Pretreatment of Lignocelollusic Biomass Based on Improving Enzymatic Hydrolysis

Lignocellulosic materials among the alternative energy resources are the most desirable resources that can be employed to produce cellulosic ethanol, but this materials due to physical and chemical structure arranges strong native recalcitrance and results in low yield of ethanol. Then, a proper pre-treatment method is required to overcome this challenge. Until now, different pre-treatment technologies have been established to enhance lignocellulosic digestibility. This paper widely describes the structure of lignocellulosic biomass and effective parameters in pre-treatment of lignocelluloses, such as cellulose crystallinity, accessible surface area, and protection by lignin and hemicellulose. In addition, an overview about the most important pretreatment processes include physical, chemical, and biological are provided. Finally, we described about the inhibitors enzymes which produced from sugar degradation during pre-treatment process and the ways to control this inhibitors.


Introduction
Bioethanol derived from biomass is regarded as a most promising alternatives to fossil fuels due to its higher octane, clean burning, higher flame speeds and higher heats of vaporizations than gasoline, which can be blended with gasoline or used sprightly as clean alcohol in dedicated vehicles (Choi et al., 2015). Anyway, in the first generation bioethanol production, expensive starch and sugar derived from sugar cane and maize are involved as feed stocks but in the second generation process, lignocellulosic materials are used, because it's cheap, abundant and renewable (Dias et al., 2013). Lignocellulosic biomass is mainly composed of cellulose (38-50%), hemicelluloses (23-32%) and lignin (15-25%), in a complex structure, which the recalcitrancy of these compositions is very high. One of the best methods to convert such biomass into soluble sugars is enzymatic hydrolysis because of its low energy demand; despite, the main problem is the low cellulose accessibility due to strong linkage of cellulose with lignin (Gupta et al., 2011). This leads to difficulties within the conversion process; therefore an appropriate pre-treatment technology ( Fig. 1) is needed to overcome this recalcitrance and makes polysaccharides comfortably feasible for enzymatic hydrolysis (Gupta et al., 2011;Singh et al., 2015;Wang et al., 2016). In other words, pre-treatment is the crucial and expensive unit process in converting lignocellulosic biomass into ethanol (Kim et al., 2016).

Fig. 1: Schematic pre-treatment of lignocellulosic biomass
A suitable pre-treatment method involves (1) breaking hydrogen bonds in crystalline cellulose (Crl), (2) breaking down across-linked matrix of hemicelluloses and lignin, and (3) improving cellulose accessible surface for following enzymatic hydrolysis (Huang et al., 2015;Si et al., 2015). Until now, various pre-treatment methods have been developed,which can be extensively categorized into physical (chipping, grinding, irradiation, steam explosion and hot water), chemical (alkaline, acid and ionic liquid), Review Article physico-chemical and biological process (Si et al., 2015;Wang et al., 2016). On the other side, regardless of the pretreatment method used, during the process many inhibitory compounds are produced, which have negative impact on microbial activity in the hydrolysis step. Generally, inhibitors are classified into three main group: (1) weak acids like levulinic, acetic and formic acids, (2) furan derivatives like 5-hydroxy-2-methyll furfural (HMF), and furfural, and (3) phenolic compounds as well (Behera et al., 2014;Kim et al., 2016). This review first present the structure of lignocellulose and effective factors in pretreatment lignocelluloses, then describes the most commonly used pre-treatment methods in improving the digestibility of lignocellulosic biomass and finally briefly review inhibitors enzymes and efficient techniques to remove components that inhibit microbial growth and enzymatic hydrolysis of lignocellulose.

Cellulose
In nature, the major abundant polysaccharide is cellulose and mostly serves as the main scaffolding constituent for plant cell wall. Cellulose is composed of linear chain of Dglucose connected by β-(1,4)glycosidic bonds together. The Cellulose strain are associated to each other to make cellulose fibril. Cellulose fibers are connected by a number of intra-and intermolecular hydrogen bonds (Li et al., 2010). Then, cellulose is insoluble in water and most organic solvents (Swatloski et al., 2002).

Hemicellulose
Hemicelluloses, mainly located in secondary cell walls, are heterogeneous branched biopolymers containing pentoses, hexoses, and urgonic acids (Gírio et al., 2010).They are relatively easy to hydrolyse due to their amorphous, branched structure, and their lower molecular weight as well (M. F. Li et al., 2010). In order to increase the cellulose digestibility, large amounts of hemicelluloses need to be removed as they cover cellulose fibrils limiting their accessibility for the enzymatic hydrolysis (Agbor et al., 2011). Hemicelluloses are somewhat sensitive to operation condition, hence, factors like retention time and temperature need to be regulated to avoid the formation of by-products like furfurals and hydroxymthyl furfurals that later inhibit the yeast fermentation process (Palmqvist and Hahn-Hägerdal, 2000).

Lignin
Lignin is a heterogeneous three-dimensional network macromolecule generally formulated from the oxidative combination of monolignols, in particular p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) (Ralph et al., 2004). Besides the three main monolignols other products such as traces of hydroxyl cinnamyl aldehydes, acetates, pcoumarates, p-hydroxybenzoates, and tyramineferulate were also observed (Boerjan et al., 2003).In various plat species, plant tissues and cell wall layers these monolignols present different abundance. In addition, there are many possible linkages among these monolignols, like b- O-4, b-5, b-1, b-b, 5-5, and 4-O-5 (Sette et al., 2011). The actual structure of the lignin macromolecule is completely complicated because of the variety of monolignols and the ''randomness" of linkages among these monolignols (Ralph et al., 2004).
Lignin content is one of the most prominent limiting parameters for making lignocellulose impressionable to microbial attack. Studies have proved that lignin reduce facilitates hydrolysis of cellulose. The mechanism by which lignin hinder cellulose hydrolysis is still not entirely known. Although, two explanations have been recommended that could be the lead to this phenomenon. Firstly, lignin is a structural polysaccharide that by covalent linking to hemicellulose imparts strength to the plant cell wall . The covalent bonding between lignin and cellulose prevents the carbohydrate to be exposed for enzymatic hydrolysis. Secondly, woody plants (woody biomass) as compare with herbaceous crops (grass biomass) is harder to treat. A comparison of lignin structures in both substrates present that the extent of cross-linking and the phenyl content lignin found in wood is far more complex than that of grassy substrates (Khalil et al., 2014;Ververis et al., 2004).

Effective parameters in pre-treatment lignocelluloses
The intrinsic features of native lignocellulosic biomass make them resistant to enzymatic attack. The purpose of pre-treatment is to change these features in order to make the materials ready for enzymatic degradation. Since lignocellulosic materials are extremely complex, their pretreatment is very complicated either. The best procedure and conditions of pre-treatment rely entirely on the type of lignocelluloses (Huang et al., 2015;Wang et al., 2016). The cellulose crystallinity (Crl), degree of cellulose polymerization (DP), accessible surface area, and lignin and hemicellulose protection are the major parameters regarded as influencing the rate of lignocelluloses degradation by the enzymes. These parameters will be discussed briefly below.

Cellulose Cristallinity (Cristallinity Index, Crl)
Cellulose Crl exists in various polymorphs and is seen in microfibrils in plant cell wall. They are formed by hydrogen bonds by long chains of (1,4) β-D glucan bonded to each other. The existence of hydrogen bonds between chains with stand microbial and enzymatic attack while breaking them increase depolymerisation (Chundawat et al., 2010). Researches have exhibited that during hydrolysis digestion of amorphous cellulose takes place before Cellulose Crl (Zhao and Chen, 2013).Cellulose Crl is a key feature of cell walls that not only accounts for the amorphous and crystalline regions of native cellulose but also reflects cellulose interactions with other wall polymers. Using Xray diffraction, the crystalline index (CrI) has been applied to detect cellulose Crl in various biomass materials (Bansal et al., 2010). It has been presented that lignocellulosics pretreatment enhance sits hydrolysability although in many cases improves the cellulose Crl fraction. This fact has been recommend to be because of the elimination or decreasing of more simply accessible amorphous cellulose after pretreatments (Hu et al., 2014;Meng and Ragauskas, 2014). In contrast, in many cases high pH pre-treatments have been displayed to have less influence and even decrease biomass crystallinity (Kumar and Wyman, 2009)

Degree of Polymerisation (DP)
The degree of polymerization (DP) is mainly related to other substrate features, such as crystallinity and it is highly variable among different plant species. Although the function of glucan chain length is not absolutely known, it is believed to influence cellulose hydrolysis (Puri, 1984). Cellulose DP is an noticeable factor in determining biomass digestibility. Pre-treatment of lignocellulose as well as enzymatic hydrolysis is a depolymerisation process of cellulose (Meng and Ragauskas, 2014).The influence of various pre-treatments on cellulose chain length has been studied presenting decreased DP in solids prepared by different pre-treatments recommended that xylan reduction had a more rigorous effect on cellulose chain length than lignin removal.(Kumar and Wyman, 2009).

Accessible Specific Surface Area
The accessible specific surface area is a critical parameter that influence the enzymatic hydrolysis of lignocellulosic materials. The particle size and porosity or pore volume present an important effect on the accessible specific surface area of lignocellulosic materials. A high enzymatic hydrolysis rate and glucose yield can be obtained with the decreasing of the lignocellulose particle size (Chen et al., 2011;Yeh et al., 2010). As lignocellulosic biomass are dispersed into nanoscale, the accessible specific surface area of lignocellulose will improve remarkable. Almost the cellulose digestibility complete in a short time because more cellulose is exposed to the enzymes (Martin-Sampedro et al., 2012). The lignocellulose digestion can be noticeable improved when the pores of the substrate are large enough to assist both large and small enzyme constituents to maintain the synergistic action of the cellulase enzyme system.

Hemicelluloses and Lignin
Hemicellulose and lignin are closely connected to cellulose through non-covalent and covalent linkages, the content and distribution of hemicellulose and lignin also show obvious influence on the lignocellulose enzymatic hydrolysis (Alvira et al., 2010;Wang et al., 2016). The presence of hemicelluloses and lignin greatly obstructs the contact of enzymes with cellulose, leading to a very low enzymatic hydrolysis rate of raw materials. Moreover, the nonproductive binding of enzymes to lignin obstructs cellulose hydrolysis as well (Jönsson and Martín, 2016;Pan et al., 2005). Lignin and hemicelluloses reduction may improve the pore size and accessible surface of the lignocellulosic materials and reduce the non-productive adsorption of enzymes to lignin. Therefore, the removal of hemicelluloses and lignin can significantly improve the digestibility of lignocellulosic materials (Mussatto et al., 2008;Puro et al., 2016;Wang et al., 2016). Many pre-treatment processes are purposed to improve the digestibility of lignocellulose materials by hemicelluloses and/or lignin reduction.

Pre-Treatment Technologies for Lignocellulose Biomass
During the last decades, different pre-treatment technologies to enhance various lignocellulosic biomass enzymatic digestion have been reported and investigated Table 1 shows the percentage of biomass digestibility by different pre-treatments in various plant species. All these pre-treatment methods are presented to decrease lignocellulose recalcitrance to enzymes mostly by decreasing the level of hemicelluloses and lignin, improving accessible surface area, cellulose CrI reduction, improving pore volume, or changing lignin structure . The past two decades have observed an enormous amount of study being done in the area of pretreatments. The main features of different pre-treatment methods on the compositions of lignocellulosic biomass and their limitation summarized in Table 2. Various methods have been occupied to examine the productivity as a pretreatment estimate. The synergistic influences on efficient lignin reduction have also been investigated on a large scale. Pre-treatment methods possibly categorized in different ways. For example it can be classified into two groups: acidic and alkaline according to the pH that is maintained during the pre-treatments process (Singh et al., 2015;. Therefore, pre-treatment technologies can be broadly categorised into physical, chemical, physicochemical and biological. In this section, we described the most used pre-treatment in the last decades.

Physical Pre-Treatments
The main purpose of physical pre-treatments are to improve the accessible specific surface area of lignocellulosic materials to enzymes by decreasing their particle size or disturbing their structural regularity. Different types of physical processes, such as mechanical comminution (chipping and grinding), irradiation (gamma rays and microwave), hot water have been used to enhance the lignocellulosic materials digestion (Puro et al., 2016;Wang et al., 2016). Steam explosion, hot water and irradiation microwave have been applied as powerful physical pretreatments in different biomass materials (Table 1) (Aguedo et al., 2015;Studer et al., 2016;Wood et al., 2014).
Chipping and grinding among the mechanical comminution processes, is preferred to disperse immense lignocellulosic materials into small pieces and promote the following pretreatment procedures. Milling can remarkably decrease the particle size and cellulose CrI, and as a result by extending the milling time enhance their enzymatic hydrolysis. Nevertheless, the energy utilization of mechanical comminution is closely related to the final particle size of lignocellulosic materials.Then, to obtain a high enzymatic hydrolysis value and fermentable sugar yield a high energy input is demanded. This makes milling technology not economically valuable (Martin-Sampedro et al., 2012;Yeh et al., 2010). Other physical pre-treatment technologies have been applied to enhance enzymatic hydrolysis of lignocellulosic biomass such as irradiation and steam explosion. In recent years generally physical pre-treatment used as assistance to chemical pre-treatment method because, physical cannot remove hemicelluloses or lignin from the lignocellulosic biomass or reduce their particle size. It was reported that by many researchers that physical pre-treatment assisted to chemical pre-treatments are more effective in lignocellulose biomass digestion than the conventional heating chemical pre-treatments (Huang et al., 2015;Kapoor et al., 2015;Ma et al., 2009;Vanholme, 2014;Wei et al., 2016)

Alkaline pretreatment
The most commonly used method for hemicelluloses and lignin removal in the lignocellulosic biomass is alkali pretreatment and it disperses bulk lignocellulosic materials into lignocellulosic fibers. The ester linkages in hemicelluloses and lignin comfortably broken down under alkaline conditions. At the relatively high temperature, the ether linkages in hemicelluloses and lignin can also be destroyed. The cleavages of these linkages considerably encourage the solubilisation of hemicelluloses and lignin, hence, enhancing the porosity of biomass. Different alkaline reagents, such as sodium hydroxide (NaOH), calcium hydroxide (CaOH2), potassium hydroxide (KOH), and aqueous ammonia, have been used to pretreat many lignocellulosic materials. Among these alkaline reagents, NaOH and CaOH2 are the most commonly used (Hendriks and Zeeman, 2009;Wang et al., 2016). Alkali pre-treatment at relatively low temperatures could lead to much higher biomass enzymatic digestibility and does not demand complicated reactors, which are appealing to be employed on-farm. Anyway, the main drawbacks are long residence time (from hours to days) and the need for neutralization of the pretreated slurry (Jia et al., 2014;Wang et al., 2015;Wu et al., 2013;Xu et al., 2012). Combination of NaOH and CaOH2 were used to increase the cost productivity of alkaline pre-treatment in switch grass. The raw material was first pretreated by NaOH, and then biomass was pretreated by Ca(OH)2 which led to glucose and xylose yields of 59.4% and 57.3%, respectively (Xu and Cheng, 2011). Moreover, Li et al. (2014) reported that mild alkali pretreatment effectively extracts G-rich lignin for high biomass digestibility in Miscanthus.

Acid and dilute-acid pre-treatments
Acid pre-treatment especially by using sulfuric acid (H2SO4) are the most used chemical pre-treatment for lignocellulosic materials where polysaccharides are hydrolysed to monosaccharaides leading to high cellulose approachability to enzymatic hydrolysis. Acid pretreatment can be presented either under low acid concentration and high temperature or under higher acid concentration and lower temperature (Chen et al., 2017). Anyway, the concentrated acid pre-treatments are lead to cause severe cellulose and monosaccharaides reduction i.e. glucose, production of inhibitors (like HMF and 2furfuralaldehyde) and oxidation of equipment, then the concentrated acids are less attractive. On the other hand, diluted acid because of lower generation of inhibitors is more desirable (C. Li et al., 2010;Xu et al., 2012;Zheng et al., 2009). There are many different diluted reagents acids, like H2SO4, HCl, H3PO4, and HNO3 which have been applied to pre-treat different lignocellulosic biomass . The influence of dilute sulphuric acid pretreatment conditions on the recovery of xylose and glucose during the pre-treatment and following enzymatic hydrolysis procedures was examined by many researches (Chen et al., 2017;Hsu et al., 2010). In different investigations reported that for enhancing biomass digestibility and enzymatic hydrolysis two-step acid and alkaline pre-treatments are more effective (Table 1) (Jia et al., 2014;Si et al., 2015;Xu et al., 2012). For instance, lately, Si et al. (2015) have reported pre-treatments with 2% NaOH and 1% H2SO4increasing biomass digestibility (100%) in hemicelluloses-rich samples via the efficient coextraction of hemicelluloses and lignin. Additionally, Jia et al. (2014) have shown that two-step 2% NaOH and 1% H2SO4 improving biomass digestibility (98%) in maize straw with reduction of cellulose and lignin.
Commonly, lignin reduction by mineral acid pre-treatment is not highly advantageous. It was reported by many studies that after pre-treated with mineral acids, lignocellulosic residues were mainly contain high contents of lignin. A subsequent delignification procedure is always demanded to further enhance lignocellulosic residues digestibility (Xu et al., 2012;Zhang et al., 2012).

Ionic liquids (ILs) pre-treatment
Ionic liquids or salts are usually constituted of large organic cations and small inorganic anions. By modifying the compounds of anions and cations the influence of ILs can be different (Gao et al., 2013).This pre-treatment has high effects on lignin degradation, enhancing cellulose Crl, increasing accessible surface area. Some of the ILs recognised such as1-allyl-3-methylimidazolium-chloride, 1-ethyl-3-methylimidazolium-acetate,1-butyl-3methylimidazolium chloride and 1-ethyl-3-methyl imidazolium diethyl phosphate have recently received much attention because of their capability in cellulose decrystallinity . The main drawbacks of ILs method in the industrial scale are very costly, recycling of pure ILs is energy-intensive and, during pre-treatment, solution becomes viscous that makes it difficult to handle. Then, for enhancement of ionic liquids solubility for lignocellulosic biomass more research is required to increase the economics of ILs pre-treatments and construct effective IL recovery proses (Gao et al., 2013;Sathitsuksanoh et al., 2015). It has reported in sugarcane bagasse and poplar wood cellulose degradation by ionic liquids occurred around 98% and 97%, relatively (Gao et al., 2013;L. Wu et al., 2014).

Biological Pretreatment
Microorganisms or enzymes have also been applied to pretreat various lignocellulosic biomss before enzymatic hydrolysis. For the degradation of lignin and hemicelluloses many different microorganisms like brown-, white-and soft-rot fungi have been selected and used (Sánchez, 2009 (Hatakka, 1983). Furthermore, white-rot fungi produce lignin-modifying enzymes, such as laccase, ligninperoxidases, and manganese peroxidases, which present prominent influence on lignin reduction(Kumar and Wyman, 2009). It was reported by Ghorbani et al. (2015) in rice straw white rot fungi (Trichoderma viride) can remove lignin about 56% and also in poplar wood white rot fungi can effectively deconstruct lignin construction (85%), resulting in enhancement of biomass enzymatic saccharification . Hatakka (1983)has presented pre-treatment of wheat straw for five weeks by white rot fungi (Pleurotusostreatus) reduced lignin in the original wheat straw about 34%, but in the un-treated samples only 12% lignin reduction occurred.

Inhibitors Enzymes and Overcome of the Inhibitors
During the pre-treatment procedure of lignocellulose many ccompounds formed which can confirm prohibitory to microbial growth and metabolism. Capability Inhibitory chemicals that can be released from cellulose, hemicellulose and lignin when employing thermos chemical treatments are aliphatic acids like acetic, formic and levulinic acid, furaldehydes like furfural and 5hydroxymethylfurfural (HMF) and, uronic acid, 4hydroxybenzoic acid, vanillic acid, vanillin, phenol, cinnam aldehyde, and formaldehyde (Jönsson and Martín, 2016). By thermal breakdown of pentose and hexose sugars furan by-products are formed. The two furan derived illustrative, furfural and HMF arise from the dissolution of pentose and hexose sugars on treating lignocellulose with diluted H2SO4. By-products of furan are harmful to the activity of particularenzymes that are engaged in microbial metabolism like hexokinase, aldolase, phosphofructokinase, triosephosphate dehydrogenase, and alcohol dehydrogenase (Behera et al., 2014;Kim et al., 2016).
On the fermentation procedure by several methods, we can control the influence of inhibitors (Jönsson and Martín, 2016; Rabemanolontsoa and Saka, 2016). Generally, to remove the negative impact of inhibitors, there can be four methods: (1) prevent the formation of inhibitors during pretreatment and hydrolysis (2) detoxify the hydrolyzate prior to fermentation; (3) develop genetically modified microorganisms that can resist attack of inhibitors (4) conversion of toxic compounds into neutral products (Rabemanolontsoa and Saka, 2016). To prevent the formation of toxic compounds, a simple strategy would be the careful choice of lignocellulosic material and the application of mild pre-treatments. This may not agree with the industrial demand as a high cellulose and subsequent sugar yield is desired which can only be obtained by implementing harsh pre-treatment methods (Kim et al., 2016;Rabemanolontsoa and Saka, 2016). Addition of chemicals to the fermentation process is one way for detoxification (Behera et al., 2014;Duque et al., 2016).

Conclusion
In order to improve fermentable carbohydrate recovery, produce effective by-product, reduce inhibitors, low-cost bioethanol procedure, and increase ethanol production various effective pre-treatment technology have been identified for lignocellulosic materials. In general, pretreatment methods are divided into four major groups i.e. physical, chemical, physic-chemical and biological. The main parameters to obtain an efficient hydrolysis are crystallinity of cellulose, its accessible surface area and M. Madadi et al. (2017) Int. J. Appl. Sci. Biotechnol. Vol 5(1): 1-11 This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT protection by lignin and hemicellulose. Despite most of the pre-treatment have many advantages, but one method could not be the best selection for all types of biomass. Therefore, basic understanding of various pre-treatment technologies, different composition of biomass feedstock and the relationship between composition of biomass feedstock and pre-treatment methods would considerably help in matching the best pre-treatment method for a specific biomass feedstock.