Lignocellulosic biomass to bioethanol, a comprehensive review with a focus on pretreatment

Full text

Lignocellulosic biomass to bioethanol, a comprehensive review
with a focus on pretreatment
Sohrab Haghighi Mood
a
, Amir Hossein Golfeshan
a
, Meisam Tabatabaei
b,
n
,
Gholamreza Salehi Jouzani
b
, Gholam Hassan Najafi
c
,
Mehdi Gholami
b
, Mehdi Ardjmand
a,b
a
Department of Chemical Engineering, South Tehran Branch, Islamic Azad University, Tehran, Iran
b
Biofuel Research Team (BRTeam), Microbial Biotechnology and Biosafety Department, Agricultural Biotechnology Research Institute of Iran (ABRII),
P.O. Box 31535-1897, Karaj, Iran
c
Tarbiat Modares University, P.O. Box 14115-336, Jalale Ale Ahmad Express Way, Nasr, Bridge, Tehran, Iran
article info
Article history:
Received 17 March 2012
Received in revised form
22 June 2013
Accepted 24 June 2013
Keywords:
Bioethanol
Biomass
Lignocellulosic wastes
Pretreatment
Combination
Genetic engineering
abstract
Pretreatment technologies are aimed to increase enzyme accessibility to biomass and yields of
fermentable sugars. In general, pretreatment methods fall into four different categories including
physical, chemical, physico-chemical, and biological. This paper comprehensively reviews the lignocel-
lulosic wastes to bioethanol process with a focus on pretreatment methods, their mechanisms,
advantages and disadvantages as well as the combinations of different pretreatment technologies.
Moreover, the new advances in plant “omics”and genetic engineering approaches to increase cellulose
composition, reduce cellulose crystallinity, produce hydrolases and protein modules disrupting plant cell
wall substrates, and modify lignin structure in plants have also been expansively presented.
Crown Copyright &2013 Published by Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2. Lignocellulosic material composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.1. Cellulose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.2. Hemicelluloses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.3. Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3. Influence of lignocellulosic biomass composition and structure on cellulose hydrolysis and bioconversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4. Pretreatment methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.1. Physical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.1.1. Milling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.1.2. Extrusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.1.3. Microwave . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.1.4. Freeze pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.2. Chemical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.2.1. Acid pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.2.2. Alkaline pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.2.3. Ionic liquid (IL) pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.2.4. Organosolv pretreatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.2.5. Ozonolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.3. Physico-chemical pretreatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.3.1. Steam explosion pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/rser
Renewable and Sustainable Energy Reviews
1364-0321/$- see front matter Crown Copyright &2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.rser.2013.06.033
n
Correspondence to: Biofuel Research Team (BRTeam), Microbial Biotechnology and Biosafety Department, Agricultural Biotechnology Research Institute of Iran (ABRII),
Fahmideh Blvd., Mahdasht Road, 31535-1897 Karaj, Iran. Tel.: +98 261 2703536; fax: +98 261 2704539.
E-mail address: meisam_tab@yahoo.com (M. Tabatabaei).
Renewable and Sustainable Energy Reviews 27 (2013) 77–93

4.3.2. Ammonia fiber explosion (AFEX) pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.3.3. CO
2
explosion pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.3.4. Liquid hot water (LHW). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.3.5. Wet oxidation (WO). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.4. Biological pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
5. Combination of pretreatment methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
5.1. Combination of alkaline and dilute acid pretreatments (ALK-DA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
5.2. Combination of alkaline and IL pretreatments (ALK-IL). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.3. Combination of dilute acid and steam explosion pretreatments (DA-SExp.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.4. Combination of supercritical CO
2
and steam explosion pretreatments (SCCO
2
-SExp.). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.5. Combination of organosolv and biological pretreatments
(bio-organosolv). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.6. Combination of biological and dilute acid pretreatments
(bio-DA)...................................................................................................... 84
5.7. Combination of biological and steam explosion pretreatment (bio-SExp.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.8. Microwave-assisted alkali pretreatment (MW-ALK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.9. Combination of dilute acid and microwave
pretreatments (DA-MW) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
5.10. Combination of IL and ultrasonic pretreatment (IL-UL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
6. Future prospective of pretreatment; genetic manipulation of energy crops. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
6.1. Increasing cellulose composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
6.2. Reduction of plant cell wall recalcitrance and cellulose crystallinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
6.3. Production of hydrolases in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6.4. Lignin modification.......................................................................................... ... 87
6.5. Protein modules disrupting plant cell wall substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
References.............................................................................................................. 89
1. Introduction
Recent economic developments in many countries all around the
globe have heightened the need for alternative energy resources due
to the well-documented drawbacks of fossil fuels: (1) their finite
supply (2) greenhouse gasses emission and global warming and
(3) increasing price and unexpected fluctuations. All these weak-
nesses have strengthened the interest in alternatives, renewable,
sustainable, and economically viable fuel such as bioethanol. Bioetha-
nol can be either mixed with gasoline or used as a sole fuel using
dedicated engines; moreover, it has higher heat of vaporization and
octane number compared to gasoline [1]. Ethanol is already blended
with gasoline and supports by vehicle manufacturers have resulted in
vehicles that can use up to an 85% ethanol–15% gasoline mixture [2].
In fact, gasoline can use bioethanol as an oxygenated fuel to increase
its oxygen content, causing better hydrocarbon oxidation and dimin-
ishing greenhouse gasses [3].
In the first generation bioethanol production, expensive starch
and sugar derived from sugar cane and maize are employed as
feedstock but in the second generation process, lignocellulosic
materials, which are cheap, abundant and renewable, are used [4].
Besides, lignocellulosic materials do not negatively affect the
human food supply chain by eliminating the food in favor of
bioethanol production [5]. Lignocelluloses are composed of cellu-
lose, hemicelluloses and lignin (Fig. 1) in an intricate structure,
which is recalcitrant to decomposition. One of the best strategies
to convert such biomass into sugars is enzymatic saccharification
due to its low energy requirement and less pollution caused; but,
the major problem is the low accessibility of cellulose because of
rigid association of cellulose with lignin [6]. This leads to difficul-
ties within the conversion process; therefore, breaking down
lignin seal in order to make cellulose more accessible to enzymatic
hydrolysis for conversion is one main aim of pretreatment (Fig. 2).
In other words, pretreatment is the crucial and costly unit process
in converting lignocellulosic materials into fuels [7].
A suitable pretreatment procedure involves (1) disrupting
hydrogen bonds in crystalline cellulose, (2) breaking down
coross-linked matrix of hemicelluloses and lignin, and finally,
(3) raising the porosity and surface area of cellulose for
subsequent enzymatic hydrolysis [8,9]. There are several pre-
treatment methods including, physical pretreatment (grinding
and milling, microwave and extrusion), chemical pretreatment
(alkali, acid, organosolv, ozonolysis and ionic liquid), physico-
chemical pretreatment (steam explosion, liquid hot water,
ammonia fiber explosion, wet oxidation and CO
2
explosion)
and biological pretreatment. On the other hand, regrdless of
the pretreatment method used, some inhibitory compounds
are produced during the process, which have negative effects
on microbial activity in the hydrolysis step. Inhibitors are
classified into three major groups: (1) weak acids such as
levulinic, acetic and formic acids, (2) furan derivatives such
as HMF (5-hydroxy-2-methyll furfural) and furfural as well as
(3) phenolic compounds [10].
The purpose of this paper is to review different pretreatment
methods for bioethanol production and to offer an in-depth
discussion on the benefits and drawbacks of each while striving
to present and highlight several combined pretreatment methods.
Moreover, the crucial role of genetic and metabolic engineering in
facilitating pretreatment and hydrolysis processes and conse-
quently in economical production of ethanol from lignocellulosic
wastes has been also discussed.
2. Lignocellulosic material composition
2.1. Cellulose
Cellulose (C6H10O5)x, the main constituent of lignocellulosic
biomass, is a polysaccharide that consists of a linear chain of
D-glucose linked by β-(1,4)-glycosidic bonds to each other. The
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–9378

Cellulose strains are associated together to make cellulose fibrils.
Cellulose fibers are linked by a number of intra- and intermole-
cular hydrogen bonds [12]. Therefore, cellulose is insoluble in
water and most organic solvents [13].
2.2. Hemicelluloses
Hemicelluloses (C5H8O4)m, located in secondary cell walls, are
heterogeneous branched biopolymers containing pentoses (β-D-
xylose, α-L-arabinose), hexoses (β-D-mannose, β-D-glucose, α-D
galactose) and/or urgonic acids (α-D-glucuronic, α-D-4-O-methyl-
galacturonic and a-D-galacturonic acids) [14]. They are relatively
easy to hydrolyze because of their amorphous, and branched
structure (with short lateral chain) as well as their lower mole-
cular weight [12]. In order to increase the digestibility of cellulose,
large amounts of hemicelluloses must be removed as they cover
cellulose fibrils limiting their availability for the enzymatic hydro-
lysis [15]. Hemicelluloses are relatively sensitive to operation
condition, therefore, parameters such as temperature and reten-
tion time must be controlled to avoid the formation of unwanted
products such as furfurals and hydroxymthyl furfurals which later
inhibit the fermentation process [10].
2.3. Lignin
Lignin [C9H10O3(OCH3)0.9–1.7]nis an aromatic polymer
synthesized from phenylpropanoid precursors. The major chemi-
cal phenylpropane units of lignin consisting primarily of syringyl,
guaiacyl and p-hydroxy phenol are linked together by a set of
linkages to make a complicated matrix [16].
3. Influence of lignocellulosic biomass composition and
structure on cellulose hydrolysis and bioconversion
As mentioned earlier, lignocellulosic biomass composition
plays a very crucial role in the performance and efficiency of both
pretreatment and biodegradation stages. Table 1 presents the
compositions of several suitable lignocellulosic biomass for
bioethanol production [8,17–27]. Utilization of cellulose in native
form, not only consumes large amount of enzyme but also results
in low cellulose enzymatic digestibility yield (o20%). Therefore,
some structural modification of lignocellulosic material (pretreat-
ment), is required and to select a suitable pretreatment technol-
ogy, recognition of the main structural limiting factors is a critical
step. These factors include (1) specific surface area, (2) cellulose
crystallinity index (CrI), (3) degree of polymerization (Dp),
Table 1
Different lignocellulosic biomass compositions (% dry basis).
Lignocellulosic biomass Cellulose glucan Hemicellulose lignin References
Xylan Arabinan Galactan Mannan Acid-insoluble lignin Acid-soluble lignin
Barley hull 33.6 30.5 6.1 0.6 Trace 19.3 ND [17]
Barley straw 33.8 21.9 13.8 [18]
Corn cobs 33.7 31.9 6.1 [18]
Corn stover 38.3 21.0 2.7 2.1 ND 17.4 [19]
Cotton stalks 14.4 14.4 21.5 [18]
Wheat straw 30.2 18.7 2.8 0.8 ND 17 [20]
Rice straw 31.1 18.7 3.6 ND ND 13.3 [21]
Rye straw 30.9 21.5 ND ND ND 22.1 3.2 [22]
Oat straw 39.4 27.1 17.5 [18]
Soya stalks 34.5 24.8 9.8 [18]
Sunflower stalks 42.1 29.7 13.4 [18]
Switchgrass 39.5 20.3 2.1 2.6 ND 17.8 4.0 [8]
Sugarcane bagasse 43.1 31.1 11.4 [23]
Sweet sorghum bagasse 27.3 13.1 1.4 ND ND 14.3 [24]
Forage sorghum 35.6 18.4 1.8 ND ND 18.2 [24]
Olive tree pruning 25.0 11.1 2.4 1.5 0.8 16.2 2.2 [25]
Poplar 43.8 14.8 ND ND ND 29.1 [26]
Spruce 43.8 6.3 ND ND 14.5 28.3 0.53 [27]
Oak 45.2 20.3 ND ND 4.2 21.0 3.3 [27]
Fig. 1. General composition of lingocellulosic biomass feedstock.
Fig. 2. Schematic pretreatment of lignocellulosic material.
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–93 79

(4) cellulose sheathing by hemicelluloses, (5) lignin content and
(6) acetyl content [28].
4. Pretreatment methods
4.1. Physical pretreatment
The objective of physical pretreatment such as milling, grind-
ing, chipping, freezing, radiation is to increase surface area and
reduce particle size of lignocellulosic materials [29]. Moreover, it
leads to decrease degree of polymerization and decrystallization of
feedstock. Combination of physical pretreatments and other pre-
treatment is usually used.
4.1.1. Milling
Milling is usually considered as the first step of pretreatment.
Ball milling, two-roll milling, hammer milling, colloid milling and
disk milling are several types of milling used in bioethanol
production processes. The final particle size achieved depends on
the type of the physical pretreatment, for example, after chipping,
milling or grinding, the particle size reduces to 10–30 mm and
0.2–2 mm, respectively [30]. The most important drawback of
milling is high energy requirement. To overcome that, wet disk
milling has been introduced as a more reasonable mechanical
pretreatment in terms of energy consumption. However, the yields
of glucose and xylose after enzymatic hydrolysis of some pre-
treated biomass by ball milling are higher than those of wet disk
milling. For instance, Hideno et al. investigated the enzymatic
hydrolysis of rice straw after being pretreated by wet disk milling
and ball milling. They reported the yields of glucose and xylose of
78.5% and 41.5%, and 89.4% and 54.3%, respectively.
Sant'Ana da Silva et al. evaluated the effectiveness of ball
milling and wet disk milling on treating sugarcane bagasse. Based
on their investigation, glucose and xylose hydrolysis yields under
optimum conditions for ball milling-treated bagasse were 78.7%
and 72.1% respectively, while maximum glucose and xylose yields
for bagasse wet milling-treated were 49.3% and 36.7%, respectively
[31,32].
4.1.2. Extrusion
Extrusion pretreatment is a thermo-physical pretreatment in
which materials undergo mixing, heating and shearing leading to
physical and chemical alteration [5,33]. High shear, rapid mixing,
short residence time, moderate barrel temperature, no furfural
and HMF formation, no washing and conditioning, adaptability to
process modification, easy scale-up, and most importantly, possi-
bility of continuous operation are considered among the advan-
tages of this method [34,36]. Furthermore, extrusion results in no
effluent and consequent effluent disposal cost and solid loss [35].
Yoo et al. [33] employed extrusion pretreatment (screw speed,
350 rpm; maximum barrel temperature, 80 1C; in-barrel moisture,
40% wet basis) for soybean hulls and achieved 94.8% glucose
conversion after enzymatic hydrolysis (glucose yield of 0.37 g/g
biomass).
4.1.3. Microwave
Microwave irradiation could be an alternative to the conventional
heating in order to alter the ultra structure of cellulose, degrade/
partially remove lignin and hemicelluloses, disrupt the silicified waxy
surface and finally enhance the enzymatic susceptibility of reducing
sugars [37,38]. Technically, conventional heating is based on superficial
heat transfer but, as for microwave, heat is generated by direct
interaction of a heated object and an applied electromagnetic field
[39].Morespecifically, microwave irradiation leads to cellulosic break-
down mainly through molecular collision due to dielectric polarization
[40]. The advantages of this method include, (1) short process time, (2)
high uniformity and selectivity (3) and less energy input than the
conventional heating [41].
4.1.4. Freeze pretreatment
A novel approach recently developed for physical pretreatment
of biomass is the freeze pretreatment and has been found to
significantly increase the enzyme digestibility of rice straw. In
spite of the high cost involved and that only a few studies have
been carried out using this pretreatment so far, its unique features
i.e. lower negative environmental impact, application of less
dangerous chemicals and high effectiveness have attracted a great
deal of attention [42].
4.2. Chemical pretreatment
4.2.1. Acid pretreatment
Acid pretreatment in particular by using sulfuric acid is the
most commonly employed chemical pretreatment for lignocellu-
losic biomass where polysaccharides (mainly hemicelluloses) are
hydrolyzed to monosaccharides leading to higher accessibility of
cellulose to enzyme hydrolysis. Acid pretreatment can be per-
formed either under low acid concentration and high temperature
or under higher acid concentration and lower temperature. [43].
Using concentrated acid is more economic as the process is
performed at low temperature [14]; however, toxicity, corrosive-
ness of equipments, acid recovery [30], degradation of monosac-
charides i.e. glucose, and the production of fermentation inhibitors
(such as furan-type inhibitors; HMF (5-hydroxy methyl- furfural)
and 2-furfuralaldehyde [44,45]), are major drawbacks preventing
the widespread application of this method [5,]. Moreover, at high
temperatures, the produced inhibitors such as furfural could also
degrade into other unwanted products e.g. formic and levulinic
acids [46]. Industrially, diluted acid is more attractive as it
generates lower amounts of fermentation inhibitors and in light
of that, numerous studies have been conducted employing this
technique. For instance, rice straw was pretreated with 1% (w/w)
sulfuric acid with 1–5 min retention time at 1601or 180 1C
followed by enzymatic hydrolysis, which resulted in the maximal
sugar yield of 83% [47]. In a different investigation using the same
acid, rape seed straw with 20% solid content was pretreated for
10 min at 180 1C and 75.12% of xylan and 63.17% of glucan were
respectively converted into xylose and glucose through enzymatic
hydrolysis [48]. Redding et al. [2] also pretreated Coastal Bermuda
grass with sulfuric acid (1.2%) at 140 1C for 30 min and total sugar
yield of 94% of the theoretical value was achieved.
Another acid used is peracetic acid which is capable of
converting lignin to soluble fragments [49]. However, it is explo-
sive in concentrated form and costly as well [50]. Rocha et al. [51]
applied a mixture of sulfuric and acetic acid to pre-treat sugarcane
bagasse. The solution used contained 1% (w/v) sulfuric acid and 1%
(w/v) acetic acid. Solid-to-liquid ratios tested were 1.5:10 and 1:10
and the pretreatment was performed in a rotary reactor for 10 min
at 190 1C. The outcome for conditions was efficient removal of
hemicellulose by above 90% and cellulose degradation was
recorded below 15%. In addition to inorganic acids, organic acids
such as maleic and fumaric acids could also be used. In a study,
hydrolysis yields when wheat straw was treated by maleic and
fumaric were compared with that of sulfuric acid and it was
revealed that wheat straw could be pretreated efficiently with
organic acids as well [52].
4.2.2. Alkaline pretreatment
Removing lignin, acetyl groups and different uronic acid sub-
stitution which inhibit the cellulose accessibility for enzymatic
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–9380

saccharification are the most crucial advantages of this pretreat-
ment [19]. Solubilization of hemicelluloses and cellulose in this
method is less than in acid or hydrothermal processes [53]. This
method is also known for causing chemical swelling of fibrous
cellulose [7], in which, saponification and salvation reactions occur
which lead to the disruption the crosslinks between hemicellu-
loses and the other components, hence, increasing the porosity of
biomass [30]. More specifically, the ester bond cross-linking lignin
and xylan are disrupted leading to delignification [54]. Compara-
tively, alkaline pretreatment is operated at lower temperatures
[55] and does not require complex reactors, which are appealing to
be employed on-farm [56]. However, the major drawbacks are
long residence time (from hours to days) and the need for
neutralization of the pretreated slurry [12,57]. Sodium hydroxide
(NaOH), potassium hydroxide (KOH), calcium hydroxide (CaOH
2
),
and ammonia are mostly used in this pretreatment method. Wan
et al. [57] pretreated soybean straw with NaOH (4–40 g/100 g dry
straw) at ambient condition and achieved glucose yield of 64.55%
and xylan removal of up to 46.37%. In another study, Costal
Bermuda grass was pretreated with 0.75% NaOH solution during
15 min, and a total reducing sugar yield of 71% was achieved. In
addition, the overall conversion efficiencies for glucan and xylan
were 90.43% and 65.11%, respectively [59].
Combination of NaOH andCaOH
2
(0.10 and 0.02 g/g raw bio-
mass, respectively), were also used in a study to enhance the cost
effectiveness of alkaline pretreatment of switchgrass at ambient
condition. Biomass was first pretreated by NaOH, and the regen-
erated biomass was then pretreated by Ca(OH)
2
which led to
glucose and xylose yields of 59.4% and 57.3%, respectively [58].
4.2.3. Ionic liquid (IL) pretreatment
Ionic liquids (ILs)-based pretreatment of lignocellulosic bio-
mass has gained much attention recently and they can dissolve
carbohydrates and lignin simultaneously. ILs (melting point
o100 1C[60]), are organic salts composed of cations and anions,
typically, large organic cations and small inorganic anions [5].
In fact, cation structure (the symmetry and the length of alkyl
substituents, the presence of hydrophobic groups, etc.) and degree
of anion charge delocalization are two major factors that can affect
physical, chemical and biological properties of ILs. Overall, cations,
anions, temperature, and time used in the pretreatment process
are the major factors affecting the interaction between the ILs used
and the lignocellulosic materials [61]. Cellulose could be dissolved
by ILs containing chloride, formate, acetate or alkylphosphonate
inions by formation of strong hydrogen bonds [65]. In another
word, hydrogen bonds are formed between the non-hydrated ions
of ILs and the sugars'hydroxyl protons leading to the degradation
of the complex network of cellulose, hemicelluloses and lignin [5].
Among ILs investigated, 1-allyl-3-methylimidazolium-chloride
([AMIM]Cl), 1-ethyl-3-methylimidazolium-acetate ([EMIM]Ac), 1-
butyl-3-methylimidazolium chloride ([BMIM]Cl) and 1-ethyl-3-
methyl imidazolium diethyl phosphate ([EMIM]DEP) have recently
received much attention due to their remarkable cellulose dis-
sulotion capability [66].
In a study, pretreatment of switchgrass using [EMIM]Ac pro-
duced a glucan content ranging from 50.4% to 67.7%. This IL was
also found capable of removing 69.2% of total switchgrass lignin at
160 1C for 3 h [8]. [BMIM]Cl has also been reported as one of the
best cellulose solvent (up to 25 wt% of cellulose) [13]. In a different
study on oil palm frond (OPF) pretreatment using [BMIM]Cl,
cellulose crystallinity reduction and improved enzymatic digest-
ibility was accomplished [60]. As for lignin, 1-butyl-3-
methylimidazolium trifluoromethanesulfonate and 1,3-dimethyli-
midazolium methylsulphate have been reported as the best
solvents [67].
Although significant changes in biomass structure after ILs
pretreatment have been reported but composition of biomass
was proven to alter slightly [60]. For example, lignin, hemicellu-
lose and cellulose percentages in OPF before and after IL treatment
were measured at 27, 36, 37 wt%, and 27, 34, 39 wt%, respectively
[60]. This could be explained by the fact that after ILs pretreat-
ment, regenerated cellulose becomes amorphous and porous,
which is much more susceptible to degradation by cellulases
[62]. The ILs'advantages in comparison with the other conven-
tional methods include, less dangerous process condition and
chemicals, remaining liquid in a wide range of temperature, being
green solvents due to low vapour pressure and finally high
thermal and chemical stability [60]. Besides, ILs are non-
derivatizing [13], require mild operational conditions; and, ionic
liquids are easily recycled. On the other hand, incompatibility of IL
with cellulase leading to the inactivation and unfolding of the
enzyme is regarded as one of their most important disadvantages
[64]. Viscosity is of great importance when applying ILs as low
viscosity causes cellulose to be dissolved at low temperature thus
lowering energy consumption. Moreover, high temperatures cause
some negative side-effects, such as deteriorated stability of ILs,
and occurrence of side-reactions [63].
Application of ILs at industrial scale is faced with three major
challenges: (1) huge amounts of currently expensive ILs is needed,
(2) recycling of pure ILs is energy-intensive and, (3) during
pretreatment, solution becomes viscous that makes it difficult to
handle [68]. Fu and Mazza [68] offered a strategy to overcome
these problems which was the application of water-mixtures of
ILs. They managed to reduce the amount of ILs required which in
turn decreased viscosity and, finally IL was recycled easily as
evaporation or reverse osmosis were not necessary.
4.2.4. Organosolv pretreatment
Organic solvents i.e. methanol, ethanol, acetone, ethylene
glycol and tetrahydrofurfuryl alcohol, with or without the addition
of a catalyst agent could be used in organosolv process [70]. The
catalysts used include organic or inorganic acids (hydrochloric and
sulfuric acids), bases (sodium hydroxide, ammonia and lime) [69].
This pretreatment is capable of breaking the internal lignin and
hemicelluloses bonds and is therefore especially efficient for high
lignin lignocellulosic biomass. Moreover, relatively pure and high-
quality lignin can be obtained as a by-product through this process
[70]. Obviously, lignin removal leads to an increased surface area
making cellulose more acceceble to enzyme [71]. The main draw-
backs of this method are low-boiling point of organic solvents,
high risk of high-pressure operation, and flammability and vola-
tility of such solvents [72]. Moreover, solvents should be recycled
in order to diminish the operation cost and prevent their inhibi-
tory effects on enzymatic hydrolysis and microorganisms [30].
Koo et al. [71] treated Liriodendron tulipifera with ethanol, and
sodium hydroxide as catalyst and managed to minize the loss of
glucan. In a different investigation, Pinus radiata was exposed to
acetone–water pretreatment (acetone: water molar ratio of 1:1) at
195 1C, pH 2.0 for 5 min and ethanol yield of about 99.5% was
achieved [73].
4.2.5. Ozonolysis
This pretreatment includes using ozone gas as an oxidant in
order to break down lignin and hemicelluloses and increase
cellulose biodegradability [74]. Being a powerful oxidant, soluble
in water and available, are major advantages of ozone gas. It also
could break down lignin and release soluble compounds of less
molecular weight such as acetic and formic acid. Williams [75]
achieved lignin degradation of 49% when corn stover was broken
down by ozonolysis. His findings were later confirmed by those of
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–93 81

Garcia-cubero et al. [22] where enzymatic hydrolysis yields of up
to 88.6% and 57% were obtained compared to 29% and 16% in non-
ozonated wheat and rye straw respectively. The most important
advantage of this method is lack of degradation by-products which
leads to less complication in the subsequent hydrolysis steps [22].
Besides, this process is carried out at ambient condition. On the
other hand, the main disadvantage is the cost of ozone used as a
large amount of ozone is employed to treat lignocellulosic materi-
als [30].
4.3. Physico-chemical pretreatment
4.3.1. Steam explosion pretreatment
This is an extensively investigated thermo-mechanochemical
method which involves the break-down of structural components
by steam-heating (thermo), shearing (mechano; due to sudden
decompression and evaporation of moisture), and auto-hydrolysis
(chemical) of glycosidic bonds. More specifically, biomass particles
are heated using pressurized steam (20–50 bar, 160-270 1C) for
several seconds to a few minutes and then the pressure is released
to atmospheric pressure, condensed moisture evaporates and
desegregation of lignocellulosic matrix takes place [76,].Itis
believed that this pretreatment causes hemicellulose hydrolysis,
lignin transformation due to high temperature and increases
crystallinity of cellulose by promoting crystallization of the amor-
phous portions.
The parameters affecting steam explosion efficiency are particle
size, temperature (T) and residence time (t) and the combined
effect of both temperature and residence time is described by
severity factor (R
0
)[77]:
ðlog R
0
ÞR
0
¼Z
t
0
exp T100
14:75

dt
The hydrolysis of hemicellulose during steam explosion pre-
treatment is accomplished by organic acids such as acetic acids
generated from hydrolysis of acetyl groups associated with the
hemicellulose and formic and levulinic acids derived from other
functional groups. Water, also acts as an acid and possesses certain
acid properties at high temperature. Finally, hemicellulose
removal from the surface of cellulose microfibrils increases
enzyme accessibility and enzymatic hydrolysis rate of cellulose
by exposing the cellulose surface [78]. However, due to the
existing acidic conditions, the degradation of sugars into furfural
and HMF might also happen during the process [79].
For some lignocellulosic biomass e.g. softwood in in order to
reach high sugar yields, addition of an acid catalyst such as H
2
SO
4
or SO
2
is a prerequisite to the steam explosion process. This is
ascribed to the low content of acetyl groups in the softwood's
hemicellulose. On the other hand, this also leads to the formation
of higher amounts of inhibitory compounds which would
adversely affect the reaction rate during the fermentation process
[80]. Therefore, lignocellulosic biomass needs to be washed by
water after pretreatment in order to remove these inhibitors.
In a study, Reczey and Zacchi [81] reported that steam explo-
sion pretreatment at 200 1C for 5 min after swelling the fibers
(Corn Stover) with 2% sulfuric acid enhanced the enzymatic
conversion (50 1C, 24 h) of cellulose to glucose by nearly four
times at 80%, compared to the untreated raw materials and that
the ethanol yield of about 90% was achieved. In a different
experiment, the optimal conditions for steam explosion pretreat-
ment (with 0.9% sulfuric acid as catalyst) of wheat straw which led
to the maximum overall sugar yield of 85% of total sugars
contained in raw material were found at 180 1C and 10 min [20].
Steam explosion due to its lower capital investment and higher
energy efficiency is among the very limited number of cost-
effective pretreatment technologies for pilot scale demonstration
and commercialized applications [77].
4.3.2. Ammonia fiber explosion (AFEX) pretreatment
In AFEX, pretreatment of biomass is conducted by using liquid
ammonia and based on the steam explosion process concept. Four
parameters including ammonia loading, water loading, reaction
temperature, and residence time can be varied in order to optimize
the AFEX pretreatment [82]. The process includes high pressure
(1.72–2.06 Mpa) and moderate temperatures (60–120 1C) for several
minutes (o30 min) followed by a sudden pressure release [83].
Rapid expansion of the ammonia gas causes cleavage of lignin-
carbohydrate complex and consequent physical disruption of biomass
fibers leading to increased digestibility of biomass [84].
In contrast with steam explosion that produces a slurry, AFEX
due to the ammonia's low boiling point, produces only solid
material. It also does not liberate any sugars directly because of
low hemicelluloses solubilizetion but opens up the structure of
lignocellulosic biomass and increases polymers surface area and
consequently, the enzymatic digestibility. AFEX pretreatment has
been demonstrated to result in higher conversion rates of different
kinds of cellulosic biomass, such as aspenwood, wheat straw,
alfalfa stems [85] switchgrass [86], rice straw [87], corn stover
[88], and has been shown to be less effective on high-lignin
content biomass such as hardwood and softwood feedstocks.
Numorous studies have been conducted to determine the
optimal AFEX conditions for different biomas sources. Li et al.
[24] reported ammonia to biomass loading of 2:1, 120% moisture
(dry weight basis), 140 1C and residence time of 5 min as optimal
to convert both forage and sweet sorghum bagasse to ethanol. As
for poplar (Populus nigra) and corn stover Balan et al. [89] found
optimal conditions as 2:1 ammonia to biomass loading, 233%
moisture (dry weight basis), and 180 1C and 1:1, ammonia to
biomass loading, 60% moisture, and 90 1C, respectively. Overall, in
order to reduce the high operation cost basically due to the high
cost of ammonia as well as environmental issues, an efficient
ammonia recovery and recycling seems to be inevitable [90].
4.3.3. CO
2
explosion pretreatment
Basically, super-critical CO
2
explosion is the same as AFEX and
steam explosion pretreatments as CO
2
molecules have a similar
size property to those of water and ammonia making them
capable of penetrating into small pores of lignocellulosic material
[92]. In contrast with steam explosion, supercritical CO
2
explosion
needs lower temperature and is also less costly in comparison
with AFEX [92], making it an ideal choice among the explosion-
type methods. Besides, CO
2
explosion possesses other advantages
such as non-toxicity and non-flammability.
Super critical CO
2
(critical temperature (Tc) of 31 1C and critical
pressure (Pc) of 7.4 MPa) possesses a liquid-like density while it
exhibits gas-like transport properties of diffusivity and viscosity
[91]. On the other hand, while CO
2
pretreatment maintains the
advantages of an acid-catalyzed process by forming carbonic acid
when CO
2
is dissolved in water, but due to the specific features of
carbonic acid, it leads to significantly less corrosiveness. Further-
more, due to the easy removal of CO
2
by depressurization,
it creates no waste products and requires no further recovery [93].
Kim and Hong [94] evaluated the effect of supercritical CO
2
pretreatment on enzymatic digestibility of aspen wood and south-
ern yellow pine with a 73% (w/w) moisture content. They argued
that aspen and southern yellow pine pretreated with supercritical
CO
2
at 21.37 Mpa and 165 1C for 30 min in comparison with the
untreated ones produced higher amounts of reducing sugar (form
14.5 and 12.8% to 84.7 and 27.3%, respectively) when underwent
the enzymatic hydrolysis process.
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–9382

4.3.4. Liquid hot water (LHW)
LHW pretreatment employs high temperatures (160–220 1C)
and pressure to keep water in liquid state and in contact with
biomass for about 15 min residence time without addition of any
chemicals or catalysts. In LHW and unlike steam explosion, rapid
decompression or expansion is not required and utilization of
pressure is only for maintaining water and preventing evapora-
tion. LHW pretreatment has been shown as an efficient method for
treating different kinds of lignocellulosic material, including corn-
cobs [95], sugarcane bagasse [96], corn stover [97], wheat straw
[98] and rye straw [99]. This method is reported to be capable of
solubilizing most of the biomass hemicellulose by 480% [96], and
consequently increasing cellulose digestibility through hemicellu-
loses removal [100]. The slurry generated through the pretreat-
ment process consists of solid (enriched cellulose and water-
insoluble materials) and liquid fraction (water and most of the
solubilized hemicelluloses) and low or no inhibitors.
In order to prevent the formation of inhibitors and sugar
degradation during the LHW, pH should be controlled between
4 and 7. Laser et al. [96] optimized the pH-controlled LHW
pretreatment of corn stover (190 1C for 15 min) and achieved
maximized hemicellulose solubilization while minimizing the
formation of inhibitors. In addition, they managed to obtain
cellulose to glucose conversion rate of 90% through the enzymatic
hydrolysis. In a different study, Perez et al. [98] also employed
LHW for treating wheat straw and reported similar findings under
optimized condition when they accomplished considerable xylose
and glucose yields of 80% and 91%, respectively.
4.3.5. Wet oxidation (WO)
Oxygen or air as catalyst and water are employed in WO
pretreatment technology. This operation usually occurs at tem-
perature above 120 1C and pressures ranging between 0.5 and
2 MPa for about o30 min [101]. It has been proven that WO is an
efficient pretreatment technique for fractionation of lignocellulosic
materials by solubilization and hydrolysis of hemicelluloses and
delignification with organic acid formed during the pretreatment
process and oxidative reactions. Formation of inhibitors such as
furfural and HMF in comparison with steam explosion and LHW is
lower in the WO pretreatment. Moreover, by using an alkali in
combination with WO, increased monosaccharide sugars produc-
tion and less inhibitory compounds generation due to decreased
acidity have been reported [102].
The best WO pretreatment conditions for rice husk were
achieved in a study conducted by Banerjee et al. [103] where they
applied a reaction temperature of 185 1C and air pressure of
0.5 MPa for 15 min. They managed to maintain 66.97% of the
cellulose in the solid fraction, while achieved 89% lignin removal.
On the other hand, 69.77% of the hemicellulose was solubilized
through the WO treatment. Similarly, the findings obtained by
Martin et al. [23] found WO an appropriate method for fractionat-
ing and consequent enzymatic hydrolysis of sugarcane bagasse.
In their investigation, alkaline WO (195 1C, 15 min) resulted in the
highest yield of solid material with nearly 70% cellulose content,
solubilization of approximately 93% of hemicelluloses and 50%
lignin removal. Furthermore, an enzymatic convertibility of cellu-
lose of around 75% was accomplished. Despite of WO's advantages,
its economic application is generally ruled out due to the high
capital cost imposed by pressure equipment and the high cost of
oxygen and catalyst used.
4.4. Biological pretreatment
Unlike the chemical and physicochemical pretreatment meth-
ods, biological or microbial pretreatment has no chemical require-
ments. It is basically an environmental friendly pretreatment
converting lignocellulosic biomass by microorganisms especially
fungi into more accessible compounds for hydrolysis and subse-
quent bioethanol production [104]. In contrast to most of the
pretreatment methods that require high capital and operational
cost, this method only takes advantage of white-, brown-, soft-rot
fungi to delignify and enhance enzymatic hydrolysis of lignocellu-
losic biomasses [105]. The highest efficiency among the biological
pretreatment methods has been achieved by lignin-degrading
white-rot fungi for the soft and brown fungi only attack cellulose.
Among the known species of white-rot fungi used to date, the
highest efficiency belongs to Phanerochaete chrysosporium due to
its high growth rate and lignin biodegradation capabilities [106].
Shi et al. [103] studied the impact of white-rot fungi P. chrysospor-
ium, on cotton stalks under two different culture conditions and
reported 19.38% and 35.53% lignin degradation in submerged
cultivation (SmC) and solid state cultivation (SSC).
In biological pretreatment, particle size, moisture content,
pretreatment time and temperature could affect lignin degrada-
tion and enzymatic hydrolysis yield. Wan and Li [107] used
Ceriporiopsis subvermispora for ethanol production on corn stover
while investigating the effect of various factors on the pretreat-
ment efficiency. The findings obtained revealed up to 31.59% lignin
degradation while maintaining 94% of cellulose during an 18-d
pretreatment. In addition, the highest glucose yield of 66.61%, was
achieved at 28 1C with the moisture content 75% and the particle
size of 5 mm. Various microbial agents could also significantly
affect the pretreatment efficiency of certain biomass. Patel et al.
[108] compared a number of fungal species when different types
of lignocellulosic waste, i.e. wheat straw, rice straw, rice husk and
bagasse were used for bioethanol production. Aspergillus niger and
Aspergillus awamori led to the highest ethanol production form
wheat and rice straw. On the other hand, the best results for rice
husk and bagasse were achieved by Aspergillus awamori and
Pleurotus sajor-caju, respectively.
Despite low energy consumption, modest environmental con-
ditions and no chemical requirement, biological pretreatment still
faces some drawbacks negatively affecting its widespread applica-
tion as a commercial pretreatment method. These include long
process time, large space requirement and the need for continuous
monitoring of microorganism growth [109].
5. Combination of pretreatment methods
As mentioned earlier various pretreatment methods have some
drawbacks limiting their applications. Combined pretreatment
methods have been recently considered as a promising approach
to overcome this challenge, by increasing efficiency of sugar
production, decreasing formation of inhibitors and shortening
process time. These would collectively result in higher bioethanol
yield and more economical process.
5.1. Combination of alkaline and dilute acid pretreatments (ALK-DA)
As mentioned previously, acid pretreatment could delignify and
increase the surface area of cellulose fiber. However, single-stage
acid pretreatment with acetic acid for instance, requires much acid
(50%, based on the initial quantity of the dry materials), so to
overcome this challenge, a pre-pretreatment could be applied to
partially remove lignin prior to this step. Alkali pretreatment has
been known to be a suitable method for delignification and
therefore, could be used in combination with acid pretreatment.
In a study, sugarcane bagasse was pretreated by alkali (NaOH)-
peracetic acid (PAA) combined pretreatment under mild condi-
tions. In short, 10% NaOH (based on the initial quantity of the dry
materials) with 3:1 liquid-to-solid ratio at 90 1C for 1.5 h was
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–93 83

utilized in the first step. Then, 10% peracetic acid was employed at
75 1C for 2.5 h to delignify the regenerated biomass obtained in
the first step. As a result, the yield of reducing sugars produced
during the enzymatic hydrolysis (120 h, cellulase loading of 15
FPU/g solid) reached as high as 92.04%. Therefore, ALK-DA pre-
treatment (e.g. alkali-peracetic acid) in comparison with akali and
acid pretreatment solely, could be performed under milder con-
ditions while leading to more effective delignification and less
carbohydrates degradation [48].
5.2. Combination of alkaline and IL pretreatments (ALK-IL)
Nguyen et al. [110] reported successful pretreatment of rice
straw with the combination of ammonia and IL ([EMIM]Ac). This
method is based on delignification effect of ammonia [109] and
solubilization effect of ILs [13] in order to increase biodigestibility
of lignocellulosic materials. The cellulose recovery and glucose
conversion in this combined pretreatment has been found higher
than those of the individual ammonia or ILs pretreatments.
Moreover, higher pretreatment efficiency could be obtained by
simplifying the sample communication, reducing the processing
time for solubilization, using less enzyme amount for hydrolysis,
and increasing the IL recycling rate for reuse.
5.3. Combination of dilute acid and steam explosion pretreatments
(DA-SExp.)
The combination of dilute acid and steam explosion pretreat-
ment (DA-SExp.) could lead to enhanced saccharification effi-
ciency. Dilute-acid hydrolysis is the first step to maximize xylose
conversion and steam explosion is the second step to break down
the lignocellulosic structure. Recently, Chen et al. [30] investigated
the application of DA-SExp. on rice straw with the first step (2%
H2SO4) performed at 165 1C for 2 min followed by the second step
performed at 180 1C for 20 min. Through the two-step pretreat-
ment, a high xylose yield was obtained, and low levels of inhibitors
were recorded. Based on their findings, a total of 75.9% xylan and
77.1% glucan were converted to xylose and glucose, respectively.
5.4. Combination of supercritical CO
2
and steam explosion
pretreatments (SCCO
2
-SExp.)
Comparison of supercritical CO
2
and combined supercritical
CO
2
and steam explosion pretreatment (SCCO2-SExp.) of wheat
straw was made by Alinia et al. [91]. The experimental results
showed that the combined method conducted in two stages i.e.
the steam-injection stage (200 1C for 15 min) and the supercritical
CO
2
explosion (12 MPa, 190 1C for 60 min), was more efficient than
the supercritical CO
2
(12 MPa, 190 1C and 30 min) alone. The
reducing sugars obtained for the supercritical CO
2
and the
SCCO
2
-SExp. pre treatments were 149.1 and 234.6 g/kg wheat
straw, respectively. Interestingly, wetting the feed prior to the
supercritical CO
2
, considerably increased this value to 208.4 g/kg
wheat straw revealing the positive effect of wetting alone on the
treatment procedure.
5.5. Combination of organosolv and biological pretreatments
(bio-organosolv)
Bio-organosolv pretreatment was investigated by Monrroy
et al. [111] to evaluate the synergic effect of the combination of
the two methods on P. radiata wood chips. They used brown rot
fungus Gloephyllum trabeum for 3 weeks on P. radiata followed by
delignificatation of the biotreated material by organosolve etha-
nol:water mixture. The results obtained revealed that the same
bioethanol yield was achieved for both experiments (i.e. bio-
organosolv and organosolv alone); however, milder conditions
(ethanol: water mixture (60/40 v/v), 185 1C, 18 min) were required
when the chips had been initially biotreated in comparison to
those of the organosolv pretreatment alone (ethanol: water
mixture (60/40 v/v), 200 1C, 32 min). Simillar findings have been
reported elsewhere when beech wood was subjected to biological
pretreatment with white rot fungi i.e. Ceriporiopsis subvermispora,
Dichomitus squalens,Pleurotus ostreatus (P. ostreatus), and Coriolus
versicolor for 2–8 weeks fallowed by organosolv (ethanolysis)
pretreatment [112]. The highest bioethanol yield was accom-
plished for C. subvermispora. Moreover, the combined pretreat-
ment increased the pretreatment turnover up to 1.6 times and
decreased the electricity requirement by 15% in comparison with
the ethanolysis pretreatment alone.
These findings were also confirmed by those of Munoz et al.
[113] where P. radiata and Acacia dealbata wood chips were
biotreated with white rot fungi C. subvermispora and Ganoderma
australe prior to organosolve pretreatment by ethanol–water
mixture. The pretreatment conditions were 27 1C and 55% relative
humidity for 30 days for the biological pretreatment and 60%
ethanol solution at 200 1C and 1 h for the organosolve pretreat-
ment. The experimental results showed an increased glucan
production and reduced lignin content of theof wood chips treated
by bio-organosolve in comparison with the sole organosolve
pretreatment.
5.6. Combination of biological and dilute acid pretreatments
(bio-DA)
Generally, acid pretreatment is a suitable method to dissolve
hemicelluloses, while, biological pretreatment with fungas is
known for disrupting the lignin–hemicellulose sheath, low energy
requirements and mild environmental conditions [,]. Nevertheless,
both pretreatment methods have some drawbacks such as being
energy-intensive, environment-unfriendly and high pressure
requirements for the acid pretreatment [114] and lack of econom-
ical feasibility and long process time for biological pretreatment.
As a result, Fuying et al. [115] published a paper investigating the
combination of biological and mild acid pretreatments (Bio-DA), in
order to enhance enzymatic hydrolysis of water hyacinth. The
combined pretreatment with Echinodontium taxodii and H2SO4
successfully led to enhanced enzymatic hedrolysis, elevated levels
of reducing sugar and ethanol yield.
5.7. Combination of biological and steam explosion pretreatment
(bio-SExp.)
Taniguchi et al. [116] used four white-rot fungi to pretreat rice
straw and among the fungi used P. ostreatus was found to be most
effective on degrading lignin; however, its impact on hemicellu-
lose and cellulose degradation was low. Therefore, in order to
decrease the considerable loss of holocellulose during the long
pretreatment time (60 days) and to increase the efficiency of
pretreatment, the combination with steam explosion (1.5 MPa for
1 min) was employed. Interestingly, the combination of the two
methods reduced the pretreatment time significantly from 60 to
36 days while approximately the same net amount of glucose yield
was achieved.
5.8. Microwave-assisted alkali pretreatment (MW-ALK)
As mentioned earlier, microwave irradiations could increase
the chemical reaction rate through their synergic effect or in fact,
leads to an explosion effect among the biomass particles. In a
study, microwave-based heating was utilized instead of the con-
ventional heating to pretreat switchgrass with an alkali. Under the
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–9384

optimum temperature of 190 1C and 50 g/L solid content for
30 min treatment time, the sugar yield recorded was 58.7 g/
100 g biomass [40]. In another study, Shengdong et al. [117]
evaluated the effect of MW-ALK pretreatment (700 W) on wheat
straw, and the results were compared with those of the conven-
tional alkali pretreatment (1% NaOH). The cellulose content of 79.6
vs. 73.5%, lignin content of 5.7 vs. 7.2%, hemicellulose content of
7.8 vs. 11.2% and reaction time of 25 vs. 60 min were recorded af ter
the MW-ALK and alkali pretreatments, respectively. In conclusion,
the MW-ALK pretreatment resulted in more lignin and hemicellu-
loses removal from wheat straw and shorter pretreatment time.
Moreover, the hydrolysis rate of the combined method was higher
than that of the conventional alkali pretreatment. Similar positive
findings were previously reported by Shengdong et al. [118]
where they applied the same combined method on a different
feedstock i.e. rice straw.
5.9. Combination of dilute acid and microwave
pretreatments (DA-MW)
In a study, the impact of dilute sulfuric acid pretreatment on
sugarcane bagasse combined with microwave heating was eval-
uated [119]. In that study, 10 g of bagasse sample (on a dry basis)
was soaked in 200 ml acid sulfuric with concentration of 1.56 wt %,
Table 2
Recombinant cell-wall-deconstructing enzymes in plants.
Plant Objective Transgenic enzyme Subcellular storage
compartment
References
Arabidopsis
thaliana
Accumulation of endonuclease Acitothermus celluluolyticus E1-CAT Apoplast [139]
RBS promoter to enhancing
endonuclease accumulation
Thermotoga maritima endoglucanase Cel5A Chloroplast [140]
Xylan hydrolysis in biomass Dictyoglomus thermophilum XynA and XynB Apoplast [141]
Tobacco Xylan hydrolysis in biomass Clostridium thermocellum XynZ Cytosol, and ER [142]
Economic production of plant-derived
enzyme
C. thermocellum CelD and CelO,T. reesei EgI, SwoI, Axe1, Xyn2 and Bgl1,
Fusarium solani PelA, PelB&PelD
Chloroplast [143]
Mass production and autohydrolysis of
endoglucanase
T. maritima endoglucanase, Cel5A, and CBM6-engineered Cel5A Cytosol, apoplast, and
chloroplast
[144]
Endoglucanase expression Thermostable E2, E3 and a E3-E2 fusion Apoplast and cytosol [145]
Production of biomass hydrolyzing
enzymes
T. fusca Thermostable cell wall-degrading enzymes Chloroplast [146]
Enhancing hydrolysis of
methylglucuronoxylans
T. maritima GH10 xylanase Xyl10B Chloroplast [147]
Endoglucanase expression A. cellulolyticus E1-CAT Chloroplast [148]
Endoglucanase expression T. fusca Cel6A gene encoding an endoglucanase Chloroplast [149]
Production of multifunctional
lignocellulosic hydrolases
[150]
Enhancing cellulase activity A. celluluolyticus E1 and E1CAT Apoplast [151]
Enhancing cellulase activity A. cellulolyticus Cel5A Targeted to the cell wall [152]
Enhancing cellulase activity A. celluluolyticus E1 ER [153]
Hemicellulose hydrolysis C. thermocellum XynZ Apoplast [154]
Enhancing cellulase activity A. celluluolyticus E1 Chloroplast [153,155]
Enhancing cellulase activity Maize β-glucosidase Chloroplast [156]
Enhancing cellulase activity A. celluluolyticus E1 CAT Chloroplast [151]
Enhancing cellulase activity T. fusca E2 and E3 Cytosol [157]
Enhancing cellulase activity T. reesei CBH1 Cytosol [158]
Enhancing cellulase activity Human β-glucosidase Cytosol [159]
Hemicellulose hydrolysis C. thermocellum XynA CAT Cytosol [160]
Enhancing cellulase activity A. cellulolyticus E1 Cytosol [88]
Potato Enhancing cellulase activity A. celluluolyticus E1 Apoplast, chloroplast,
vacuole
[161]
Enhasing xylan hydrolysis Streptomyces olivaceoviridis XynB Apoplast [162]
Enhancing cellulase activity T. fusca E2 Cytosol [163]
Enhancing cellulase activity T. fusca E3 Cytosol [157]
Hemicellulose hydrolysis S. olivaceoviridis XynB Cytosol [164]
Alfalfa Enhancing cellulase activity T. fusca E2 and E3 Cytosol [157]
Rice Rice biomass hydrolysis A. celluluolyticus E1-CAT Apoplast [165]
Hemicellulose hydrolysis C. thermocellum XynA CAT Cytosol [160]
Barley Hemicellulose hydrolysis Rumen Neocallimastix patriciarum XynA Cytosol [166]
Sugar cane Sugar cane biomass hydrolysis CBH I, CBH II, EG Tissue specific
expression
[167]
Maize Hydrolysis of maize biomass A. cellulolyticus E1 ER and mitochondria [168]
Corn stover biomass hydrolysis XynA, XynB, Nasutitermes Takasagoensis EGA, A. cellulolyticus EGB,
Aspergillus niger AccA, AccB, AccA/AccB
Apoplast [169]
Hydrolysis of maize biomass A. celluluolyticus E1CAT Apoplast [170]
Hydrolysis of maize biomass A. cellulolyticus E1, T. reesei CBH I, exo-cellulase and bovine rumen
Butyrivibrio fibrisolvens cellobiase
ER, apoplast, vacuole [171]
Enhancing cellulase activity A. cellulolyticus Cel5A Targeted to the cell wall [152]
Hemicellulose hydrolysis Xylanase Chloroplasts [172]
Hydrolysis of maize biomass A. celluluolyticus E1-CAT Apoplast [173]
Sengon Hydrolysis xyloglucan bounds Poplar cellulose Apoplast [174]
AccA, AccB, AccA/B: feruloyl esterase A, B, A/B, CAT: catalytic domain, CBH1, CBHII: celluobiohydrolase, EGA and EGB: endo-β-1,4-glucanase, cel: endocellulase, E1, E2 and E3:
endoglucanases (endocellulases), ER: endoplasmic reticulum, PelA, PelB and PelD: pectate lyase, XynA, XynB and XynZ: xylanases (hemicellulases).
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–93 85

blended in a reactor and subjected to heating by microwave at
three different reaction temperatures of 130, 160, 190 1C and two
heating times of 5 and 10 min. At 190 1C, highest biomass
fragmentation and swelling as well as complete hemicellulose
degradation were achieved. However, increasing time had no
significant effect on the contents of the constituents.
5.10. Combination of IL and ultrasonic pretreatment (IL-UL)
The application of ultrasound as a pretreatment method instead
of the conventional heating pretreatment could lead to the enhance-
ment of the saccharification ratio. In a study, Ninomiya et al. [120]
evaluated the synergic effect of the combination of ultrasonic
pretreatment and different ILs i. e. [BMIM]Cl, [AMIM]Cl, [EMIM]Cl,
[EMIM]DEP, and [EMIM]Ac. In their report, the application of con-
ventional heating at 110 1C for 120 min for kenaf powders pretreated
in firsr four ILs mentioned above resulted in the cellulose sacchar-
ification ratio of about 20%. This was significantly less than the ratio
of 60–95% obtained when the conventional heating was replaced by
the ultrasonic pretreatment in the same ILs at 25 1Cfor120min.
Surprisingly, the cellulose saccharification ratio of kenaf powder in
[EMIM]Acwasashighas86%afteronly15minoftheultrasonic
pretreatment at 25 1C, compared to only 47% in the case of thermal
pretreatment in the same IL.
6. Future prospective of pretreatment; genetic manipulation
of energy crops
It has been estimated that about 18–20% of the total projected
cost for biological production of lignocellulosic ethanol can be
attributed to pretreatment, more than for any other single steps
[121,122]. Genetic and metabolic engineering could also play a
crucial role in facilitating pretreatment and hydrolysis processes
and consequently in economical production of ethanol from
lignocellulosic wastes. Currently, different omicses tools (func-
tional genomics, metagenomics, transcriptomics, proteomics and
metabolomics), high throughput sequencing and genetic engineer-
ing strategies are used to enhance pretreatment and hydrolysis of
lignocelulosic biomasses. In this section a summary of different
omices and genetic engineering (upstream) approaches used to
enhance economic plant biomass hydrolysis is reported.
Recent advances in understanding the biochemical machinery
used by different plants in cell wall structure and their biochemical
characteristics, such as cellulose, hemicelluloses and lignin offers
new avenues for the development of new biological-based processes
for biomass conversion to bioethanol at industrial scale. Whole
genome sequences of different plants, such as maize, rice, sugar
cane and barley has accelerated this process [123,124]. As discussed
earlier, cellulose-crystallinity and lignin cross linking are known as
major barriers critically making biomass pretreatment and enzyme
digestion expensive [124]. Therefore, altering plant cell wall structure
toward appropriate traits for bioethanol production, such as high
cellulose content, low lignin content and recalcitrance as well as high
hydrolase activity, is a key step for improving biomass quality. It is
important to note that because of the diversity in plant cell wall
structure and the complexity of its function, the genetic modification
of plant cell walls could unexpectedly lead to alteration of plant cell
growth and development [125].Jensenetal.,[126], studied the
genetic variation in degradability of some wheat varieties straw and
the improvement potentials through plant breeding. They showed
that degradability of cereal straw was not correlated with grain yield,
and therefore straw degradability may be improved through breed-
ing without any serious negative effects on grain yield. Also, Xie and
Peng [124] reported increasing biomass degradability of rice through
mutagenesis.
Previously, mutation breeding and marker assisted selection
methodologies have been widely used in plant improvement
programs for crop yield, plant biotic and abiotic resistance, as well
as quality. Recently, the genetic engineering of the selected energy
crops and their cellulosic biomass has been taken in account for
further increasing biomass yield and bioethanol production at
large scale. Brereton et al. [127] studied the identified quantitative
trait loci (QTL) associated with enzymatic saccharification yield in
willow varieties. They found significant natural variation in glu-
cose yields from willow stem biomass, and four enzyme-derived
glucose QTL were mapped onto chromosomes V, X, XI, and XVI,
indicating that enzymatic saccharification yields are under sig-
nificant genetic influence.
These strategies rely on genetic modification of plant cell walls
by specifically altering wall polymer inter-linking and cellulose
crystallinity, reducing lignin and phenolic acid ester levels,
increasing specific hemicelluloses contents, and adding foreign
cellulase enzymes and/or other wall proteins. To achieve the above
goals, selection of appropriate genes is an initial and crucial step,
and the related genetic manipulation approach should then be
considered [128]. Fortunately, genetic engineering of most food
crop and woody plant species, such as rice, corn, wheat, barley,
sorghum, poplar, willow, switch grass has been well established,
using either Agrobacterium tumefaciens or gene-gun-mediated
gene transfer [129]. The most important plant genetic engineering
strategies to enhance economic pretreatment and hydrolysis of
lignocellulosic biomass are presented below.
6.1. Increasing cellulose composition
One of the most important strategies to reduce the pretreatment
cost is by increasing cellulose content of plant biomass. So far, more
than 1000 genes have been found to be related to plant cell wall
biosynthesis, degradation and regulations and the search for finding
new candidate genes involved in cell wall and cellulose biosynthesis
is on-going [125]. Two major gene superfamilies cesAandcsl
involved in cellulose biosynthesis have been identified in rice, maize
and other crops, which could be considered for biomass enhance-
ment [124]. In addition to these two genes, some other genes, such as
Korrigan,Cobra and Kobito,involvedincellwallsynthesiscouldalso
be used for energy crop modification [130].Importantly,asmajor
transcription factors are identified for regulating secondary cell wall
synthesis in Arabidopsis, it is possible to directly improve quantity
and quality of biomass by altering the expression time and level of
these genes in energy crops. Coleman et al. [131] showed that
overexpression of the gene SuSy in poplar led to cellulose content
increase by 2–6% without any negative consequences on plant
growth habits. It has been suggested that the overexpression of this
gene in other plants may also significantly enhance the cellulose
contentoftheplantbiomass[124].
6.2. Reduction of plant cell wall recalcitrance and cellulose
crystallinity
One of the challenges faced during the pretreatment of ligno-
cellulosic biomasses is plant cell wall recalcitrance due to the
extreme complexity of the cell-wall matrix, which causes difficulty
in biomass degradation. The plant cell wall recalcitrance is a direct
function of cellulose crystallinity leading to plant resistance to
biotic and abiotic stresses. Plant cell wall recalcitrance also
depends on the types of hemicelluloses and the ratio of lignin
monomers [14]. There are two major hemicelluloses in grasses: β-
1,3-β-1,4-glucan and β-1,4-linked xylose backbone with single
arabinose and glucuronic acid side chains [132]. To overcome
this barrier for economic bioethanol production, it is feasible
to genetically introduce some special microorganism-derived
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–9386

cellulose binding proteins and enzymes into cell walls. Fry et al.
[133] managed to reduce recalcitrance by increasing the ratio of β-
1,3-β-1,4-glucan backbone by over-expressing cslF and cslH genes
that have been characterized to catalyze β-1,3-β-1,4-glucan bio-
synthesis. In addition to this, it was also found possible to down-
regulate three glycosyltransferase (TaGT) proteins participating in
the β-1,4-linked xylose polymer synthesis, and as result to reduce
the level of plant biomass recalcitrance [134].
On the other hand, reduced cellulose crystallinity could be
achieved by the expression of cellobiose dehydrogenase in feed-
stock crops. Previously, it has been reported that cellobiose
dehydrogenase in a crude mixture of cellulases could increase
solubility of crystalline cellulose [135].
This may be because of preventing the re-condensation of
glycosidic bonds of cellulose chains that have been nicked by
endocellulases and consequently changing the structure of cellu-
lose, hemicellulose and lignin by creating hydroxyl radicals. In
addition, a number of studies showed that transferring α-
glucosidase and β-glucosidase into plants increased the cellulose
solubility and facilitated the conversion of plant tissues into
fermentable sugars [136,137].
6.3. Production of hydrolases in plants
During the bioconversion process of lignocellulosic biomass to
bioethanol, the hydrolysis of cell wall lignocellulose is synergisti-
cally catalyzed by cellulases, including endoglucanases, exogluca-
nases and β-glucosidases [7,55,110 ]. At present, these plant cell-
wall hydrolyzing enzymes are expensively produced in microbial
bioreactors for commercial use. On the other hand, plants are
already used industrially for the production of enzymes and other
proteins, carbohydrates, lipids, industrial polymers and pharma-
ceuticals. Moreover, expertise is available for plant genetic trans-
formation, farming of transgenic crops as well as harvesting,
transporting and processing the plant matter. Cell-wall hydrolyz-
ing enzymes can potentially be produced in all feedstock crops
that are to be used for cellulosic ethanol production.
The plant-based production of these enzymes has a crucial
advantage i.e. growing transgenic plants in the field requires a
much lower energy input than that of the microbial production of
these enzymes. Since many of the cell-wall hydrolyzing enzymes
identified so far are of bacterial origin, codon alteration of the
coding region is usually necessary to ensure efficient expression in
plants; this is a straightforward procedure that is widely used for
the heterologous expression of microbial proteins in eukaryotes.
Another potential concern is misfolding of the enzymes in their
new environment [138]. Over the past years, there have been
several attempts to express microbial cellulase genes in plants, and
to determine their hydrolysis activity in the transgenic plants, such
as tobacco, maize, potato, sugar cane, barley, arabidopsis, sengon,
rice and alfalfa (Table 2)[88,139–174 ]. Interestingly, no visible side-
effect on plant growth and biomass yield was observed [124].
Gadab et al. [175] expressed the catalytic domain of the thermo-
stable 1,4-b-endoglucanase (E1) of A. cellulolyticus in corn and
proved that this crop could be used as a bioreactor for cellulose-
degrading enzymes.
When expressed in plants, accumulation of the cell-wall
hydrolyzing enzymes in subcellular compartments is preferred
over their accumulation in cytosol. When targeted for accumula-
tion in subcellular compartments, including the apoplast, vacuole,
endoplasmic reticulum, Golgi apparatus, and microbodies such as
liposomes and peroxisomes, these enzymes are more likely to
display correct folding and activity, glycosylation, reduced degra-
dation and increased stability, as compared to production and
accumulation in the cytosol [138,176]. The subcellular targeting of
heterologously-expressed hydrolyzing enzymes is important for
several reasons; first such targeting keeps the foreign enzymes
away from cytoplasmic metabolic activities, avoiding potential
damage. Secondly, it also allows higher levels of enzyme accumu-
lation and can increase enzyme stability through reduced expo-
sure to proteases. Finally, targeting can also enable better folding
of proteins in subcellular compartments where there are molecu-
lar chaperones, and keeps the cell-wall hydrolyzing enzymes away
from host cell walls.
Several microbial hydrolyzing enzymes have already been
produced in plants through subcellular targeting. However, most
previous research has been performed on tobacco and alfalfa,
which are not biofuel crops. Recently, a great deal of research
endeavors on biofuel crops, such as maize, potato, barley, rice and
sugar cane has been started. As indicated above, the cytosol might
not be an ideal location for the accumulation of heterologous
molecules because of potential interference with metabolic activ-
ities. Therefore, the apoplast has been selected in many cases,
assuming that this compartment is the most spacious and thus,
capable of accumulating large quantities of heterologous proteins
[139,172]
In addition to corn, the E1 enzyme originally obtained from
Acidothermus cellulolyticus has been produced in rice at almost 5%
plant total soluble protein (TSP), but these levels need to be
increased to about 10% TSP for complete hydrolysis without the
need for addition of microbially produced endoglucanase. It has
been shown that expressing just the catalytic domain of these
enzymes results in a higher level of expression [168]. Another
method of increasing the level of enzyme production is to
genetically engineer the chloroplast genome instead of the nuclear
genome. Because the chloroplast genome of most flowering plants
is maternally inherited, chloroplast transgenesis also provides the
benefit of transgene containment, which is important for crops
with out-crossing wild relatives. Genetic transformation of chlor-
oplast genomes is now possible in most dicotyledonous crops,
including poplar [88,165 ,173 ]. In arabidopsis, when a heterologous
fungal xylanase was targeted to either the chloroplast, the peroxi-
some or both of these compartments, the dual compartment
targeted xylanase accumulated 160% of that targeted to the
chloroplast alone and 240% of that targeted to the peroxisome
alone [168,172].
6.4. Lignin modification
Three precursors, including paracoumaryl, coniferyl and sinapyl
alcohols are involved in lignin biosynthesis. The structure of
lignins is composed of guaiacyl (35%–49%), syringyl (40%–61%)
and hydroxycinnamates (4%–15%) units. The ferulic acid and
coumaric acid are also present in plant cell walls [177,124]. The
most important factors in the level of lignocellulose biodegrada-
tion are the rate of lignins and phenolic acids esters, and the ratio
of coniferyl lignin to syringyl lignin. The rate of esterified phenolic
acids, including the ferulic and p-coumaric acids, are also key
factors which limit biodegradation of nonlignified cell walls in
grasses [178,179].
To decrease the lignin level in the lignocellulosic biomass, at
first it is necessary to select and characterize the major genes
involved in lignin biosynthesis pathways. If a gene is a house-
keeper, it should be partially silenced by RNA interference (RNAi)
technology rather than totally knocked out by antisense [124].
Several lignin metabolism key enzymes, such as cinnamate 4-
hydroxylase (C4H); hydroxycinnamoyl CoA: shikimate hydroxy-
cinnamoyl transferase (HCT); coumaroyl shikimate 3-hydroxylase
(C3H); caffeoyl CoA 3-O-methyltransferase (CCoAOMT); ferulate 5-
hydroxylase (F5H); or caffeic acid 3-O-methyltransferase (COMT),
have been characterized in dicot plants, so down regulation of
these key enzymes in order to modify the chemical structures of
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–93 87

lignin components and/or reduce plant lignin content is a poten-
tially promising way to reduce pretreatment costs in bioethanol
production [179,180 ].
The research works dedicated to change lignin features in
plants are summarized in Table 3 [155,179,181–201]. Down-
regulation of lignin biosynthesis enzymes was initially performed
using antisense oligonucleotides; however, RNAi technology has
been also used for this purpose [155]. Down-regulation of some
key enzymes, such as C3H, cytochrome P450 enzyme, cinnamyl
alcohol dehydrogenase (CAD), O-methyl transferase (OMT) in
different plants such as alfalfa, wheat, barley, corn, populous sp.,
Pinus radiata and tobacco resulted in modification of lignin residue
composition and increased in situ digestibility (Table 4).
Shifting energy from lignin biosynthesis to polysaccharide
synthesis is another strategy to reduce the lignin content of
biomass. For example, down-regulation of 4-coumarate CoA ligase
(4CL), coniferaldehyde 5-hydroxylase (CAld5H) and cinnamoyl
CoA reductase (CCR) in transgenic plants (corn, aspen, switch
Table 3
Plant genetic engineering to change lignin features.
Plant Aim Gene Reference
Wheat Lignin reduction Down regulating cinnamoyl-CoA reductase and low phytic acid [181]
Barley Lignin reduction Down regulating cinnamoyl-CoA reductase and low phytic acid [181]
Alfalfa Lignin reduction and enhance
hydrolysis
Down regulation of the caffeic acid O-methyltransferase [182]
Lignin reduction Cytochrome P450 enzyme [183]
Lignin reduction Cinnamyl alcohol dehydrogenase (CAD) [184]
Lignin reduction and increase
fermentable sugars
Cinnamate 4-hydroxylase (C4H); hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT);
coumaroyl shikimate 3-hydroxylase (C3H); caffeoyl CoA 3-O-methyltransferase (CCoAOMT); ferulate
5-hydroxylase (F5H); or caffeic acid 3-O-methyltransferase (COMT)
[185]
[179]
Lignin reduction Down-regulation of coumarate 3-hydroxylase (C3H) [155]
Lignin reduction Cinnamoyl CoA reductase (CCR) or cinnamyl alcohol dehydrogenase (CAD) [186]
Tobacco Lignin reduction Down-regulation of Leucaena leucocephala cinnamoyl CoA reductase (LlCCR) gene [187]
Biomass increase O-methyl transferase (OMT) [188]
Lignin reduction and xylose and
glucose increase
Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) [189]
Pinus
radiata
Lignin reduction Caffeoyl CoA 3-O-methyltransferase (CCoAOMT) [190]
Sweet
sorghum
Lignin reduction Down-regulation caffeoyl CoA-O-methyltransferae (CCoAOMT) [191]
Poplar Change the structure of lignin Overexpression of the ferulate 5-hydroxylase (F5H) gene [192]
Arabidobsis Change the structure of lignin Mutation in the gene encoding caffeic acid O-methyltransferase (comt) with over-expression of ferulate
5-hydroxylase (F5H1)
[193]
Change the structure Catabolic enzyme LigD [194]
Increase 5-hydroxy-guaiacyl
units
Over-expressing ferulate 5-hydroxylase and downregukating lacking caffeic acid O-methyltransferase. [195]
Switchgrass Lignin reduction Silencing (RNAi) of 4-coumarate:coenzyme A ligase (4CL) [196]
Populus sp. Lignin reduction Cinnamyl alcohol dehydrogenase (CAD) [197]
Maize Lignin reduction O-methyl transferase (OMT) [198]
Lignin reduction O-methyl transferase (OMT) [199]
Aspen Lignin reduction 4-coumarate CoA ligase (4CL) [200]
Lignin reduction CAld5H and 4CL [201]
Change the structure of lignin Catabolic enzyme LigD [194]
Table 4
Effect of different pretreatment methods on the chemical composition and structure of lignocellulosic biomass and their limitation.
Pretreatment
method
Increase
specific
surface
Cellulose de-
crystallization
Hemicellulose removal
and solubilization
Lignin
removal
Inhibitor
compounds
formation
Drawback and disadvantages
Physical ++
a
++ –––High energy consumption
Acid ++ –++ + ++ Equipment corrosion, degrading produce sugar,
neutralization of pretreated slurry
Alkaline ++ –++++/–Long pretreatment resident time, neutralization of
pretreated slurry
Ionic liquid ++ ++ + + –High cost of ionic liquid
Organosolv ++ ND ++ ++ –Recovery and recycle of solvent by evaporation, high cost
Ozonolysis ++ ND –++ –Large amount of ozone requirement, expensive process
Steam
explosion
++ –++ +/–++ Incomplete disruption of lignin–carbohydrate matrix,
formation of toxic component
AFEX ++ ++ + ++ +/–High pressure requirement, low efficiency for high lignin
content biomass, high cost of ammonia
Co
2
explosion ++ –++ –– High pressure requirement, does not affect on lignin and
hemicelloluse
Wet
oxidation
++ + ++ ++ +/–High cost of oxygen and catalyst
LHW ++ ND ++ +/–+High temperature, need to add alkaline to control PH
Biological ++ + +/–++ –Low hydrolysis rate, large space requirement, watchful
control condition of microorganism growth
a
++: high effect; +: moderate effect; +/–: low effect; N.D: not determined.
S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–9388

grass, arabidopsis) resulted in a decrease in lignin content and a
concomitant increase in xylose and glucose associated with the
cell wall [179,196]. For efficient and sustainable production of kraft
pulp and the other biomass-derived products such as bioethanol,
Ishikawa et al. [194] successfully transfered the ligD gene into
arabidopsis and hybrid aspen and managed to generate transgenic
plants whose lignin could be easily removed from hollocellulose
fraction under alkaline conditions.
6.5. Protein modules disrupting plant cell wall substrates
Some protein modules, such as expancins and swollenin, have
been recognized to disrupt plant cell-wall substrates, potentially
by increasing the accessibility and efficiency of hydrolases.
Previously, the important role of expansins in loosening the cell
wall to allow expansion and growth, and subsequently increase in
cellulose deconstruction efficiency has been documented
[202,203]. In addition, it was shown that ‘swollenin’has a
cellulose-binding domain and an expansin-like domain, which
together have a disrupting effect on crystalline carbohydrates
[204]. Transferring or over-expression of these genes in the
cellulosic biomass crops might provide another route towards
modifying cell walls and decreasing the need for expensive
pretreatment processes.
7. Conclusion
Lignocellulosic biomass as an available and cheap source is
gaining popularity as a source of fermentable sugars for liquid fuel
production. One of the most expensive steps of bioethanol
production from such biomass is pretreatment followed by enzy-
matic treatment. Extensive research has been carried out in order
to increase fermentable carbohydrate recovery, decrease inhibitors
produced from sugar degradation during pretreatment process,
diminish utilization of chemical materials and energy input,
produce valuable by-products and decrease cost of bioethanol
process. In general, pretreatment technologies are divided into
four major groups i.e. physical, chemical, physic-chemical and
biological. Although each method has some advantages, one
method could not be the choice for all types of biomass. Effects
of different pretreatment methods on the chemical composition
and structure of lignocellulosic biomass and their limitation are
presented in Table 4. Fundamental understanding of various
pretreatment technologies, different composition of biomass feed-
stock and the relationship between composition of biomass feed-
stock and pretreatment methods would significantly help in
matching the best pretreatment method/combinations for a spe-
cific biomass feedstock. On the other hand, recent advances in
functional genomics, metagenomics, genetic and metabolic engi-
neering imply that the future of economic bioethanol production
form biomass will strongly depend on achievements in artificially-
designed plants, containing high levels of cellulose while capable
of producing hydrolases.
Acknowledgment
The authors would like to thank Dr. Mohammad A. Nikbakht for
his comments on the manuscript.
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