Bioresource Technology 100 (2009) 1018

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Pretreatments to enhance the digestibility of lignocellulosic biomass

A.T.W.M. Hendriks *,1, G. Zeeman 1
Wageningen University, Department of Agrotechnology and Food Sciences, Sub-Department of Environmental Technology, P.O. Box 8129, 6700 EV Wageningen, The Netherlands

a r t i c l e

i n f o

Article history:
Received 11 July 2006
Received in revised form 18 May 2008
Accepted 20 May 2008
Available online 2 July 2008
Keywords:
Pretreatment
Lignocellulose
Digestibility
Fermentation
Bioenergy

a b s t r a c t
Lignocellulosic biomass represents a rather unused source for biogas and ethanol production. Many factors, like lignin content, crystallinity of cellulose, and particle size, limit the digestibility of the hemicellulose and cellulose present in the lignocellulosic biomass. Pretreatments have as a goal to improve the
digestibility of the lignocellulosic biomass. Each pretreatment has its own effect(s) on the cellulose, hemicellulose and lignin; the three main components of lignocellulosic biomass. This paper reviews the different effect(s) of several pretreatments on the three main parts of the lignocellulosic biomass to improve its
digestibility. Steam pretreatment, lime pretreatment, liquid hot water pretreatments and ammonia based
pretreatments are concluded to be pretreatments with high potentials. The main effects are dissolving
hemicellulose and alteration of lignin structure, providing an improved accessibility of the cellulose for
hydrolytic enzymes.
2008 Elsevier Ltd. All rights reserved.

1. Introduction

2.1. Cellulose

For a long time research is being done to enhance the digestibility of lignocellulosic biomass for mainly the efcient conversion of
(hemi-) cellulose to ethanol, methane and, in the last years, also to
hydrogen. It is however not clear which characteristics of the lignocellulosic biomass are important, to determine a successful pretreatment. Further more additional problems, like production of
recalcitrant or inhibitory products, are to be solved.
A lot of literature is written about different pretreatment methods
to enhance the digestibility of lignocellulosic material. The objective
of this review is to nd out which characteristics of lignocellulosic
biomass determine which pretreatment method will be successful
and attractive to apply. This will be done by explaining the composition of lignocellulosic material, giving an overview of the methane
and ethanol production process, summarizing the effects of different
pretreatment methods on lignocellulosic biomass and the consequences of these effects on ethanol and methane production. Moreover, additional problems will be analyzed, and nally, conclusions
with respect to promising pretreatment techniques and needed future research are made. Hydrogen production is left out in this paper,
because it is still in the R&D phase (Reith et al., 2003).

Cellulose exists of D-glucose subunits, linked by b-1,4 glycosidic

bonds (Fengel and Wegener, 1984).
The cellulose in a plant consists of parts with a crystalline
(organized) structure, and parts with a, not well-organized, amorphous structure. The cellulose strains are bundled together and
form so called cellulose brils or cellulose bundles. These cellulose
brils are mostly independent and weakly bound through hydrogen bonding (Laureano-Perez et al., 2005).

2. The composition of lignocellulosic material

Lignocellulosic material consists of mainly three different types
of polymers, namely cellulose, hemicellulose and lignin, which are
associated which each other (Fengel and Wegener, 1984).
* Corresponding author. Tel.: +31 648758229; fax: +31 246412268.
E-mail address: Hendriks.Alexander@gmail.com (A.T.W.M. Hendriks).
1
Tel.: +31 317483339; fax: +31 317482108.
0960-8524/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2008.05.027

2.2. Hemicellulose
Hemicellulose is a complex carbohydrate structure that consists
of different polymers like pentoses (like xylose and arabinose),
hexoses (like mannose, glucose and galactose), and sugar acids.
The dominant component of hemicellulose from hardwood and
agricultural plants, like grasses and straw, is xylan, while this is
glucomannan for softwood (Fengel and Wegener, 1984; Saha,
2003).
Hemicellulose has a lower molecular weight than cellulose, and
branches with short lateral chains that consist of different sugars,
which are easy hydrolyzable polymers (Fengel and Wegener,
1984). Hemicellulose serves as a connection between the lignin
and the cellulose bers and gives the whole cellulosehemicelluloselignin network more rigidity (Laureano-Perez et al., 2005).
The solubility of the different hemicellulose compounds is in
descending order: mannose, xylose, glucose, arabinose, and galactose. The solubilitys increase with increasing temperature. The solubilitys of higher molecular polymers could not be predicted,
because of unknown melting points (Gray et al., 2003). The solubilization of hemicellulose compounds into the water starts around

A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 1018

180 C under neutral conditions according to Bobleter (1994). Garrote et al. (1999) however mention that already from 150 C parts
of the hemicellulose solubalize. The solubilization of lignocellulose
components not only depends on temperature, but also on other
aspects like moisture content and pH (Fengel and Wegener, 1984).
The xylan of hemicellulose can be extracted quite well in an
acid or alkaline environment, while glucomannan can hardly be
extracted in an acid environment and needs a stronger alkaline
environment than xylan to be extracted (Balaban and Ucar, 1999;
Fengel and Wegener, 1984; Lawther et al., 1996b). Xylan appears
to be the part that can be extracted the most easily.
Of cellulose, hemicellulose and lignin the hemicelluloses are the
most thermal-chemically sensitive (Levan et al., 1990; Winandy,
1995). During thermalchemical pretreatment rstly the side
groups of hemicellulose react, followed by the hemicellulose backbone (Sweet and Winandy, 1999).

11

the hydrolysis step. The conversion of (hemi) cellulose to monomeric sugars can be done chemically by acids or enzymatically
by addition of cellulases (enzymes responsible for the hydrolysis
of cellulose). The produced monomeric hexoses (six carbon sugars)
can be fermented to ethanol quite easily, while the fermentation of
pentoses (ve carbon sugars) is only done by a few strains. Volatile
products are also not easily fermented to ethanol. A problem
occurring during the fermentation is that the formed product ethanol is an inhibitor for the yeasts/bacteria that perform the fermentation. This puts a limit to the concentration of fermentable
sugars. Latter is not the case for methane production as methane
escapes from the water phase. After the fermentation the ethanol
has to be recovered from the fermentation broth by distillation
(Mosier et al., 2005b).
Furfural and other inhibitors like (probably) soluble lignin compounds also form a problem for the fermentation step, because
such compounds can inhibit, or even stop the fermentation (Laser,
2001; Laser et al., 2002).

2.3. Lignin
Lignin is, after cellulose and hemicellulose, one of the most
abundant polymers in nature and is present in the cellular wall.
It is an amorphous heteropolymer consisting of three different
phenylpropane units (p-coumaryl, coniferyl and sinapyl alcohol)
that are held together by different kind of linkages. The main purpose of lignin is to give the plant structural support, impermeability, and resistance against microbial attack and oxidative stress.
The amorphous heteropolymer is also non-water soluble and
optically inactive; all this makes the degradation of lignin very
tough (Fengel and Wegener, 1984).
Lignin, just like hemicellulose, normally starts to dissolve into
water around 180 C under neutral conditions (Bobleter, 1994).
The solubility of the lignin in acid, neutral or alkaline environments depends however on the precursor (p-coumaryl, coniferyl,
sinapyl alcohol or combinations of them) of the lignin (Grabber,
2005).
3. Methane production by anaerobic digestion
The production of methane from lignocellulosic material can
consist of three phases, namely pretreatment, anaerobic hydrolysis
and methane production, and post-treatment of the liquid fraction.
A product separation step is not needed during the methane production step, because methane is, under normal conditions, a gas
and will separate itself from the liquid fraction.
Pretreatment can be done to improve the hydrolysis yield and
total methane yield. The hydrolysis of the lignocellulose and
conversion to methane is in general done in one reactor by a consortium of micro-organisms. Often only the second phase, hydrolysis and methane production is executed, for energy production and
recovery.
The advantage of using a mixture of micro-organisms is that almost all products, like pentoses, hexoses, volatile products and
even sometimes inhibiting compounds like furfural and soluble lignin compounds (in not too high concentrations) can be converted
to methane after a period of adaptation (Benjamin et al., 1984;
Fox et al., 2003; Noike and Niigata Eng., 2001).
4. Ethanol production by fermentation
The production of ethanol from lignocellulosic material consists
of mainly ve different steps, namely pretreatment, (enzymatic)
hydrolysis, fermentation, product separation, and post-treatment
of the liquid fraction. The pretreatment is necessary to improve
the rate of production and the total yield of monomeric sugars in

5. Factors limiting the hydrolysis

The (enzymatic) hydrolysis of lignocellulose is limited by several factors. Several researchers conclude that crystallinity of cellulose is just one of the factors. Other factors are degree of
polymerization (DP), moisture content, available surface area and
lignin content (Chang and Holtzapple, 2000; Koullas et al., 1992;
Laureano-Perez et al., 2005; Puri, 1984). Chang and Holtzapple
(2000) however mention that crystallinity affects the 1-h enzymatic hydrolysis, but not the 3-d enzymatic hydrolysis. Cauleld
and Moore (1974) mention that decreased particle size and increased available surface rather than crystallinity affect the rate
and extent of the hydrolysis. Zhang and Lynd (2004) mention that
a slower conversion of crystalline cellulose as compared to amorphous cellulose, would increase the percentage crystallinity of
the hydrolyzed biomass. This trend however has not been conrmed by research (Zhang and Lynd, 2004). Other researchers like
Grethlein (1985), Grous et al. (1986) and Thompson et al. (1992)
conclude that the pore size of the substrate in relation to the size
of the enzymes is the main limiting factor in the enzymatic hydrolysis of (lignocellulosic) biomass. Removal of hemicellulose increases the mean pore size of the substrate and therefore
increases the probability of the cellulose to get hydrolyzed (Gregg
and Saddler, 1996; Grethlein, 1985; Palonen et al., 2004). Drying of
pretreated lignocellulose can cause a collapse in pore structure,
resulting in a decreased (enzymatic) hydrolysability (Grous et al.,
1986). Zhang and Lynd (2004) mention that cellulases can get
trapped in the pores if the internal area is much larger than the
external area, which is the case for many lignocellulosic biomasses.
Lignin limits the rate and extend of (enzymatic) hydrolysis by acting as a shield, preventing the digestible parts of the substrate to be
hydrolyzed (Chang and Holtzapple, 2000).
6. Mechanical pretreatment
6.1. Process description and mode of action
Milling (cutting the lignocellulosic biomass into smaller pieces)
is a mechanical pretreatment of the lignocellulosic biomass. The
objective of a mechanical pretreatment is a reduction of particle
size and crystallinity. The reduction in particle size leads to an increase of available specic surface and a reduction of the degree of
polymerization (DP) (Palmowski and Muller, 1999). The milling
causes also shearing of the biomass.
The increase in specic surface area, reduction of DP, and the
shearing, are all factors that increase the total hydrolysis yield of

12

A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 1018

the lignocellulose in most cases by 525% (depends on kind of biomass, kind of milling, and duration of the milling), but also reduces
the technical digestion time by 2359% (thus an increase in hydrolysis rate) (Delgens et al., 2002; Hartmann et al., 1999). A particle
size reduction below 40 mesh however has little effect on the
hydrolysis yield as well as hydrolysis rate of the biomass (Chang
and Holtzapple, 2000).
6.2. Consequences for ethanol and methane production
Milling causes both an increased methane (525%; Delgens et
al., 2002) and ethanol yield and also increases the hydrolysis rate.
As no production of inhibitors (like furfural and HMF (hydroxymethylfurfural)) are produced, milling is suited for both methane
and ethanol production. It has however a high energy requirement
(Cowling and Kirk, 1976; Ramos, 2003) and was found therefore
not economically feasible as pretreatment in 1987 (Fan et al.,
1987). Taking into account the high energy requirements of milling
and the continuous rise of the energy prices, it is likely that milling
is still not economically feasible.

7. Thermal pretreatment
7.1. General thermal processes in lignocellulose
During this pretreatment the lignocellulosic biomass is heated.
If the temperature increases above 150180 C, parts of the lignocellulosic biomass, rstly the hemicelluloses and shortly after that
lignin, will start to solubalize (Bobleter, 1994; Garrote et al., 1999).
The composition of the hemicellulose backbone and the branching
groups determine the thermal, acid and alkali stability of the hemicellulose (see Section 2.2). From the two dominant components of
hemicelluloses (xylan and glucomannan), the xylans are thermally
the least stable, but the difference with the glucomannans is only
small. Above 180 C an exothermal reaction (probably solibilization) of the hemicellulose starts (Beall and Eickner, 1970; Domansky and Rendos, 1962). This temperature of 180 C is probably just
an indication of the temperature at which an exothermal reaction
of the hemicellulose starts, because the thermal reactivity of lignocellulosic biomass depends largely on its composition (Fengel and
Wegener, 1984; Hon and Shiraishi, 1991).
During thermal processes a part of the hemicellulose is hydrolyzed and forms acids. These acids are assumed to catalyze the further hydrolysis of the hemicellulose (Gregg and Saddler, 1996). Liu
and Wyman (2003) and Zhu et al. (2004, 2005) conclude that other,
so far unknown, factors than the catalyzing effect of in situ formed
acids play a role in the solubilization of hemicellulose.
Thermal pretreatment with temperatures of 160 C and higher,
causes, besides the solubilization of hemicellulose, also the solubilization of lignin. The produced compounds are almost always phenolic compounds and have in many cases an inhibitory or toxic
effect on bacteria, yeast and methanogens/archae (Gossett et al.,
1982). These soluble lignin compounds are very reactive and will,
if not removed quickly, recondensate en precipitate on the biomass
(Liu and Wyman, 2003). Especially too severe pretreatment conditions promote the condensation and precipitation of soluble lignin
compounds, sometimes even with soluble hemicellulosic compounds like furfural and HMF (Bobleter and Concin, 1979; Lora
and Wayman, 1978; Negro et al., 2003).
Heat pretreatment in which soluble (hemi) cellulose and lignin compounds are formed, has a risk of formation of phenolic
and heterocyclic compounds, like vanillin, vanillin alcohol, furfural and HMF, especially in acidic environments (Ramos, 2003).
These formed compounds can be inhibitory. When the solid concentration was 3% or higher and the temperature exceeded

220 C during a 2 min pretreatment time, ethanol production

was almost completely inhibited, due to formation of furfural
and other compounds (probably soluble lignin compounds)
(Laser et al., 2002).
Temperatures of 250 C and higher should be avoided during
pretreatment, as unwanted pyrolysis reactions start to take place
at such temperatures (Brownell et al., 1986).
According to Weimer et al. (1995) thermal pretreatment can
also cause an increase of the Crystallinity Index (CrI) of cellulose,
though no increase was observed when the CrI was already high.
7.2. Process description and mode of action
7.2.1. Steam pretreatment/steam explosion (ST/SE)
During steam pretreatment the biomass is put in a large vessel and steam with a high temperature (temperatures up to
240 C) and pressure, is applied for a few minutes. After a set
time, the steam is released and the biomass is quickly cooled
down. The objective of a steam pretreatment/steam explosion
is to solubalize the hemicellulose to make the cellulose better
accessible for enzymatic hydrolysis and to avoid the formation
of inhibitors.
The difference between steam pretreatment and steam
explosion pretreatment is the quick depressurization and cooling down of the biomass at the end of the steam explosion pretreatment, which causes the water in the biomass to explode.
In this article only steam pretreatment will be discussed, as
the impact of the change in structure of the biomass, caused
by the explosion, on the digestibility is still doubted (Brownell
et al., 1986).
During steam pretreatment parts of the hemicellulose hydrolyze and form acids, which could catalyze the further hydrolysis
of the hemicellulose. This process, in which the in situ formed acids
catalyze the process itself, is called auto-cleave steam pretreatment. The role of the acids, is probably however not to catalyze
the solubilization of the hemicellulose, but to catalyze the hydrolysis of the soluble hemicellulose oligomers (Bobleter et al., 1991;
Mok and Antal, 1992).
A common term used in steam pretreatment is the so called
severity factor (log R0), which is a measure for the severity of
the pretreatment. In this severity factor the temperature of the
pretreatment and the duration of the pretreatment are combined
in the following way:
log R0 = log(t  e((T 100)/14.75)); with t in minutes and T in degrees Celsius (Overend and Chornet, 1987).
During steam pretreatment the moisture content of the biomass
inuences the needed pretreatment time. The higher the moisture
content, the longer the optimum steam pretreatment times (Brownell et al., 1986).
Low pressure steam pretreatment (2 bars, 120 C, and pretreatment times up to 300 min) did not have a large effect on the composition of the wheat straw according to Lawther et al. (1996a),
though no enzymatic conversion step was carried out for determining the effect on the digestibility.
7.2.2. Liquid hot water (LHW)
A different thermal pretreatment is the liquid hot water
pretreatment. In this case liquid hot water (LHW) is used instead of steam. The objective of the liquid hot water is to solubalize mainly the hemicellulose to make the cellulose better
accessible and to avoid the formation of inhibitors. To avoid
the formation of inhibitors, the pH should be kept between 4
and 7 during the pretreatment. Maintaining the pH between 4
and 7 minimizes the formation of monosaccharides, and therefore also the formation of degradation products that can further
catalyze hydrolysis of the cellulosic material during pretreat-

A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 1018

ment (Kohlmann et al., 1995; Mosier et al., 2005a; Weil et al.,

1997). If catalytic degradation of sugars occurs it results in a
series of reactions that are difcult to control and result in
undesirable side products. By keeping the pH between 4 and
7 the autocatalytic formation of fermentation inhibitors are
avoided during the pretreatment.
A difference between the LHW and steam pretreatment is the
amount and concentration of solubilized products. In a LHW pretreatment the amount of solubilized products is higher while the
concentration of these products is lower compared to steam pretreatment (Bobleter, 1994). This is probably caused by the higher
water input in LHW pretreatment compared to steam pretreatment. The yield of solubilized (monomeric) xylan is generally also
higher for LHW pretreatment; though this result diminishes when
the solid concentration increases, because (monomeric) xylan is
then further degraded by hydrolytic reactions to, for example, xylose and furfural (Laser et al., 2002).
Yang and Wyman (2004) discovered that ow through systems removed more hemicellulose and lignin from corn stover
than batch systems did, at the same severity factors. Moreover,
the addition of an external acid during the ow through process
caused higher hemicellulose and lignin removal, while in batch
tests, the addition of an external acid caused less lignin removal;
at increased reaction times at temperatures above 200 C Klasonlignin (acid-insoluble lignin) removal decreased. It was concluded that the external acid caused the lignin to solubalize
faster, but also to condensate faster. The higher hemicellulose
and lignin removal with the addition of an external acid during
ow through experiments is in conict with the conclusions of
Bobleter et al. (1991), Jacobsen and Wyman (2002) and Liu and
Wyman (2003), which state that acids have no real effect or
are not the only factor affecting the solubilization of hemicellulose and lignin and that there should be other reasons for the
solubilization of hemicellulose and lignin during ow through
experiments.
7.3. Consequences for ethanol and methane production
7.3.1. Steam pretreatment/steam explosion
Steam pretreatment includes a risk on production of compounds, like furfural, HMF, and soluble phenolic compounds. These
compounds are inhibiting the ethanol fermentation and methane
production. The methane producing bacteria are however capable
of adapting, at least to a certain concentration, to such compounds.
The adaption to these compounds, and sometimes even conversion,
by anaerobic bacteria after a certain period, is demonstrated by
Benjamin et al. (1984), Fox et al. (2003), and Noike and Niigata
Eng. (2001). Grous et al. (1986) reported a six-fold increase in enzymatic digestibility of the biomass after steam pretreatment. A way
to minimize the hemicellulose degradation during steam pretreatment is by separating the biomass from the condensate during the
pretreatment (Allen et al., 2001), by keeping the pH between 5 and
7 during the pretreatment by the addition of an external alkali (Li et
al., 2005; Weil et al., 1998), or by applying a two-step steam pretreatment. It is however not clear if the higher ethanol or methane
yield out weighs the additional costs of a second pretreatment step
(Shahbazi et al., 2005; Sderstrm et al., 2002).
The positive effect of steam pretreatment is mostly due to removal of a large part of the hemicellulose, causing an increase of
cellulose ber reactivity, probably because the cellulose is easier
accessible for the enzymes (Converse et al., 1989; Grohman et al.,
1986; Laser et al., 2002).
Some yeast species can also convert pentoses to ethanol (Kuyper et al., 2005). Consequently the degradation of pentoses to, for
example, furfural during the steam pretreatment results in a loss
of carbon for the ethanol production.

13

Steam pretreatment includes a risk on condensation and

precipitation of soluble lignin components, making the biomass less
digestible, reducing the ethanol as well as the methane production.
7.3.2. Liquid hot water
Liquid hot water has the major advantage that the solubilized
hemicellulose and lignin products are present in lower concentrations, when compared to steam pretreatment, due to higher water
input. Due to these lower concentrations the risk on degradation
products like furfural and the condensation and precipitation of
lignin compounds is reduced. Weil et al. (1998) had a 2- to 5-fold
increase in enzymatic hydrolysis of their substrate after LHW
pretreatment.

8. Acid pretreatment
8.1. Process description and mode of action
Pretreatment of lignocellulose with acids at ambient temperature are done to enhance the anaerobic digestibility. The objective
is to solubilize the hemicellulose, and by this, making the cellulose
better accessible.
The pretreatment can be done with dilute or strong acids. The
main reaction that occurs during acid pretreatment is the hydrolysis of hemicellulose, especially xylan as glucomannan is relatively
acid stable. Solubilized hemicelluloses (oligomers) can be subjected to hydrolytic reactions producing monomers, furfural,
HMF and other (volatile) products in acidic environments (Fengel
and Wegener, 1984; Ramos, 2003). During acid pretreatment solubilized lignin will quickly condensate and precipitate in acidic
environments (Liu and Wyman, 2003; Shevchenko et al., 1999).
The solubilization of hemicellulose and precipitation of solubilized
lignin are more pronounced during strong acid pretreatment compared to dilute acid pretreatment.
Xiao and Clarkson (1997) showed that the addition of nitric acid
during acid pretreatment has a tremendous effect on the solubilization of lignin of newspaper.
8.2. Consequences for ethanol and methane production
The advantage of acid pretreatment is the solubilization of
hemicellulose and by this, making the cellulose more easily accessible for the enzymes. There is however a risk on the formation of
volatile degradation products and this carbon is in many cases lost
for the conversion to ethanol. Volatile products however can be
converted to methane. The condensation and precipitation of solubilized lignin components is an unwanted reaction, as it decreases
digestibility.
Strong acid pretreatment is for the ethanol production not
attractive, because there is a risk on the formation of inhibiting
compounds. Dilute acid pretreatment however is considered as
one of the promising pretreatment methods, because secondary
reactions during the pretreatment can be prohibited in dilute acid
pretreatment.
For methane production acid pretreatment is more attractive
than for ethanol production, because methanogens can handle
compounds like furfural and HMF to a certain concentration and
with an acclimatization period. For both the ethanol as well as
the methane production the chance on soluble lignin components
is a risk, because soluble lignin compounds are often inhibiting for
both processes. Methanogens are however capable of adapting to
such inhibiting compounds (Benjamin et al., 1984; Xiao and Clarkson, 1997).
When sulfuric or nitric acids are used in the acidic pretreatment, the methane production during anaerobic treatment will

14

A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 1018

be reduced as a result of reduction of sulphate and nitrate to

respectively H2S and N2.

10. Oxidative pretreatment

10.1. Process description and mode of action

9. Alkaline pretreatment
9.1. Process description and mode of action
During alkaline pretreatment the rst reactions taking place are
solvation and saphonication. This causes a swollen state of the biomass and makes it more accessible for enzymes and bacteria. At
strong alkali concentrations dissolution, peeling of end-groups,
alkaline hydrolysis and degradation and decomposition of dissolved polysaccharides can take place. Loss of polysaccharides is
mainly caused by peeling and hydrolytic reactions (Fengel and
Wegener, 1984). This peeling is an advantage for later conversion,
but, because lower molecular compounds are formed as a result,
the risk on degradation and loss of carbon, in the form of carbon
dioxide, also increases.
Xylan can be selectively removed with aqueous potassium
hydroxide. The temperature is kept low during extraction (room
temperature or lower) to prevent peeling (Hon and Shiraishi,
2001). Glucomannans and xylans can both be subject of the peeling
reaction. This itself is not a problem, but the higher the monomeric
hemicellulose fraction, the lower the total recovery of the hemicellulose (Laser et al., 2002). The monomeric forms of hemicellulose
are probably easily degradable to other (volatile) compounds and
for example furfural, which leads to losses of digestible substrate
for the ethanol process (Bobleter, 1994).
An important aspect of alkali pretreatment is that the biomass
on itself consumes some of the alkali. The residual alkali concentration after the alkali consumption by the biomass is the alkali
concentration left over for the reaction (Gossett et al., 1982). Pavlostathis and Gossett (1985) for example found during their experiments an alkali consumption of approximately 3 g NaOH/100 g TS.
Lime works remarkably better than sodium hydroxide (Gossett et
al., 1982).
Alkali extraction can also cause solubilization, redistribution
and condensation of lignin and modications in the crystalline
state of the cellulose. These effects can lower or counteract the positive effects of lignin removal and cellulose swelling (Gregg and
Saddler, 1996).
Another important aspect of alkaline pretreatment is the
change of the cellulose structure to a form that is denser and thermodynamically more stable than the native cellulose (Pettersen,
1984).

An oxidative pretreatment consists of the addition of an oxidizing compound, like hydrogen peroxide or peracetic acid, to the biomass, which is suspended in water. The objective is to remove the
hemicellulose and lignin to increase the accessibility of the cellulose. During oxidative pretreatment several reactions can take
place, like electrophilic substitution, displacement of side chains,
cleavage of alkyl aryl ether linkages or the oxidative cleavage of
aromatic nuclei (Hon and Shiraishi, 2001).
In many cases the used oxidant is not selective and therefore
losses of hemicellulose and cellulose can occur. A high risk on
the formation of inhibitors exists, as lignin is oxidized and soluble
aromatic compounds are formed.
Teixeira et al. (1999) have investigated the use of peracetic acid
at ambient temperatures as a pretreatment method for hybrid poplar and sugar cane bagasse. Peracetic acid is very lignin selective
and no signicant carbohydrate losses occurred. The enzymatic
hydrolysis of the cellulose increased from 6.8% (untreated) to a
maximum of about 98% (pretreated) at a 21% peracetic acid pretreatment. Teixeira et al. (1999) showed similar results for digestibility at using a mixture of NaOH and peracetic acid. The yields
during experiments carried out at ambient temperature were higher as compared to higher temperatures.
Gould (1984) demonstrated the use of H2O2 for delignication
with a maximum at pH 11.5. No substantial delignication occurred below a pH of 10.0. Hydrogen peroxide had no real effect
on the enzymatic digestibility at a pH 12.5 or higher. According
to Gould (1984) the hydrogen peroxide concentration should be
at least 1% and the weight ratio between H2O2 and biomass should
be 0.25 for a good delignication. The delignication is probably
caused by the hydroxyl ion (HO), which is a degradation product
of hydrogen peroxide with a maximum at pH 11.511.6. About half
of the lignin was solubilized in this way (temperature around 25 C
and a duration of 1824 h) (Gould, 1984).

10.2. Consequences for ethanol and methane production

During an oxidative pretreatment often a lot of sugars get lost,
because of non-selective oxidation. Also soluble lignin compounds
are formed, which can be inhibiting in the subsequent conversion
step of the (hemi) cellulose to ethanol or methane.

11. Combinations, ammonia and carbon dioxide pretreatment

9.2. Consequences for ethanol and methane production
11.1. Thermal pretreatment in combination with acid pretreatment
Alkaline pretreatment causes hemicellulose and parts of lignin
to solubalize. The removal of hemicellulose has a positive effect
on the degradability of cellulose. There is however often a loss of
hemicellulose to degradation products and the solubilized lignin
components often have an inhibitory effect. Gossett et al. (1982)
for example concluded that alkaline heat treated lignin in concentrations over 1 g/l gave a major inhibitory effect to the methanogenic micro-organisms. This is probably caused by the products
formed from the lignin during the alkaline heat pretreatment.
The loss of fermentable sugars and production of inhibitory compounds makes the alkaline pretreatment less attractive for the ethanol production. The production of inhibitors is less severe for
methanogens as compared to yeasts for ethanol production. Methanogens are (often) capable of adapting to such compounds. Pavlostathis and Gossett (1985) mentioned a 100% increase in methane
production after an alkaline pretreatment of wheat straw.

A way to improve the effect of thermal steam or LHW pretreatment is to add an external acid. This addition of an external acid
catalyzes the solubilization of the hemicellulose, lowers the optimal pretreatment temperature and gives a better enzymatic
hydrolysable substrate (Brownell et al., 1986; Gregg and Saddler,
1996). The lignocellulose is often impregnated (soaked) with SO2
or H2SO4. During steam pretreatment the SO2 is converted to
H2SO4 in the rst 20 seconds of the process; after that, the catalytic hydrolysation of the hemicellulose starts. Another important
point is that gradual removal of hemicellulose and lignin can trigger reorientation of cellulose to a more crystalline form (Gregg
and Saddler, 1996). Latter is true for every pretreatment that
gradually removes hemicellulose and lignin. The effect of the
added acid is however still not clear. Tengborg et al. (1998)
showed a severe inhibition in the ethanol production step at a

A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 1018

severity factor of 3 and higher with the addition of an external

acid. This is in line with the conclusion of Grohmann et al.
(1985) that during steam pretreatment at temperatures of
160 C and higher with 0.5% sulfuric acid addition an appreciable
production of furfural occurs.
Sderstrm et al. (2002) investigated the use of a two-step
steam pretreatment of softwood with SO2. The rst step was carried out at lower severities (log R0 2.664.24) for the recovery of
hemicellulose sugars and the second step was done at higher
severities (log R0 2.954.24) to improve the digestibility of the cellulose. The highest hemicellulose recovery in the rst pretreatment
step was achieved at a severity of about 3 and the highest sugar
yield in the second pretreatment step at a severity between 3.5
and 4.3. However, pretreatments at temperatures of 220 C did
not give satisfactory yields because of the formation of inhibiting
compounds. Wu et al. (1999) got the highest amount of monomeric sugars from hemicellulose at a severity factor of approximately 3 (175 C, 7.5 min, 4,5% SO2). At higher severities the
monomeric sugars were probably degraded to furfural and other
compounds.
11.2. Thermal pretreatment in combination with alkaline
pretreatment
Another way to improve the thermal pretreatment is to add an
external alkali instead of an acid to the process. A very common
alkaline thermal pretreatment is lime pretreatment. This pretreatment is usually carried out at temperatures of 100150 C with
lime addition of approximately 0.1 g Ca(OH)2 g substrate 1 (Chang
et al., 2001). Chang and Holtzapple (2000) attribute the effectiveness of lime pretreatment to the opening of the acetyl valve and
partly opening the lignin valve, making the substrate more accessible to hydrolysis. According to Kaar and Holtzapple (2000)
lime pretreatment (with heating) is sufcient to increase the
digestibility of low-lignin containing biomass, but not for highlignin containing biomass. Chang et al. (2001) mention that lime
pretreatment of switchgrass and corn stover did not inhibit the
enzymatic saccharication and fermentation steps. Pretreated
softwood however was washed before the enzymatic saccharication and fermentation step to prevent possible inhibiting by (the
large amount of) solubilized lignin. A positive effect of lime is that
it is relatively cheap and safe (Gandi et al., 1997) and that the calcium can be regained as insoluble calcium carbonate by the reaction with carbon dioxide. This calcium carbonate can be converted
to lime again with the lime kiln technology (Chang et al., 1998).
Fox et al. (2003) reported an improvement in methane production with a factor of 3 to 4.5 after pretreating newsprint waste with
alkaline heat pretreatment.
11.3. Thermal pretreatment in combination with oxidative
pretreatment
Ando et al. (1988) mentions that the saccharication of cedar,
soaked in peracetic acid and steam treated at 231 C for 10 min,
was directly proportional to the amount of peracetic acid adsorbed
in the chips.
Wet-oxidation is another oxidative pretreatment method,
which uses oxygen as oxidator. The soluble sugars produced during
wet-oxidation pretreatment of wheat straw are mainly polymers
opposite to the monomers produced during steaming or acid
hydrolysis as pretreatment. Phenolic monomers are no end products during wet-oxidation but are further degraded to carboxylic
acids. Also the production of furfural and HMF was low during
wet-oxidation, but part of the hemicellulose was lost by reaction
to carbon dioxide and water (Klinke et al., 2002).

15

11.4. Thermal pretreatment in combination with alkaline oxidative

pretreatment
According to Chang et al. (2001) thermal lime pretreatment is
not capable of removing enough lignin of high-lignin biomass to
enhance the enzymatic digestibility and therefore oxygen as oxidant must be included during the pretreatment. A low sugar degradation was observed, probably as a result of the relative low
temperature of 150 degrees, applied during the pretreatment.
The enzymatic digestibility of the treated biomass was 13 times
higher than for the untreated biomass. The pretreated biomass
was however washed to remove the probably produced inhibiting
soluble lignin compounds (Chang et al., 2001). After the oxidative
lime pretreatment about 21% of the added lime could be recovered
by carbon dioxide carbonation (Chang et al., 2001b).
11.5. Ammonia and carbon dioxide pretreatment
Other applied pretreatments are ammonia and carbon dioxide
pretreatment. The objective of the ammonia pretreatment (also
called AFEX pretreatment). The ammonia pretreatment is conducted with ammonia loadings around 1:1 (kg ammonia/kg dw
biomass) at temperatures ranging from ambient temperature with
a duration of 1060 days, to temperatures of up to 120 C with a
duration of several minutes (Alizadeh et al., 2005; Kim and Lee,
2005). Alizadeh et al. (2005) reported a six-fold increased enzymatic hydrolysis yield and a 2.5-fold ethanol yield after pretreatment. Bariska (1975) and Kim and Lee (2005) mention swelling
of the cellulose and delignication as the responsible factors for
the increased yield.
Carbon dioxide pretreatment is conducted with high-pressure
carbon dioxide at high temperatures of up to 200 C with a duration of several minutes. Explosive steam pretreatment with highpressure carbon dioxide causes the liquid to be acidic and this acid
hydrolyses especially the hemicellulose (Puri and Mamers, 1983).
Carbon dioxide is also applied as supercritical carbon dioxide
(35 C, 73 Bars), increasing the glucose yield of bagasse with 50
70% (Zheng et al., 1998), 14% for yellow pine and 70% for aspen
(Kim and Hong, 2001). This is probably caused by increase in pore
size.
12. Overview of effects of pretreatments on lignocellulose
In chapter six up to and including chapter eleven the effects of
several pretreatments on the physical/chemical composition or
structure of lignocellulose is reported. Table 1 summarizes the
most important effects of the different pretreatment methods,
discussed in this paper. The table suggests that increasing the
surface area is one of the major approaches of a pretreatment by
solubilization of the hemicellulose and/or lignin and/or altering
the lignin. The importance of surface area is mentioned by Zeng
et al. (2007).

13. Conclusion and nal discussion

The biodegradability of lignocellulosic biomass is limited by
several factors like crystallinity of cellulose, available surface area,
and lignin content. Pretreatments have an effect on one or more of
these aspects, as showed in Table 1. Several factors are mentioned
to have a positive effect on the overall economy of the process. It is
for example favourable to avoid the production of inhibitors
(Ramos, 2003), because detoxication of the liquid fraction showed
to be costly and/or ineffective (Gregg and Saddler, 1996; Von Sivers
et al., 1994); to leave the lignin with the substrate and remove it
after the hydrolysis of the (hemi) cellulose to minimize the overall

16

A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 1018

Table 1
Effects of the different pretreatments on the physical/chemical composition or structure of lignocellulose (adapted from Mosier et al. (2005b))

Mechanical
ST/SE
LHW (batch)
LHW (ow through)
Acid
Alkaline
Oxidative
Thermal + acid
Thermal + alkaline (lime)
Thermal + oxidative
Thermal + alkaline + oxidative
Ammonia (AFEX)
CO2 explosion

Increase accessible
surface area

Decrystallization
cellulose

+
+
+
+
+
+
+
+
+
+
+
+
+

+
ND
ND

ND
ND
ND
ND
ND
+

Solubilization
hemicellulose
+
+
+
+

Solubilization
lignin

Formation furfural/
HMF

Alteration lignin
structure

+
+
+
+
+
+
+
+

+/
+/
+/
+/
+/
+/
+/
+

+ = major effect.
= minor effect.
ND = not determined.

costs of the process for ethanol production (Shevchenko et al.,

1999); low water, energy and alkali/acid use and a pretreatment
which can be performed continuously, which is very attractive
for industrial appliance (Schmidt and Thomsen, 1998).
It can be concluded that pretreatments like concentrated acids,
wet oxidation, solvents and metal complexes are effective, but too
expensive compared to the value of glucose (Fan et al., 1987; Mosier et al., 2005b). Steam pretreatment, lime pretreatment, LHW
systems and ammonia based pretreatments are the ones that,
according to the factors determining the economic effectiveness
mentioned above, and the effects of the pretreatments (Table 1),
have a high potential. An economical evaluation of ve different
pretreatment technologies (dilute acid, hot water, ammonia ber
explosion (AFEX), ammonia recycle percolation (ARP), and lime)
is presented by Eggeman and Elander (2005) in the special issue
Coordinated development of leading biomass pretreatment technologies (Wyman et al., 2005). The main conclusion is that there
is only little differentiation between the projected economic performances of the pretreatment options, but that optimizing enzyme blends for each pretreatment approach and conditioning
requirements of hydrolysates at process-relevant sugar concentrations resulting from each pretreatment, may result in greater differentiation in projected process economics.
Not much research on the carbon dioxide explosion based pretreatments has been done, so it is difcult to judge if this pretreatment is a potential one ore not.
The effect of the pretreatments is however very dependent on
the biomass composition and operating conditions. All these pretreatments have their advantages and disadvantages and future research is needed for optimization.
An important aspect is the choice of the bio-fuel to produce. The
differences between the ethanol and methane conversion efciencies are not large anymore, as nowadays also yeast strains capable
of converting C5 and C6 sugars to ethanol are available (Kuyper et
al., 2005). A disadvantage of the ethanol production, compared to
methane production, is the low tolerance for inhibiting compounds
like furfural and HMF by the yeasts.
The higher tolerance for inhibiting compounds, the simple one
step process, and the use of a direct-methane fuel cell in for example stationary power generation, could favour the production of
CH4 in stead of ethanol (Murray et al., 1999). The performance of
a direct-methane fuel cell is comparable with that of a hydrogen
based fuel cell (Murray et al., 1999).
An aspect, which earns more attention, is the production of
ethanol by thermophilic bacteria instead of yeasts. Thermophilic
bacteria can produce an optimal mixture of enzymes for the

hydrolysis, while such optimal enzyme mixture is difcult to produce articially (Schwarz, 2001). The production of the enzymes
by the thermophilic bacteria does not consume a lot of extra energy (Ng et al., 1977), the enzyme hydrolyses rate can be comparable with that of the enzymes of Trichoderma reesei (Lynd et al.,
1989). These bacteria can be adapted to higher ethanol concentrations (Baskaran et al., 1995; Bender et al., 1985; Herrero and
Gomez, 1980; Klapatch et al., 1994; Larsen et al., 1997; Rani et
al., 1996; Tailliez et al., 1989a, 1989b), and are maybe even capable of handling compounds like furfural and HMF or adapt to
them. Thermophilic conditions decrease the risk on infection
(Payton, 1984; Wiegel, 1980), and could create the possibility
of continuous distillation (Payton, 1984; Zeikus, 1980; Wiegel,
1980).
Acknowledgements
The authors would like to thank T. Fernandes, C. Pabon, K.
Grolle, V. de Wilde, and B. Willemsen for their experimental discussion and assistance throughout the investigation.
References
Alizadeh, H., Teymouri, F., Gilbert, T.I., Dale, B.E., 2005. Pretreatment of
switchgrass by ammonia ber explosion (AFEX). Appl. Biochem. Biotechnol.,
11331141.
Allen, S.G., Schulman, Deborah, Lichwa, J., Antal Jr., M.J., 2001. A comparison of
aqueous and dilute-acid single-temperature pretreatment of yellow poplar
sawdust. Ind. Eng. Chem. Res. 40, 23522361.
Ando, S., Kakimoto, T., Itoh, K., Arai I., Kiyoto, K., Hanai, S., 1988. Increased
digestibility of cedar by pretreatment with peracetic acid and steam explosion.
Biotechnol. Bioeng. 31, 802804.
Balaban, M., Ucar, G., 1999. The effect of the duration of alkali treatment on the
solubility of polyoses. Turk. J. Agric. Forest. 23, 667671.
Bariska, M., 1975. Collapse phenomena in beechwood during and after NH3impregnation. Wood Sci. Technol. 9, 293306.
Baskaran, S., Ahn, H.-J., Lynd, L.R., 1995. Investigation of the ethanol tolerance of
Clostridium thermosaccharolyticum in continuous culture. Biotechnol. Progr. 11,
276281.
Beall, F.C., Eickner, H.W., 1970. Thermal degradation of wood components: a review
of the Literature. U.S.D.A. Forest service, research paper FPL 130.
Bender, J., Vatcharapijarn, Y., Jeffries, T.W., 1985. Characteristics and adaptability of
some new isolates of Clostridium thermocellum. Appl. Environ. Microbiol. 49 (3),
475477.
Benjamin, M.M., Woods, S.L., Ferguson, J.F., 1984. Anaerobic toxicity and
biodegradability of pulp mill waste constituents. Water Res. 18 (5), 601607.
Bobleter, O., 1994. Hydrothermal degradation of polymers derived from plants.
Prog. Polym. Sci. 19, 797841.
Bobleter, O., Concin, R., 1979. Degradation of poplar lignin by hydrothermal
treatment. Cellul. Chem. Technol. 13, 583593.
Bobleter, O., Bonn, G., Prutsch, W., 1991. Steam explosion-hydrothermolysisorganosolv. A comparison. In: Focher, B., Marzetti, A., Crescenzi, V. (Eds.),
Steam explosion techniques. Gordon and Breach, Philadelphia, pp. 5982.

A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 1018

Brownell, H.H., Yu, E.K.C., Saddler, J.N., 1986. Steam-explosion pretreatment of
wood: effect of chip size, acid, moisture content and pressure drop. Biotechnol.
Bioeng. 28, 792801.
Cauleld, D., Moore, W.E., 1974. Effect of varying crystallinity of cellulose on
enzymic hydrolysis. Wood Sci. 6 (4), 375379.
Chang, V.S., Holtzapple, M.T., 2000. Fundamental factors affecting enzymatic
reactivity. Appl. Biochem. Biotechnol., 537.
Chang, V.S., Nagwani, M., Holtzapple, M.T., 1998. Lime pretreatment of crop
residues bagasse and wheat straw. Appl. Biochem. Biotechnol. 74, 135159.
Chang, V.S., Kaar, W.E., Burr, B., Holtzapple, M.T., 2001. Simultaneous
saccharication and fermentation of lime-treated biomass. Biotechnol. Lett.
23 (16), 13271333.
Chang, V.S., Nagwani, M., Kim, C., Holtzapple, T., 2001b. Oxidative lime
pretreatment of high-lignin biomass. Appl. Biochem. Biotechnol. 94, 128.
Converse, A.O., Kwarteng, I.K., Grethlein, H.E., Ooshima, H., 1989. Kinetics of
thermochemical pretreatment of lignocellulosic materials. Appl. Biochem.
Biotechnol. 21, 111116.
Cowling, E.B., Kirk, T.K., 1976. Properties of cellulose and lignocellulosic materials as
substrates for enzymatic conversion process. Biotechnol. Bioeng. Symp. 6, 95
123.
Delgens, J.P., Penaud, V., Moletta, R., 2002. Pretreatments for the enhancement of
anaerobic digestion of solid wastes Chapter 8. In: Biomethanization of the
Organic Fraction of Municipal Solid Wastes. IWA Publishing, pp. 201228.
Domansky, R., Rendos, F., 1962. On the pyrolysis of wood and its components. Holz
Roh Werkst. 29, 473476.
Eggeman, T., Elander, R.T., 2005. Process and economic analysis of pretreatment
technologies. Bioresour. Technol. 96, 20192025.
Fan, L.T., Gharpuray, M.M., Lee, Y.H., 1987. Cellulose hydrolysis. Springer-Verlag,
Berlin, Germany.
Fengel, D., Wegener, G., 1984. Wood: Chemistry, Ultrastructure, Reactions. De
Gruyter, Berlin.
Fox, M.H., Noike, T., Ohki, T., 2003. Alkaline subcritical-water treatment and alkaline
heat treatment for the increase in biodegradability of newsprint waste. Water
Sci. Technol. 48 (4), 7784.
Gandi, J., Holtzapple, M.T., Ferrer, A., Byers, F.M., Turner, N.D., Nagwani, M., Chang,
S., 1997. Lime treatment of agricultural residues to improve rumen digestibility.
Anim. Feed Sci. Technol. 68, 195211.
Garrote, G., Dominguez, H., Parajo, J.C., 1999. Hydrothermal processing of
lignocellulosic materials. Holz Roh Werkst. 57, 191202.
Gossett, J.M., Stuckey, D.C., Owen, W.F., Mccarty, P.L., 1982. Heat treatment and
anaerobic digestion of refuse. J. Environ. Eng. Div. 108, 437454.
Gould, J.M., 1984. Alkaline peroxide delignication of agricultural residues to
enhance enzymatic saccharication. Biotechnol. Bioeng. 26, 4652.
Grabber, J.H., 2005. How do lignin composition, structure, and cross-linking affect
degradability? A review of cell wall model studies. Crop Sci. 45,
820831.
Gray, M.C., Converse, A.O., Wyman, C.E., 2003. Sugar monomer and oligomer
solubility. Data and predictions for application to biomass hydrolysis. Appl.
Biochem. Biotechnol., 179193.
Gregg, D., Saddler, J.N., 1996. A techno-economic assessment of the pretreatment
and fractionation steps of a biomass-to-ethanol process. Appl. Biochem.
Biotechnol., 711727.
Grethlein, H.E., 1985. The effect of pore size distribution on the rate of enzymatic
hydrolysis of cellulose substrates. Bio/Technol. 3, 155160.
Grohmann, K., Torget, R., Himmel, M., 1985. Optimization of dilute acid
pretreatment of biomass. Biotechnol. Bioeng. Symp. 15, 5980.
Grohman, K., Torget, R., Himmel, M., 1986. Dilute acid pretreatment of biomass at
high solids concentration, Biotechnol. Bioeng. Symp. 17, 135151.
Grous, W.R., Converse, A.O., Grethlein, H.E., 1986. Effect of steam explosion
pretreatment on pore size and enzymatic hydrolysis of poplar. Enzyme
Microbiol. Technol. 8, 274280.
Hartmann, H., Angelidaki, I., Ahring, B.K., 1999. Increase of anaerobic degradation of
particulate organic matter in full-scale biogas plants by mechanical maceration.
In: Mata-Alvarez, J., Tilche, A., Cecchi, F. (Eds), Proceedings of the Second
International Symposium on Anaerobic Digestion of Solid Wastes, Barcelona,
vol. 1, pp 129-136.
Herrero, A.A., Gomez, R.F., 1980. Development of ethanol tolerance in Clostridium
thermocellum effect of growth temperature. Appl. Environ. Microbiol. 40 (3),
571577.
Hon, D.N.S., Shirashi, N., 1991. Wood and Cellulosic Chemistry. Marcel Dekker, New
York.
Hon, D.N.S., Shiraishi, N., 2001. Wood and Cellulosic Chemistry, second ed. Dekker,
New York.
Jacobsen, S.E., Wyman, C.E., 2002. Xylose monomer and oligomer yields for
uncatalyzed hydrolysis of sugarcane bagasse hemicellulose at varying solids
concentration. Ind. Eng. Chem. Res. 41, 14541461.
Kaar, W.E., Holtzapple, M.T., 2000. Using lime pretreatment to facilitate the
enzymatic hydrolysis of corn stover. Biomass Bioenergy 18 (3),
189199.
Kim, K.H., Hong, J., 2001. Supercritical CO2 pretreatment of lignocellulose enhances
enzymatic cellulose hydrolysis. Bioresour. Technol. 77, 139144.
Kim, T.H., Lee, Y.Y., 2005. Pretreatment of corn stover by soaking in aqueous
ammonia. Appl. Biochem. Biotechnol., 11191132.
Klapatch, T.R., Hogsett, D.A.L., Baskaran, S., Pal, S., 1994. Organism development and
characterization for ethanol production using thermophilic bacteria. Appl.
Biochem. Biotechnol., 209223.

17

Klinke, H.B., Ahring, B.K., Schmidt, A.S., Thomsen, A.B., 2002. Characterization of
degradation products from alkaline wet oxidation of wheat straw. Bioresour.
Technol. 82 (1), 1526.
Kohlmann, K.L., Westgate, P.J., Sarikaya, A., Velayudhan, A., Weil, J., Hendrickson, R.,
Ladisch, M.R. 1995. Enhanced enzyme activities on hydrated lignocellulosic
substrates. BTEC paper 127. In: 207th American Chemical Society National
Meeting, ACS Symposium series No. 618. Enzymatic Degradation of Insoluble
Carbohydrates, pp. 237255.
Koullas, D.P., Christakopoulos, P., Kekos, D., Macris, B.J., Koukios, E.G., 1992.
Correlating the effect of pretreatment on the enzymatic hydrolysis of straw.
Biotechnol. Bioeng. 39, 113116.
Kuyper, M., Toirkens, M.J., Diderich, J.A., Winkler, A.A., Van Dijken, J.P., Pronk, J.T.,
2005. Evolutionary engineering of mixed-sugar utilization by a xylosefermenting Saccharomyces cerevisiae strain . FEMSYeast Res. 5 (10),
925934.
Larsen, L., Nielsen, P., Ahring, B.K., 1997. Thermoanaerobacter mathranii sp. nov., an
ethanol-producing, extremely thermophylic anaerobic bacterium from a hot
spring in Iceland. Arch. Microbiol. 168, 114119.
Laser, M., 2001. Hydrothermal pretreatment of cellulosic biomass for bioconversion
to ethanol. Ph. D. Thesis, Thayer School of Engineering, Dartmouth College.
Laser, M., Schulman, D., Allen, S.G., Lichwa, J., Antal Jr, M.J., Lynd, L.R., 2002. A
comparison of liquid hot water and steam pretreatments of sugar cane bagasse
for bioconversion to ethanol. Bioresour. Technol. 81, 3344.
Laureano-Perez, L., Teymouri, F., Alizadeh, H., Dale, B.E., 2005. Understanding
factors that limit enzymatic hydrolysis of biomass. Appl. Biochem. Biotechnol.,
10811099.
Lawther, J.M., Sun, R., Banks, W.B., 1996a. Effect of steam treatment on the chemical
composition of wheat straw. Holzforschung 50, 365371.
Lawther, J.M., Sun, R., Banks, W.B., 1996b. Effects of extraction conditions and alkali
type on yield and composition of wheat straw hemicellulose. J. Appl. Polym. Sci.
60, 18271837.
Levan, S.L., Ross, R.J., Winandy, J.E., 1990. Effects of re retardant chemicals on
bending properties of wood at elevated temperatures. Research Paper FPL-RP498. Madison, WI: U.S. Department of agriculture, Forest service, Forest
Products Laboratory, p. 24
Li, J., Henriksson, G., Gellerstedt, G., 2005. Carbohydrate reactions during hightemperature steam treatment of Aspen wood. Appl. Biochem. Bioeng. 125, 175
188.
Liu, C., Wyman, C.E., 2003. The effect of ow rate of compressed hot water on xylan,
lignin and total mass removal from corn stover. Ind. Eng. Chem. Res. 42, 5409
5416.
Lora, J.H., Wayman, M., 1978. Delignication of hardwoods by autohydrolysis and
extraction. Tappi 61, 4750.
Lynd, L.R., Grethlein, H.E., Wolkin, R.H., 1989. Fermentation of cellulosic substrates
in batch and continuous culture by Clostridium thermocellum. Appl. Environ.
Microbiol. 55 (12), 31313139.
Mok, W.S.-L., Antal, M.J.A., 1992. Uncatalyzed solvolysis of whole biomass
hemicellulose by hot compressed liquid water. Ind. Eng. Chem. Res. 31, 1157
1161.
Mosier, N., Hendrickson, R., Ho, N., Sedlak, M., Ladisch, M.R., 2005a. Optimization of
pH controlled liquid hot water pretreatment of corn stover. Bioresour. Technol.
96, 19861993.
Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M.,
2005b. Features of promising technologies for pretreatment of lignocellulosic
biomass. Bioresour. Technol. 96, 673686.
Murray, E.P., Tsai, T., Barnett, S.A., 1999. A direct-methane fuel cell with a ceriabased anode. Lett. Nature 400, 649651.
Negro, M.J., Manzanares, P., Oliva, J.M., Ballesteros, I., Ballesteros, M., 2003. Changes
in various physical chemical parameters of Pinus Pinaster wood after steam
explosion pretreatment. Biomass Bioenergy 25, 301308.
Ng, T.K., Weimer, P.J., Zeikus, J.G., 1977. Cellulolytic and physiological properties of
Clostridium thermocellum. Arch. Microbiol. 114, 17.
Noike, T., Niigata Engineering, 2001. Micromolecularization of undegradable
organic substances in methane fermentation. In: Development of the
Waste Treatment System for Recycling Society. Waste Research Foundation,
Tokyo.
Overend, R.P., Chornet, E., 1987. Fractionation of lignocellulosis by steamaqueous
pretreatments. Philos. Trans. R. Soc. London A321, 523536.
Palmowski, L., Muller, J., 1999. Inuence of the size reduction of organic waste on
their anaerobic digestion. In: II International Symposium on Anaerobic
Digestion of Solid Waste. Barcelona 1517 June, pp. 137144.
Palonen, H., Thomsen, A.B., Tenkanen, M., Schmidt, A.S., Viikari, L., 2004. Evaluation
of wet oxidation pretreatment for enzymatic hydrolysis of softwood. Appl.
Biochem. Biotechnol. 117, 117.
Pavlostathis, S.G., Gossett, J.M., 1985. Alkaline treatment of wheat straw for
increasing anaerobic biodegradability. Biotechnol. Bioeng. 27, 334344.
Payton, M.A., 1984. Production of ethanol by thermophilic bacteria. Trends
Biotechnol. 2, 153158.
Pettersen, R.C., 1984. The chemical composition of wood (chapter 2). In: Rowell,
R.M. (Ed.), The chemistry of solid wood, Advances in Chemistry Series, vol. 207.
American Chemical Society, Washington, DC, p. 984.
Puri, V.P., 1984. Effect of crystallinity and degree of polymerization of cellulose on
enzymatic saccharication. Biotechnol. Bioeng. 26, 12191222.
Puri, V.P., Mamers, H., 1983. Explosive pretreatment of lignocellulosic residues with
high-pressure carbon dioxide for the production of fermentation substrates.
Biotechnol. Bioeng. 25, 31493161.

18

A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 1018

Ramos, L.P., 2003. The chemistry involved in the steam treatment of lignocellulosic
materials. Quim. Nova. 26 (6), 863871.
Rani, K.S., Swamy, M.V., Sunitha, D., Haritha, D., Seenayya, G., 1996. Improved
ethanol tolerance and production in strains of Clostridium thermocellum. World
J. Microbiol. Biotechnol. 12, 5760.
Reith, J.H., Wijffels, R.H., Barten, H., 2003. Bio-methane & Bio-hydrogen. Status and
perspectives of biological methane and hydrogen production. Production of the
Dutch biological hydrogen production, The Hague.
Saha, B.C., 2003. Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30, 279
291.
Schmidt, A.S., Thomsen, A.B., 1998. Optimization of wet oxidation pre-treatment of
wheat straw. Bioresour. Technol. 64, 139151.
Schwarz, W.H., 2001. The cellulosome and cellulose degradation by anaerobic
bacteria. Appl. Microbiol. Biotechnol. 56, 634649.
Shahbazi, A., Li, Y., Mims, M.R., 2005. Application of sequential aqueous steam
treatments to the fractionation of softwood. Appl. Biochem. Biotechnol., 973
987.
Shevchenko, S.M., Beatson, R.P., Saddler, J.N., 1999. The nature of lignin from steam
explosion/enzymatic hydrolysis of softwood. Appl. Biochem. Biotechnol., 867
876.
Sderstrm, J., Pilcher, L., Galbe, M., Zacchi, G., 2002. Two-step pretreatment of
softwood with SO2 impregnation for ethanol production. Appl. Biochem.
Biotechnol., 521.
Sweet, M.S., Winandy, J.E., 1999. Inuence of degree of polymerization of cellulose
and hemicellulose on strength loss in re-retardant-treated southern pine.
Holzforschung 53 (3), 311317.
Tailliez, P., Girard, H., Longin, R., Beguin, P., Millet, J., 1989a. Cellulose fermentation
by an asporogenous mutant and an ethanol-tolerant mutant of Clostridium
thermocellum. Appl. Environ. Microbiol. 55 (1), 203206.
Tailliez, P., Girard, H., Millet, J., Beguin, P., 1989b. Enhanced cellulose fermentation
by an asporogenous and ethanol-tolerant mutant of Clostridium thermocellum.
Appl. Environ. Microbiol. 55 (1), 207211.
Teixeira, L.C., Linden, J.C., Schroeder, H.A., 1999. Alkaline and peracetic acid
pretreatments of biomass for ethanol production. Appl. Biochem. Biotechnol.,
1934.
Tengborg, C., Stenberg, K., Galbe, M., Zacchi, G., Larsson, S., Palmqvist, E., HahnHgerdal, B., 1998. Comparison of SO2 and H2SO4 impregnation of softwood
prior to steam pretreatment on ethanol production. Appl. Biochem. Biotechnol.,
315.
Thompson, D.N., Chen, H.-C., Grethlein, H.E., 1992. Comparison of pretreatment
methods on the basis of available surface area. Bioresour. Technol. 39, 155163.
Von Sivers, N., Zacchi, G., Olsson, L., Hahn-Hgerdal, B., 1994. Cost analysis of
ethanol production from willow using recombinant escherichia coli. Biotechnol.
Progr. 10, 555560.

Weil, J.R., Brewer, M., Hendrickson, R., Sarikaya, A., Ladisch, M.R., 1997. Continuous
pH monitoring during pretreatment of yellow poplar wood sawdust by pressure
cooking in water. Appl. Biochem. Biotechnol. 68, 2140.
Weil, J.R., Sarikaya, A., Rau, S.L., Goebz, J., Lasisch, C.M., Brwer, M., Hendrickson, R.,
Tadisch, M.R., 1998. Pretreatment of corn ber by pressure cooking in water.
Appl. Biochem. Biotechnol. 73, 117.
Weimer, P.J., Hackney, J.M., French, A.D., 1995. Effects of chemical treatments and
heating on the crystallinity of celluloses and their implications for evaluating
the effect of crystallinity on cellulose biodegradation. Biotechnol. Bioeng. 48,
169178.
Wiegel, J., 1980. Formation of ethanol by bacteria. A pledge for use of extreme
thermophilic anaerobic bacteria in industrial ethanol fermentation processes.
CMLS 36 (12), 14341446.
Winandy, J.E., 1995. Effects of re retardant treatments after 19 months of exposure
at 150F (66C). Res. Note FPL-RN-0264. U.S. Department of agriculture, Forest
Service, Forest Products Laboratory, Madison, WI., p. 13
Wu, M.M., Chang, K., Gregg, D.J., Boussaid, A., Beatson, R.P., Saddler, J.N., 1999.
Optimization of steam explosion to enhance hemicellulose recovery and
enzymatic hydrolysis of cellulose in softwoods. Appl. Biochem. Biotechnol.,
4754.
Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M., Ladisch, M.R., Lee, Y.Y., 2005.
Coordinated development of leading biomass pretreatment technologies.
Bioresour. Technol. 96, 19591966.
Xiao, W., Clarkson, W.W., 1997. Acid solubilization of lignin and bioconversion of
treated newsprint to methane. Biodegradation 8, 6166.
Yang, B., Wyman, C.E., 2004. Effect of xylan and lignin removal by batch and ow
through pretreatment on the enzymatic digestibility of Corn Stover Cellulose.
Biotechnol. Bioeng. 86 (1), 8895.
Zeikus, J.G., 1980. Chemical and fuel production by anaerobic bacteria. Annu. Rev.
Microbiol. 34, 423464.
Zeng, M., Mosier, N.S., Huang, C.P., Sherman, D.M., Ladisch, M.R., 2007. Microscopic
examination of changes of plant cell structure in corn stover due to cellulase
activity and hot water pretreatment. Biotechnol. Bioeng. 97 (2).
Zhang, Y.H.P., Lynd, L.R., 2004. Toward an aggregated understanding of enzymatic
hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol. Bioeng. 88
(7), 797824.
Zheng, Y., Lin, H.M., Tsao, G.T., 1998. Pretreatment for cellulose hydrolysis by carbon
dioxide explosion. Biotechnol. Progr. 14, 890896.
Zhu, Y., Lee, Y.Y., Elander, R.T., 2004. Dilute-acid pretreatment of corn stover using a
high-solids percolation reactor. Appl. Biochem. Biotechnol. 117, 103114.
Zhu, Y., Lee, Y.Y., Elander, R.T., 2005. Optimaization of dilute-acid pretreatment of
corn stover using a high solids percolation reactor. Appl. Biochem. Biotechnol.,
10451054.