pretreatments to enhance the digestibility of lignocellulosic biomass

Bioresource Technology 100 (2009) 10–18

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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 a b s t r a c t

Article history:Received 11 July 2006Received in revised form 18 May 2008Accepted 20 May 2008Available online 2 July 2008

Keywords:PretreatmentLignocelluloseDigestibilityFermentationBioenergy

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.05.027

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Lignocellulosic biomass represents a rather unused source for biogas and ethanol production. Many fac-tors, like lignin content, crystallinity of cellulose, and particle size, limit the digestibility of the hemicel-lulose and cellulose present in the lignocellulosic biomass. Pretreatments have as a goal to improve thedigestibility of the lignocellulosic biomass. Each pretreatment has its own effect(s) on the cellulose, hemi-cellulose and lignin; the three main components of lignocellulosic biomass. This paper reviews the differ-ent effect(s) of several pretreatments on the three main parts of the lignocellulosic biomass to improve itsdigestibility. Steam pretreatment, lime pretreatment, liquid hot water pretreatments and ammonia basedpretreatments are concluded to be pretreatments with high potentials. The main effects are dissolvinghemicellulose and alteration of lignin structure, providing an improved accessibility of the cellulose forhydrolytic enzymes.

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1. Introduction 2.1. Cellulose

For a long time research is being done to enhance the digestibil-ity of lignocellulosic biomass for mainly the efficient conversion of(hemi-) cellulose to ethanol, methane and, in the last years, also tohydrogen. It is however not clear which characteristics of the lig-nocellulosic biomass are important, to determine a successful pre-treatment. Further more additional problems, like production ofrecalcitrant or inhibitory products, are to be solved.

A lot of literature is written about different pretreatment methodsto enhance the digestibility of lignocellulosic material. The objectiveof this review is to find out which characteristics of lignocellulosicbiomass determine which pretreatment method will be successfuland attractive to apply. This will be done by explaining the composi-tion of lignocellulosic material, giving an overview of the methaneand ethanol production process, summarizing the effects of differentpretreatment methods on lignocellulosic biomass and the conse-quences of these effects on ethanol and methane production. More-over, additional problems will be analyzed, and finally, conclusionswith respect to promising pretreatment techniques and needed fu-ture research are made. Hydrogen production is left out in this paper,because it is still in the R&D phase (Reith et al., 2003).

2. The composition of lignocellulosic material

Lignocellulosic material consists of mainly three different typesof polymers, namely cellulose, hemicellulose and lignin, which areassociated which each other (Fengel and Wegener, 1984).

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Cellulose exists of D-glucose subunits, linked by b-1,4 glycosidicbonds (Fengel and Wegener, 1984).

The cellulose in a plant consists of parts with a crystalline(organized) structure, and parts with a, not well-organized, amor-phous structure. The cellulose strains are ‘bundled’ together andform so called cellulose fibrils or cellulose bundles. These cellulosefibrils are mostly independent and weakly bound through hydro-gen bonding (Laureano-Perez et al., 2005).

2.2. Hemicellulose

Hemicellulose is a complex carbohydrate structure that consistsof different polymers like pentoses (like xylose and arabinose),hexoses (like mannose, glucose and galactose), and sugar acids.The dominant component of hemicellulose from hardwood andagricultural plants, like grasses and straw, is xylan, while this isglucomannan for softwood (Fengel and Wegener, 1984; Saha,2003).

Hemicellulose has a lower molecular weight than cellulose, andbranches 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 ligninand the cellulose fibers and gives the whole cellulose–hemicellu-lose–lignin network more rigidity (Laureano-Perez et al., 2005).

The solubility of the different hemicellulose compounds is indescending order: mannose, xylose, glucose, arabinose, and galact-ose. The solubility’s increase with increasing temperature. The sol-ubility’s of higher molecular polymers could not be predicted,because of unknown melting points (Gray et al., 2003). The solubi-lization of hemicellulose compounds into the water starts around

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A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 10–18 11

180 �C under neutral conditions according to Bobleter (1994). Gar-rote et al. (1999) however mention that already from 150 �C partsof the hemicellulose solubalize. The solubilization of lignocellulosecomponents not only depends on temperature, but also on otheraspects like moisture content and pH (Fengel and Wegener, 1984).

The xylan of hemicellulose can be extracted quite well in anacid or alkaline environment, while glucomannan can hardly beextracted in an acid environment and needs a stronger alkalineenvironment than xylan to be extracted (Balaban and Ucar, 1999;Fengel and Wegener, 1984; Lawther et al., 1996b). Xylan appearsto be the part that can be extracted the most easily.

Of cellulose, hemicellulose and lignin the hemicelluloses are themost thermal-chemically sensitive (Levan et al., 1990; Winandy,1995). During thermal–chemical pretreatment firstly the sidegroups of hemicellulose react, followed by the hemicellulose back-bone (Sweet and Winandy, 1999).

2.3. Lignin

Lignin is, after cellulose and hemicellulose, one of the mostabundant polymers in nature and is present in the cellular wall.It is an amorphous heteropolymer consisting of three differentphenylpropane units (p-coumaryl, coniferyl and sinapyl alcohol)that are held together by different kind of linkages. The main pur-pose of lignin is to give the plant structural support, impermeabil-ity, and resistance against microbial attack and oxidative stress.The amorphous heteropolymer is also non-water soluble andoptically inactive; all this makes the degradation of lignin verytough (Fengel and Wegener, 1984).

Lignin, just like hemicellulose, normally starts to dissolve intowater around 180 �C under neutral conditions (Bobleter, 1994).The solubility of the lignin in acid, neutral or alkaline environ-ments 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 canconsist of three phases, namely pretreatment, anaerobic hydrolysisand methane production, and post-treatment of the liquid fraction.A product separation step is not needed during the methane pro-duction step, because methane is, under normal conditions, a gasand will separate itself from the liquid fraction.

Pretreatment can be done to improve the hydrolysis yield andtotal methane yield. The hydrolysis of the lignocellulose andconversion to methane is in general done in one reactor by a con-sortium of micro-organisms. Often only the second phase, hydroly-sis and methane production is executed, for energy production andrecovery.

The advantage of using a mixture of micro-organisms is that al-most all products, like pentoses, hexoses, volatile products andeven sometimes inhibiting compounds like furfural and soluble lig-nin compounds (in not too high concentrations) can be convertedto 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 consistsof mainly five different steps, namely pretreatment, (enzymatic)hydrolysis, fermentation, product separation, and post-treatmentof the liquid fraction. The pretreatment is necessary to improvethe rate of production and the total yield of monomeric sugars in

the hydrolysis step. The conversion of (hemi) cellulose to mono-meric sugars can be done chemically by acids or enzymaticallyby addition of cellulases (enzymes responsible for the hydrolysisof cellulose). The produced monomeric hexoses (six carbon sugars)can be fermented to ethanol quite easily, while the fermentation ofpentoses (five carbon sugars) is only done by a few strains. Volatileproducts are also not easily fermented to ethanol. A problemoccurring during the fermentation is that the formed product eth-anol is an inhibitor for the yeasts/bacteria that perform the fer-mentation. This puts a limit to the concentration of fermentablesugars. Latter is not the case for methane production as methaneescapes from the water phase. After the fermentation the ethanolhas to be recovered from the fermentation broth by distillation(Mosier et al., 2005b).

Furfural and other inhibitors like (probably) soluble lignin com-pounds also form a problem for the fermentation step, becausesuch compounds can inhibit, or even stop the fermentation (Laser,2001; Laser et al., 2002).

5. Factors limiting the hydrolysis

The (enzymatic) hydrolysis of lignocellulose is limited by sev-eral factors. Several researchers conclude that crystallinity of cellu-lose is just one of the factors. Other factors are degree ofpolymerization (DP), moisture content, available surface area andlignin 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 enzy-matic hydrolysis, but not the 3-d enzymatic hydrolysis. Caulfieldand Moore (1974) mention that decreased particle size and in-creased available surface rather than crystallinity affect the rateand extent of the hydrolysis. Zhang and Lynd (2004) mention thata slower conversion of crystalline cellulose as compared to amor-phous cellulose, would increase the percentage crystallinity ofthe hydrolyzed biomass. This trend however has not been con-firmed by research (Zhang and Lynd, 2004). Other researchers likeGrethlein (1985), Grous et al. (1986) and Thompson et al. (1992)conclude that the pore size of the substrate in relation to the sizeof the enzymes is the main limiting factor in the enzymatic hydro-lysis of (lignocellulosic) biomass. Removal of hemicellulose in-creases the mean pore size of the substrate and thereforeincreases the probability of the cellulose to get hydrolyzed (Greggand Saddler, 1996; Grethlein, 1985; Palonen et al., 2004). Drying ofpretreated 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 gettrapped in the pores if the internal area is much larger than theexternal area, which is the case for many lignocellulosic biomasses.Lignin limits the rate and extend of (enzymatic) hydrolysis by act-ing as a shield, preventing the digestible parts of the substrate to behydrolyzed (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. Theobjective of a mechanical pretreatment is a reduction of particlesize and crystallinity. The reduction in particle size leads to an in-crease of available specific surface and a reduction of the degree ofpolymerization (DP) (Palmowski and Muller, 1999). The millingcauses also shearing of the biomass.

The increase in specific surface area, reduction of DP, and theshearing, are all factors that increase the total hydrolysis yield of

12 A.T.W.M. Hendriks, G. Zeeman / Bioresource Technology 100 (2009) 10–18

the lignocellulose in most cases by 5–25% (depends on kind of bio-mass, kind of milling, and duration of the milling), but also reducesthe technical digestion time by 23–59% (thus an increase in hydro-lysis rate) (Delgenés et al., 2002; Hartmann et al., 1999). A particlesize reduction below 40 mesh however has little effect on thehydrolysis yield as well as hydrolysis rate of the biomass (Changand Holtzapple, 2000).

6.2. Consequences for ethanol and methane production

Milling causes both an increased methane (5–25%; Delgenés etal., 2002) and ethanol yield and also increases the hydrolysis rate.As no production of inhibitors (like furfural and HMF (hydroxym-ethylfurfural)) are produced, milling is suited for both methaneand ethanol production. It has however a high energy requirement(Cowling and Kirk, 1976; Ramos, 2003) and was found thereforenot economically feasible as pretreatment in 1987 (Fan et al.,1987). Taking into account the high energy requirements of millingand the continuous rise of the energy prices, it is likely that millingis 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 150–180 �C, parts of the ligno-cellulosic biomass, firstly the hemicelluloses and shortly after thatlignin, will start to solubalize (Bobleter, 1994; Garrote et al., 1999).The composition of the hemicellulose backbone and the branchinggroups determine the thermal, acid and alkali stability of the hemi-cellulose (see Section 2.2). From the two dominant components ofhemicelluloses (xylan and glucomannan), the xylans are thermallythe least stable, but the difference with the glucomannans is onlysmall. Above 180 �C an exothermal reaction (probably solibiliza-tion) of the hemicellulose starts (Beall and Eickner, 1970; Doman-sky and Rendos, 1962). This temperature of 180 �C is probably justan indication of the temperature at which an exothermal reactionof the hemicellulose starts, because the thermal reactivity of ligno-cellulosic biomass depends largely on its composition (Fengel andWegener, 1984; Hon and Shiraishi, 1991).

During thermal processes a part of the hemicellulose is hydro-lyzed and forms acids. These acids are assumed to catalyze the fur-ther hydrolysis of the hemicellulose (Gregg and Saddler, 1996). Liuand Wyman (2003) and Zhu et al. (2004, 2005) conclude that other,so far unknown, factors than the catalyzing effect of in situ formedacids 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 solubi-lization of lignin. The produced compounds are almost always phe-nolic compounds and have in many cases an inhibitory or toxiceffect 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 condi-tions promote the condensation and precipitation of soluble lignincompounds, sometimes even with soluble hemicellulosic com-pounds like furfural and HMF (Bobleter and Concin, 1979; Loraand Wayman, 1978; Negro et al., 2003).

Heat pretreatment in which soluble (hemi) cellulose and lig-nin compounds are formed, has a risk of formation of phenolicand heterocyclic compounds, like vanillin, vanillin alcohol, furfu-ral and HMF, especially in acidic environments (Ramos, 2003).These formed compounds can be inhibitory. When the solid con-centration was 3% or higher and the temperature exceeded

220 �C during a 2 min pretreatment time, ethanol productionwas almost completely inhibited, due to formation of furfuraland other compounds (probably soluble lignin compounds)(Laser et al., 2002).

Temperatures of 250 �C and higher should be avoided duringpretreatment, as unwanted pyrolysis reactions start to take placeat such temperatures (Brownell et al., 1986).

According to Weimer et al. (1995) thermal pretreatment canalso cause an increase of the Crystallinity Index (CrI) of cellulose,though no increase was observed when the CrI was already high.


7.2.1. Steam pretreatment/steam explosion (ST/SE)During steam pretreatment the biomass is put in a large ves-

sel and steam with a high temperature (temperatures up to240 �C) and pressure, is applied for a few minutes. After a settime, the steam is released and the biomass is quickly cooleddown. The objective of a steam pretreatment/steam explosionis to solubalize the hemicellulose to make the cellulose betteraccessible for enzymatic hydrolysis and to avoid the formationof inhibitors.

The difference between ‘steam’ pretreatment and ‘steamexplosion’ pretreatment is the quick depressurization and cool-ing down of the biomass at the end of the steam explosion pre-treatment, which causes the water in the biomass to ‘explode’.In this article only steam pretreatment will be discussed, asthe impact of the change in structure of the biomass, causedby the explosion, on the digestibility is still doubted (Brownellet al., 1986).

During steam pretreatment parts of the hemicellulose hydro-lyze and form acids, which could catalyze the further hydrolysisof the hemicellulose. This process, in which the in situ formed acidscatalyze the process itself, is called ‘auto-cleave’ steam pretreat-ment. The role of the acids, is probably however not to catalyzethe solubilization of the hemicellulose, but to catalyze the hydroly-sis 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 ofthe pretreatment. In this severity factor the temperature of thepretreatment and the duration of the pretreatment are combinedin the following way:

‘‘log R0 = log(t � e((T�100)/14.75))”; with ‘t’ in minutes and ‘T’ in de-grees Celsius (Overend and Chornet, 1987).

During steam pretreatment the moisture content of the biomassinfluences the needed pretreatment time. The higher the moisturecontent, the longer the optimum steam pretreatment times (Brow-nell et al., 1986).

Low pressure steam pretreatment (2 bars, 120 �C, and pretreat-ment times up to 300 min) did not have a large effect on the com-position of the wheat straw according to Lawther et al. (1996a),though no enzymatic conversion step was carried out for deter-mining 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 in-stead of steam. The objective of the liquid hot water is to sol-ubalize mainly the hemicellulose to make the cellulose betteraccessible and to avoid the formation of inhibitors. To avoidthe formation of inhibitors, the pH should be kept between 4and 7 during the pretreatment. Maintaining the pH between 4and 7 minimizes the formation of monosaccharides, and there-fore also the formation of degradation products that can furthercatalyze hydrolysis of the cellulosic material during pretreat-


ment (Kohlmann et al., 1995; Mosier et al., 2005a; Weil et al.,1997). If catalytic degradation of sugars occurs it results in aseries of reactions that are difficult to control and result inundesirable side products. By keeping the pH between 4 and7 the autocatalytic formation of fermentation inhibitors areavoided during the pretreatment.

A difference between the LHW and steam pretreatment is theamount and concentration of solubilized products. In a LHW pre-treatment the amount of solubilized products is higher while theconcentration of these products is lower compared to steam pre-treatment (Bobleter, 1994). This is probably caused by the higherwater input in LHW pretreatment compared to steam pretreat-ment. The yield of solubilized (monomeric) xylan is generally alsohigher for LHW pretreatment; though this result diminishes whenthe solid concentration increases, because (monomeric) xylan isthen further degraded by hydrolytic reactions to, for example, xy-lose and furfural (Laser et al., 2002).

Yang and Wyman (2004) discovered that flow through sys-tems removed more hemicellulose and lignin from corn stoverthan batch systems did, at the same severity factors. Moreover,the addition of an external acid during the flow through processcaused higher hemicellulose and lignin removal, while in batchtests, the addition of an external acid caused less lignin removal;at increased reaction times at temperatures above 200 �C Kla-son–lignin (acid-insoluble lignin) removal decreased. It was con-cluded that the external acid caused the lignin to solubalizefaster, but also to condensate faster. The higher hemicelluloseand lignin removal with the addition of an external acid duringflow through experiments is in conflict with the conclusions ofBobleter et al. (1991), Jacobsen and Wyman (2002) and Liu andWyman (2003), which state that acids have no real effect orare not the only factor affecting the solubilization of hemicellu-lose and lignin and that there should be other reasons for thesolubilization of hemicellulose and lignin during flow throughexperiments.


7.3.1. Steam pretreatment/steam explosionSteam pretreatment includes a risk on production of com-

pounds, like furfural, HMF, and soluble phenolic compounds. Thesecompounds are inhibiting the ethanol fermentation and methaneproduction. The methane producing bacteria are however capableof 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 byBenjamin et al. (1984), Fox et al. (2003), and Noike and NiigataEng. (2001). Grous et al. (1986) reported a six-fold increase in enzy-matic digestibility of the biomass after steam pretreatment. A wayto minimize the hemicellulose degradation during steam pretreat-ment is by separating the biomass from the condensate during thepretreatment (Allen et al., 2001), by keeping the pH between 5 and7 during the pretreatment by the addition of an external alkali (Li etal., 2005; Weil et al., 1998), or by applying a two-step steam pre-treatment. It is however not clear if the higher ethanol or methaneyield out weighs the additional costs of a second pretreatment step(Shahbazi et al., 2005; Söderström et al., 2002).

The positive effect of steam pretreatment is mostly due to re-moval of a large part of the hemicellulose, causing an increase ofcellulose fiber reactivity, probably because the cellulose is easieraccessible for the enzymes (Converse et al., 1989; Grohman et al.,1986; Laser et al., 2002).

Some yeast species can also convert pentoses to ethanol (Kuy-per et al., 2005). Consequently the degradation of pentoses to, forexample, furfural during the steam pretreatment results in a lossof carbon for the ethanol production.

Steam pretreatment includes a risk on condensation andprecipitation of soluble lignin components, making the biomass lessdigestible, reducing the ethanol as well as the methane production.

7.3.2. Liquid hot waterLiquid hot water has the major advantage that the solubilized

hemicellulose and lignin products are present in lower concentra-tions, when compared to steam pretreatment, due to higher waterinput. Due to these lower concentrations the risk on degradationproducts like furfural and the condensation and precipitation oflignin compounds is reduced. Weil et al. (1998) had a 2- to 5-foldincrease in enzymatic hydrolysis of their substrate after LHWpretreatment.

8. Acid pretreatment


Pretreatment of lignocellulose with acids at ambient tempera-ture are done to enhance the anaerobic digestibility. The objectiveis to solubilize the hemicellulose, and by this, making the cellulosebetter accessible.

The pretreatment can be done with dilute or strong acids. Themain reaction that occurs during acid pretreatment is the hydroly-sis of hemicellulose, especially xylan as glucomannan is relativelyacid stable. Solubilized hemicelluloses (oligomers) can be sub-jected to hydrolytic reactions producing monomers, furfural,HMF and other (volatile) products in acidic environments (Fengeland Wegener, 1984; Ramos, 2003). During acid pretreatment solu-bilized lignin will quickly condensate and precipitate in acidicenvironments (Liu and Wyman, 2003; Shevchenko et al., 1999).The solubilization of hemicellulose and precipitation of solubilizedlignin are more pronounced during strong acid pretreatment com-pared to dilute acid pretreatment.

Xiao and Clarkson (1997) showed that the addition of nitric acidduring acid pretreatment has a tremendous effect on the solubili-zation of lignin of newspaper.


The advantage of acid pretreatment is the solubilization ofhemicellulose and by this, making the cellulose more easily acces-sible for the enzymes. There is however a risk on the formation ofvolatile degradation products and this carbon is in many cases lostfor the conversion to ethanol. Volatile products however can beconverted to methane. The condensation and precipitation of solu-bilized lignin components is an unwanted reaction, as it decreasesdigestibility.

Strong acid pretreatment is for the ethanol production notattractive, because there is a risk on the formation of inhibitingcompounds. Dilute acid pretreatment however is considered asone of the promising pretreatment methods, because secondaryreactions during the pretreatment can be prohibited in dilute acidpretreatment.

For methane production acid pretreatment is more attractivethan for ethanol production, because methanogens can handlecompounds like furfural and HMF to a certain concentration andwith an acclimatization period. For both the ethanol as well asthe methane production the chance on soluble lignin componentsis a risk, because soluble lignin compounds are often inhibiting forboth processes. Methanogens are however capable of adapting tosuch inhibiting compounds (Benjamin et al., 1984; Xiao and Clark-son, 1997).

When sulfuric or nitric acids are used in the acidic pretreat-ment, the methane production during anaerobic treatment will


be reduced as a result of reduction of sulphate and nitrate torespectively H2S and N2.

9. Alkaline pretreatment


During alkaline pretreatment the first reactions taking place aresolvation and saphonication. This causes a swollen state of the bio-mass 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 dis-solved polysaccharides can take place. Loss of polysaccharides ismainly caused by peeling and hydrolytic reactions (Fengel andWegener, 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 carbondioxide, also increases.

Xylan can be selectively removed with aqueous potassiumhydroxide. The temperature is kept low during extraction (roomtemperature or lower) to prevent peeling (Hon and Shiraishi,2001). Glucomannans and xylans can both be subject of the peelingreaction. This itself is not a problem, but the higher the monomerichemicellulose fraction, the lower the total recovery of the hemicel-lulose (Laser et al., 2002). The monomeric forms of hemicelluloseare probably easily degradable to other (volatile) compounds andfor example furfural, which leads to losses of digestible substratefor the ethanol process (Bobleter, 1994).

An important aspect of alkali pretreatment is that the biomasson itself consumes some of the alkali. The residual alkali concen-tration after the alkali consumption by the biomass is the alkaliconcentration left over for the reaction (Gossett et al., 1982). Pav-lostathis and Gossett (1985) for example found during their exper-iments an alkali consumption of approximately 3 g NaOH/100 g TS.Lime works remarkably better than sodium hydroxide (Gossett etal., 1982).

Alkali extraction can also cause solubilization, redistributionand condensation of lignin and modifications in the crystallinestate of the cellulose. These effects can lower or counteract the po-sitive effects of lignin removal and cellulose swelling (Gregg andSaddler, 1996).

Another important aspect of alkaline pretreatment is thechange of the cellulose structure to a form that is denser and ther-modynamically more stable than the native cellulose (Pettersen,1984).


Alkaline pretreatment causes hemicellulose and parts of ligninto solubalize. The removal of hemicellulose has a positive effecton the degradability of cellulose. There is however often a loss ofhemicellulose to degradation products and the solubilized lignincomponents often have an inhibitory effect. Gossett et al. (1982)for example concluded that alkaline heat treated lignin in concen-trations over 1 g/l gave a major inhibitory effect to the methano-genic micro-organisms. This is probably caused by the productsformed from the lignin during the alkaline heat pretreatment.The loss of fermentable sugars and production of inhibitory com-pounds makes the alkaline pretreatment less attractive for the eth-anol production. The production of inhibitors is less severe formethanogens as compared to yeasts for ethanol production. Meth-anogens are (often) capable of adapting to such compounds. Pavlo-stathis and Gossett (1985) mentioned a 100% increase in methaneproduction after an alkaline pretreatment of wheat straw.

10. Oxidative pretreatment


An oxidative pretreatment consists of the addition of an oxidiz-ing compound, like hydrogen peroxide or peracetic acid, to the bio-mass, which is suspended in water. The objective is to remove thehemicellulose and lignin to increase the accessibility of the cellu-lose. During oxidative pretreatment several reactions can takeplace, like electrophilic substitution, displacement of side chains,cleavage of alkyl aryl ether linkages or the oxidative cleavage ofaromatic nuclei (Hon and Shiraishi, 2001).

In many cases the used oxidant is not selective and thereforelosses of hemicellulose and cellulose can occur. A high risk onthe formation of inhibitors exists, as lignin is oxidized and solublearomatic compounds are formed.

Teixeira et al. (1999) have investigated the use of peracetic acidat ambient temperatures as a pretreatment method for hybrid pop-lar and sugar cane bagasse. Peracetic acid is very lignin selectiveand no significant carbohydrate losses occurred. The enzymatichydrolysis of the cellulose increased from 6.8% (untreated) to amaximum of about 98% (pretreated) at a 21% peracetic acid pre-treatment. Teixeira et al. (1999) showed similar results for digest-ibility at using a mixture of NaOH and peracetic acid. The yieldsduring experiments carried out at ambient temperature were high-er as compared to higher temperatures.

Gould (1984) demonstrated the use of H2O2 for delignificationwith a maximum at pH 11.5. No substantial delignification oc-curred below a pH of 10.0. Hydrogen peroxide had no real effecton the enzymatic digestibility at a pH 12.5 or higher. Accordingto Gould (1984) the hydrogen peroxide concentration should beat least 1% and the weight ratio between H2O2 and biomass shouldbe 0.25 for a good delignification. The delignification is probablycaused by the hydroxyl ion (HO�), which is a degradation productof hydrogen peroxide with a maximum at pH 11.5–11.6. About halfof the lignin was solubilized in this way (temperature around 25 �Cand a duration of 18–24 h) (Gould, 1984).


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

11. Combinations, ammonia and carbon dioxide pretreatment

11.1. Thermal pretreatment in combination with acid pretreatment

A way to improve the effect of thermal steam or LHW pretreat-ment is to add an external acid. This addition of an external acidcatalyzes the solubilization of the hemicellulose, lowers the opti-mal pretreatment temperature and gives a better enzymatichydrolysable 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 toH2SO4 in the first 20 seconds of the process; after that, the cata-lytic hydrolysation of the hemicellulose starts. Another importantpoint is that gradual removal of hemicellulose and lignin can trig-ger reorientation of cellulose to a more crystalline form (Greggand Saddler, 1996). Latter is true for every pretreatment thatgradually removes hemicellulose and lignin. The effect of theadded acid is however still not clear. Tengborg et al. (1998)showed a severe inhibition in the ethanol production step at a


severity factor of 3 and higher with the addition of an externalacid. This is in line with the conclusion of Grohmann et al.(1985) that during steam pretreatment at temperatures of160 �C and higher with 0.5% sulfuric acid addition an appreciableproduction of furfural occurs.

Söderström et al. (2002) investigated the use of a two-stepsteam pretreatment of softwood with SO2. The first step was car-ried out at lower severities (log R0 2.66–4.24) for the recovery ofhemicellulose sugars and the second step was done at higherseverities (log R0 2.95–4.24) to improve the digestibility of the cel-lulose. The highest hemicellulose recovery in the first pretreatmentstep was achieved at a severity of about 3 and the highest sugaryield in the second pretreatment step at a severity between 3.5and 4.3. However, pretreatments at temperatures of 220 �C didnot give satisfactory yields because of the formation of inhibitingcompounds. Wu et al. (1999) got the highest amount of mono-meric sugars from hemicellulose at a severity factor of approxi-mately 3 (175 �C, 7.5 min, 4,5% SO2). At higher severities themonomeric sugars were probably degraded to furfural and othercompounds.

11.2. Thermal pretreatment in combination with alkalinepretreatment

Another way to improve the thermal pretreatment is to add anexternal alkali instead of an acid to the process. A very commonalkaline thermal pretreatment is lime pretreatment. This pretreat-ment is usually carried out at temperatures of 100–150 �C withlime addition of approximately 0.1 g Ca(OH)2 g substrate�1 (Changet al., 2001). Chang and Holtzapple (2000) attribute the effective-ness of lime pretreatment to the opening of the ‘acetyl valve’ andpartly opening the ‘lignin valve’, making the substrate more acces-sible to hydrolysis. According to Kaar and Holtzapple (2000)lime pretreatment (with heating) is sufficient to increase thedigestibility of low-lignin containing biomass, but not for high-lignin containing biomass. Chang et al. (2001) mention that limepretreatment of switchgrass and corn stover did not inhibit theenzymatic saccharification and fermentation steps. Pretreatedsoftwood however was washed before the enzymatic saccharifica-tion and fermentation step to prevent possible inhibiting by (thelarge amount of) solubilized lignin. A positive effect of lime is thatit is relatively cheap and safe (Gandi et al., 1997) and that the cal-cium can be regained as insoluble calcium carbonate by the reac-tion with carbon dioxide. This calcium carbonate can be convertedto lime again with the lime kiln technology (Chang et al., 1998).

Fox et al. (2003) reported an improvement in methane produc-tion with a factor of 3 to 4.5 after pretreating newsprint waste withalkaline heat pretreatment.

11.3. Thermal pretreatment in combination with oxidativepretreatment

Ando et al. (1988) mentions that the saccharification of cedar,soaked in peracetic acid and steam treated at 231 �C for 10 min,was directly proportional to the amount of peracetic acid adsorbedin the chips.

Wet-oxidation is another oxidative pretreatment method,which uses oxygen as oxidator. The soluble sugars produced duringwet-oxidation pretreatment of wheat straw are mainly polymersopposite to the monomers produced during steaming or acidhydrolysis as pretreatment. Phenolic monomers are no end prod-ucts during wet-oxidation but are further degraded to carboxylicacids. Also the production of furfural and HMF was low duringwet-oxidation, but part of the hemicellulose was lost by reactionto carbon dioxide and water (Klinke et al., 2002).

11.4. Thermal pretreatment in combination with alkaline oxidativepretreatment

According to Chang et al. (2001) thermal lime pretreatment isnot capable of removing enough lignin of high-lignin biomass toenhance the enzymatic digestibility and therefore oxygen as oxi-dant must be included during the pretreatment. A low sugar deg-radation was observed, probably as a result of the relative lowtemperature of 150 degrees, applied during the pretreatment.The enzymatic digestibility of the treated biomass was 13 timeshigher than for the untreated biomass. The pretreated biomasswas however washed to remove the probably produced inhibitingsoluble lignin compounds (Chang et al., 2001). After the oxidativelime pretreatment about 21% of the added lime could be recoveredby carbon dioxide carbonation (Chang et al., 2001b).

11.5. Ammonia and carbon dioxide pretreatment

Other applied pretreatments are ammonia and carbon dioxidepretreatment. The objective of the ammonia pretreatment (alsocalled AFEX pretreatment). The ammonia pretreatment is con-ducted with ammonia loadings around 1:1 (kg ammonia/kg dwbiomass) at temperatures ranging from ambient temperature witha duration of 10–60 days, to temperatures of up to 120 �C with aduration of several minutes (Alizadeh et al., 2005; Kim and Lee,2005). Alizadeh et al. (2005) reported a six-fold increased enzy-matic hydrolysis yield and a 2.5-fold ethanol yield after pretreat-ment. Bariska (1975) and Kim and Lee (2005) mention swellingof the cellulose and delignification as the responsible factors forthe increased yield.

Carbon dioxide pretreatment is conducted with high-pressurecarbon dioxide at high temperatures of up to 200 �C with a dura-tion of several minutes. Explosive steam pretreatment with high-pressure carbon dioxide causes the liquid to be acidic and this acidhydrolyses 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 poresize.

12. Overview of effects of pretreatments on lignocellulose

In chapter six up to and including chapter eleven the effects ofseveral pretreatments on the physical/chemical composition orstructure of lignocellulose is reported. Table 1 summarizes themost important effects of the different pretreatment methods,discussed in this paper. The table suggests that increasing thesurface area is one of the major approaches of a pretreatment bysolubilization of the hemicellulose and/or lignin and/or alteringthe lignin. The importance of surface area is mentioned by Zenget al. (2007).

13. Conclusion and final discussion

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

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

Increase accessiblesurface area

Decrystallizationcellulose

Solubilizationhemicellulose

Solubilizationlignin

Formation furfural/HMF

Alteration ligninstructure

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

+ = 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 pretreatmentwhich can be performed continuously, which is very attractivefor industrial appliance (Schmidt and Thomsen, 1998).

It can be concluded that pretreatments like concentrated acids,wet oxidation, solvents and metal complexes are effective, but tooexpensive compared to the value of glucose (Fan et al., 1987; Mo-sier et al., 2005b). Steam pretreatment, lime pretreatment, LHWsystems and ammonia based pretreatments are the ones that,according to the factors determining the economic effectivenessmentioned above, and the effects of the pretreatments (Table 1),have a high potential. An economical evaluation of five differentpretreatment technologies (dilute acid, hot water, ammonia fiberexplosion (AFEX), ammonia recycle percolation (ARP), and lime)is presented by Eggeman and Elander (2005) in the special issue‘Coordinated development of leading biomass pretreatment tech-nologies’ (Wyman et al., 2005). The main conclusion is that thereis only little differentiation between the projected economic per-formances of the pretreatment options, but that optimizing en-zyme blends for each pretreatment approach and conditioningrequirements of hydrolysates at process-relevant sugar concentra-tions resulting from each pretreatment, may result in greater dif-ferentiation in projected process economics.

Not much research on the carbon dioxide explosion based pre-treatments has been done, so it is difficult to judge if this pretreat-ment is a potential one ore not.

The effect of the pretreatments is however very dependent onthe biomass composition and operating conditions. All these pre-treatments have their advantages and disadvantages and future re-search is needed for optimization.

An important aspect is the choice of the bio-fuel to produce. Thedifferences between the ethanol and methane conversion efficien-cies are not large anymore, as nowadays also yeast strains capableof converting C5 and C6 sugars to ethanol are available (Kuyper etal., 2005). A disadvantage of the ethanol production, compared tomethane production, is the low tolerance for inhibiting compoundslike furfural and HMF by the yeasts.

The higher tolerance for inhibiting compounds, the simple onestep process, and the use of a direct-methane fuel cell in for exam-ple stationary power generation, could favour the production ofCH4 in stead of ethanol (Murray et al., 1999). The performance ofa direct-methane fuel cell is comparable with that of a hydrogenbased fuel cell (Murray et al., 1999).

An aspect, which earns more attention, is the production ofethanol by thermophilic bacteria instead of yeasts. Thermophilicbacteria can produce an optimal mixture of enzymes for the

hydrolysis, while such optimal enzyme mixture is difficult to pro-duce artificially (Schwarz, 2001). The production of the enzymesby the thermophilic bacteria does not consume a lot of extra en-ergy (Ng et al., 1977), the enzyme hydrolyses rate can be compa-rable with that of the enzymes of Trichoderma reesei (Lynd et al.,1989). These bacteria can be adapted to higher ethanol concen-trations (Baskaran et al., 1995; Bender et al., 1985; Herrero andGomez, 1980; Klapatch et al., 1994; Larsen et al., 1997; Rani etal., 1996; Tailliez et al., 1989a, 1989b), and are maybe even capa-ble of handling compounds like furfural and HMF or adapt tothem. Thermophilic conditions decrease the risk on infection(Payton, 1984; Wiegel, 1980), and could create the possibilityof 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 dis-cussion and assistance throughout the investigation.

References

Alizadeh, H., Teymouri, F., Gilbert, T.I., Dale, B.E., 2005. Pretreatment ofswitchgrass by ammonia fiber explosion (AFEX). Appl. Biochem. Biotechnol.,1133–1141.

Allen, S.G., Schulman, Deborah, Lichwa, J., Antal Jr., M.J., 2001. A comparison ofaqueous and dilute-acid single-temperature pretreatment of yellow poplarsawdust. Ind. Eng. Chem. Res. 40, 2352–2361.

Ando, S., Kakimoto, T., Itoh, K., Arai I., Kiyoto, K., Hanai, S., 1988. Increaseddigestibility of cedar by pretreatment with peracetic acid and steam explosion.Biotechnol. Bioeng. 31, 802–804.

Balaban, M., Ucar, G., 1999. The effect of the duration of alkali treatment on thesolubility of polyoses. Turk. J. Agric. Forest. 23, 667–671.

Bariska, M., 1975. Collapse phenomena in beechwood during and after NH3-impregnation. Wood Sci. Technol. 9, 293–306.

Baskaran, S., Ahn, H.-J., Lynd, L.R., 1995. Investigation of the ethanol tolerance ofClostridium thermosaccharolyticum in continuous culture. Biotechnol. Progr. 11,276–281.

Beall, F.C., Eickner, H.W., 1970. Thermal degradation of wood components: a reviewof the Literature. U.S.D.A. Forest service, research paper FPL 130.

Bender, J., Vatcharapijarn, Y., Jeffries, T.W., 1985. Characteristics and adaptability ofsome new isolates of Clostridium thermocellum. Appl. Environ. Microbiol. 49 (3),475–477.

Benjamin, M.M., Woods, S.L., Ferguson, J.F., 1984. Anaerobic toxicity andbiodegradability of pulp mill waste constituents. Water Res. 18 (5), 601–607.

Bobleter, O., 1994. Hydrothermal degradation of polymers derived from plants.Prog. Polym. Sci. 19, 797–841.

Bobleter, O., Concin, R., 1979. Degradation of poplar lignin by hydrothermaltreatment. Cellul. Chem. Technol. 13, 583–593.

Bobleter, O., Bonn, G., Prutsch, W., 1991. Steam explosion-hydrothermolysis-organosolv. A comparison. In: Focher, B., Marzetti, A., Crescenzi, V. (Eds.),Steam explosion techniques. Gordon and Breach, Philadelphia, pp. 59–82.


Brownell, H.H., Yu, E.K.C., Saddler, J.N., 1986. Steam-explosion pretreatment ofwood: effect of chip size, acid, moisture content and pressure drop. Biotechnol.Bioeng. 28, 792–801.

Caulfield, D., Moore, W.E., 1974. Effect of varying crystallinity of cellulose onenzymic hydrolysis. Wood Sci. 6 (4), 375–379.

Chang, V.S., Holtzapple, M.T., 2000. Fundamental factors affecting enzymaticreactivity. Appl. Biochem. Biotechnol., 5–37.

Chang, V.S., Nagwani, M., Holtzapple, M.T., 1998. Lime pretreatment of cropresidues bagasse and wheat straw. Appl. Biochem. Biotechnol. 74, 135–159.

Chang, V.S., Kaar, W.E., Burr, B., Holtzapple, M.T., 2001. Simultaneoussaccharification and fermentation of lime-treated biomass. Biotechnol. Lett.23 (16), 1327–1333.

Chang, V.S., Nagwani, M., Kim, C., Holtzapple, T., 2001b. Oxidative limepretreatment of high-lignin biomass. Appl. Biochem. Biotechnol. 94, 1–28.

Converse, A.O., Kwarteng, I.K., Grethlein, H.E., Ooshima, H., 1989. Kinetics ofthermochemical pretreatment of lignocellulosic materials. Appl. Biochem.Biotechnol. 21, 111–116.

Cowling, E.B., Kirk, T.K., 1976. Properties of cellulose and lignocellulosic materials assubstrates for enzymatic conversion process. Biotechnol. Bioeng. Symp. 6, 95–123.

Delgenés, J.P., Penaud, V., Moletta, R., 2002. Pretreatments for the enhancement ofanaerobic digestion of solid wastes Chapter 8. In: Biomethanization of theOrganic Fraction of Municipal Solid Wastes. IWA Publishing, pp. 201–228.

Domansky, R., Rendos, F., 1962. On the pyrolysis of wood and its components. HolzRoh Werkst. 29, 473–476.

Eggeman, T., Elander, R.T., 2005. Process and economic analysis of pretreatmenttechnologies. Bioresour. Technol. 96, 2019–2025.

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. DeGruyter, Berlin.

Fox, M.H., Noike, T., Ohki, T., 2003. Alkaline subcritical-water treatment and alkalineheat treatment for the increase in biodegradability of newsprint waste. WaterSci. Technol. 48 (4), 77–84.

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, 195–211.

Garrote, G., Dominguez, H., Parajo, J.C., 1999. Hydrothermal processing oflignocellulosic materials. Holz Roh Werkst. 57, 191–202.

Gossett, J.M., Stuckey, D.C., Owen, W.F., Mccarty, P.L., 1982. Heat treatment andanaerobic digestion of refuse. J. Environ. Eng. Div. 108, 437–454.

Gould, J.M., 1984. Alkaline peroxide delignification of agricultural residues toenhance enzymatic saccharification. Biotechnol. Bioeng. 26, 46–52.

Grabber, J.H., 2005. How do lignin composition, structure, and cross-linking affectdegradability? A review of cell wall model studies. Crop Sci. 45,820–831.

Gray, M.C., Converse, A.O., Wyman, C.E., 2003. Sugar monomer and oligomersolubility. Data and predictions for application to biomass hydrolysis. Appl.Biochem. Biotechnol., 179–193.

Gregg, D., Saddler, J.N., 1996. A techno-economic assessment of the pretreatmentand fractionation steps of a biomass-to-ethanol process. Appl. Biochem.Biotechnol., 711–727.

Grethlein, H.E., 1985. The effect of pore size distribution on the rate of enzymatichydrolysis of cellulose substrates. Bio/Technol. 3, 155–160.

Grohmann, K., Torget, R., Himmel, M., 1985. Optimization of dilute acidpretreatment of biomass. Biotechnol. Bioeng. Symp. 15, 59–80.

Grohman, K., Torget, R., Himmel, M., 1986. Dilute acid pretreatment of biomass athigh solids concentration, Biotechnol. Bioeng. Symp. 17, 135–151.

Grous, W.R., Converse, A.O., Grethlein, H.E., 1986. Effect of steam explosionpretreatment on pore size and enzymatic hydrolysis of poplar. EnzymeMicrobiol. Technol. 8, 274–280.

Hartmann, H., Angelidaki, I., Ahring, B.K., 1999. Increase of anaerobic degradation ofparticulate organic matter in full-scale biogas plants by mechanical maceration.In: Mata-Alvarez, J., Tilche, A., Cecchi, F. (Eds), Proceedings of the SecondInternational 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 Clostridiumthermocellum effect of growth temperature. Appl. Environ. Microbiol. 40 (3),571–577.

Hon, D.N.S., Shirashi, N., 1991. Wood and Cellulosic Chemistry. Marcel Dekker, NewYork.

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 foruncatalyzed hydrolysis of sugarcane bagasse hemicellulose at varying solidsconcentration. Ind. Eng. Chem. Res. 41, 1454–1461.

Kaar, W.E., Holtzapple, M.T., 2000. Using lime pretreatment to facilitate theenzymatic hydrolysis of corn stover. Biomass Bioenergy 18 (3),189–199.

Kim, K.H., Hong, J., 2001. Supercritical CO2 pretreatment of lignocellulose enhancesenzymatic cellulose hydrolysis. Bioresour. Technol. 77, 139–144.

Kim, T.H., Lee, Y.Y., 2005. Pretreatment of corn stover by soaking in aqueousammonia. Appl. Biochem. Biotechnol., 1119–1132.

Klapatch, T.R., Hogsett, D.A.L., Baskaran, S., Pal, S., 1994. Organism development andcharacterization for ethanol production using thermophilic bacteria. Appl.Biochem. Biotechnol., 209–223.

Klinke, H.B., Ahring, B.K., Schmidt, A.S., Thomsen, A.B., 2002. Characterization ofdegradation products from alkaline wet oxidation of wheat straw. Bioresour.Technol. 82 (1), 15–26.

Kohlmann, K.L., Westgate, P.J., Sarikaya, A., Velayudhan, A., Weil, J., Hendrickson, R.,Ladisch, M.R. 1995. Enhanced enzyme activities on hydrated lignocellulosicsubstrates. BTEC paper 127. In: 207th American Chemical Society NationalMeeting, ACS Symposium series No. 618. Enzymatic Degradation of InsolubleCarbohydrates, pp. 237–255.

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, 113–116.

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 xylose-fermenting Saccharomyces cerevisiae strain . FEMSYeast Res. 5 (10),925–934.

Larsen, L., Nielsen, P., Ahring, B.K., 1997. Thermoanaerobacter mathranii sp. nov., anethanol-producing, extremely thermophylic anaerobic bacterium from a hotspring in Iceland. Arch. Microbiol. 168, 114–119.

Laser, M., 2001. Hydrothermal pretreatment of cellulosic biomass for bioconversionto 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. Acomparison of liquid hot water and steam pretreatments of sugar cane bagassefor bioconversion to ethanol. Bioresour. Technol. 81, 33–44.

Laureano-Perez, L., Teymouri, F., Alizadeh, H., Dale, B.E., 2005. Understandingfactors that limit enzymatic hydrolysis of biomass. Appl. Biochem. Biotechnol.,1081–1099.

Lawther, J.M., Sun, R., Banks, W.B., 1996a. Effect of steam treatment on the chemicalcomposition of wheat straw. Holzforschung 50, 365–371.

Lawther, J.M., Sun, R., Banks, W.B., 1996b. Effects of extraction conditions and alkalitype on yield and composition of wheat straw hemicellulose. J. Appl. Polym. Sci.60, 1827–1837.

Levan, S.L., Ross, R.J., Winandy, J.E., 1990. Effects of fire retardant chemicals onbending properties of wood at elevated temperatures. Research Paper FPL-RP-498. Madison, WI: U.S. Department of agriculture, Forest service, ForestProducts Laboratory, p. 24

Li, J., Henriksson, G., Gellerstedt, G., 2005. Carbohydrate reactions during high-temperature steam treatment of Aspen wood. Appl. Biochem. Bioeng. 125, 175–188.

Liu, C., Wyman, C.E., 2003. The effect of flow 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. Delignification of hardwoods by autohydrolysis andextraction. Tappi 61, 47–50.

Lynd, L.R., Grethlein, H.E., Wolkin, R.H., 1989. Fermentation of cellulosic substratesin batch and continuous culture by Clostridium thermocellum. Appl. Environ.Microbiol. 55 (12), 3131–3139.

Mok, W.S.-L., Antal, M.J.A., 1992. Uncatalyzed solvolysis of whole biomasshemicellulose 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 ofpH controlled liquid hot water pretreatment of corn stover. Bioresour. Technol.96, 1986–1993.

Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M.,2005b. Features of promising technologies for pretreatment of lignocellulosicbiomass. Bioresour. Technol. 96, 673–686.

Murray, E.P., Tsai, T., Barnett, S.A., 1999. A direct-methane fuel cell with a ceria-based anode. Lett. Nature 400, 649–651.

Negro, M.J., Manzanares, P., Oliva, J.M., Ballesteros, I., Ballesteros, M., 2003. Changesin various physical chemical parameters of Pinus Pinaster wood after steamexplosion pretreatment. Biomass Bioenergy 25, 301–308.

Ng, T.K., Weimer, P.J., Zeikus, J.G., 1977. Cellulolytic and physiological properties ofClostridium thermocellum. Arch. Microbiol. 114, 1–7.

Noike, T., Niigata Engineering, 2001. Micromolecularization of undegradableorganic substances in methane fermentation. In: Development of theWaste Treatment System for Recycling Society. Waste Research Foundation,Tokyo.

Overend, R.P., Chornet, E., 1987. Fractionation of lignocellulosis by steam–aqueouspretreatments. Philos. Trans. R. Soc. London A321, 523–536.

Palmowski, L., Muller, J., 1999. Influence of the size reduction of organic waste ontheir anaerobic digestion. In: II International Symposium on AnaerobicDigestion of Solid Waste. Barcelona 15–17 June, pp. 137–144.

Palonen, H., Thomsen, A.B., Tenkanen, M., Schmidt, A.S., Viikari, L., 2004. Evaluationof wet oxidation pretreatment for enzymatic hydrolysis of softwood. Appl.Biochem. Biotechnol. 117, 1–17.

Pavlostathis, S.G., Gossett, J.M., 1985. Alkaline treatment of wheat straw forincreasing anaerobic biodegradability. Biotechnol. Bioeng. 27, 334–344.

Payton, M.A., 1984. Production of ethanol by thermophilic bacteria. TrendsBiotechnol. 2, 153–158.

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 onenzymatic saccharification. Biotechnol. Bioeng. 26, 1219–1222.

Puri, V.P., Mamers, H., 1983. Explosive pretreatment of lignocellulosic residues withhigh-pressure carbon dioxide for the production of fermentation substrates.Biotechnol. Bioeng. 25, 3149–3161.


Ramos, L.P., 2003. The chemistry involved in the steam treatment of lignocellulosicmaterials. Quim. Nova. 26 (6), 863–871.

Rani, K.S., Swamy, M.V., Sunitha, D., Haritha, D., Seenayya, G., 1996. Improvedethanol tolerance and production in strains of Clostridium thermocellum. WorldJ. Microbiol. Biotechnol. 12, 57–60.

Reith, J.H., Wijffels, R.H., Barten, H., 2003. Bio-methane & Bio-hydrogen. Status andperspectives of biological methane and hydrogen production. Production of theDutch 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 ofwheat straw. Bioresour. Technol. 64, 139–151.

Schwarz, W.H., 2001. The cellulosome and cellulose degradation by anaerobicbacteria. Appl. Microbiol. Biotechnol. 56, 634–649.

Shahbazi, A., Li, Y., Mims, M.R., 2005. Application of sequential aqueous steamtreatments 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 steamexplosion/enzymatic hydrolysis of softwood. Appl. Biochem. Biotechnol., 867–876.

Söderström, J., Pilcher, L., Galbe, M., Zacchi, G., 2002. Two-step pretreatment ofsoftwood with SO2 impregnation for ethanol production. Appl. Biochem.Biotechnol., 5–21.

Sweet, M.S., Winandy, J.E., 1999. Influence of degree of polymerization of celluloseand hemicellulose on strength loss in fire-retardant-treated southern pine.Holzforschung 53 (3), 311–317.

Tailliez, P., Girard, H., Longin, R., Beguin, P., Millet, J., 1989a. Cellulose fermentationby an asporogenous mutant and an ethanol-tolerant mutant of Clostridiumthermocellum. Appl. Environ. Microbiol. 55 (1), 203–206.

Tailliez, P., Girard, H., Millet, J., Beguin, P., 1989b. Enhanced cellulose fermentationby an asporogenous and ethanol-tolerant mutant of Clostridium thermocellum.Appl. Environ. Microbiol. 55 (1), 207–211.

Teixeira, L.C., Linden, J.C., Schroeder, H.A., 1999. Alkaline and peracetic acidpretreatments of biomass for ethanol production. Appl. Biochem. Biotechnol.,19–34.

Tengborg, C., Stenberg, K., Galbe, M., Zacchi, G., Larsson, S., Palmqvist, E., Hahn-Hägerdal, B., 1998. Comparison of SO2 and H2SO4 impregnation of softwoodprior to steam pretreatment on ethanol production. Appl. Biochem. Biotechnol.,3–15.

Thompson, D.N., Chen, H.-C., Grethlein, H.E., 1992. Comparison of pretreatmentmethods on the basis of available surface area. Bioresour. Technol. 39, 155–163.

Von Sivers, N., Zacchi, G., Olsson, L., Hahn-Hägerdal, B., 1994. Cost analysis ofethanol production from willow using recombinant escherichia coli. Biotechnol.Progr. 10, 555–560.

Weil, J.R., Brewer, M., Hendrickson, R., Sarikaya, A., Ladisch, M.R., 1997. ContinuouspH monitoring during pretreatment of yellow poplar wood sawdust by pressurecooking in water. Appl. Biochem. Biotechnol. 68, 21–40.

Weil, J.R., Sarikaya, A., Rau, S.L., Goebz, J., Lasisch, C.M., Brwer, M., Hendrickson, R.,Tadisch, M.R., 1998. Pretreatment of corn fiber by pressure cooking in water.Appl. Biochem. Biotechnol. 73, 1–17.

Weimer, P.J., Hackney, J.M., French, A.D., 1995. Effects of chemical treatments andheating on the crystallinity of celluloses and their implications for evaluatingthe effect of crystallinity on cellulose biodegradation. Biotechnol. Bioeng. 48,169–178.

Wiegel, J., 1980. Formation of ethanol by bacteria. A pledge for use of extremethermophilic anaerobic bacteria in industrial ethanol fermentation processes.CMLS 36 (12), 1434–1446.

Winandy, J.E., 1995. Effects of fire retardant treatments after 19 months of exposureat 150F (66C). Res. Note FPL-RN-0264. U.S. Department of agriculture, ForestService, 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 andenzymatic hydrolysis of cellulose in softwoods. Appl. Biochem. Biotechnol.,47–54.

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, 1959–1966.

Xiao, W., Clarkson, W.W., 1997. Acid solubilization of lignin and bioconversion oftreated newsprint to methane. Biodegradation 8, 61–66.

Yang, B., Wyman, C.E., 2004. Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of Corn Stover Cellulose.Biotechnol. Bioeng. 86 (1), 88–95.

Zeikus, J.G., 1980. Chemical and fuel production by anaerobic bacteria. Annu. Rev.Microbiol. 34, 423–464.

Zeng, M., Mosier, N.S., Huang, C.P., Sherman, D.M., Ladisch, M.R., 2007. Microscopicexamination of changes of plant cell structure in corn stover due to cellulaseactivity and hot water pretreatment. Biotechnol. Bioeng. 97 (2).

Zhang, Y.H.P., Lynd, L.R., 2004. Toward an aggregated understanding of enzymatichydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol. Bioeng. 88(7), 797–824.

Zheng, Y., Lin, H.M., Tsao, G.T., 1998. Pretreatment for cellulose hydrolysis by carbondioxide explosion. Biotechnol. Progr. 14, 890–896.

Zhu, Y., Lee, Y.Y., Elander, R.T., 2004. Dilute-acid pretreatment of corn stover using ahigh-solids percolation reactor. Appl. Biochem. Biotechnol. 117, 103–114.

Zhu, Y., Lee, Y.Y., Elander, R.T., 2005. Optimaization of dilute-acid pretreatment ofcorn stover using a high solids percolation reactor. Appl. Biochem. Biotechnol.,1045–1054.

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