added to the same process unit, and glucose is is the most costly step. Such a pretreatment method must
immediately consumed by the fermenting microorganism be simple and must avoid high consumption of expensive
[66]. It has shown that higher ethanol yields can be obtained chemicals and high energy demands. Furthermore, poly-
using SSF compared with SHF. SSF is an efficient process saccharides from the algae biomass should be hydrolyzed
for ethanol production because it reduces the number of unit directly without sugar degradation, which might produce
operations needed [7]. fermentation inhibitors. Among the different methods, the
use of dilute sulfuric acid is currently one of the most
effective and includes the most promising technologies
4.3.3. Simultaneous saccharification and for industrial applications. To further decrease the cost of
cofermentation the pretreatment step in the algae feedstocks conversion
While there are a variety of yeast and bacteria that will to ethanol, it is essential to minimize sugar losses, to in-
ferment six-carbon sugars (hexose), most cannot easily crease solids concentration as high as possible, and to keep
ferment five-carbon sugars (pentose), which limits ethanol low reactors and associated equipment costs. Additionally,
production from cellulosic biomass. Researchers are using from a basic research point of view, one approach that is
genetic engineering to design microorganisms that can receiving more attention is the study of the effects of
efficiently ferment both five-carbon and six-carbon sugars pretreatment at a more fundamental level. The composition
to ethanol at the same time. SSCF represents the hydrolysis of algae cell wall is very complex, and research at cellular,
and cofermentation of pentose and hexose sugars in one ultrastructural, and even molecular levels could contribute
vessel [86]. Peralta-Ruíz et al. [87] used the software to understanding the diverse catalytic reactions acting on
ASPEN PLUS 7.1 to simulate technological routes based biomass as well as the consequences of pretreatments. This
on experimental information; results of simulation shows knowledge should be applied to achieve an integrated and
that route SSCF presents a bioethanol yield of 23.6%, efficient biomass conversion process to ethanol. Last but
which is the highest of the routes evaluated, and SSF route not the least, biological treatment is an economically efficient
gives a yield of 20.1% and SHF 18.5%. method; if we can find specific marine bacteria to each kind
of algae, it will be a technical breakthrough that will acceler-
4.4. Distillation ate the commercial development of algae bioethanol.
Moreover, the efficiency of bioethanol production from
Distillation technologies that will allow the economic re- microalgae and macroalgae is also different. Because
covery of dilute volatile products from streams containing microalgae contain high capacity of vegetable oils, up to
a variety of impurities have been developed and commer- now, biodiesel production from microalgae still would ide-
cially demonstrated. But algae bioethanol has not been in ally be the main product. However, diversified biofuels
commercial production, and the unit of distillation is sel- production from microalgae is necessary to improve the
dom discussed. overall energy balance. One of the successful examples is
to use the microalgae biomass (after lipid extraction) for
bioethanol production because high concentrations of car-
5. CONCLUSIONS AND FUTURE bohydrates still remain in the biomass. This is a win–win
PERSPECTIVE strategy in re-utilizing the waste to produce another source
of energy, which greatly amplifies the sustainability of
Although the raw materials of lignocellulosic bioethanol microalgae biofuels. Akin to microalgae, macroalgae also
are cost effective, renewable, and abundant, the production have the ability to grow at a fast rate and yield huge
of the second-generation bioethanol is costly because of amounts of biomass. The high yields are due to the fact that
the high recalcitrance of lignocellulosic raw materials. So macroalgae require less energy for the production of
as the sustainable development of modern society is supporting tissue than land plants, and they have the capa-
hindered seriously by the energy crisis and environmental bility to take up nutrients over their entire surface. So
pollution, the algal biomass as a raw material for bioethanol from macroalgae will be popular in the future.
bioethanol production is undoubtedly a sustainable and The next significant technical barrier is cost of enzymes.
eco-friendly source for renewable biofuel production. Close cooperation and investment should be developed

Int. J. Energy Res. (2014) © 2014 John Wiley & Sons, Ltd.
DOI: 10.1002/er
K. Li, S. Liu and X. Liu An overview of algae bioethanol production

with the aim of increasing the effectiveness and specificity 11. John RP, Anisha GS, Nampoothiri KM, Pandey A. Micro
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Int. J. Energy Res. (2014) © 2014 John Wiley & Sons, Ltd.
DOI: 10.1002/er