Sugarcane Bagasse Pretreatment Methods for Ethanol Production Sugarcane Bagasse Pretreatment Methods for Ethanol Production

Lignocellulosic biomass such as sugarcane bagasse (SCB) is a renewable and abundant source for ethanol production. Sugarcane bagasse is composed of cellulose, hemicellulose, lignin, extractives, and several inorganic materials. Pretreatment methods of SCB are necessary for the successful conversion of SCB to ethanol. Each pretreatment process has a specific effect on the cellulose, hemicellulose, and lignin fraction. The conversion of SCB to ethanol typically consists of four main steps: pretreatment, enzymatic hydrolysis, fermentation, and distillation. Hence, different pretreatment methods should be chosen according to the process design for the following hydrolysis, fermentation, and distillation steps. There are many types of pretreatments such as physical, chemical, physico-chemical, and biological pretreatments. This chapter reviews the chemical and physico-chemical pretreatment methods of SCB which are often used by many research- ers for ethanol production. Different chemical and physico-chemical pretreatment meth ods of SCB are introduced and discussed based on relevance to the sugar yield, lignin removal, and cellulose content after pretreatment.


Introduction
According to the latest report produced by the United Nations Food and Agricultural Organization, there are 10 largest sugarcane producing countries in the world in 2018. The 10 countries are Brazil, India, China, Thailand, Pakistan, Mexico, Colombia, Indonesia, Philippines, and United States. About 540 million metric tons per year of sugarcane bagasse are produced globally [1]. Table 1 presents sugarcane bagasse production annually for several countries. Sugarcane bagasse is the solid residue obtained after extraction of the juice from sugar cane (Saccharum officinarum) and can be a potential substrate for ethanol production since it has high sugar content and is a renewable, cheap, and readily available feedstock.
Sugarcane bagasse is mainly composed of cellulose (33-36%), hemicellulose (28-30%), and lignin (17-24%). Cellulose is the most abundant polysaccharide polymer which comprised of a linear chain of β(1 → 4) linked D-glucose units that generates crystalline regions and consequently increases resistance to the hydrolytic process. Hemicellulose is the second most abundant polysaccharide after cellulose and is a short and highly branched polymers which comprised of pentose (xylose and arabinose) and hexose (mannose, glucose, and galactose) sugars. It possesses a heteropolysaccharide composition that varies according to the source. Sugarcane bagasse hemicellulose is composed of heteroxylans, with a predominance of xylose. Hence, it can be chemically hydrolyzed more easily than cellulose. Lignins are complex phenylpropanoid polymers formed by the polymerization of aromatic alcohols. The combination of the cellulose-hemicellulose-lignin matrix is conferring resistance to enzymatic and chemical degradation [10,11]. Bagasse could represent the main lignocellulosic biomass in many tropical countries since it is available at the sugar factory without additional cost and contains high sugar and low lignin content [12].
Production of bioethanol from SCB has a major advantage, like its less carbon intensive, than fossil fuel which reduces air pollution [13]. The bioethanol produced from lignocellulosic materials is named as second-generation (2G) ethanol or cellulosic ethanol, while the first generation ethanol is produced from sucrose (juice extracted from sugarcane, sugarbeet, or sweet sorghum) or starch (typically extracted from grains) [14]. The second-generation ethanol production from lignocellulosic biomass has been considered to be the biofuel with the greatest potential to replace oil-based fuels ( [15,16], and it can be produced from various lignocellulosic biomasses such as wood, agricultural, or forest residues. Typically, bioethanol can be produced in a four-step process, that is, pretreatment, enzymatic hydrolysis, fermentation, and distillation (Figure 1), where hydrolysis and fermentation may be combined. Currently, bioethanol is produced mostly in U.S and Brazil ( Table 2) [17].

Country
Sugarcane bagasse production (million metric ton/year) References

Pretreatment
The main objective of the pretreatments is to break down the lignin structure and disrupt the crystalline structure of cellulose for enhancing enzymes accessibility to the cellulose during the hydrolysis step [18]. These pretreatments may be biological, chemical, and physical processes that are used individually, combined, and/or sequentially [19,20]. The natural structure of lignocellulosic material is extremely recalcitrant to enzymatic hydrolysis. Therefore, the pretreatment step is required for efficient enzymatic hydrolysis of cellulose by removal of lignin and hemicellulose, reduction of cellulose crystallinity and increase the porosity of the biomass [21]. Each pretreatment has a different effect on the cellulose, hemicellulose, and lignin fraction.
It is necessary to choose suitable pretreatment methods for SCB since different lignocellulosic materials have different physico-chemical characteristics [22]. An efficient pretreatment should (1) improve the formation of fermentable sugars, (2) avoid the loss or degradation of carbohydrates, (3) avoid the formation of inhibitory by-products, and (4) be cost-effective [23]. According to Puligundla et al. [24], an ideal pretreatment should be economically efficient, Figure 1. A four-step process for ethanol production from biomass.

Country
Bioethanol production (million gallon) low energy consumption, and producing less or no residues. High digestibility of cellulose and versatility of feedstock are also important in the pretreatment process. In addition, other factors such as low sugar decomposition, low water or high solids, and low chemical consumption during the process should be considered. Besides that, the pretreatment should be performed at low operational risk and safe.

Dilute acid pretreatment
There are two types of acid pretreatments either using concentrated acid or diluted acids. Concentrated acid hydrolysis can be performed at a low temperature (30-60°C) using acid with the concentration around 40-80%. High sugar yield can be obtained using this method, however, requires large volumes of acid which are toxic and corrosive. Thus, corrosion resistant reactors are needed if concentrated acid is employed. Furthermore, the acid concentration must be recovered after hydrolysis to make the process economically feasible [10]. The development of effective acid recovery technologies has made this process renewed its interest [25].
On the other hand, dilute acid hydrolysis is the most widely used and has been considered to be one of the treatment methods with greater potential for wide-scale application. This process can be performed using diluted acids in the range of 0.5-6% and high temperatures from 120-170°C, with variable treatment times from minutes up to an hour.
Dilute acid pretreatment has received numerous research interests, and it has been successfully developed for pretreatment of lignocellulosic biomass. Dilute acid pretreatments are normally used to degrade the hemicellulosic fraction and increase the biomass porosity, improving the enzymatic hydrolysis of cellulose. The dilute acid pretreatment is important to weaken the glycosidic bond in the hemicellulose and lignin-hemicellulose bond and the lignin bond. This will lead to the dissolution of the sugar in the hemicellulose and also increase the porosity of the plant cell wall for effective enzyme digestibility [26]. Acid pretreatment is a very commonly used technology for biomass to ethanol conversion due to its low cost and the fact that the used acids are easily available. However, acid pretreatments can cause side effects such as the formation of furan and short chain aliphatic acid derivatives, which are considered strong inhibitors in microbial fermentation [27,28].
Several different acids used in pretreatments of SCB, including dilute sulfuric acid [29][30][31][32][33][34][35], dilute hydrochloric acid [36], dilute phosphoric acid [32,37], and dilute nitric acid [38], have been reported. High hydrolysis yields have been obtained when lignocellulosic biomass was pretreated with dilute sulfuric acid compared with hydrochloric, phosphoric, and nitric acid [22]. Sulfuric (H 2 SO 4 ) and phosphoric (H 3 PO 4 ) acids are widely used for acid pretreatment since they are relatively inexpensive and efficient in hydrolyzing lignocellulose. H 3 PO 4 also gives less negative impact on the environment compared to H 2 SO 4 , meanwhile hydrochloric (HCl) acid had better penetration to biomass and more volatile and easier to recover than H 2 SO 4 [39]; similarly, nitric acid (HNO 3 ) possesses good cellulose to sugar conversion rates [40]. However, both acids are expensive compared to H 2 SO 4 . Sulfuric acid is the most commonly used acid in the pretreatment of SCB [41,42]. Table 2 shows the yield of sugar at different types of acid pretreatment of SCB. Table 3, the acid concentration used in the range of 0.5-6.0%, temperature 120-170°C and time is around 10 to 300 min. Dilute acid at moderate temperature effectively removes most of the hemicelluloses and recovers as dissolved sugars.
The physical structure and chemical composition of the substrate as well as the treatment conditions are important factors for the effectiveness of alkaline pretreatment. In general, alkaline pretreatment is more effective on hardwood, herbaceous crops, and agricultural residues with a low lignin content than on softwood with a high lignin content [61]. Although hydroxides are not expensive, the drawback of this process is that it consumes a lot of water for washing the sodium (or calcium) salts that incorporate into the biomass so that the treatment of a large amount of  salts becomes a challenging issue for alkaline pretreatment. In addition, some enzyme inhibitors can be generated during lignin depolymerization [62]. In comparison with other pretreatment technologies, alkali pretreatment usually uses lower temperatures and pressures, even ambient conditions. Pretreatment time, however, is recorded in terms of hours such as 24 hours or days that are much longer than other pretreatment processes [63].
Alkaline pretreatments differ from acid pretreatments so that they are more efficient in lignin removal, substantially increasing cellulose digestibility, even after removing only part of the lignin. The hydrolysis of ester linkages between hemicellulose residues and lignin promotes an increase of porosity in the biomass, and as a result, cellulose and hemicellulose become more accessible to enzyme action [10,64]. As this pretreatment results in a large fraction of both cellulose and hemicellulose to remain intact, it has the potential for hydrolysis of a much larger fraction of the pretreated biomass, releasing glucose from cellulose and additional pentose sugars from hemicellulose. In addition, this occurs in an environment free of strong acids and fermentation inhibitors. Under these conditions, the degradation of sugars is minimal [65]. Sodium hydroxide shows the greatest lignin degradation when compared to other alkalis, such as sodium carbonate, ammonium hydroxide, calcium hydroxide, and hydrogen peroxide.
Lime (calcium hydroxide) pretreatment is another attractive alkali pretreatment technology due to the low formation of fermentation inhibitors, which increases pH and provides a lowcost alternative for lignin solubilization where the process is removing approximately 33% of lignin and 100% of acetyl groups. Even though the action of lime is slower than other pretreatments, lime is much cheaper than other alkalis and has low toxicity to the environment and safe handling [66]. The effectiveness of lime pretreatment in improving sugarcane bagasse susceptibility to enzymatic hydrolysis was studied by Rabelo et al. [54]. The result showed that lime pretreatment improved the enzymatic digestibility of SCB.

Organosolv pretreatments
The organosolv process is a delignification process, with varying simultaneous hemicellulose solubilization. The organosolv process uses organic or aqueous organic solvent mixtures with or without an acid or alkali catalysts to extract lignin from lignocellulosic biomass. Numerous organic solvent mixtures including methanol, ethanol, acetone, ethylene glycol, triethylene glycol, and tetrahydrofurfuryl alcohol have been used. The advantages of ethanol as a solvent are that it is produced in many biorefineries. It is easily replenished and recycled as a solvent for the pretreatment process. Ethanol is also inexpensive and less toxic to humans compared to other solvents such as methanol [67].
The ethanol organosolv process is among the chemical pretreatment being studied for the conversion of SCB to ethanol. In this pretreatment, high degrees of delignification can be achieved for SCB following ethanol organosolv pretreatment using formic acid as a catalyst. The degree of delignification increased with increasing pretreatment temperature. The maximum degree of delignification of sugarcane bagasse reached 80% at 210°C [68]. Mesa et al. [69] reported that the combination of a dilute-acid pretreatment followed by the organosolv pretreatment with NaOH at a temperature of 195°C for 60 min using 30% (v/v) was an efficient technique for SCB fractionation for the subsequent use on the enzymatic hydrolysis process, since yielded a residual solid material containing 67.3% (w/w) glucose. Novo et al. [70] showed that one of the best pretreatment conditions for lignin removal from SCB by the organosolv method could be achieved at 190°C and 150 min.
Beside ethanol, glycerol is an excellent solvent for organosolv pretreatment [71]. Glycerol, a high-boiling-point organic solvent derived from the oleochemical industry as a by-product has become very attractive. Martıń et al. [72] studied the effect of glycerol pretreatment on the main components of SCB. The result shows that the glycerol acted more selectively on lignin than on xylan where cellulose was almost completely recovered in the pretreated solids, accounting for 72% (g/g) of the pretreated substrate. Meanwhile, Novo et al. [70] reported that the glycerol pretreatment attained good cellulose preservation (>91%) and 80% lignin removal. However, Zhang et al. [73] found that >96% of the cellulose was recovered, whereas the lignin and hemicellulose removal were almost 60 and 80%, respectively, when SCB was treated with an acid-catalyzed glycerol organosolv pretreatment.

Steam explosion pretreatment
Steam explosion is one of the most efficient methods to deconstruct the plant cell wall macromolecular organization [19,74]. This process occurs both chemically and physically by revealing the lignocellulosic materials to high temperatures ranging from 160 to 260°C for reaction times varying from 2 to 30 min in the saturated steam either in the absence or presence of an exogenous acid or basic catalyst. The steam is able to expand the cell wall of the polysaccharide fiber and destroys cell structure into small pieces and breaks down the lignin network. This process would increase the accessibility of the enzyme to cellulose by exposing internal cellulose surface, which acetyl groups of hemicellulose can be hydrolyzed to acetic acid [75,76].
The physical forces cause partial hemicellulose solubilization and lignin reorganization. The major variables that affect steam explosion pretreatment efficacy include biomass origin, particle size, temperature, residence time, and moisture content [77,78].
When pretreatment is performed in the presence of an acid catalyst such as sulfuric (H 2 SO 4 ) or phosphoric (H 3 PO 4 ) acids, the need for time and temperature decreases substantially depending on the strength of the acid and its actual concentration in relation to the dry mass of the biomass. In addition, this process can remove hemicelluloses almost completely, whereas lignin is modified to a deeper extend, thus making the cellulosic materials more susceptible to enzymatic or acid hydrolysis [27,74,79]. There are several advantages of steam explosion pretreatment which includes lower environmental impact, cost-effectiveness, greater energy efficiency, and less or no chemical usage [22]. Also, to obtain the same particle size of the substrate, steam explosion method requires a 70% lower energy consumption compared to the conventional mechanical process [10]. The main drawbacks of steam explosion pretreatment are the partial degradation of hemicelluloses and the formation of toxic components that could affect the enzymatic hydrolysis and fermentation process [76].

Liquid hot water
According to Sánchez et al. [80], liquid hot water (LHW) pretreatment is performed at various temperatures from 160 to 240°C in the liquid state with water instead of steam. The LHW process primarily maximizes the solubilization of hemicellulose, partial removal of lignin, and making cellulose more accessible to the enzyme. In addition, the formation of the undesirable side products in liquid fraction can be reduced due to solubilized hemicellulose mostly appears in oligomers forms [18]. The LHW pretreatment cleaves hemicellulose linkages and liberates various acids during the process. These acids help to hydrolyze hemicellulose to monomeric sugars, which can be subsequently degraded to aldehydes (i.e., furfural from five carbon sugars and HMF from six carbon sugars). LHW has a great potential to be chosen as a pretreatment step in the biorefinery process as it can be considered as a green technology [81].
During high temperature pretreatment processes, water molecules penetrate the biomass cell wall and hydrate cellulose, with the partial removal of hemicellulose and minor amount of lignin [82]. The advantage of using the neutral method compared to the dilute-acid and alkaline catalyzed pretreatments is to avoid the chemical use in excess, because pH close to neutral does not cause corrosion from occurring, and the formation of excess furans during sugar degradation reactions can be eluded. [83]. However, sugar release yields from LHW pretreated biomass are lower than diluted acid pretreated biomass, otherwise higher pretreatment temperature and longer residence time are required for comparable performance [84]. The LHW has a few advantages compared to other pretreatment methods such as no additional catalysts or chemicals, operates at relatively moderate temperature, high hemicelluloses recovery, low levels of inhibitory by-products and cost-effective [85]. Table 5 presents the comparison between the cellulose content before and after pretreatment of LHW and steam explosion. The temperature range used in LHW is around 170-200°C, whereas in steam explosion the temperature is in the range of 180-195°C. Compared to the untreated SCB, cellulose content increased in pretreated SCB for both LHW and steam explosion pretreatments. The LHW pretreatment of SCB led to an excellent preservation of glucan (cellulose) fraction [88]. Meanwhile, steam explosion with and aid of H 2 SO 4 acid during pretreatment also increases the cellulose content in the pretreated SCB [91]. The increment of cellulose in pretreated SCB is related to the lignin removal during the pretreatment process either in LHW or steam explosion.

Biological pretreatment
Biological pretreatment of lignocellulosic biomass is considered as an efficient, ecofriendly, and cheap alternative [94]. The biological pretreatment of lignocellulosic biomass is usually performed using cellulolytic and hemicellulolytic microorganisms. The commonly used microorganisms are filamentous fungi which are ubiquitous and can be isolated from the soil, living plants or lignocellulosic waste materials [95]. White-rot fungi have been reported as the most effective microorganisms for the pretreatment of most of the lignocellulosic materials [96]. These microorganisms degrade lignin through the action of lignin-degrading enzymes such as peroxidases and laccases [97]. Brown-rot fungi mainly attack cellulose, while white and soft rot fungi attack both cellulose and lignin [10]. Table 6 shows the type of fungal species commonly used in biological pretreatment. The biological pretreatment appears to be a promising technique and has very apparent advantages, including low-capital cost, low energy requirement, no chemical requirement, and mild environmental conditions. However, the main disadvantages are the long incubation time, low efficiency, considerable loss of carbohydrate requirement of careful control of growth conditions, and space restrain its applications [98].
Jiraprasertwong et al. [99] investigated the effect of different microbial strains on biological pretreatment of SCB for enzymatic hydrolysis. The results showed that the pretreatment with the white-rot fungus gave the highest glucose concentration around two-fold higher when compared with the others. Hernández et al. [100] reported that SCB pretreated with Pycnoporus sanguineus promotes better lignin decay, glucose release, and hydrolysis yields. Studies by Khuong et al. [101] have shown that the initial moisture content of the bagasse was found to affect biological delignification by MG-60, and the 75% moisture content was LSR: liquid solid ratio. suitable for selective lignin degradation and subsequent ethanol production when white-rot fungus Phlebia sp. MG-60 was applied to sugarcane bagasse.

Conclusions
There are several pretreatment methods available for SCB; however, the final choice for the selection of pretreatment methods depends upon the effective delignification or hemicellulose removal, low sugar loss, time savings, being economic, and causing less environmental pollution. Each pretreatment method has its own advantages and disadvantages. Instead of performing the chemical pretreatment alone, it is good to combine the pretreatment with other physico-chemical pretreatment such as steam explosion in order to improve the sugar yield and increase the lignin removal from SCB. The combination of pretreatment is a promising method to improve enzymatic hydrolysis and ethanol production from SCB.

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