métodos de pré-tratamento na fermentação etanólica.pdf

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Pretreatment Methods for Bioethanol Production

Zhaoyang Xu &Fang Huang

Received: 21 January 2014 /Accepted: 15 June 2014 /Published online: 28 June 2014# Springer Science+Business Media New York 2014

Abstract Lignocellulosic biomass, such as wood, grass, agricultural, and forest residues,are potential resources for the production of bioethanol. The current biochemical processof converting biomass to bioethanol typically consists of three main steps: pretreatment,enzymatic hydrolysis, and fermentation. For this process, pretreatment is probably themost crucial step since it has a large impact on the efficiency of the overall bioconver-sion. The aim of pretreatment is to disrupt recalcitrant structures of cellulosic biomass tomake cellulose more accessible to the enzymes that convert carbohydrate polymers intofermentable sugars. This paper reviews several leading acidic, neutral, and alkalinepretreatments technologies. Different pretreatment methods, including dilute acid pre-treatment (DAP), steam explosion pretreatment (SEP), organosolv, liquid hot water

(LHW), ammonia fiber expansion (AFEX), soaking in aqueous ammonia (SAA), sodiumhydroxide/lime pretreatments, and ozonolysis are intensively introduced and discussed.In this minireview, the key points are focused on the structural changes primarily incellulose, hemicellulose, and lignin during the above leading pretreatment technologies.

Keywords Pretreatment. Cellulose . Hemicellulose . Lignin

Abbreviations

AFEX Ammonia fiber expansionCrI Crystallinity indexCS Combined severityDAP Dilute acid pretreatmentDP Degree of polymerizationHW HardwoodHMF 5-HydroxymethylfurfuralLCC Lignin-carbohydrate complex

Appl Biochem Biotechnol (2014) 174:4362DOI 10.1007/s12010-014-1015-y

Z. Xu (*)

College of Materials Science and Engineering, Nanjing Forestry University, Jiangsu 210037, PeoplesRepublic of Chinae-mail: [email protected]

F. Huang (*)School of Chemistry and Biochemistry, Institute of Paper Science and Technology, Georgia Institute ofTechnology, Atlanta, GA 30332, USAe-mail: [email protected]


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LHW Liquid hot waterLODP Leveling-off degree of polymerizationNMR Nuclear magnetic resonanceSAA Soaking in aqueous ammonia

SEP Steam explosion pretreatmentSW SoftwoodXRD X-ray diffraction

Introduction

In order to cope with the growing demand for energy, the depletion of fossil fuel resources andenvironmental concerns raised by fossil fuel use, countries wishing to limit their energydependence on petroleum exporting countries are developing alternative energy sources, suchas bioethanol produced from renewable biomass [14]. Cellulosic bioethanol is regarded as

one of the most promising renewable biofuels in the transportation sector for the next comingfew decades [5]. Current production of bioethanol relies on sugars that are obtained fromstarch-based agricultural crops by using first-generation conversion technologies [6]. Nowa-days, bioethanol produced from lignocellulosic biomass using second-generation technologieshas become a promising alternative, mainly because lignocellulosic raw materials do notcompete with food crops or productive agricultural land and they are also less expensiveand more abundant than conventional agricultural feedstocks [7,8].

The biological process of converting biomass to bioethanol typically consists of threemain steps: pretreatment, enzymatic hydrolysis, and fermentation (Fig. 1). During the

whole process, pretreatment is the most crucial step since it has a large impact on theefficiency of the overall bioconversion. In lignocellulosic biomass, cellulose and hemi-cellulose are densely packed together with lignin, which serves several plant functionsincluding protection against enzymatic hydrolysis [9]. The aim of pretreatment is todisrupt recalcitrant structures of cellulosic biomass to make cellulose more accessible tothe enzymes that convert carbohydrate polymers into fermentable sugars. During thepretreatment, the removal of lignin and hemicellulose depends on the pretreatmenttechnology, process conditions, and severity. For example, acidic chemical pretreatmentremoves most of hemicellulose, and lignin is condensed when pretreatment temperaturereaches above 160 C [10]. On the contrary, the ammonia fiber expansion (AFEX)

pretreatment does not significantly remove hemicellulose.Numerous pretreatment strategies have been developed to enhance the reactivity of cellu-

lose and to increase the yield of fermentable sugars. Typical goals of pretreatment include (a)producing highly digestible solids to enhance sugar yields during enzyme hydrolysis, (b)avoiding the degradation of sugars including those derived from hemicellulose, and (c)minimizing the formation of inhibitors for subsequent fermentation steps [11].

The pretreatment methods can be classified into different categories according to variouscriteria [2,12]. It is common to differentiate pretreatment methods that are efficient at high,low, or neutral pH. Basically, low pH methods require addition of acids to increase the

hydrolytic capacity while higher pH methods need pH-adjusting agents such as sodiumhydroxide or ammonia. The neutral pH methods, mainly liquid hot water (LHW) pretreatment,simply apply water in the process. However, the substrate medium in the neutral methods isweakly acidic due to the release of organic acids from the biomass during the pretreatments.Another way to do the grouping is based on the principal mechanism acting during pretreat-ment. As such, the methods can be classified as physical, physicochemical, and chemicalpretreatments. In this review, mainly the first classification is used. The acidic pretreatments

44 Appl Biochem Biotechnol (2014) 174:4362


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include dilute acid pretreatment (DAP), steam explosion pretreatment (SEP), and organosolvpretreatment. The neutral pretreatment is LHW, and the sodium hydroxide/lime pretreatmentand AFEX are classified into the alkaline pretreatments (Fig.1). Besides the above methods,pretreatment of biomass with high pressure ozone (ozonolysis) was recently developed in

bioethanol production [13].In this review, a brief process description is first given with recent developments for eachpretreatment, followed by discussion of the technologys advantages and disadvantages.Different feedstocks, including herbaceous/agricultural residues, hardwood and softwoodbiomasses, applied in these technologies are highlighted. The key points are focused on thestructural changes primarily in cellulose, hemicellulose, and lignin during the above leadingpretreatment technologies.

Fig. 1 Schematic flow sheet for the bioconversion of biomass to bioethanol

Appl Biochem Biotechnol (2014) 174:4362 45


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Lignocellulose Compositions

The main components of the lignocellulosic biomass are cellulose, hemicellulose, lignin, and aremaining smaller part (extractives and ash). The composition of lignocellulose highly depends

on its source. There is a significant variation of the lignin and (hemi)cellulose content oflignocellulose depending on whether it is derived from hardwood (HW), softwood (SW), orherbaceous/agricultural residue biomass. Table 1 summarizes the typical composition oflignocellulose encountered in the most common sources of biomass.

In the chemical aspects, cellulose is a linear chain homopolymer consisting of (1-4)--D-glucopyranosyl units with a varying degree of polymerization (DP) up to ~10,000[17]. The cellulose chain has a tendency to form intramolecular and intermolecularhydrogen bonds through hydroxyl groups on its glucose units, which promote celluloseaggregations and lead to a supramolecular structure with crystalline and amorphousdomains. Hemicellulose consists of a broad class of mixed heteroglycans of pentoses

and hexanoses (mainly xylose and mannose) that are linked together and frequently havebranching and substitution groups. Lignin is an irregular polyphenolic biopolymerconstructed of phenylpropanoid monomers with various degrees of methoxylation thatare biosynthesized into a complex and highly heterogeneous aromatic macromolecule.Generally, the plant cell wall microstructure is regarded to be a matrix of lignin andpolysaccharides intimately associating with each other [18,19].

Acidic Pretreatment

The processes and mode of action are discussed for different acidic pretreatment methodsincluding DAP, SEP, and organosolv pretreatment.

Dilute Acid Pretreatment

Process Description

Among the numerous pretreatment techniques, dilute acid pretreatment has been shown as aleading pretreatment process that is currently under commercial development. Dilute acid

Table 1 Typical lignocellulosic biomass composition (% dry basis)

Cellulose Hemicellulose Lignin Reference

Pine 43.3 20.5 28.3 [5]

Spruce 45.0 22.9 27.9 [5]

Douglas fir 44.0 19.2 30.0 [5]

Poplar 44.7 18.5 26.4 [4]

Eucalyptus 49.5 13.1 27.7 [4]Corn stover 36.8 30.6 23.1 [5]

Miscanthus 52.1 25.8 12.6 [14]

Wheat straw 44.1 23.8 20.5 [15]

Switchgrass 33.5 26.1 17.4 [16]



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pretreatment can significantly reduce lignocellulosic recalcitrance by disrupting the compositematerial linkage, such as the covalent bonds [20]. The most widely used and tested approaches inDAP are based on dilute sulfuric acid since it is inexpensive and effective [21, 22]. However, nitricacid [23], hydrochloric acid [24], and phosphoric acid [25] have also been examined. In addition, it

was shown that SO2 was also an efficient acid catalyst in the dilute acid pretreatment, especially forsoftwood [26]. The DAP is usually performed over a temperature range of 120 to 210 C, with acidconcentration typically less than 4 wt% and residence time from several minutes to an hour [27].Inthe DAP pretreatment, the combined severity (CS) is used for an easy comparison of pretreatmentconditions and for facilitation of process control, which relates to the experimental effects oftemperature, residence time, and acid concentration [28]. Lower CS is beneficial for the hemicel-lulose to hydrolyze to oligomers and monomers while higher CS could further convert thesemonomers to furfurals and 5-hydroxymethylfurfural (HMF), which are inhibitors for the subse-quent enzymatic hydrolysis [29]. In order to maximize the efficiency of pretreatments, severalstudies have proposed a two-step procedure for dilute acid pretreatment of softwoods [21, 30]. The

conditions in the first step are less severe and serve to hydrolyze the hemicelluloses resulting in ahigh recovery of hemicellulose-derived fermentable sugars in the pretreatment effluent. The solidmaterial recovered from the first step is treated again under more severe conditions which promotesthe enzymatic digestibility of cellulose fibers.

Mode of Action

One of the main reactions that occurs during acid pretreatment is the hydrolysis of hemicel-lulose. Hemicelluloses are hydrolyzed to fermentable sugars during DAP [12]. Solubilized

hemicelluloses (oligomers) can be subjected to hydrolytic reactions producing monomers,furfural, HMF, and other (volatile) products in acidic environments [31,32]. Recently, Hu andSannigrahi et al. [33,34] have demonstrated that pseudo-lignin can be generated solely fromcarbohydrates without a significant contribution from lignin during DAP especially under highseverity pretreatment conditions. Further analysis indicates that pseudo-lignin is a polymericmaterial that is present on the surface of pretreated biomass as spherical droplets andstructurally has carbonyl, aromatic, methoxy, and aliphatic functionalities.

During DAP pretreatments, the hydrolyzation of cellulose and subsequent solubilization ofglucose most often results in an increase of cellulose crystallinity index (CrI) in biomass, asshown in Table2. Foston et al. [33,35] suggested that the majority of the increase of cellulose

crystallinity is primarily due to localized hydrolyzation and removal of cellulose from theamorphous regions. Cao et al. [36] observed that the CrI of poplar cellulose remained almostunchanged during the early DA pretreatment (0.35.4 min), then a slight increase during theDAP. In addition, the partial hydrolyzation of cellulose in DAP also leads to a reduction ofcellulose DP (Table2) especially at high severity pretreatment conditions, which increases theenzymatic digestibility of cellulose. The DP of cellulose from different substrates usuallydecreases gradually until reaching a nominal value, namely, the leveling-off degree of poly-merization (LODP) throughout the course of pretreatment [37]. The initial DP reduction phaseperiod is believed to represent the hydrolysis of the reactive amorphous region of cellulose,

whereas the slow plateau rate phase corresponds to the hydrolysis of the slow reactingcrystalline fraction of cellulose, to some extent.In contrast, DAP does not lead to significant delignification [44]. Recent studies have

revealed an increase in the degree of condensation of lignin during the dilute acid pretreatment.The increase in degree of condensation is accompanied by a decrease in -O-4 linkages whichare fragmented and subsequently recondensed during the high temperature acid-catalyzedreactions [45]. In addition, studies also indicated that lignin balls (or lignin droplets) were



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formed during DAP. These lignin droplets were proposed to originate from lignin and possiblelignin carbohydrates complexes [34,46,47].

DAP not only alternates the lignocellulosic biomass chemical structures but also changesthe anatomical structure of plant cell wall, especially the pore structures. The specific surfacearea and the mean pore size of the plant cell wall are influential structural features related tocellulose accessibility to cellulases during the enzymatic hydrolysis [48,49]. Several studieshave indicated that the breakdown and loosening of the lignocellulosic structure by DAP

increases the specific surface area, pore volume, and pore size of the biomass [50,51].

Advantage and Disadvantage

DA pretreatment can facilitate high enzymatic deconstruction reaction rates and significantlyimprove hemicellulose and cellulose hydrolysis by varying the severity of the pretreatment [3].A drawback of this method is the formation of many inhibiting by-products and pH neutral-ization requirements for downstream processes [52]. In addition, the corrosive nature of thispretreatment mandates expensive materials of construction [53]. Furthermore, most of thereported work used bioresources with significant size reduction, which consume large amounts

of energy, and future studies will need to examine the reactivity of DAP with larger biomasschip sizes [53].

Steam Explosion Pretreatment

Process Description

SEP is one of the most common pretreatments applied to fractionate biomass componentsand increase its chemical and biological reactivity. In this method, biomass is treated

with high-pressure saturated steam, and then, the pressure is swiftly reduced, whichmakes the materials undergo an explosive decompression. Steam explosion is typicallyinitiated at a temperature of 160260 C (corresponding pressure, 0.694.83 MPa) forseveral seconds to a few minutes before the material is exposed to atmospheric pressure[54]. Addition of an acid catalyst such as SO2 or preferably H2SO4 because it isinexpensive to steam explosion can significantly increase its hemicellulose sugar yields[55]. SEP applies physicochemical pretreatment for lignocellulosic biomass. The

Table 2 Cellulose CrI and DP before and after DAP pretreatments

Substrate Pretreatmentconditions

CrI (%) beforepretreatment

CrI (%) afterpretreatment

DP beforepretreatment

DP afterpretreatment

Corn stover[38] 180 C, 3 wt%H2SO4, 90 s 50.3a

52.5a

7,300b

2,600b

Poplar [38] 190 C, 2 wt%H2SO4, 70 s

49.9a 50.6a 3,500b 600b

Loblollypine [39]

180 C, 1.0 wt%H2SO4, 30 min

55.1c 59.8c 3,642d 1,326d

aCrI was measured by X-ray diffraction (XRD) method [40]b DP was measured by viscometric method [41]c CrI was measured by solid-state nuclear magnetic resonance (NMR) technique [42]d DP was measured by gel-permeation chromatography (GPC) technique [43]



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in the risk of condensation and precipitation of soluble lignin components making the biomassless digestible. Other limitation of this process is the formation of fermentation inhibitors athigher temperatures [67].

Organosolv Pretreatment

Process Description

In the organosolv pretreatment, numerous organic or aqueous solvent mixtures can beutilized, including methanol, ethanol, acetone, ethylene glycol, and tetrahydrofurfurylalcohol, in order to solubilize lignin and hemicellulose, providing treated cellulosesuitable for enzymatic hydrolysis [68]. Although several organic solvents can be appliedin the organosolv pretreatments, the low molecular weight alcohols such as ethanol andmethanol are favored solvents mainly due to their lower boiling points. The preferred

conditions of organosolv process is generally in the following ranges: a cooking tem-perature of 180195 C, a cooking time of 3090 min, an ethanol concentration of 3570 % (w/v), and a liquor to solid ratio ranging from 4:1 to 10:1. The pH of the liquorranges from 2 to 4. In some studies, these mixtures are combined with acid catalysts(HCl, H2SO4, oxalic, or salicylic) to break hemicellulose bonds and lignin linkages [69].A high yield of xylose can usually be obtained with the addition of acid. However, thisacid addition can be avoided for a satisfactory delignification by increasing processtemperature (above 185 C) [70]. Usually, in the organosolv pretreatment, high ligninremoval (>70 %) and minimum cellulose loss (less than 2 %) could be achieved [71].

Mode of Action

During the organosolv pretreatment, the largest component, cellulose, is partially hydro-lyzed into smaller fragments that largely remain insoluble. Sannigrahi et al. [72] revealedthe degree of cellulose crystallinity increases and the relative proportion ofparacrystalline and amorphous cellulose decreases after the organosolv pretreatment ofLoblolly pine. The second largest component, hemicellulose, is hydrolyzed mostly intosoluble components, such as oligosaccharides, monosaccharides, and acetic acid. Aceticacid can act as catalyst for the rupture of lignin-carbohydrate complex [68]. Some of the

pentose sugars are subsequently dehydrated under the operating conditions to formfurfural [54]. The third major polymer component, lignin, is hydrolyzed into lowermolecular weight fragments that dissolve in the aqueous ethanol liquor. In addition,the depolymerization of lignin occurs primarily through cleavage of-O-4 linkages [73].Moreover, lignin condensation was also observed in organosolv pretreatment [74]. Inaddition, the hydrolysis of hemicellulose and degradation of lignin in organosolv pre-treatment also lead to the increase of cellulose surface accessibility [75].


Compared with other pretreatments, organosolv pretreatment has some advantages as follows:(1) organic solvents are always easy to recover by distillation and recycled for pretreatment; (2)the chemical recovery in organosolv pulping processes can isolate lignin as a solid materialand carbohydrates as a syrup, both of which show promise as chemical feedstocks [7678]. Itseems that organosolv pretreatment is feasible for biorefinery of lignocellulosic biomass,which considers the utilization of all the biomass components. However, there are inherent



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drawbacks to the organosolv pretreatment. Organic solvents are always expensive, so it shouldbe recovered as much as possible, but this causes increase of energy consumption. In addition,organosolv pretreatment must be performed under extremely tight and efficient control due tothe volatility/flammability of organic solvents which increase capital cost. Moreover, removal

of solvents from the system is necessary since the solvents might be inhibitory to enzymatichydrolysis and fermentative microorganisms [3].

Neutral Pretreatment

The liquid hot water (LHW) pretreatment is generally regarded as the neutral pretreatmentsince the water (pH neutral) is used as pretreatment media.

Process Description

LHW pretreatment, also named as autohydrolysis, is a hydrothermal pretreatment. Thetemperatures applied in the pretreatment can range from 160 to 240 C and over lengthsof time ranging from a few minutes up to an hour [79,80]. LHW pretreatment results insolubilization of hemicelluloses and increase of cellulose digestibility in the enzymatichydrolysis. LHW pretreatment can be performed in batch and flow-through reactors. Inbatch reactors, the slurry of biomass and water is heated to the desired temperature andheld at the pretreatment conditions for the desired residence time before being cooled. Ina flow-through reactor, hot water is made to pass over a stationary bed of lignocelluloses

[81]. Generally, the flow-through process is regarded to be the more effective forremoving hemicellulose than the batch technique. This is because, in part, the largeamount of water used in a flow-through reactor quickly dilutes and removes organicacids, which lowers the organic acid concentrations and minimizes the time for them toact on the solid hemicellulose.

Mode of Action

During LHW, water acts as a weak acid and releases the hydronium ion, which causesdepolymerization of hemicellulose by selective hydrolysis of glycosidic linkages, liber-

ating O-acetyl group and other acid moieties from hemicellulose to form acetic anduronic acids. The release of these acids has been postulated to catalyze the hydrolysis ofhemicelluloses and oligosaccharides from hemicelluloses [8285]. LHW pretreatmentresults in preserving most of the cellulose in solid form, and the amount of glucanretained is greater than in DAP [86]. Usually, the extent of cellulose hydrolysis in LHWis less than in DAP, which is mainly due to the milder acid conditions. Similar to theDAP pretreatment, several researchers have reported that the CrI of cellulose increasedafter LHW pretreatment because the amorphous cellulose is more reactive than crystal-line cellulose [87, 88]. The degradation of hemicellulose and cellulose will also form

furfural and HMF, respectively, during LHW pretreatment as occurred in DAP. However,the quantities of sugar degradation products generated are lower than DAP [89,90] andwould not significantly inhibit the fermentation process if LHW pretreatment is per-formed under 220 C [83]. Moreover, LHW can maximize the solubilization of thehemicellulose fraction while minimizing the formation of monosaccharides and thesubsequent sugar degradation products by maintaining the pH between 4 and 7 [91,92]. Usually, the pH can be controlled by adding some bases (i.e., NaOH or KOH) into



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LHW pretreatment process with its role to maintain the pH value not as a catalyst inalkaline pretreatment [93,94]. In addition, the organic acids released from the biomassalso lead to the acidolysis of lignin and decrease the -O-4 linkage content in ligninduring the LHW pretreatment [95]. Moreover, the removal of hemicellulose and the

cleavage of lignin in LHW result in the increase of cellulose accessibility [96].


The major advantage of LHW pretreatment is that no additives such as acid catalysts arerequired, minimizing the formation of inhibitory products. This also eliminates the need forfinal washing step or neutralization because the pretreatment solvent is water. However, theLHW pretreatment is regarded as energy demanding because of higher pressures and largeamounts of water supplied to the system.

Application of Acidic and Neutral Pretreatment

Table3shows the applications of different acidic pretreatments on herbaceous/agriculturalresidues, hardwood, and softwood biomasses. It should be noted that the cellulose conversionyields cited in this paper were based on cellulose fraction in the pretreated biomass, ascalculated in the literature [97]. Generally, the acidic pretreatments are effective for theherbaceous/agricultural residues (i.e., corn stover, wheat straw, andmiscanthus) and hardwood(i.e., poplar and olive tree) while less effective for the softwood (i.e., radiata pine, Loblollypine, Douglas fir, and Lodgepole pine), which is mainly due to its high lignin content in the

biomass [34]. Among LHW, DAP, SEP, and organosolv pretreatments, the organosolv pre-treatment yields the highest cellulose conversion (>80 %).

Alkaline Pretreatment

Alkaline pretreatments have received numerous studies as another major chemical pretreat-ment technology besides acidic pretreatments. Alkaline pretreatments can be divided into twogroups based on the chemical used: (1) pretreatments that use sodium or calcium hydroxideand (2) pretreatments that use ammonia [107]. The process description and alternations of

major chemical components for these two types of alkaline pretreatments are discussed in thefollowing sections.

Sodium Hydroxide and Lime Pretreatment

Process Description

Both sodium hydroxide and lime pretreatments have been shown to be effectively enhancingcellulose digestibility [108111]. However, lime has received much more attentions than

sodium hydroxide since it is inexpensive (about 6 % cost of sodium hydroxide) [112], hasimproved handling, and can be recovered easily by using carbonated wash water [113]. Incomparison with other pretreatment technologies, the sodium hydroxide and lime pretreat-ments usually use lower temperatures and pressures, even at ambient conditions. Pretreatmenttime, however, is recorded in terms of hours or days which are much longer than otherpretreatment processes. In the alkaline pretreatment, the residual alkali could be reused throughthe chemical recycle/recovery process, which may make the system more complex due to the



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Table3

Applications

ofacidicandneutralpretreatments

fordifferentbiomasses

Pretreatmentmethod

Substrate

Pretreatmentconditions

Celluloseconversionyield(%)

Enz

ymeloadings

DAP

Cornstover[49]

140C,1.0wt%

H2SO4,40min

82.3

in72h

15FPU/gforcellulasefromCelluclast

1.5Land

2

6.25CBU/gfor-glucosidasefrom

Novozyme188

Olivetree[98]

210C,1.40wt%

H2SO4,10min

76.5

in72h


1.5Land

1

5CBU/gfor-glucosidasefromN

ovozyme188

Loblollypine[39]

180C,1.0wt%

H2SO4,30min

35in72h


1.5Land

4

0IU/gfor-glucosidasefromNov

ozyme188

SEP

Wheatstraw[99]

190C,10min

85in72h


1.5Land

1

2.6IU/gfor-glucosidasefromNovozyme188

Poplar[100]

220C,4min

60in72h


1.5Land

1

2.6IU/gfor-glucosidasefromNovozyme188

Douglasfir[49]

4.5w

t%SO2,195C,4.5min

54.2

in72h

20FPU/gcellulaseenzymeand35CB

U/gfor

-glucosidase

Organosolv

pretreatment

Miscanthus[101]

170C,80min,1.2wt%

H2SO4,50%ethanol

78in48h


1.5Land

4


ozyme188

Poplar[102]

180C,60min,1.25wt%

H2SO4,50%ethanol

97in48h


1.5Land

4


ozyme188

Lodgepolepine[103]

170C,80min,1.1wt%

H2SO4,65%ethanol

97in48h

20FPU/gforcellulasefromSpezyme

CPand

4


ozyme188

LHW

Cornstover[104]

190C,15min

69.6

in72h

15FPU/gforcellulasefromSpezyme

CPand

6


ozyme188

Poplar[105]

200C,10min

52in72h

15FPU/gcellulasefromSpezymeCP

and

4

0CBU-glucosidasefromNovozyme188

Radiatapine[106]

200C,30min

27in72h

20FPUC-30cellulasefromNovozym

e



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need for chemical recovery [94,114]. In order to increase the pretreatment efficiency, usually,the particle size needs to be reduced to 10 mm or less [113].

Mode of Action

The sodium hydroxide and lime pretreatments are basically a delignification process, in whicha significant amount of hemicellulose is solubilized as well. The major effect is the removal oflignin from the biomass, thus improving the reactivity of the remaining polysaccharides. Inaddition, the alkaline pretreatment could swell cell wall and improve cell wall accessibility forthe subsequent enzymatic hydrolysis. The proposed reaction mechanism is believed to besaponification of intermolecular ester bonds cross-linking hemicellulose and lignin [94]. Thesaponification leads to the cleavage of lignin-carbohydrate complex (LCC) linkages, and theexpose of cellulose microfibrils can increase enzymatic digestibility of cellulose. Acetylgroups and various uronic acid substitutes are also removed by alkali, thereby reducing steric

hindrance of hydrolytic enzymes and increasing the accessibility of carbohydrates to enzymes[109]. Furthermore, the degraded hemicellulose could also form furfural and HMF in thehydrolysates, but the amount is typically lower than that with DAP [115]. In addition, alkalinepretreatment decreases the DP of cellulose (Table 4) and causes swelling of cellulose, leading toan increase in its internal surface area [116]. This makes cellulose more accessible for enzymesin the subsequent hydrolysis stage. In terms of cellulose crystallinity change during thepretreatment, research indicated that the amorphous regions suffered greater peeling reactionsthan the crystalline regions, and the occurrence of the peeling actions of the amorphous regionsleads to an increase of cellulose crystallinity (Table4)[108]. During the alkaline pretreatment,

lignin suffered delignification, which has similarities to soda chemical pulping technologies[113,117]. In addition, recent studies indicated that the alkali pretreatment could also increasethe fiber porosity due to the disruption of biomass structures [118,119].


Compared with DAP, alkaline pretreatments have some practical operational advantagesincluding lower reaction temperatures and pressures. A significant disadvantage of alkalinepretreatment is the conversion of alkali into irrecoverable salts and/or the incorporation of saltsinto the biomass during the pretreatment reactions so that the treatment of a large amount of

salts becomes a challenging issue for alkaline pretreatment [94]. In addition, during the limepretreatment, the precipitation of calcium oxalate on the heat exchanger and process streams isanother issue to be considered since it may cause severe problems in the plants [ 120].

Table 4 Cellulose CrI and DP before and after lime pretreatments

Substrate Pretreatment conditions CrI (%) beforepretreatment

CrI (%) afterpretreatment

DP beforepretreatment

DP afterpretreatment

Corn

stover

55 C, 0.5:1 Ca(OH)2to

biomass, 4 weeks

50.3a 56.2a 7,300b 3,200b

Poplar 65 C, 0.5:1 Ca(OH)2tobiomass, 4 weeks

49.9a 54.5a 3,500b 1,600b

From Kumar et al. [38]aCrI was measured by X-ray diffraction (XRD) method [40]b DP was measured by viscometric method [41]



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Ammonia Fiber Expansion Pretreatment

Process Description

The AFEX pretreatment, also known as ammonia fiber explosion pretreatment, is anotherphysicochemical process, much like SEP, in which the biomass material is subjected to liquidanhydrous ammonia under high pressures and moderate temperatures and is then rapidlydepressurized. This swift pressure release leads to a rapid expansion of the ammonia gas thatcauses swelling and physical disruption of biomass fibers [121]. The AFEX is usuallyperformed to treat moist biomass (0.12 g H2O/g dry biomass) with liquid ammonia (0.32 g NH3/g dry biomass) while heating (60100 C) the biomass-water-ammonia mixture for aperiod of time (560 min) before rapidly releasing the pressure [122]. AFEX can be carried outin either a batch or a flow-through reactor. The major parameters influencing the AFEXprocess are ammonia loading, temperature, pressure, moisture content of biomass, and resi-

dence time [123]. During the AFEX pretreatment, about 95 % of the ammonia can berecovered in the gas phase and recycled, with a small amount of ammonia that remains inthe lignocellulosics which might serve as a nitrogen source for the microbes in the fermenta-tion process [124].

Mode of Action

During the AFEX, the physicochemical treatment induces the disruption in the lignin-carbohydrate linkage, hemicellulose hydrolysis, and ammonolysis of glucuronic cross-linked

bonds, and partial decrystallization of the cellulose structure, all leading to a higher accessiblesurface area for enzymatic attack [123]. In AFEX pretreatment, the removal of acetyl groupsfrom hemicellulose results in the formation of acetamide and acetic acid, but it is reported thatAFEX removes the least amount of acetyl groups from lignocellulosic material compared toother leading pretreatment technologies [38]. In addition, the ammonia also causes a series ofammonolysis (amide formation) and hydrolysis reactions (acid formation) in the presence ofwater, which cleave LCC ester linkages [125]. The major effect of AFEX on cellulose is thedecrystallization, which is mainly due to the generation of more amorphous portions incellulose during this process [38]. These chemical structural changes lead to the formationof highly porous structures on the fiber cell wall, which greatly enhance enzyme accessibility

to the cellulosic microfibrils [125].


The important advantages of AFEX pretreatment include lower moisture content, lowerformation of sugar degradation products due to moderate conditions, almost complete recoveryof solid material and high cellulose digestibility in the subsequent enzymatic hydrolysis. Oneconcern for this process is the cost of ammonia and the need for recycling technologies [94].

Soaking in Aqueous Ammonia PretreatmentAnother type of process utilizing ammonia is soaking aqueous ammonia (SAA), whichappears as an interesting alternative since it is performed at lower temperature (3075 C)[2]. The main purpose of SAA pretreatment is the removal of lignin, which is the physical andchemical barrier that inhibits accessibility of enzyme to the cellulose substrate [126]. It isregarded as a valuable pretreatment methodology due to the retention of the hemicellulose



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fraction and removal of lignin after pretreatment [127]. SAA has been shown to be able toremove 74 % of the lignin from corn stover while retaining >85 % of the xylan and nearly100 % of the glucan [128]. This allows easy downstream utilization of sugars in a single co-fermentation process in which net sugar yield was increased due to the presence of hemicel-

luloses [129]. SAA utilizing lower temperatures and less extreme pHs reduces the associatedchemical and energy costs and may also reduce the formation of carbohydrate degradationproducts. However, the SAA pretreated biomass pretreated shows relatively lower enzymaticdigestibility due to its mild pretreatment conditions [130,131].

Application of alkaline pretreatment

Table5shows the applications of different alkaline pretreatments on herbaceous/agriculturalresidues, hardwood, and softwood biomasses. Among the numerous types of biomass, soft-woods are generally recognized as being much more refractory than hardwoods or herbaceous/

agricultural residues in the alkaline pretreatment process. This is, in part, due to the fact thatsoftwoods have a more rigid structure and contains more lignin [132].

Ozonolysis

Ozonolysis of biomass is usually carried out at low temperature (2030 C), and the ozoneflow rate ranged from 0.5 to 0.8 L/min. The ozone consumption (% dry wt. of biomass) is 2

Table 5 Application of alkaline pretreatment for different biomasses

Pretreatment method Substrate Pretreatmentconditions

Celluloseconversion yield (%)

Enzyme loadings

NaOH orCa(OH)2

pretreatment

Cornstover [133]

55 C, 7.3 wt%Ca(OH)2, 4 weeks

98 in 96 h 15 FPU/g for cellulasefrom Spezyme CPand 40 CBU/g for-glucosidase from

Novozyme 188

Birch [134] 80 C, 7.0 wt%NaOH, 2 h

80 in 96 h 20 FPU/g for cellulasefrom Celluclast 1.5 L

and 50 IU/g for-glucosidase from

Novozyme 188

Spruce [134] 80 C, 7.0 wt%NaOH, 2 h

24 in 96 h 20 FPU/g for cellulasefrom Celluclast 1.5 Land 50 IU/g for-glucosidase from

Novozyme 188

AFEXpretreatment

Cornstover [49]

90 C, 1:1 ammoniato biomass loading,60 % moisture

content, 5 min

76.6 in 72 h 15 FPU/g for cellulasefrom Celluclast 1.5 Land 26.25 CBU/g for

-glucosidase fromNovozyme 188

Poplar [135] 180 C, 2:1 ammoniato biomass loading,233 % moisturecontent, 30 min

70 in 72 h 15 FPU/g for cellulasefrom Celluclast 1.5 Land 26.25 CBU/g for-glucosidase from

Novozyme 188



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7 %[115,136]. Ozone treatment is one way of reducing the lignin content of lignocellulosicwastes. This results in an increase of thein vitrodigestibility of the treated material, and unlikeother chemical treatments, it does not produce toxic residues. Ozone can be used to degradelignin and hemicellulose in many lignocellulosic materials such as wheat straw [137], bagasse,green hay, peanut, pine [138], cotton straw [139], and poplar sawdust [140]. Research

indicated [141] that ozone is highly reactive toward compounds incorporating conjugateddouble bonds and functional groups with high electron densities. Therefore, the moiety, mostlikely to be oxidized in ozonization of lignocellulosic materials, is lignin due to its high contentof C=C bonds. Thus, during the ozonolysis, the degradation is mainly limited to lignin. Ozoneattacks lignin releasing soluble compounds of less molecular weight, mainly organic acidssuch as formic and acetic acids [141]. The main advantages linked to this process are the lackof any degradation products that might interfere with subsequent hydrolysis or fermentationand the reactions occurring at ambient temperature and normal pressure. Furthermore, the factthat ozone can be easily decomposed by using a catalytic bed or increasing the temperaturemeans that processes can be designed to minimize environmental pollution. A drawback of

ozonolysis is that a large amount of ozone is required, which can make the process expensiveand less applicable [142]. However, recently, Hu et al. [13] demonstrated that a lower charge ofozone could be used to enhance the enzymatic digestibility of cellulose, if the ozone-treatedbiomass was not washed and thein situgenerated acids were employed in a subsequent DAP.

Conclusions and Perspectives

Most of the pretreatment technologies that have been described herein are effective on one or

more factors that contribute to lignocellulosic recalcitrance, as shown in Table6. From thework that has been presented, it is clear that each pretreatment method has its own merits anddisadvantages and consequences on the enzymatic hydrolysis. Recently, researchers appliedsome combinations of different pretreatment methods, such as alkaline pretreatment followedby steam pretreatment [143,144] and organosolv pretreatment coupled with steam explosion[143], to improve the biomass digestibility. Although the results are promising compared withthe single pretreatment, the extra equipment and operation cost will compromise these

Table 6 Effect of different chemical pretreatment technologies on the structure of lignocellulose

Pretreatmentmethod

Increase ofaccessible surfacearea

Cellulosedecrystallization

Hemicellulosesolubilization

Ligninremoval

Generation ofinhibitorcompounds

Ligninstructurealteration

DAP H L H L H H

SEP H L M L H M

Organosolv H L H H H H

LHW H L M L L M

NaOH/Ca(OH)2

H L M M L H

AFEX H H L L L H

SAA H L L H L L

Ozonolysis H L M H L H

From Alvira et al., Brodeur et al., and Li et al. [2,11,20]

Hhigh effect,Mmoderate effect,L low effect



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processes. In addition, advanced transgenic techniques were reported to alternate the chemicalstructures of lignocellulosic biomass with the aim to reduce their recalcitrance in the pretreat-ments [145,146]. However, these techniques are still in development, and significant researchneeds to be done before the commercialization.

Despite much research that has been dedicated to understanding the chemistry and theplant cell wall structure changes during various pretreatment technologies, the insuffi-cient knowledge of cell wall structure, ultrastructure, and pretreatment effects still limitsthe economics and effectiveness of pretreatment. For instance, the biological and chem-ical properties of plants are very complex in terms of composition, structure, andultrastructure [147]. Although researchers have put significant effort into optimizingthe pretreatment effectiveness, the fundamental science behind these optimizations isstill not fully understood. Furthermore, there has been a lack of mechanistic understand-ing of the ultrastructural and physicochemical changes occurring within the cell wall atthe molecular level and the cellular/tissue scale during various pretreatment technologies.

It is thus essential to understand the effects of pretreatment on plant cell walls at a morefundamental level, in order to develop a cost-effective pretreatment technology withmaximum fermentable sugar recovery; minimum inhibitor production and energy input;low demand of post-pretreatment processes; and low capital costs for reactors, water, andchemicals. In addition, advances in the analytical chemistry would provide useful tools toinvestigate the cell wall deconstruction and understand the recalcitrance during thepretreatment process [148,149].

Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No.31300483), Natural Science Foundation of Jiangsu Province of China (Grant No. BK20130971), and the PriorityAcademic Program Development of Jiangsu Higher Education Institutions (PAPD).

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