Biotechnology Advances 29 (2011) 675685

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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Biomass pretreatment: Fundamentals toward application

Valery B. Agbor a, Nazim Cicek a, Richard Sparling b, Alex Berlin c, David B. Levin a,
a
Department of Biosystems Engineering, University of Manitoba, E2-376 EITC, Winnipeg, Manitoba, Canada R3T 5V6
b
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
c
Lignol Innovations Ltd, 1014705 Wayburne Drive, Burnaby, British Columbia, Canada, V5G 3L1

a r t i c l e i n f o a b s t r a c t

Article history: Development of sustainable energy systems based on renewable biomass feedstocks is now a global effort.
Received 12 July 2010 Lignocellulosic biomass contains polymers of cellulose, hemicellulose, and lignin, bound together in a
Received in revised form 11 February 2011 complex structure. Liquid biofuels, such as ethanol, can be made from biomass via fermentation of sugars
Accepted 9 May 2011
derived from the cellulose and hemicellulose within lignocellulosic materials, but the biomass must be
Available online 23 May 2011
subjected to pretreatment processes to liberate the sugars needed for fermentation. Production of value-
Keywords:
added co-products along-side biofuels through integrated biorenery processes creates the need for
Biomass selectivity during pretreatment. This paper presents a survey of biomass pretreatment technologies with
Cellulose emphasis on concepts, mechanism of action and practicability. The advantages and disadvantages, and the
Lignocellulose potential for industrial applications of different pretreatment technologies are the highlights of this paper.
Pretreatment 2011 Elsevier Inc. All rights reserved.
Physicochemical
Biorenery

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675
2. Bioprocessing of lignocelluloses and bre analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676
2.1. Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676
2.2. Hemicellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676
2.3. Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677
3. Biomass types, properties, and recalcitrance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677
3.1. Efcacy of pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677
3.2. Assessment of pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677
4. Pretreatment methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678
4.1. Physical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678
4.1.1. Comminution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678
4.1.2. Biological pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678
4.1.3. Chemical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678
4.1.4. Physicochemical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679
5. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683
Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683

1. Introduction or after a conversion process (ASTM 2002). The use of biomass as

Biomass has been dened to be any material, excluding fossil fuel,
which was a living organism that can be used as a fuel either directly
Corresponding author. Tel.: + 204 474 7429; fax: + 204 474 7512.
E-mail address: levindb@cc.umanitoba.ca (D.B. Levin).
source of fuel dates back to Peking Man, 460,000 to 230,000 years ago
(Binford and Ho 1985), and the use of plant biomass as a fuel for heat,
light, and food preparation has been central to the evolution of our
species (Gowlett 2006). Although such empirical forms of biomass-
derived fuels are still in use today, the bioprocessing options (aerobic

0734-9750/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.05.005
676 V.B. Agbor et al. / Biotechnology Advances 29 (2011) 675685
and anaerobic fermentation) to produce liquid transportation fuels is
of paramount importance to any sustainable energy development
scheme. The global rise in energy consumption, predicted increase in
energy demands in the near future, the depletion of low extraction
cost fossil fuel reserves, and climate change have converged to create
an urgent need to develop more sustainable energy systems based on
renewable lignocellulosic biomass.
Traditional production of bioethanol involves batch fermentation
of sugars derived from sugarcane (in Brazil) or from starch from grain
(predominantly corn in the US and eastern Canada, and wheat in the
prairie provinces of Canada), followed by ethanol recovery from the
fermentation broths using distillation (Khanal 2008). Biofuels
produced from these processes are referred to as rst generation
biofuels.
The long-term viability of grain-based ethanol production,
however, is in question (Groom et al. 2008; Searchinger et al. 2008;
Simpson et al. 2008). Issues concerning the impact of expanded corn
production for fuel ethanol in the US, for example, include poor
energy balance (energy output of the biofuel/energy inputs required
during production) and negative impacts on regional water resources,
biodiversity, and soil quality (Groom et al. 2008; Simpson et al. 2008).
Estimates suggest that in the USA, starch based ethanol output will
reach a maximum between 12 and 15 billion gallons per year, one
tenth of the projected 140 billion gallons per year, required to impact
use of petroleum in the states (Jessen 2006). Moreover, corn-based
ethanol production may actually result in increased greenhouse gas
emissions (Searchinger et al. 2008). Grain-based fuel ethanol
production also creates a moral dilemma over the use of agricultural
crops and/or land for fuel vs. food (Brown 2006; Sun and Cheng 2002).
Based on protein weights it takes anywhere from 40100 times more
enzymes to breakdown cellulose than starch, yet the cost of enzyme
production is not substantially different (Merino and Cherry 2007).
Cellulosic materials are particularly attractive as feedstocks for
biofuel production because of their relatively low cost, great
abundance, sustainable supply (Lynd et al. 2002). Cellulose is the
most abundant biopolymer on earth (O'Sullivan 1996; Zhang and
Lynd 2006) and biofuel production from cellulosic biomass has
become the major focus of intensive research and development (Lynd
et al. 1999). Raw biomass is composed mostly of cellulose, hemi-
celluloses, lignin, and proteins. Lignocellulosic material often requires
pretreatment to liberate the sugars contained within cellulose bres
embedded in the hetero-matrix of plant cell walls.
The process that converts any source of lignocellulosic biomass
from its native form, which is recalcitrant to hydrolysis with cellulase
enzyme systems, into a form which enzymatic hydrolysis is effective
is referred to as pretreatment in bioprocess engineering (Lynd et al.
2002). An ideal pretreatment step by this denition should render
lignocellulosics completely susceptible to the action of cellulases.
Various types of pretreatment have been developed, but key
bottleneck in biofuel production is the initial conversion of biomass
to sugars. New biotechnological solutions for the decomposition of
lignocellulosic biomass are required to improve the production
efciencies and reduce the costs of cellulosic biofuel production
(Lynd et al. 2008). This paper reviews developments in biomass
pretreatment technologies with emphasis on concepts, strategies, and
practicality for industrial applications.
2. Bioprocessing of lignocelluloses and bre analysis
Lignocellulosic materials consist mainly of three polymers:
cellulose, hemicellulose and lignin. These polymers are associated
with each other in a hetero-matrix to different degrees and varying
relative composition depending on the type, species and even source
of the biomass (Carere et al. 2008; Chandra et al. 2007; Fengel and
Wegener 1984). The relative abundance of cellulose, hemicellulose,
and lignin are inter alia, key factors in determining the optimum
energy conversion route for each type of lignocellulosic biomass
(Mckendry 2002). Lignocellulosic feedstocks require aggressive
pretreatment to yield a substrate easily hydrolyzed by commercial
cellulolytic enzymes, or by enzyme producing microorganisms, to
release sugars for fermentation.
2.1. Cellulose
Cellulose is the main constituent of plant cell wall conferring
structural support and is also present in bacteria, fungi, and algae.
When existing as unbranched, homopolymer, cellulose is a polymer of
-D-glucopyranose moieties linked via -(1,4) glycosidic bonds with
well documented polymorphs (Fig. 1). The degree of polymerization
of cellulose chains in nature ranges from 10,000 glucopyranose units
in wood to 15,000 in native cotton. The repeating unit of the cellulose
chain is the disaccharide cellobiose as oppose to glucose in other
glucan polymers (Desvaux 2005; Fengel and Wegener 1984). The
cellulose chains (20300) are a grouped together to form microbrils,
which are bundled together to form cellulose bres. The cellulose
microbrils are mostly independent but the ultrastructure of cellulose
is largely due to the presence of covalent bonds, hydrogen bonding
and Van der Waals forces. Hydrogen bonding within a cellulose
microbril determines straightness of the chain but interchain
hydrogen bonds might introduce order (crystalline) or disorder
(amorphous) into the structure of the cellulose (Laureano-Perez et al.
2005).
2.2. Hemicellulose
Hemicellulose is the second most abundant polymer (2050% of
Lignocellulose biomass) and differs from cellulose in that it is not
chemically homogeneous. Hemicelluloses are branched, heteroge-
nous polymers of pentoses (xylose, arabinose), hexoses (mannose,
glucose, galactose) and acetylated sugars. They have lower molecular
weight compared to cellulose and branches with short lateral chains
that are easily hydrolysed (Fengel and Wegener 1984; Saha 2003).
Hemicelluloses differ in composition. Hemicelluloses in agricul-
tural biomass like straw and grasses are composed mainly of xylan
while softwood hemicelluloses contain mainly glucomannan. In many
plants xylans are heteropolysaccharides with backbone chains of 1,4-
linked -D-xylopyranose units. In addition to xylose, xylan may
contain arabinose, glucuronic acid, or its 4-O-methyl ether, acetic acid,
ferulic and p-coumaric acids. Xylan can be extracted easily in an acid
or alkaline environment while extraction of glucomannan requires a
Fig. 1. Interconversion of polymorphs of cellulose. Cellulose I, is native or natural
cellulose. Cellulose II obtained from cellulose I by solubilisation in a solvent followed by
precipitation by dilution in water i.e. regeneration. Or by mercerization, which involves
the swelling of native cellulose bres in concentrated sodium hydroxide upon removal
swelling agent. Cellulose III1 and III11 are formed by treatment with ammonia or amines
from celluloses I and II, respectively. Polymorphs IV1 and IV11 are derived from III1 and
III11 respectively by heating to 206 C in glycerol (O'Sullivan, 1996).
 V.B. Agbor et al. / Biotechnology Advances 29 (2011) 675685
stronger alkaline environment (Balaban and Ucar 1999; Fengel and
Wegener 1984).
Among the key components of lignocellulosics, hemicelluloses are
the most thermo-chemically sensitive (Hendricks and Zeeman 2009;
Levan et al. 1990). Hemicelluloses within plant cell walls are thought
to coat cellulose-brils and it has been proposed that at least 50% of
hemicellulose should be removed to signicantly increase cellulose
digestibility. Nevertheless, severity parameters must be carefully
optimized to avoid the formation of hemicellulose degradation
products such as furfurals and hydroxymethyl furfurals which have
been reported to inhibit the fermentation process (Palmqvist and
Hahn-Hgerdal 2000a; b). For this reason, pretreatment severity
conditions are usually a compromise to maximize sugar recovery and
depending upon what type of pretreatment method is used
hemicellulose could be obtained either as a solid fraction or a
combination of both solid and liquid fractions (Chandra et al. 2007).
2.3. Lignin
Lignin is the third most abundant polymer in nature. It is present in
plant cell walls and confers a rigid, impermeable, resistance to
microbial attack and oxidative stress. Lignin is an amorphous
heteropolymer network of phenyl propane units (p-coumaryl,
coniferyl and sinapyl alcohol) held together by different linkages
(Hendricks and Zeeman 2009). Lignin is generally accepted as the
glue that binds the different components of lignocellulosic biomass
together, thus making it insoluble in water. Because of its close
association with cellulose microbrils, lignin has been identied as a
major deterrent to enzymatic and microbial hydrolysis of lignocellu-
losic biomass (Avgerinos and Wang 1983). Chang and Holtzapple,
(Chang and Holtzapple 2000) showed that biomass digestibility is
enhanced with increasing lignin removal. In addition to being a
physical barrier, the detrimental effects of lignin include: i) non-
specic adsorption of hydrolytic enzymes to sticky lignin; ii)
interference with, and non-productive binding of cellulolytic enzymes
to lignin-carbohydrates complexes; and iii) toxicity of lignin de-
rivatives to microorganisms.
Different feedstocks contain different amount of lignin that must
be removed via pretreatment to enhance biomass digestibility. The
lignin is believed to melt during pretreatment and coalesces upon
cooling such that its properties are altered; it can subsequently be
precipitated (Brownell and Saddler 1987; Converse 1993; Lynd et al.
2002). Delignication (extraction of lignin by chemicals) causes
biomass swelling, disruption of lignin structure, increases in internal
surface area, and increased accessibility of cellulolytic enzymes to
cellulose bers. Although not all pretreatments result in substantial
delignication, the structure of lignin may be altered without
extraction due to changes in the chemical properties of the lignin.
The pretreated biomass becomes more digestible than the raw
biomass even though it may have approximately the same lignin
content as non-pretreated biomass.
3. Biomass types, properties, and recalcitrance
Different types of biomass, such as woody plants, herbaceous
plants, grasses, aquatic plants, agricultural crops and residues,
municipal solid waste and manures, contain different amounts of
cellulose, hemicellulose, lignin, and extractives (Chandra et al. 2007).
Generally plant biomass contain 4050% cellulose (with exception to
a few plants, such as cotton and hemp bast-bre that are made up of
80% cellulose), 2040% hemicellulose, 2030% lignin by weight
(Chandra et al. 2007; Mckendry 2002). Biomass recalcitrance to
bioprocessing is directly related to the inherent properties of the
biomass source. Properties such as lignin content, cellulose accessi-
bility to cellulase (CAC), and cellulose crystallinity (CC) determine the
overall digestibility of the biomass.
677
The complexity of a given biomass type is reected in the
relationship between its structural and carbohydrate components. The
factors that contribute to biomass recalcitrance include: crystallinity
and degree of polymerization of cellulose; Accessible surface area (or
porosity); protection of cellulose by lignin; cellulose sheathing by
hemicellulose; and bre strength (Mosier et al. 2005a, b). It is this
variability that accounts for differences in the digestibility/hydrolysis of
a given biomass feedstock. Removal of lignin enhances biomass
digestibility up to the point where the effect of lignin present is no
longer sufcient to limit enzymatic hydrolysis or microbial digestibility
(Chang and Holtzapple 2000; Draude et al. 2001; Jeoh et al. 2007). It has
also been shown that that highly crystalline cellulose is less accessible to
cellulase attack than amorphous cellulose (Chang and Holtzapple 2000),
and that cellulose accessibility to cellulase is one of the most important
(rate limiting) factors in enzymatic hydrolysis when the affect of lignin
is minimized (Jeoh et al. 2007).
Pointing out that the difculty is not in achieving good sugar and
ethanol yields, but in obtaining good yields at low energy input, Zhu
and Pan suggested that bio-based research efforts targeting woody
biomass should be focused on upstream activity such as size reduction
and physicochemical pretreatment to improve direct microbial
utilisation of polysaccharides and sugar yields following enzymatic
saccharication (Zhu et al. 2010; Zhu and Pan 2010).
3.1. Efcacy of pretreatment
There are a number of key features for the effective pretreatment
of lignocellulosic biomass. The pretreatment process should have a
low capital and operational cost. It should be effective on a wide range
and loading of lignocellulosic material and should result in the
recovery of most the lignocellulosic components in a useable form in
separate fractions. The need for preparation/handling or precondi-
tioning steps prior to pretreatment such as size reduction should be
minimized. It should produce no or limited amounts of sugar and
lignin degradation products that inhibit the growth of fermentative
microorganisms or the action of hydrolytic enzymes, and it should have
a low energy demand or be performed in a manner that energy invested
could be used for other purposes such as secondary heating (Chandra et
al. 2007). Other features such as regeneration/cost of catalyst,
generation of higher-value lignin co-products, and obtaining hemicel-
lulose sugars in the liquid phase to reduce the need for the use of
hemicellulases in subsequent enzymatic hydrolysis forms the basis of
comparison of different pretreatment options (Galbe and Zacchi 2007;
Mosier et al. 2005b). All these features are considered in order that
pretreatment results balance against their impact cost on downstream
processing steps and the trade-off with operational cost, capital cost and
biomass cost (Lynd et al. 1996; Mosier et al. 2005b).
Many pretreatment methods have been studied, and are still in
development. It is difcult to evaluate and compare pretreatment
technologies because they involve upstream and downstream
processing cost, capital investment, chemical recycling and waste
treatment systems (Jeoh et al. 2007). However, as an integrated part
of an industrial system or biorenery, mass balance analysis can be
used to validate the pretreatment efcacy of a process with any given
feedstock. This necessitates thorough economic analysis to determine
the best pretreatment method suitable for a particular local feedstock
for an industrial process especially with regards to co-location with
existing plants where inexpensive power, steam or default treatment
is available (Aden et al. 2002).
3.2. Assessment of pretreatment
In theory, the ideal pretreatment process produces a disrupted,
hydrated substrate that is easily hydrolyzed but avoids the formation
of sugar degradation products and fermentation inhibitors. Assess-
ment of biomass pretreatment processes depends on a parameter
678 V.B. Agbor et al. / Biotechnology Advances 29 (2011) 675685
called the severity factor, which is dened as the combined effect of
temperature, acidity, and duration of pretreatment. Studies on
biomass pretreatment have used the severity factor for comparing
pretreatment results even though it does not provide an accurate
measure of the severity. In other words it is used for rough estimates
(Galbe and Zacchi 2007). Pre-tretreatment assessment is conducted
by i) analyzing the sugars (both monomers and oligomers) released in
the liquid and the carbohydrate content of the water insoluble solids
(WIS) after pretreatment. This gives the total amount of recoverable
carbohydrates; ii) enzymatic hydrolysis of either the washed or
unwashed WIS; iii) fermentation of the pretreated liquid to assess its
fermentibility directly or diluted to target concentration and
inhibition of fermenting microorganisms with respect to growth; iv)
fermentation of either the slurry or washed WIS for biofuels
production; v) assessment of additional biotechnological potential
of the pretreated fractions such as value added products. For example,
Ammonia Fibre/Freeze Explosion pretreated (AFEX) WIS can be used
as a nutritional supplement in cattle feed.
4. Pretreatment methods
A rough classication of pretreatment based on pH divides it into
acidic, alkaline and neutral pretreatments (Galbe and Zacchi 2007).
This classication is focused on chemical pretreatment only. It does
not encompass other pretreatment methods such as physical or
biological pretreatments. A generalized classication of pretreatment
methods groups them into; physical, chemical, biological and multiple
or combinatorial pretreatment. In combinatorial pretreatment
methods, physical parameters such as temperature or pressure or a
biological step are combined with chemical treatments and are
termed physicochemical or biochemical pretreatment methods. AFEX
is a good example of a physicochemical method (Sun and Cheng
2002), and Bioorganosolv is a good example of a biochemical method
for biomass pretreatment (Itoh et al. 2003). Combinatorial pretreat-
ment strategies are generally more effective in enhancing the biomass
digestibility, and often employed in designing leading pretreatment
technologies.
4.1. Physical pretreatment
4.1.1. Comminution
Coarse size reduction, chipping, shredding, grinding, milling are
amongst the different mechanical size reduction methods that have
been used to enhance the digestibility of lignocellulosic biomass
(Palmowski and Muller 1999). These treatments increase the
available specic surface area, and reduce both the degree of
polymerization (DP) and cellulose crystallinity (Sun and Cheng
2002). Harvesting and preconditioning reduces lignocellulosic bio-
mass from logs to coarse sizes of about 1050 mm, chipping reduces
the biomass size to 1030 mm while grinding and milling can reduce
the particle size to 0.22 mm. However, it has been shown that further
reduction of biomass particle size below 40 mesh (0.400 mm) has
little effect on the rates and yields of biomass hydrolysis (Chang et al.
1997). The objective of chipping is to reduce heat and mass transfer
limitations. Grinding and milling are more effective at reducing the
particle size and cellulose crystallinity than chipping probably as
result of the shear forces generated during milling. The type and
duration of milling as well as the kind of biomass determine the
increase in specic surface area, nal DP and the net reduction in
cellulose crystallinity. Vibratory ball milling is more effective than
ordinary ball milling in reducing cellulose crystallinity of spruce and
aspen chips. Disk milling which produces bres, is more efcient in
enhancing cellulose hydrolysis than hammer milling which produces
ner bundles (Zhua et al. 2009).
The energy requirements of mechanical Comminution of lignocel-
lulosic biomass depend on both biomass characteristics and the nal
particle size required. Hardwoods require more energy input than
agricultural residues (Cadoche and Lpez 1989). Size reduction is
used in most research studies of hydrolysis but with little information
provided about the characteristics of the substrate and energy
consumed during the process (Nguyen 2000; Zhu et al. 2005).
Studies have shown that milling increases biogas, bioethanol and
biohydrogen yields (Delgenes et al. 2002). Taking into account the
high energy requirement of milling on an industrial scale and the rise
in energy demands, it is unlikely that milling is still economically
feasible (Hendricks and Zeeman 2009). However, because milling can
be performed before or after chemical pretreatment, recent studies
shows that milling after chemical pretreatment will signicantly
reduce: i) milling energy consumption, ii) reduce cost of solid liquid
separation because the pretreated chips can be easily separated, iii)
eliminate energy intensive mixing of pretreatment slurries, iv) liquid
to solid ratio and v) did not result in the production of fermentation
inhibitors (Zhu et al. 2010; Zhua et al. 2009).
Other forms of physical pretreatment that have been suggested
involve the use of gamma rays (Takacs et al. 2000) that cleave the -
1,4 glycosidic bonds, thus giving a larger surface area and lower
crystallinity. This method will undoubtedly be very expensive on a
large scale with huge environmental and safety concerns.
4.1.2. Biological pretreatment
Biological pretreatments have been mostly been associated with
the action of fungi capable of producing enzymes that can degrade
lignin, hemicellulose, and polyphenols. White- and soft-rot fungi have
been reported to degrade lignocellulose material, with White-rot
being the most effective at biological pretreatment of biomass (Lee
1997; Sun and Cheng 2002). Brown-rot fungi primarily attack
cellulose, whereas white and soft-rot attack both lignin and cellulose
via the production of enzymes such as lignin peroxidases, polyphenol
oxidases, maganesse-dependent peroxidases, and laccases that
degrade the lignin. Hatakka et al. (1993, 1994) reported the selective
delignication of wood and wheat straw by selected white-rot fungi
such as; Phanerochaete chrysosporium, Phlebia radiata, Dichmitus
squalens, Rigidosporus lignosus, and Jungua separabilima. Lignin
depolymerization by these fungi takes weeks to achieve signicant
result but can be very selective and efcient (Hatakka 1994; Hatakka
et al. 1993).
The rate of biological pretreatment however, is too slow for
industrial purposes. The residence time of 1014 days, the require-
ment of careful growth conditions, and the large amount of space to
perform biological pretreatments are the disadvantages that make
this method of pretreatment less attractive on an industrial scale. The
fact that some of the carbohydrate fraction is consumed by the
microorganism could also be a disadvantage. Biological pretreatment
could be exploited as a rst step default pretreatment in combination
with another pretreatment method or on its own if the biomass has
low lignin content. Magnusson et al. (2008) reported biohydrogen
production from barley hulls contaminated by Fusarium head blight
(CBH), compared with biohydrogen production from normal barley
hulls (BH) using Clostridium thermocellum (Magnusson et al. 2008).
The rate of biohydrogen production from CBH was higher than that of
BH even though the nal yields where approximately the same,
suggesting that the cellulose in CBH was more available to the bacteria
compared than in BH. Analysing the impact of biological pretreatment
with white-rot fungi in a combine pretreatment of beech, Itoh et al.
(2003), stated that biological pretreatment saved 15% of the
electricity needed for ethanolysis of beech wood undergoing
bioorganosolv pretreatment (Itoh et al. 2003).
4.1.3. Chemical pretreatment
Some chemicals such as acids, alkali, organic solvents, and ionic
liquids have been reported to have signicant effect on the native
structure of lignocellulosic biomass (Fengel and Wegener 1984;
 V.B. Agbor et al. / Biotechnology Advances 29 (2011) 675685
Swatloski et al. 2002). Pretreatment with alkali such as NaOH, KOH, Ca
(OH)2, hydrazine and anhydrous ammonia cause swelling of biomass,
which increases the internal surface area of the biomass, and
decreases both the degree of polymerization, and cellulose crystal-
linity. Alkaline pretreatment disrupts the lignin structure and breaks
the linkage between lignin and the other carbohydrate fractions in
lignocellulosic biomass, thus making the carbohydrates in the hetero-
matrix more accessible. The reactivity of remaining polysaccharides
increases as the lignin is removed. Acetyl and other uronic acid
substitutions on hemicellulose that lessen the accessibility of enzymes
to cellulose surface are also removed by alkali pretreatments
(Chandra et al. 2007; Chang and Holtzapple 2000; Galbe and Zacchi
2007; Mosier et al. 2005b). However, most of the alkali is consumed.
Alkali pretreatment is most effective with low lignin content biomass
like agricultural residues but becomes less effective as lignin content
of the biomass increases.
Solutions of dilute (b4 wt.%) sulfuric acid, hydrochloric acid, and
phosphoric acid have been also used to hydrolyze biomass (Mcmillan
1994; Nguyen 2000; Torget et al. 1990). Concentrated acids are not
preferred because they are corrosive and must be recovered to make
the pretreatment economically feasible (Sivers and Zacchi 1995).
Dilute acid pretreatment in hydrolysis of hemicellulose to its
monomeric units, rendering the cellulose more available. Acid
pretreatment may require the use of an alkali to neutralize the
hydrolysate.
4.1.4. Physicochemical pretreatment
This category includes the vast majority of pretreatment technol-
ogies such as Steam pretreatment (or Steam Explosion), Liquid Hot
Water pretreatment, Wet Oxidation pretreatment, Ammonia Fibre/
Freeze Explosion, Ammonia Recycle Percolation, Aqueous Ammonia
pretreatment and Organosolv pretreatment. Recently the used of Ionic
Liquids and Cellulose Solvent-based Lignocellulose Fractionation have
been proposed (Zhu et al. 2006; Zhu et al. 2009). These forms of
pretreatment exploit the use of conditions and compounds that affect
the physical and chemical properties of biomass.
4.1.4.1. Steam pretreatment (SP). Concept: steam pretreatment (also
referred to as steam-explosion) is the most extensively studied and
commonly applied physicochemical method of biomass pretreatment.
SP was previously called steam explosion because of the believe that
an explosive action on the bres was necessary for them to become
hydrolysable. The term autohydrolysis has also been used as a
synonym for SP describing the changes that occur during SP (Chandra
et al. 2007; Mcmillan 1994; Saddler et al. 1993).
Process description and mode of action: in this process physically
pretreated (chipped, ground or simply raw preconditioned) biomass
is usually treated with high pressure saturated steam at temperatures
of about 160240 C and pressures between 0.7 and 4.8 MPa. The
pressure is held for several seconds to a few minutes to promote
hemicellulose hydrolysis and then released. Hemicellulose is the
predominant fraction of the carbohydrates solubilized in the liquid
phase during pretreatment, while the lignin is transformed as result of
the high temperature. The cellulose in the solid fraction becomes
more accessible hence increasing the digestibility of the lignocellu-
losic feedstock.
Hydrolysis of hemicellulose is thought to be mediated by the acetic
acid generated from acetyl groups associated with hemicellulose and
other acids released during pretreatment, that may further catalyse
the hemicellulose hydrolysis resulting in release of glucose and xylose
monomers hence the use of the term autohydrolysis (Mosier et al.
2005b; Weil et al. 1997). At high severity (270 C, 1 min) SP results in
optimal hemicellulose solubilization but lower temperature and
longer residence time (190 C, 10 min) have been shown to be more
favourable because they avoid the formation of sugar degradation
products that inhibit subsequent fermentation (Wright 1998).
679
SP can be effectively enhanced by addition of H2SO4, CO2 or SO2 as
a catalyst. The use of acid catalyst increases the recovery of
hemicellulose sugars, decreases the production of inhibitory com-
pounds and improve the enzymatic hydrolysis on the solid residue
(Mosier et al. 2005b; Sun and Cheng 2002).
Advantage of SP: the advantages of SP are that it is an attractive
process because it makes limited use of chemicals, it does not result in
excessive dilution of the resulting sugars; and it requires low energy
input with no recycling or environmental cost.
Disadvantage of SP: the disadvantages of SP are incomplete
destruction of lignin-carbohydrate matrix resulting in the risk of
condensation and precipitation of soluble lignin components making
the biomass less digestible, destruction of a portion of the xylan in
hemicellulose and possible generation of fermentation inhibitors at
higher temperatures, and the need to wash the hydrolysate, which
may decrease overall saccharication yields by 2025% of initial dry
matter due to removal of soluble sugars.
Application: SP is effective for the pretreatment of hardwoods and
agricultural residues, but less effective for softwoods, in which case
the use of an acid catalyst is especially important. SP is near
commercialization and has been tested in pilot-scale at NREL pilot
plant in Golden, SEKAB pilot plant in Sweden, the Italian steam
explosion programme in Trisaia southern Italy and by a demonstra-
tion-scale ethanol plant at Iogen in Ottawa, Canada. However,
uncatalysed SP is applied commercially in the Masonite process for
the production of bre-board and other products (Avella and Scoditti
1998; Galbe and Zacchi 2007; Mosier et al. 2005a; Mosier et al.
2005b).
4.1.4.2. Liquid hot water pretreatment (LHW). Concept: solvolysis,
hydrothermolysis, aqueous fractionation, and aquasolv are all terms
that have been used to describe the use of liquid hot water (LHW) for
biomass pretreatment. LHW pretreatment is similar to SP but uses
water in the liquid state at elevated temperatures instead of steam.
LHW pretreatment results in hemicellulose hydrolysis and removal of
lignin, rendering cellulose in the biomass more accessible while
avoiding the formation of fermentation inhibitors that occur at higher
temperatures (Yang and Wyman 2004).
Process description and mode of action: in this process, lower
temperatures (optimum between 180190 C for corn stover) and
low dry matter- about (18%) content are used leading to more poly
and oligosaccharide production. The temperature of 160190 C for
pH controlled LHW pretreatment and 170230 C have been reported
depending on the severity of the pretreatment (Bobleter 1994;
Wyman et al. 2005). Depending on the direction of the ow of the
water and biomass into the reactor, LHW pretreatment has been
performed in three types of reactor congurations; co-current,
counter current and ow through reactor conguration. The water
and biomass are brought in contact for up to 15 min at temperatures
of 200230 C. The hot water cleaves hemiacetal linkages thus
liberating acids during biomass hydrolysis, which facilitates the
breakage of ether linkages in biomass. (Antal 1996). Mosier et al.
(2005b) stated that the cleavage of O-acetly groups and uronic acid
substitutions on the hemicellulose could help or hinder LHW
pretreatment, because the release of these acids helps to catalyse
the formation and removal of oligosaccharides, or further hydrolyze
hemicellulose to monomeric sugars, which can be subsequently
degraded to aldehydes (i.e. furfural from pentoses and 5-hydroxy-
methyl furfural (HMF) from hexoses) that inhibit microbial fermen-
tation (Palmqvist and Hahn-Hgerdal 2000a). The formation of
monosaccharides and the subsequent degradation products that
further catalyze hydrolysis of cellulosic material during LHW
pretreatment can be minimized by maintaining the pH between 4
and 7 (Kohlmann et al. 1995).
The ow through reactor conguration in which hot water is
passed over a stationary bed of lignocellulose was reported by Yang
680 V.B. Agbor et al. / Biotechnology Advances 29 (2011) 675685
and Wang (2004) to be the more effective conguration for removing
hemicellulose and lignin at same severity (Yang and Wyman 2004).
High lignin solubilization impedes hemicellulose recovery (Mok and
AntalJr 1992, 1994). A catalyst such as an acid can be added making
the process similar to dilute acid pretreatment, however, catalytic
degradation of sugars results in undesirable side products. During
pretreatment the pH and pKa of water is affected by temperature, so
KOH is used to maintain the pH above 5 and below 7 to minimize the
formation of monosaccharides that are degraded to fermentation
inhibitors (Mosier et al. 2005b; Weil et al. 1998).
Advantages of LHW pretreatment: the major advantage of LHW is
that lower temperatures are used, minimizing the formation of
degradation products. This eliminates the need for a nal washing
step or neutralisation because the pretreatment solvent here is water.
The low cost of the solvent is also an advantage for large scale
application.
Disadvantages of LWH pretreatment: the amount of solubilized
product is higher, while the concentration of these products is lower
compared to SP (Bobleter 1994). Down-stream processing is also
more energy demanding because of the large volumes of water
involved.
Application: LHW pretreatment has been tested at lab scale, and pH
controlled LHW pretreatment is considered for large-scale pretreat-
ment of corn bre. Successful pretreatment of corn bre slurry in a
163 L/min reactor with a 20 min residence time has been reported
(Mosier et al. 2005a), illustrating that possibility of scaling-up LHW
pretreatment and its practicability in pretreating large quantities of
corn bre.
4.1.4.3. Dilute acid pretreatment (DA). Concept: this is method of
biomass pretreatment has received extensive research (Mcmillan
1994), both for the possibility of its application as a replacement for
enzymatic hydrolysis of cellulose and as a lignocellulosic biomass
pretreatment step to enhance biomass digestibility (Lee et al. 1999;
Nguyen 2000). As a lignocellulosic biomass pretreatment method, DA
pretreatment with high reaction rates and improved cellulose
hydrolysis has been successfully developed for biomass pretreatment
while conventional dilute acid based cellulose hydrolysis has become
an unpopular concept with little research efforts to revive its use to
hydrolyze cellulose (Esteghalian et al. 1997; Sun and Cheng 2002).
Hydrochloric acid, nitric acid, phosphoric acid, and sulfuric acid have
been tested for use in biomass pretreatment. Dilute sulfuric acid is
commonly used as the acid of choice (Kim et al. 2000; Mosier et al.
2005b; Nguyen 2000; Torget et al. 1992). Dilute sulfuric acid is mixed
with biomass to solubilize hemicellulose therefore increasing the
accessibility of the cellulose in the biomass. This mixture can be
heated directly with the use of steam as in SP, or indirectly via the
vessel walls of the reactor.
Process description and mode of action: an aqueous solution of
substrate is heated to the desired temperature and pretreated using
preheated sulfuric acid (concentrations of b4 wt.%) in a stainless-steel
reactor (Esteghalian et al. 1997; Torget et al. 1990). In this case DA
pretreatment begins upon addition of the acid. DA pretreatment is
carried at temperature ranging from 140 to 215 C. The residence time
ranges from a few seconds to minutes depending on the temperature
of the pretreatment. In another method preconditioned and physi-
cally pretreated biomass in a wire mesh is submerged in a circulating
bath of dilute sulfuric acid. The bath is then heated to desired
temperatures to effect pretreatment at different severities. A two
stage dilute acid pretreatment to maximize sugar recovery and
enhance biomass digestibility was reported by Nguyen (2000). A low
severity (low temperature, low acid concentration) DA pretreatment
was used in the rst stage to promote hemicellulose hydrolysis/
recovery and a high severity second stage was used to hydrolyze a
portion of the remaining cellulose to glucose in the second stage
(Nguyen 2000).
The acid in DA pretreatment releases oligomers and monomeric
sugars by affecting the reactivity of the biomass carbohydrate
polymers polymers. Depending on the combined severity of the
pretreatment the sugars can be converted to aldehydes such as
furfural and HMF. In a ow through DA pretreatment process, the
biomass is mixed or brought in contact with dilute sulfuric acid of less
than 0.1% to hydrolyze hemicellulose in biomass compared to the
typical 0.6 to 3.0% for DA. A Glucose yield of greater than 80%, at a
sulfuric acid equivalent of 0.05 wt.% was reported. (Mok and AntalJr
1992).
Advantages of DA pretreatment: DA pretreatment can achieve high
reaction rates and signicantly improve hemicellulose and cellulose
hydrolysis by varying the severity of the pretreatment (Sun and
Cheng 2002), so the concept of combined severity can be conveniently
applied (Nguyen et al. 2000).
Disadvantages of DA: feedstock subjected to DA pretreatment may
be a little harder to ferment because of the presence of fermentation
inhibitors (Palmqvist and Hahn-Hgerdal 2000a; b). DA pretreatment
costs more than most other physicochemical pretreatment methods,
such as SP and AFEX, especially the two-stage DA pretreatment.
Corrosion caused by DA pretreatment with sulfuric acid mandates
expensive construction material and the counter cost of nitric acid
negates the possibility of using it as a less corrosive replacement for
sulfuric acid to reduce containment cost (Brink 1994; Mosier et al.
2005b).
Application: the addition of dilute sulfuric acid to cellulosic
material has been used to commercially produce furfural which is
puried by distillation and marketed (Zeitsch 2000). DA has been
applied in the pretreatment of corn residues and short rotation woody
and herbaceous crops (Torget et al. 1990; Torget et al. 1992). The
technique is being developed by NREL for subsequent commercial
application. A potential two-stage DA pretreatment of softwoods
chips with objective of enhancing the digestibility of feedstocks with
high lignin content was proposed for large scale application (Nguyen
et al. 2000).
4.1.4.4. Ammonia bre/freeze explosion (AFEX), Ammonia recycle
percolation (ARP) and Soaking aqueous ammonia (SAA). Concept:
AFEX, ARP and SAA are alkaline pretreatment methods that use liquid
ammonia to pretreat biomass. As a physicochemical process, AFEX is
similar to SE operating at high pressure but it is conducted at ambient
(b90 C) temperatures, while ARP is carried-out at higher tempera-
tures. SAA is a modied version of AFEX utilizing aqueous ammonia to
treat biomass in a batch reactor at moderate temperatures (2560 C)
to reduce the liquid through-put during pretreatment (Kim and Lee
2005a). At ambient temperatures the duration could be up to10
60 days while at higher temperatures (150190 C) the effect of
ammonia is rapid and the duration of pretreatment is reduced to
minutes (Alizadeh et al. 2005; Kim et al. 2000).
In the AFEX, and ARP processes the lignocellulosic material is
exposed to ammonia at a given temperature and high pressure which
causes swelling and phase change in cellulose crystallinity of biomass
in addition to the alteration and removal of lignin. This increases the
reactivity of the remaining carbohydrates after pretreatment. The
pretreated biomass is easily hydrolysable with close to theoretical
yields after enzymatic hydrolysis at low enzyme loadings compared to
pretreated biomass from other PPMs (Foster et al. 2001; Holtzapple et
al. 1991; Kim and Lee 2002; Vlasenko et al. 1997).
Process description and mode of action: in AFEX pretreatment,
biomass is brought in contact with anhydrous liquid ammonia loading
in a ratio of 1:1 to 1:2 (12 kg of ammonia/kg of dry biomass) for 10
60 min at 6090 C and pressures above 3 MPa. In a closed vessel
under pressure, the biomass and ammonia mix is heated for about
30 min to the desired temperature. After holding the target
temperature for about 5 min, the vent valve is opened rapidly to
explosively relieve the pressure. The rapid release causes evaporation
 V.B. Agbor et al. / Biotechnology Advances 29 (2011) 675685
of the ammonia (which is volatile at atmospheric pressure) and a
concomitant drop in temperature of the system (Alizadeh et al. 2005;
Dale and Moreira 1982). The chemical effect of ammonia under
pressure causes the cellulosic biomass to swell, thus increasing the
accessible surface area while decrystalizing cellulose. This results in a
phase change in the crystal structure of cellulose I to cellulose III
(Mosier et al. 2005b; O'Sullivan 1996). A small amount of hemicel-
lulose is solubilized in the oligomeric form during AFEX pretreatment.
The lignin distribution in the biomass remains comparatively the
same after AFEX pretreatment, but the lignin structure is rigorously
altered resulting in increase water-holding capacity and digestibility.
The combined physical and chemical changes markedly increase the
susceptibility of the pretreated lignocellulosic biomass to subsequent
enzymatic hydrolysis (Dale et al. 1984; Dale and Moreira 1982; Galbe
and Zacchi 2007; Holtzapple et al. 1991; Kim and Lee 2005a). The mild
process conditions (temperature b100 C and pH b12) minimize the
formation sugar degradation products and fermentation inhibitors. It is
well established that AFEX pretreatment can signicantly improve the
saccharication rates of herbaceous plants, agricultural residues, and
municipal solid waste with roughly 90% of the theoretical hydrolysis of
AFEX pretreated coastal Bermuda grass (Holtzapple et al. 1992;
Meshartree et al. 1988; Vlasenko et al. 1997).
ARP is another physicochemical pretreatment that uses ammonia
percolating in a ow through mode through a packed bed reactor. In
this process aqueous ammonia (515%) passes through biomass in a
ow through column reactor at elevated temperatures (150180 C)
and a ow rate of 15 ml/min with residence time of 1090 min, after
which the ammnonia is recycled or recovered (Kim et al. 2000; Kim et
al. 2003; Yoon et al. 1995). Under these conditions, there is
simultaneous lignin removal and hemicellulose is hydrolysed, with
a concomitant decrease in cellulose crystallinity. In an attempt to
optimize the process and establish a continuous process, by dropping
the liquid ammonia throughput, Kim et al. (2006), reported a low-
liquid ARP (LLARP) using one reactor void volume with 15 wt.%
ammonia (Kim et al. 2006). The digestibility of the LLARP pretreated
biomass under optimum condition was at least 86% of the theoretical
maximum even at the lowest enzyme loading of 7.5 FPU/g glucan.
Both AFEX and ARP are effective pretreatment for herbaceous plants,
agricultural residues and MSW. ARP pretreatment is effective on
hardwoods also (Iyer et al. 1996; Kim and Lee 2005b).
Advantage of AFEX/ARP: there are several advantages to AFEX/ARP
pretreatment processes, which include i) the hydrolyzate from AFEX/
ARP is compatible with fermentation organisms without the need for
conditioning (Teymouri et al. 2005); ii) the ability to reduce, recover,
and recycle the ammonia used in both AFEX/ARP makes the
establishment of a continuous industrial process more feasible; iii)
the moderate temperatures (b100 C), pH (b12.0), and short
residence time used in AFEX mostly and ARP compared to other
physicochemical processes minimizes the formation of fermentation
inhibitors; iv) ammonia pretreatments have a high selectivity for
reaction with lignin. ARP pretreatment is an efcient and selective
delignication method reducing 7085% of corn stover lignin within
20 min of pretreatment and removing 4060% hemicellulose while
leaving the cellulose intact (Kim et al. 2003); v) ammonia is volatile
and superheated ammonia vapor at 200 C is used to strip the residual
ammonia in the pretreated biomass. The evaporated ammonia is then
withdrawn from the system by a pressure controller for recovery. This
eliminates the need for water washing step and increase total sugar
yields; vi) with close to 99% recovery, residual ammonia could serve
as a nitrogen source during fermentation; and vii) ammonia is a
widely used commodity chemical and cost about one-forth the price
of sulfuric acid on a molar basis (Kim et al. 2003) implying the AFEX/
ARP pretreatment is cheaper than DAP.
Disadvantages of AFEX/ARP: The disadvantages of the AFEX/ARP
processes include: i) they are somewhat ineffective in the pretreat-
ment of high lignin containing lignocellulosic biomass such as news
681
paper (1830% lignin), and softwoods (McMillan 1994); ii) the cost of
ammonia basically drives the process and its application on large
scale; and iii) environmental concerns with the stench of ammonia
also have a negative impact on pilot as well as industrial scale
applications.
Application: AFEX and ARP have not been reported beyond lab
scale use. It is possible however that AFEX and ARP pretreated
biomass could be used as feed for cattle serving as a source of essential
nutrients. This is a potential application of AFEX/ARP to produce value
added products for IFB.
4.1.4.5. Lime pretreatment (LP) + wet oxidative pretreatment (WOP).
Concept: lime pretreatment is a low cost alkaline physicochemical
pretreatment that has been reported to enhance the digestibility of
lignocellulosic biomass (Chang et al. 1998). The pretreatment utilizes
aqueous Ca(OH)2 at low temperatures and pressures as a pretreat-
ment agent to solubilize hemicellulose and lignin (Chang et al. 1997).
Using 0.1 g Ca(OH)2/g biomass, LP can be conducted within a wide
temperature range 25130 C. At ambient temperatures (25 C) the LP
could take weeks and at high temperatures (120C) only 2 h was
required for the pretreatment of switch grass, solubilising 26% xylan
and 2933% lignin (Chang et al. 1997).
The effectiveness of LP has been attributed to opening of acetyl
valves and lignin valves, i.e. deactylation and partial delignication
(Chang and Holtzapple 2000). Oxidative factors come into play when
oxygen is introduced at high pressures to enhance the pretreatment.
LP of wheat straw at 85 C for 3 h (Chang et al. 1998) and poplar wood
at 150 C for 6 h using 14 atm oxygen (Chang et al. 2001b) resulted in
better yield than without oxygen. WOP involves the addition of an
oxidizing agent like oxygen, water, or hydrogen peroxide to biomass
suspended in a liquid to enhance hemicellulose and lignin removal.
Advantage of LP + WOP: there are several advantages of LP and
WPO. As an alkali pretreatment, lime (calcium hydroxide) has the
additional benets of low reagent cost, safety, and ease of recovery as
calcium carbonate compared with sodium, potassium, and ammoni-
um hydroxide even though NaOH has receive the most attention
(Sharmas et al. 2002). LP performed at temperatures below 100 C
avoids the huge energy demands required to maintain high thermal
steady state conditions during pretreatment and use of pressured
vessels. Huge piles of biomass could simply be pretreated without the
need for a special vessel or using a simple design for pilot plants
(Chang et al. 1997; Wyman et al. 2005). Recalcitrant biomass is
rendered digestible as result of the combined action of the alkali and
oxygen. About 80% of the lignin in high-lignin lignocellulose material
such as hardwoods (e.g. poplar wood) is removed during oxidative LP
(Chang et al. 2001a; Wyman et al. 2005).
Disadvantage LP + WOP: there are several disadvantages to LP and
WOP. LP is not very effective for removing lignin in high lignin
biomass such as Softwood (Chang et al. 2001a). The action of lime is
slower than ammonia and most often the oxidizing agent is not very
selective as a result losses in hemicellulose and cellulose may occur.
The use of huge volumes of water in the washing step (510 g H2O/g
biomass) and the potential need for neutralisation makes down-
stream processing difcult and also increase the cost of scaling up LP.
The oxidation of lignin to other soluble aromatic compounds is a risk
due to the possibility of the formation of inhibitors (Hendricks and
Zeeman 2009).
Application: lime pretreatment has not been applied beyond lab
scale investigation. Conversely, oxidative effect of adding water, air, or
oxygen could be exploited in an industrial pretreatment process to
enhance biomass digestibility by percolating oxygen through a pile of
biomass.
4.1.4.6. Organosolv pretreatment (OP). Concept: organosolv pretreat-
ment is similar in concept to organosolv pulping but with lower
degree of delignication. OP can occur in a variety of organic or
682 V.B. Agbor et al. / Biotechnology Advances 29 (2011) 675685

aqueous-organic solvent mixture with or without a catalyst such as
HCL or H2SO4. Organic acids including oxalic acid, acetylsalicylic acid,
and salicylic acids can also be used as catalyst to the solubilisation of
hemicellulose and extraction of lignin with organic solvents or their
aqueous solutions (Sun and Cheng 2002).
Process description and mode of action: most OP are conducted at
high temperatures (100250 C) using low boiling point solvents
(methanol and ethanol), high boiling point alcohols (ethylene glycol,
glycerol, tetrahydrofurfuryl alcohol) and other classes of organic
compounds including ethers, ketones, phenols, organic acids, and
dimethyl sulfoxide (Thring et al. 1990). OP with alcohol removes
lignin extensively and results in almost complete hemicellulose
solubilisation by: i) Hydrolyzing the internal lignin bonds, as well as
the ether and 4-O-methylglucuronic acids ester bonds between lignin
and hemicellulose and ii) Hydrolyzing glycosidic bonds in hemicel-
lulose, and partially in cellulose depending on process conditions
(Zhao et al. 2009).
OP with organic acids proceeds via the dissociation of partial
hydrogen ion to accelerate delignication/hydrolysis of cellulose and
dissolution of the lignin fragments (McDonough 1993). The addition
of a catalyst is not necessary at high temperatures (N185 C) because
it is believed that organic acids from the biomass will act as catalyst to
rupture the lignin-carbohydrate complex (Duff and Murray 1996).
Delignication kinetics vary with the solvent used during pretreat-
ment. Wood delignication kinetics with aqueous ethanol occurred in
3 phases (initial, principal and residual) while delignication of sugar
cane bagasses occurred in the principal and residual phase only
(Curvelo and Pereira 1995) reecting the extent of lignin distribution
within various substrates. Solvents need to be drained and recovered
via evaporation and condensation, and recycled to reduce costs. This is
very necessary because the solvent may be inhibitory to growth of
microorganisms, subsequent enzymatic hydrolysis, and fermentation
or anaerobic digestion. Fig. 2 shows an alternative conceptual design
of biorenery platform based on the organosolv pretreatment.
Advantages of OP: OP is very a selective pretreatment method
yielding three separate fractions: dry lignin, an aqueous hemicellulose
stream, and a relatively very pure cellulose fraction (Duff and Murray
1996). The multi-product extraction phase capability of OP as a result
of the high selectivity is the most remarkable advantage of OP as part
of the IFB. Process development focused on the optimization,
integrated utilization of the biomass components is very applicable
with OP. This is recommended for process development rather than
the utilization of carbohydrates only (Zhao et al. 2009). Organosolv
lignin is sulfur free with high purity and low molecular weight. It can
be used as fuel to power pretreatment plant or further puried to
obtain high quality lignin which is used a substitute for polymeric
materials such: phenolic powders resins, polyurethane, polyisocyante
foams and expoxy resins that used for the manufacture of bioplastics
(Zhang 2008). It is the only physicochemical pretreatment method
that is very effective for the pretreatment of high-lignin lignocellulose
materials, such as soft woods. OP can be combined with other
pretreatment method to obtain a clean and effective biomass
fractionation process or a multi-stage pretreatment process for
more recalcitrant biomass as means to improve pretreatment yield.
Ethanol delignication combined with previous SE was conducted by
Hongzhang and Liying (2007) as a clean process for the wheat
fractionation (Hongzhang and Liying 2007). Other examples include
Bioorganosolv pretreatment (Itoh et al. 2003) and RASH organosolv
referring to a combination of rapid-steam hydrolysis (RASH) and
organosolv pretreatment (Rughani and McGinnis 1989). Organic
solvents are easily recovered by distillation and recycled for reuse as a
means to reduce chemical consumption and the cost of OP. OP is one
of the few pretreatments process that does not require signicant size
reduction of feedstock to achieve satisfactory cellulose conversion
making the process less energy intensive especially with the
pretreatment of woody biomass (Pan et al. 2005; Silverstein et al.
2007; Zhua et al. 2009).
Disadvantages of OP: the cost of chemicals and sometimes catalyst
makes OP more expensive than other leading pretreatment processes.
Side reactions such as acid catalyzed degradation of monosaccharides
into furfural and 5-HMF that are inhibitory to fermentation microor-
ganisms, have been associated with organosolv alcohol pretreatment.
Using volatile organic liquid at high temperature necessitates using of
containment vessels thus; no digester leaks can be tolerated due to
inherent re, explosion hazards, environmental and health and safety
concerns.

Lignin &
Hemicellulose
derived by-products
Solvent
Recirculation

Preconditioned Modified Solvent & Co-products

Colored e.g. Acetic acid
Lignocellulosic Solvent based By-product
Liquor Furfurals
Biomass Pretreatment Recovery

SHF Residual
Pretreated
SSF, SSCF Biomass &
Solid
CBP or DMC Lignin

Power Generation

Hydrogen,
Ethanol &
Methane

Fig. 2. A schematic outline of a multi-product biorenery platform based on a modied solvent based pretreatment capable of selectively purifying cellulose, solubilising
hemicelluloses and precipitating lignin; e.g. organosolv pretreatment. SHF = Separate hydrolysis and fermentation; SSF = Simultaneous saccharication and fermentation;
SSCF = Simultaneous saccharication and co-fermentation; CBP = Consolidated Bioprocessing, DMC = Direct microbial conversion. Dotted arrow represents potential power
generation options available to plants producing excess amounts of biofuels and residual biomass that could be used to generate power to run the plant over the course of time.
 V.B. Agbor et al. / Biotechnology Advances 29 (2011) 675685
Application: the organosolv process has already been applied in
organosolv pulping with a higher degree of delignication in the pulp
and paper industry. The Lignol energy process for the biorening of
wood to ethanol based on ethanol pretreatment has been well
developed and applied to various species of softwoods since 2001
(Zhao et al. 2009). The technology based on OP could also be used for
the production of other value added chemicals and liquid fuels (Arato
et al. 2005).
4.1.4.7. Carbon dioxide (CO2) explosion pretreatment (CDE). CO2
explosion involves the use of supercritical CO2 (SCCO2) under
pressure to enhance the digestibility of lignocellulosic biomass. In this
process SCCO2 is delivered to biomass placed in a high pressure
vessel (Kim and Hong 2001), or delivered at high pressure (1000
4000 psi) (Zheng et al. 1995). The vessel is heated to the desired
temperature and held for a designated length of time, usually several
minutes when conducted at high temperatures up to 200 C
(Hendricks and Zeeman 2009). CO2 penetrates the biomass at high
pressure and it is believed that once dissolved in water, CO2 will
form carbonic acid, which helps in the hydrolysis of hemicellulose.
The release of the pressurized gas results in the disruption of the
biomass native structure increasing the accessible surface area
(Zheng et al. 1995). CDE pretreatment is not effective on biomass
with no moisture content but enhances the hydrolytic yield of
moisture containing biomass with the yield increasing propor-
tionately with the moisture content of the unprocessed feedstock
(Kim and Hong 2001).
The low cost of carbon dioxide as a pretreatment solvent, no
generation of toxins, the use of low temperatures and high solids
capacity are attractive features of CDE pretreatment. However, the
high cost of equipment that can withstand high pressure conditions of
CDE pretreatment is a strong limitation to the application of this
process on a large scale. Moreover the effects on biomass carbohy-
drate components have yet to be elucidated.
4.1.4.8. Ionic liquid pretreatment (IL). A new approach on physico-
chemical pretreatment of biomass is the use of Ionic liquids (ILs). ILs
are new class of solvents with low melting points, (b100 C), high
polarities, high thermal stabilities, negligible vapour pressure, con-
sisting entirely of ions (cations and anions) (Wasserscheid and Keim
2000; Zavrel et al. 2009). Most ILs used in biomass fractionation are
imidazonium salts. Studies show that 1-allyl-3-methylimidazonium
chloride (AMIMCl) and 1-butyl-3-methylimidazonium chloride
(BMIMCl) can be used effectively as a non-derivatising solvent for
the dissolution of cellulose at temperatures below 100 C (Zhang and
Lynd 2006; Zhu et al. 2006). A possible dissolution mechanism
suggests that these ionic liquids compete with lignocellulosic
components for hydrogen bonding, thus disrupting its three di-
mensions network (Moultrop et al. 2005).
The cellulose fraction can be recovered by the addition of water,
ethanol or acetone. The solvent can be recovered and reused by
various methods such as pervaporation, reverse osmosis, salting out
and ionic exchange. ILs are being screened for their potential to
enhance the digestibility of lignocellulosic biomass (Zavrel et al.
2009). In general ILs are considered be environmentally friendly (Pu
et al. 2007), nonderivatising, nonvolatile, thermostable, single-
component solvent for cellulose with potential applications in
cellulose fractionation and dissolution in the IFB. However, details of
processing lignocellulosic biomass with ionic liquids are still under
investigation. Cellulose and organic-solvent-based lignocellulose
fractionation (COSLIF) and sulphite pretreatment to overcome
recalcitrance of lignocellulosics (SPORL) are amongst pretreatment
methods being developed in coordinated efforts to overcome biomass
recalcitrance.
683
5. Conclusion
Biomass pretreatment remains a key bottleneck in the bioproces-
sing of lignocellulosics for biofuels and other bioproducts. Although
some pretreatment methods show apparent advantages, it is unlikely
that one method will become the method of choice for all biomass, at
least, not for all feedstocks. Co-digestion of non-food cellulosic
biomass feedstocks from different pretreatment technologies as a
substrate quality optimization attempt is worth investigating.
Bioprocessing of pretreated feedstocks without complete saccchar-
ication to sugar monomers is advantageous for direct microbial
conversion employing cellulolytic microorganisms. Such cellulolytic
members of the genus Clostridium, cellulolytic yeasts, and recombi-
nant microorganisms bearing natural and engineered celluloytic
genes are being developed as candidates for consolidated bioproces-
sing (CBP). These microorganisms thrive on cellulodextrins, but
exhibit limited growth in the presence of sugar monomer substrates
such as glucose (Lynd et al. 2005; Zhang and Lynd 2005). Under these
conditions, complete sacccharication is a no longer necessary thus
reducing the overall processing cost.
Hence, biorening interests dene the extent of pretreatment for
any given feedstock. Like most established industrial processes, a
possible key step towards bioprocessing of lignocellulosics on an
industrial scale is nding or creating markets for by-products of
biomass pretreatment technologies. Multiple or combinatorial pre-
treatments have the ability to enhance biomass digestibility and
operate under various conditions to maximise selective product
recovery, while minimizing the formation of inhibitory carbohydrate
degradation products.
The classication of pretreatment as the second most expensive
unit cost in the conversion of lignocellulosic biomass to ethanol (NREL
2002), calls for analysis of pretreatment process dynamics and their
by-products as means to reduce cost in designing a cost competitive
process. In Canada, softwood is pretreated by Lignol Innovations
(Burnaby, British Columbia) via OP, extracting high quality lignin for
the production of value-added products, such as resins, adhesives, and
coatings currently derived from petroleum rening. The by-products
of the Lignol Innovation OP process are delignied wood bers and a
hemicellulose hydrolysate, which can be used for the production of
biofuels and other value added products, respectively Fig. 2. In the
USA, AFEX pretreated spent grains and lignocellulosics are being
considered as feed for livestock at DOE bioenergy research centres.
Although industrial production of biofuels has preceded a detail
understanding of pretreatment, optimisation of integrated biorening
processes requires intense coordinated research that will, delineate
pretreatment chemistries and their impacts on feedstocks as well as
fermentation yields of fuels and co-products.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
This is supported by the Natural Sciences and Engineering
Research Council of Canada (NSERC) through a Strategic Network
grant (Hydrogen Canada), and by Husky Energy through an NSERC
Collaborative Research and Development (CRD) grant fund.
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