Appl Biochem Biotechnol DOI 10.1007/s12010-014-1015-y

Pretreatment Methods for Bioethanol Production Zhaoyang Xu & Fang Huang

Received: 21 January 2014 / Accepted: 15 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 process of converting biomass to bioethanol typically consists of three main steps: pretreatment, enzymatic hydrolysis, and fermentation. For this process, pretreatment is probably the most crucial step since it has a large impact on the efficiency of the overall bioconversion. The aim of pretreatment is to disrupt recalcitrant structures of cellulosic biomass to make cellulose more accessible to the enzymes that convert carbohydrate polymers into fermentable sugars. This paper reviews several leading acidic, neutral, and alkaline pretreatments technologies. Different pretreatment methods, including dilute acid pretreatment (DAP), steam explosion pretreatment (SEP), organosolv, liquid hot water (LHW), ammonia fiber expansion (AFEX), soaking in aqueous ammonia (SAA), sodium hydroxide/lime pretreatments, and ozonolysis are intensively introduced and discussed. In this minireview, the key points are focused on the structural changes primarily in cellulose, hemicellulose, and lignin during the above leading pretreatment technologies. Keywords Pretreatment . Cellulose . Hemicellulose . Lignin Abbreviations AFEX Ammonia fiber expansion CrI Crystallinity index CS Combined severity DAP Dilute acid pretreatment DP Degree of polymerization HW Hardwood HMF 5-Hydroxymethylfurfural LCC Lignin-carbohydrate complex

Z. Xu (*) College of Materials Science and Engineering, Nanjing Forestry University, Jiangsu 210037, People’s Republic of China e-mail: [email protected] F. Huang (*) School of Chemistry and Biochemistry, Institute of Paper Science and Technology, Georgia Institute of Technology, Atlanta, GA 30332, USA e-mail: [email protected]

Appl Biochem Biotechnol

LHW LODP NMR SAA SEP SW XRD

Liquid hot water Leveling-off degree of polymerization Nuclear magnetic resonance Soaking in aqueous ammonia Steam explosion pretreatment Softwood X-ray diffraction

Introduction In order to cope with the growing demand for energy, the depletion of fossil fuel resources and environmental concerns raised by fossil fuel use, countries wishing to limit their energy dependence on petroleum exporting countries are developing alternative energy sources, such as bioethanol produced from renewable biomass [1–4]. Cellulosic bioethanol is regarded as one of the most promising renewable biofuels in the transportation sector for the next coming few decades [5]. Current production of bioethanol relies on sugars that are obtained from starch-based agricultural crops by using first-generation conversion technologies [6]. Nowadays, bioethanol produced from lignocellulosic biomass using second-generation technologies has become a promising alternative, mainly because lignocellulosic raw materials do not compete with food crops or productive agricultural land and they are also less expensive and more abundant than conventional agricultural feedstocks [7, 8]. The biological process of converting biomass to bioethanol typically consists of three main 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 the efficiency of the overall bioconversion. In lignocellulosic biomass, cellulose and hemicellulose are densely packed together with lignin, which serves several plant functions including protection against enzymatic hydrolysis [9]. The aim of pretreatment is to disrupt recalcitrant structures of cellulosic biomass to make cellulose more accessible to the enzymes that convert carbohydrate polymers into fermentable sugars. During the pretreatment, the removal of lignin and hemicellulose depends on the pretreatment technology, process conditions, and severity. For example, acidic chemical pretreatment removes most of hemicellulose, and lignin is condensed when pretreatment temperature reaches 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 cellulose 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 various criteria [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 sodium hydroxide 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 is weakly 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 pretreatment. As such, the methods can be classified as physical, physicochemical, and chemical pretreatments. In this review, mainly the first classification is used. The acidic pretreatments

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

include dilute acid pretreatment (DAP), steam explosion pretreatment (SEP), and organosolv pretreatment. The neutral pretreatment is LHW, and the sodium hydroxide/lime pretreatment and 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 each pretreatment, followed by discussion of the technology’s advantages and disadvantages. Different feedstocks, including herbaceous/agricultural residues, hardwood and softwood biomasses, applied in these technologies are highlighted. The key points are focused on the structural changes primarily in cellulose, hemicellulose, and lignin during the above leading pretreatment technologies.

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Lignocellulose Compositions The main components of the lignocellulosic biomass are cellulose, hemicellulose, lignin, and a remaining 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 of lignocellulose depending on whether it is derived from hardwood (HW), softwood (SW), or herbaceous/agricultural residue biomass. Table 1 summarizes the typical composition of lignocellulose encountered in the most common sources of biomass. In the chemical aspects, cellulose is a linear chain homopolymer consisting of (14)-β-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 intermolecular hydrogen bonds through hydroxyl groups on its glucose units, which promote cellulose aggregations and lead to a supramolecular structure with crystalline and amorphous domains. Hemicellulose consists of a broad class of mixed heteroglycans of pentoses and hexanoses (mainly xylose and mannose) that are linked together and frequently have branching and substitution groups. Lignin is an irregular polyphenolic biopolymer constructed of phenylpropanoid monomers with various degrees of methoxylation that are biosynthesized into a complex and highly heterogeneous aromatic macromolecule. Generally, the plant cell wall microstructure is regarded to be a matrix of lignin and polysaccharides intimately associating with each other [18, 19].

Acidic Pretreatment The processes and mode of action are discussed for different acidic pretreatment methods including DAP, SEP, and organosolv pretreatment. Dilute Acid Pretreatment Process Description Among the numerous pretreatment techniques, dilute acid pretreatment has been shown as a leading 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 Miscanthus

36.8 52.1

30.6 25.8

23.1 12.6

[5] [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 composite material linkage, such as the covalent bonds [20]. The most widely used and tested approaches in DAP are based on dilute sulfuric acid since it is inexpensive and effective [21, 22]. However, nitric acid [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 for softwood [26]. The DAP is usually performed over a temperature range of 120 to 210 °C, with acid concentration typically less than 4 wt% and residence time from several minutes to an hour [27]. In the DAP pretreatment, the combined severity (CS) is used for an easy comparison of pretreatment conditions and for facilitation of process control, which relates to the experimental effects of temperature, residence time, and acid concentration [28]. Lower CS is beneficial for the hemicellulose to hydrolyze to oligomers and monomers while higher CS could further convert these monomers to furfurals and 5-hydroxymethylfurfural (HMF), which are inhibitors for the subsequent enzymatic hydrolysis [29]. In order to maximize the efficiency of pretreatments, several studies 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 a high recovery of hemicellulose-derived fermentable sugars in the pretreatment effluent. The solid material recovered from the first step is treated again under more severe conditions which promotes the enzymatic digestibility of cellulose fibers. Mode of Action One of the main reactions that occurs during acid pretreatment is the hydrolysis of hemicellulose. 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 and Sannigrahi et al. [33, 34] have demonstrated that pseudo-lignin can be generated solely from carbohydrates without a significant contribution from lignin during DAP especially under high severity pretreatment conditions. Further analysis indicates that pseudo-lignin is a polymeric material that is present on the surface of pretreated biomass as spherical droplets and structurally has carbonyl, aromatic, methoxy, and aliphatic functionalities. During DAP pretreatments, the hydrolyzation of cellulose and subsequent solubilization of glucose most often results in an increase of cellulose crystallinity index (CrI) in biomass, as shown in Table 2. 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 the amorphous regions. Cao et al. [36] observed that the CrI of poplar cellulose remained almost unchanged during the early DA pretreatment (0.3−5.4 min), then a slight increase during the DAP. In addition, the partial hydrolyzation of cellulose in DAP also leads to a reduction of cellulose DP (Table 2) especially at high severity pretreatment conditions, which increases the enzymatic digestibility of cellulose. The DP of cellulose from different substrates usually decreases gradually until reaching a nominal value, namely, the leveling-off degree of polymerization (LODP) throughout the course of pretreatment [37]. The initial DP reduction phase period 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 reacting crystalline 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 β-O-4 linkages which are fragmented and subsequently recondensed during the high temperature acid-catalyzed reactions [45]. In addition, studies also indicated that lignin balls (or lignin droplets) were

Appl Biochem Biotechnol Table 2 Cellulose CrI and DP before and after DAP pretreatments Substrate

Pretreatment conditions

CrI (%) before pretreatment

CrI (%) after pretreatment

DP before pretreatment

DP after pretreatment

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

Loblolly pine [39]

180 °C, 1.0 wt% H2SO4, 30 min

55.1c

59.8c

3,642d

1,326d

a

CrI was measured by X-ray diffraction (XRD) method [40]

b

DP was measured by viscometric method [41] CrI was measured by solid-state nuclear magnetic resonance (NMR) technique [42]

c d

DP was measured by gel-permeation chromatography (GPC) technique [43]

formed during DAP. These lignin droplets were proposed to originate from lignin and possible lignin carbohydrates complexes [34, 46, 47]. DAP not only alternates the lignocellulosic biomass chemical structures but also changes the anatomical structure of plant cell wall, especially the pore structures. The specific surface area and the mean pore size of the plant cell wall are influential structural features related to cellulose accessibility to cellulases during the enzymatic hydrolysis [48, 49]. Several studies have 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 significantly improve 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 neutralization requirements for downstream processes [52]. In addition, the corrosive nature of this pretreatment mandates expensive materials of construction [53]. Furthermore, most of the reported 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 biomass chip sizes [53]. Steam Explosion Pretreatment Process Description SEP is one of the most common pretreatments applied to fractionate biomass components and increase its chemical and biological reactivity. In this method, biomass is treated with high-pressure saturated steam, and then, the pressure is swiftly reduced, which makes the materials undergo an explosive decompression. Steam explosion is typically initiated at a temperature of 160–260 °C (corresponding pressure, 0.69–4.83 MPa) for several 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 is inexpensive to steam explosion can significantly increase its hemicellulose sugar yields [55]. SEP applies physicochemical pretreatment for lignocellulosic biomass. The

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mechanical effects are caused by a sudden decompression to separate the fibers in the biomass [2]. The most important factors affecting the effectiveness of steam explosion are temperature, residence time, and the combined effect of both temperature (T) and time (t), which is described by the severity factor (R0) as follows [56]: R0 ¼ texp½ðT −100Þ=14:75Š Higher temperatures result in an increased removal of hemicelluloses from the solid fraction and an enhanced cellulose digestibility; they also promote higher sugar degradation. Previous studies indicated the optimal conditions for maximum sugar yield with a severity factor (R0) between 3.0 and 4.5 [56]. Mode of Action During the SEP pretreatment, the steam penetrates the plant cell wall, causing hemicellulose degradation and lignin transformation due to high temperature, thus increasing the potential of cellulose hydrolysis [27]. The external acid (H2SO4 or SO2) or the acetic acid released from acetylated hemicellulose serves to catalyze and further hydrolyze hemicellulose in the biomass. In addition, water itself also possesses certain acid properties at high temperature, thereby contributes to the hydrolysis hemicellulose during the SEP [57]. In general, the hemicelluloses are dissolved as oligosaccharides after acid hydrolysis, which are partially further hydrolyzed to monosaccharides. Under high temperature and acid conditions, these monosaccharides can be degraded into inhibitor compounds (i.e., furfural and HMF) for the subsequent enzymatic hydrolysis [58]. In addition, the acids also lead to the acidolysis of lignin and the cleavages of β-O-4 linkage, which are similar to the LHW and DAP pretreatments [59]. As steam explosion pretreatment conditions get more severe, the condensation and repolymerization reactions will take place between the decomposition products of hemicelluloses, leading to the formation of pseudo-lignin, as observed in DAP pretreatment [60, 61]. During the SEP pretreatment, the structure changes of cellulose are mainly on the degree of polymerization and degree of crystallinity. Asada et al. [62] observed that the DP of cellulose decreased significantly as the steaming time increased until reaching a minimum value of about 700 when examining the bagasse SEP pretreatment, which was due to the depolymerization of cellulose at a relatively short steaming time. The steam explosion pretreatment also affects cellulose crystallinity. After the SEP pretreatment, the increase of cellulose crystallinity was reported by several researchers in the literature [60, 63, 64]. Generally, this increase was attributed to the selective hydrolysis of amorphous portions [63] and transformation of some amorphous portions into crystalline portions in the cellulose structure [65]. Furthermore, cellulose can be also degraded and form HMF during SEP; the mechanism of HMF formation in steam explosion pretreatment is similar to that in DAP [34]. In addition, after the SEP pretreatment, the accessible pore volume and accessible surface area of the pretreated fibers are progressively increased, which are probably the result of hemicellulose extraction and lignin redistribution [66]. Advantage and Disadvantage SEP pretreatment is an attractive process because it makes limited use of chemicals and it does not result in excessive dilution of the resulting sugars. Moreover, it requires low energy input. The disadvantage of SEP is its incomplete destruction of lignin-carbohydrate matrix resulting

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in the risk of condensation and precipitation of soluble lignin components making the biomass less digestible. Other limitation of this process is the formation of fermentation inhibitors at higher temperatures [67]. Organosolv Pretreatment Process Description In the organosolv pretreatment, numerous organic or aqueous solvent mixtures can be utilized, including methanol, ethanol, acetone, ethylene glycol, and tetrahydrofurfuryl alcohol, in order to solubilize lignin and hemicellulose, providing treated cellulose suitable for enzymatic hydrolysis [68]. Although several organic solvents can be applied in the organosolv pretreatments, the low molecular weight alcohols such as ethanol and methanol are favored solvents mainly due to their lower boiling points. The preferred conditions of organosolv process is generally in the following ranges: a cooking temperature of 180–195 °C, a cooking time of 30–90 min, an ethanol concentration of 35– 70 % (w/v), and a liquor to solid ratio ranging from 4:1 to 10:1. The pH of the liquor ranges 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, this acid addition can be avoided for a satisfactory delignification by increasing process temperature (above 185 °C) [70]. Usually, in the organosolv pretreatment, high lignin removal (>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 hydrolyzed into smaller fragments that largely remain insoluble. Sannigrahi et al. [72] revealed the degree of cellulose crystallinity increases and the relative proportion of paracrystalline and amorphous cellulose decreases after the organosolv pretreatment of Loblolly pine. The second largest component, hemicellulose, is hydrolyzed mostly into soluble components, such as oligosaccharides, monosaccharides, and acetic acid. Acetic acid 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 form furfural [54]. The third major polymer component, lignin, is hydrolyzed into lower molecular weight fragments that dissolve in the aqueous ethanol liquor. In addition, the depolymerization of lignin occurs primarily through cleavage of β-O-4 linkages [73]. Moreover, lignin condensation was also observed in organosolv pretreatment [74]. In addition, the hydrolysis of hemicellulose and degradation of lignin in organosolv pretreatment also lead to the increase of cellulose surface accessibility [75]. Advantage and Disadvantage 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 material and carbohydrates as a syrup, both of which show promise as chemical feedstocks [76–78]. It seems 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 should be 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 to the 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 enzymatic hydrolysis and fermentative microorganisms [3].

Neutral Pretreatment The liquid hot water (LHW) pretreatment is generally regarded as the neutral pretreatment since the water (pH neutral) is used as pretreatment media. Process Description LHW pretreatment, also named as autohydrolysis, is a hydrothermal pretreatment. The temperatures applied in the pretreatment can range from 160 to 240 °C and over lengths of time ranging from a few minutes up to an hour [79, 80]. LHW pretreatment results in solubilization of hemicelluloses and increase of cellulose digestibility in the enzymatic hydrolysis. LHW pretreatment can be performed in batch and flow-through reactors. In batch reactors, the slurry of biomass and water is heated to the desired temperature and held at the pretreatment conditions for the desired residence time before being cooled. In a 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 for removing hemicellulose than the batch technique. This is because, in part, the large amount of water used in a flow-through reactor quickly dilutes and removes organic acids, which lowers the organic acid concentrations and minimizes the time for them to act on the solid hemicellulose. Mode of Action During LHW, water acts as a weak acid and releases the hydronium ion, which causes depolymerization of hemicellulose by selective hydrolysis of glycosidic linkages, liberating O-acetyl group and other acid moieties from hemicellulose to form acetic and uronic acids. The release of these acids has been postulated to catalyze the hydrolysis of hemicelluloses and oligosaccharides from hemicelluloses [82–85]. LHW pretreatment results in preserving most of the cellulose in solid form, and the amount of glucan retained is greater than in DAP [86]. Usually, the extent of cellulose hydrolysis in LHW is less than in DAP, which is mainly due to the milder acid conditions. Similar to the DAP pretreatment, several researchers have reported that the CrI of cellulose increased after LHW pretreatment because the amorphous cellulose is more reactive than crystalline 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] and would not significantly inhibit the fermentation process if LHW pretreatment is performed under 220 °C [83]. Moreover, LHW can maximize the solubilization of the hemicellulose fraction while minimizing the formation of monosaccharides and the subsequent 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 in alkaline pretreatment [93, 94]. In addition, the organic acids released from the biomass also lead to the acidolysis of lignin and decrease the β-O-4 linkage content in lignin during the LHW pretreatment [95]. Moreover, the removal of hemicellulose and the cleavage of lignin in LHW result in the increase of cellulose accessibility [96]. Advantage and Disadvantage The major advantage of LHW pretreatment is that no additives such as acid catalysts are required, minimizing the formation of inhibitory products. This also eliminates the need for final washing step or neutralization because the pretreatment solvent is water. However, the LHW pretreatment is regarded as energy demanding because of higher pressures and large amounts of water supplied to the system. Application of Acidic and Neutral Pretreatment Table 3 shows the applications of different acidic pretreatments on herbaceous/agricultural residues, hardwood, and softwood biomasses. It should be noted that the cellulose conversion yields cited in this paper were based on cellulose fraction in the pretreated biomass, as calculated in the literature [97]. Generally, the acidic pretreatments are effective for the herbaceous/agricultural residues (i.e., corn stover, wheat straw, and miscanthus) and hardwood (i.e., poplar and olive tree) while less effective for the softwood (i.e., radiata pine, Loblolly pine, 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 pretreatment yields the highest cellulose conversion (>80 %).

Alkaline Pretreatment Alkaline pretreatments have received numerous studies as another major chemical pretreatment technology besides acidic pretreatments. Alkaline pretreatments can be divided into two groups based on the chemical used: (1) pretreatments that use sodium or calcium hydroxide and (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 the following sections. Sodium Hydroxide and Lime Pretreatment Process Description Both sodium hydroxide and lime pretreatments have been shown to be effectively enhancing cellulose digestibility [108–111]. However, lime has received much more attentions than sodium hydroxide since it is inexpensive (about 6 % cost of sodium hydroxide) [112], has improved handling, and can be recovered easily by using carbonated wash water [113]. In comparison with other pretreatment technologies, the sodium hydroxide and lime pretreatments usually use lower temperatures and pressures, even at ambient conditions. Pretreatment time, however, is recorded in terms of hours or days which are much longer than other pretreatment processes. In the alkaline pretreatment, the residual alkali could be reused through the chemical recycle/recovery process, which may make the system more complex due to the

LHW

Organosolv pretreatment

190 °C, 15 min

200 °C, 10 min

200 °C, 30 min

Corn stover [104]

Poplar [105]

Radiata pine [106]

170 °C, 80 min, 1.1 wt% H2SO4, 65 % ethanol

Lodgepole pine [103]

20 FPU/g for cellulase from Celluclast 1.5 L and 40 IU/g for β-glucosidase from Novozyme 188

180 °C, 60 min, 1.25 wt% H2SO4, 50 % ethanol

Poplar [102]

27 in 72 h

52 in 72 h

69.6 in 72 h

97 in 48 h

78 in 48 h 97 in 48 h

170 °C, 80 min, 1.2 wt% H2SO4, 50 % ethanol

Miscanthus [101]

20 FPU/g cellulase enzyme and 35 CBU/g for β-glucosidase 20 FPU/g for cellulase from Celluclast 1.5 L and 40 IU/g for β-glucosidase from Novozyme 188

54.2 in 72 h

4.5 wt% SO2, 195 °C, 4.5 min

Douglas fir [49]

20 FPU C-30 cellulase from Novozyme

15 FPU/g cellulase from Spezyme CP and 40 CBU β-glucosidase from Novozyme 188

15 FPU/g for cellulase from Spezyme CP and 65 IU/g for β-glucosidase from Novozyme 188

20 FPU/g for cellulase from Spezyme CP and 40 IU/g for β-glucosidase from Novozyme 188

15 FPU/g for cellulase from Celluclast 1.5 L and 12.6 IU/g for β-glucosidase from Novozyme 188

60 in 72 h

220 °C, 4 min

Poplar [100]

20 FPU/g for cellulase from Celluclast 1.5 L and 40 IU/g for β-glucosidase from Novozyme 188

35 in 72 h

15 FPU/g for cellulase from Celluclast 1.5 L and 12.6 IU/g for β-glucosidase from Novozyme 188

15 FPU/g for cellulase from Celluclast 1.5 L and 15 CBU/g for β-glucosidase from Novozyme 188

76.5 in 72 h

85 in 72 h

15 FPU/g for cellulase from Celluclast 1.5 L and 26.25 CBU/g for β-glucosidase from Novozyme 188

82.3 in 72 h

190 °C, 10 min

180 °C, 1.0 wt% H2SO4, 30 min

Loblolly pine [39]

Enzyme loadings

Cellulose conversion yield (%)

Wheat straw [99]

210 °C, 1.40 wt% H2SO4, 10 min

Olive tree [98]

SEP

140 °C, 1.0 wt% H2SO4, 40 min

Corn stover [49]

DAP

Pretreatment conditions

Substrate

Pretreatment method

Table 3 Applications of acidic and neutral pretreatments for different biomasses

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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 which a significant amount of hemicellulose is solubilized as well. The major effect is the removal of lignin from the biomass, thus improving the reactivity of the remaining polysaccharides. In addition, the alkaline pretreatment could swell cell wall and improve cell wall accessibility for the subsequent enzymatic hydrolysis. The proposed reaction mechanism is believed to be saponification of intermolecular ester bonds cross-linking hemicellulose and lignin [94]. The saponification leads to the cleavage of lignin-carbohydrate complex (LCC) linkages, and the expose of cellulose microfibrils can increase enzymatic digestibility of cellulose. Acetyl groups 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 the hydrolysates, but the amount is typically lower than that with DAP [115]. In addition, alkaline pretreatment decreases the DP of cellulose (Table 4) and causes swelling of cellulose, leading to an increase in its internal surface area [116]. This makes cellulose more accessible for enzymes in the subsequent hydrolysis stage. In terms of cellulose crystallinity change during the pretreatment, research indicated that the amorphous regions suffered greater peeling reactions than the crystalline regions, and the occurrence of the peeling actions of the amorphous regions leads to an increase of cellulose crystallinity (Table 4) [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 increase the fiber porosity due to the disruption of biomass structures [118, 119]. Advantage and Disadvantage Compared with DAP, alkaline pretreatments have some practical operational advantages including lower reaction temperatures and pressures. A significant disadvantage of alkaline pretreatment is the conversion of alkali into irrecoverable salts and/or the incorporation of salts into 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 lime pretreatment, the precipitation of calcium oxalate on the heat exchanger and process streams is another 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 (%) before pretreatment

CrI (%) after pretreatment

DP before pretreatment

DP after pretreatment

Corn 55 °C, 0.5:1 Ca(OH)2 to biomass, 4 weeks stover

50.3a

56.2a

7,300b

3,200b

Poplar

49.9a

54.5a

3,500b

1,600b

65 °C, 0.5:1 Ca(OH)2 to biomass, 4 weeks

From Kumar et al. [38] a

CrI was measured by X-ray diffraction (XRD) method [40]

b

DP was measured by viscometric method [41]

Appl Biochem Biotechnol

Ammonia Fiber Expansion Pretreatment Process Description The AFEX pretreatment, also known as ammonia fiber explosion pretreatment, is another physicochemical process, much like SEP, in which the biomass material is subjected to liquid anhydrous ammonia under high pressures and moderate temperatures and is then rapidly depressurized. This swift pressure release leads to a rapid expansion of the ammonia gas that causes swelling and physical disruption of biomass fibers [121]. The AFEX is usually performed to treat moist biomass (0.1–2 g H2O/g dry biomass) with liquid ammonia (0.3– 2 g NH3/g dry biomass) while heating (60–100 °C) the biomass-water-ammonia mixture for a period of time (5–60 min) before rapidly releasing the pressure [122]. AFEX can be carried out in either a batch or a flow-through reactor. The major parameters influencing the AFEX process are ammonia loading, temperature, pressure, moisture content of biomass, and residence time [123]. During the AFEX pretreatment, about 95 % of the ammonia can be recovered in the gas phase and recycled, with a small amount of ammonia that remains in the lignocellulosics which might serve as a nitrogen source for the microbes in the fermentation process [124]. Mode of Action During the AFEX, the physicochemical treatment induces the disruption in the lignincarbohydrate linkage, hemicellulose hydrolysis, and ammonolysis of glucuronic cross-linked bonds, and partial decrystallization of the cellulose structure, all leading to a higher accessible surface area for enzymatic attack [123]. In AFEX pretreatment, the removal of acetyl groups from hemicellulose results in the formation of acetamide and acetic acid, but it is reported that AFEX removes the least amount of acetyl groups from lignocellulosic material compared to other leading pretreatment technologies [38]. In addition, the ammonia also causes a series of ammonolysis (amide formation) and hydrolysis reactions (acid formation) in the presence of water, which cleave LCC ester linkages [125]. The major effect of AFEX on cellulose is the decrystallization, which is mainly due to the generation of more amorphous portions in cellulose during this process [38]. These chemical structural changes lead to the formation of highly porous structures on the fiber cell wall, which greatly enhance enzyme accessibility to the cellulosic microfibrils [125]. Advantage and Disadvantage The important advantages of AFEX pretreatment include lower moisture content, lower formation of sugar degradation products due to moderate conditions, almost complete recovery of solid material and high cellulose digestibility in the subsequent enzymatic hydrolysis. One concern for this process is the cost of ammonia and the need for recycling technologies [94]. Soaking in Aqueous Ammonia Pretreatment Another type of process utilizing ammonia is soaking aqueous ammonia (SAA), which appears as an interesting alternative since it is performed at lower temperature (30–75 °C) [2]. The main purpose of SAA pretreatment is the removal of lignin, which is the physical and chemical barrier that inhibits accessibility of enzyme to the cellulose substrate [126]. It is regarded as a valuable pretreatment methodology due to the retention of the hemicellulose

Appl Biochem Biotechnol

fraction and removal of lignin after pretreatment [127]. SAA has been shown to be able to remove 74 % of the lignin from corn stover while retaining >85 % of the xylan and nearly 100 % of the glucan [128]. This allows easy downstream utilization of sugars in a single cofermentation process in which net sugar yield was increased due to the presence of hemicelluloses [129]. SAA utilizing lower temperatures and less extreme pHs reduces the associated chemical and energy costs and may also reduce the formation of carbohydrate degradation products. However, the SAA pretreated biomass pretreated shows relatively lower enzymatic digestibility due to its mild pretreatment conditions [130, 131]. Application of alkaline pretreatment Table 5 shows the applications of different alkaline pretreatments on herbaceous/agricultural residues, hardwood, and softwood biomasses. Among the numerous types of biomass, softwoods 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 that softwoods have a more rigid structure and contains more lignin [132].

Ozonolysis Ozonolysis of biomass is usually carried out at low temperature (20–30 °C), and the ozone flow 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

NaOH or Ca(OH)2 pretreatment

AFEX pretreatment

Pretreatment conditions

Cellulose Enzyme loadings conversion yield (%)

Corn 55 °C, 7.3 wt% stover [133] Ca(OH)2, 4 weeks

98 in 96 h

15 FPU/g for cellulase from Spezyme CP and 40 CBU/g for β-glucosidase from Novozyme 188

Birch [134]

80 °C, 7.0 wt% NaOH, 2 h

80 in 96 h

20 FPU/g for cellulase from Celluclast 1.5 L and 50 IU/g for β-glucosidase from Novozyme 188

Spruce [134]

80 °C, 7.0 wt% NaOH, 2 h

24 in 96 h

20 FPU/g for cellulase from Celluclast 1.5 L and 50 IU/g for β-glucosidase from Novozyme 188

Corn stover [49]

90 °C, 1:1 ammonia 76.6 in 72 h to biomass loading, 60 % moisture content, 5 min

15 FPU/g for cellulase from Celluclast 1.5 L and 26.25 CBU/g for β-glucosidase from Novozyme 188

Poplar [135]

180 °C, 2:1 ammonia 70 in 72 h to biomass loading, 233 % moisture content, 30 min

15 FPU/g for cellulase from Celluclast 1.5 L and 26.25 CBU/g for β-glucosidase from Novozyme 188

Appl Biochem Biotechnol Table 6 Effect of different chemical pretreatment technologies on the structure of lignocellulose Cellulose Hemicellulose Lignin Generation of Pretreatment Increase of method accessible surface decrystallization solubilization removal inhibitor compounds area

Lignin structure alteration

DAP

H

L

H

L

H

H

SEP

H

L

M

L

H

M

Organosolv

H

L

H

H

H

H

LHW NaOH/ Ca(OH)2

H H

L L

M M

L M

L L

M 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] H high effect, M moderate effect, L low effect

7 % [115, 136]. Ozone treatment is one way of reducing the lignin content of lignocellulosic wastes. This results in an increase of the in vitro digestibility of the treated material, and unlike other chemical treatments, it does not produce toxic residues. Ozone can be used to degrade lignin 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 conjugated double bonds and functional groups with high electron densities. Therefore, the moiety, most likely to be oxidized in ozonization of lignocellulosic materials, is lignin due to its high content of C=C bonds. Thus, during the ozonolysis, the degradation is mainly limited to lignin. Ozone attacks lignin releasing soluble compounds of less molecular weight, mainly organic acids such as formic and acetic acids [141]. The main advantages linked to this process are the lack of any degradation products that might interfere with subsequent hydrolysis or fermentation and the reactions occurring at ambient temperature and normal pressure. Furthermore, the fact that ozone can be easily decomposed by using a catalytic bed or increasing the temperature means 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 expensive and less applicable [142]. However, recently, Hu et al. [13] demonstrated that a lower charge of ozone could be used to enhance the enzymatic digestibility of cellulose, if the ozone-treated biomass was not washed and the in situ generated 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 Table 6. From the work that has been presented, it is clear that each pretreatment method has its own merits and disadvantages and consequences on the enzymatic hydrolysis. Recently, researchers applied some combinations of different pretreatment methods, such as alkaline pretreatment followed by steam pretreatment [143, 144] and organosolv pretreatment coupled with steam explosion [143], to improve the biomass digestibility. Although the results are promising compared with the single pretreatment, the extra equipment and operation cost will compromise these

Appl Biochem Biotechnol

processes. In addition, advanced transgenic techniques were reported to alternate the chemical structures of lignocellulosic biomass with the aim to reduce their recalcitrance in the pretreatments [145, 146]. However, these techniques are still in development, and significant research needs to be done before the commercialization. Despite much research that has been dedicated to understanding the chemistry and the plant cell wall structure changes during various pretreatment technologies, the insufficient knowledge of cell wall structure, ultrastructure, and pretreatment effects still limits the economics and effectiveness of pretreatment. For instance, the biological and chemical properties of plants are very complex in terms of composition, structure, and ultrastructure [147]. Although researchers have put significant effort into optimizing the pretreatment effectiveness, the fundamental science behind these optimizations is still not fully understood. Furthermore, there has been a lack of mechanistic understanding of the ultrastructural and physicochemical changes occurring within the cell wall at the 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 more fundamental level, in order to develop a cost-effective pretreatment technology with maximum fermentable sugar recovery; minimum inhibitor production and energy input; low demand of post-pretreatment processes; and low capital costs for reactors, water, and chemicals. In addition, advances in the analytical chemistry would provide useful tools to investigate the cell wall deconstruction and understand the recalcitrance during the pretreatment 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 Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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