**3.1 Acid pretreatment**

In this pretreatment, acids are used to pretreat lignocellulosic biomass. The generation of inhibitory products in the acid pretreatment renders it less attractive for pretreatment option. Furfurals, aldehydes, 5-hydroxymethylfurfural, and phenolic acids are the inhibitory compounds that are generated in huge amount in acid pretreatment. There are two types of acid treatments based on the type of end application. One treatment type is of short duration, i.e., 1–5 min, but high temperature > 180°C is used, and the second treatment type is of long duration, i.e., 30–90 min, and low temperature < 120°C is utilized. Due to hydrolysis by acid

**23**

*Different Pretreatment Methods of Lignocellulosic Biomass for Use in Biofuel Production*

treatment, separate step of hydrolysis of biomass can be skipped, but to remove acid, washing is required before the fermentation of sugars [43, 47]. For acid pretreatment, such reactors are required that show resistance to corrosive, hazardous, and toxic acids; therefore, acid pretreatment is very expensive. Flow through, percolation, shrinking-bed, counter current rector, batch, plug flow are different types of rectors that have been developed. For enhancing economic feasibility of acid pretreatment, recovery of concentrated acid at the end of the treatment is an

To treat lignocellulosic biomass, concentrated acids are also used. Most commonly used acids are sulfuric acid or hydrochloric acid. In order to improve the process of hydrolysis for releasing fermentable sugars from lignocellulosic biomass, acid pretreatment can be given. For poplar, switch grass, spruce, and corn stover, sulfuric acid pretreatment is commonly used. Reducing sugars of 19.71 and 22.93% were produced as a result of the acid pretreatment of Bermuda grass and rye, respectively. In percolation reactor, pretreatment of rice straw was carried out in two stages using aqueous ammonia and dilute sulfuric acid. When ammonia is used, 96.9% reducing sugar yield was obtained, while 90.8% yield was obtained in case of utilization of dilute acid. *Eulaliopsis binate* is a perennial grass and yielded 21.02% sugars, 3.22% lignin, and 3.34% acetic acid, and inhibitors in very less amount are produced when treated with dilute sulfuric acid [48, 49]. At 4 wt% concentration of sulfuric acid, pretreatment is preferred because of less cost and more effectiveness of the process. Dilute sulfuric acid causes biomass hydrolysis and then further breakdown of xylose into furfural is achieved. High temperature favors hydrolysis by dilute sulfuric acid [50]. Removal of hemicellulose is important to increase glucose yield from cellulose, and dilute sulfuric acid is very effective to achieve this purpose. It is necessary for an economical biomass conversion to achieve high xylan-to-xylose ratio. One-third of the total carbohydrate is xylan in most lignocellulosic materials. There are two types of dilute acid pretreatments, one is characterized by high temperature, continuous flow process for low solid loadings, and the other one is with low temperature, batch process and high solid loadings. Temperature and solid loadings for the first type are >160°C and 5–10%, respectively, and for the second type, temperature and solid loadings are around<160°C

Besides sulfuric acid and hydrochloric acid, other acids like oxalic acid and maleic acid are also used for the pretreatment of lignocellulosic biomass. Oxalic and maleic acids have high pKa value and solution pH as compared to sulfuric acid. Because of having two pKa values, dicarboxylic acids hydrolyze biomass more efficiently than sulfuric acid and hydrochloric acid. Other advantages include less toxicity to yeast, no odor, more range of pH and temperature for hydrolysis, and no hampering of glycolysis. Maleic acid has khyd/kdeg, due to which hydrolysis of cellulose to glucose is preferred over glucose breakdown. Effects of oxalic, sulfuric, and maleic acid pretreatment on biomass at the same combined severity factor (CSF) were determined [53]. The use of maleic acid produces high concentration of

Apart from acids, few bases are also used for pretreatment of biomass. Lignin contents greatly affected the result of alkaline treatment. As compared to other pretreatment methods, alkali treatment requires less pressure and temperature and ambient condition, but alkali pretreatment needs time in days and hours. Degradation of sugar in alkali treatment is less than that by acid treatment, and also the removal and recovery of caustic salt are possible and easy in case of alkali

*DOI: http://dx.doi.org/10.5772/intechopen.84995*

and 10–40%, respectively [51, 52].

xylose and glucose as compared to oxalic acid.

**3.2 Alkali pretreatment**

important step.

#### *Different Pretreatment Methods of Lignocellulosic Biomass for Use in Biofuel Production DOI: http://dx.doi.org/10.5772/intechopen.84995*

treatment, separate step of hydrolysis of biomass can be skipped, but to remove acid, washing is required before the fermentation of sugars [43, 47]. For acid pretreatment, such reactors are required that show resistance to corrosive, hazardous, and toxic acids; therefore, acid pretreatment is very expensive. Flow through, percolation, shrinking-bed, counter current rector, batch, plug flow are different types of rectors that have been developed. For enhancing economic feasibility of acid pretreatment, recovery of concentrated acid at the end of the treatment is an important step.

To treat lignocellulosic biomass, concentrated acids are also used. Most commonly used acids are sulfuric acid or hydrochloric acid. In order to improve the process of hydrolysis for releasing fermentable sugars from lignocellulosic biomass, acid pretreatment can be given. For poplar, switch grass, spruce, and corn stover, sulfuric acid pretreatment is commonly used. Reducing sugars of 19.71 and 22.93% were produced as a result of the acid pretreatment of Bermuda grass and rye, respectively. In percolation reactor, pretreatment of rice straw was carried out in two stages using aqueous ammonia and dilute sulfuric acid. When ammonia is used, 96.9% reducing sugar yield was obtained, while 90.8% yield was obtained in case of utilization of dilute acid. *Eulaliopsis binate* is a perennial grass and yielded 21.02% sugars, 3.22% lignin, and 3.34% acetic acid, and inhibitors in very less amount are produced when treated with dilute sulfuric acid [48, 49]. At 4 wt% concentration of sulfuric acid, pretreatment is preferred because of less cost and more effectiveness of the process. Dilute sulfuric acid causes biomass hydrolysis and then further breakdown of xylose into furfural is achieved. High temperature favors hydrolysis by dilute sulfuric acid [50]. Removal of hemicellulose is important to increase glucose yield from cellulose, and dilute sulfuric acid is very effective to achieve this purpose. It is necessary for an economical biomass conversion to achieve high xylan-to-xylose ratio. One-third of the total carbohydrate is xylan in most lignocellulosic materials. There are two types of dilute acid pretreatments, one is characterized by high temperature, continuous flow process for low solid loadings, and the other one is with low temperature, batch process and high solid loadings. Temperature and solid loadings for the first type are >160°C and 5–10%, respectively, and for the second type, temperature and solid loadings are around<160°C and 10–40%, respectively [51, 52].

Besides sulfuric acid and hydrochloric acid, other acids like oxalic acid and maleic acid are also used for the pretreatment of lignocellulosic biomass. Oxalic and maleic acids have high pKa value and solution pH as compared to sulfuric acid. Because of having two pKa values, dicarboxylic acids hydrolyze biomass more efficiently than sulfuric acid and hydrochloric acid. Other advantages include less toxicity to yeast, no odor, more range of pH and temperature for hydrolysis, and no hampering of glycolysis. Maleic acid has khyd/kdeg, due to which hydrolysis of cellulose to glucose is preferred over glucose breakdown. Effects of oxalic, sulfuric, and maleic acid pretreatment on biomass at the same combined severity factor (CSF) were determined [53]. The use of maleic acid produces high concentration of xylose and glucose as compared to oxalic acid.

### **3.2 Alkali pretreatment**

Apart from acids, few bases are also used for pretreatment of biomass. Lignin contents greatly affected the result of alkaline treatment. As compared to other pretreatment methods, alkali treatment requires less pressure and temperature and ambient condition, but alkali pretreatment needs time in days and hours. Degradation of sugar in alkali treatment is less than that by acid treatment, and also the removal and recovery of caustic salt are possible and easy in case of alkali

*Biomass for Bioenergy - Recent Trends and Future Challenges*

manure and sludge, 80% methane from manure and twofold increase in methane production from sludge were recorded in the study. A PEF system was designed and developed by Kumar et al. [45] that consisted of high-voltage power supply, switch circuit, a function generator, and sample holder. Neutral red dye was used to study the changes in the structure of cellulose by PEF pretreatment. Function generator drives the transistor present in the switching circuit; when pulse is applied by function generator to the switching circuit, switching circuit is turned on. Switching circuit is then transferred to the high voltage across the sample holder. So, by using function generator pulses of desired shape, width and high voltage can be applied to the sample. By using this setup, effects were observed on switch grass and wood. Results showed that at ≥8 kV/cm, switch grass showed high neutral red uptake. At low field strength, structural changes are less likely to occur. Electroporation through pulsed electric field is greatly affected by two parameters, pulse duration and electric field strength. Irreversible electroporation at >4 kV/cm with pulse duration in millisecond and ≥ 10 kV/cm with microsecond pulse duration was observed in *Chlorella vulgaris* which showed that pulse duration with a difference in micro- and milliseconds range can effect electroporation. Pulse electric field can increase hydrolysis rate by exposing cellulose to catalytic agents

In this pretreatment, acids are used to pretreat lignocellulosic biomass. The generation of inhibitory products in the acid pretreatment renders it less attractive for pretreatment option. Furfurals, aldehydes, 5-hydroxymethylfurfural, and phenolic acids are the inhibitory compounds that are generated in huge amount in acid pretreatment. There are two types of acid treatments based on the type of end application. One treatment type is of short duration, i.e., 1–5 min, but high temperature > 180°C is used, and the second treatment type is of long duration, i.e., 30–90 min, and low temperature < 120°C is utilized. Due to hydrolysis by acid

**22**

[40, 41, 46] (**Figure 5**).

**Figure 5.**

*Pulse electric field (Intechopen.com).*

**3.1 Acid pretreatment**

**3. Chemical pretreatments**

treatment. Ammonium, sodium, calcium, and potassium hydroxides are used for alkaline pretreatment, but among these sodium hydroxide is the most commonly used alkaline pretreatment agent, whereas calcium hydroxide is the cheapest yet effective among all other alkali agents for pretreatment. By neutralizing calcium with carbon dioxide, calcium can be recovered easily in form of insoluble calcium carbonate. Using lime kiln technology, calcium hydroxide can be regenerated. Apparatus required for alkali pretreatment is basically temperature controller, a tank, CO2 scrubber, water jacket, manifold for water and air, pump, tray, frame, temperature sensor, and heating element. The first step of pretreatment consists of making lime slurry with water. The next step is spraying of this slurried lime on biomass; after spray, store the biomass for hours or, in some case, days. Contact time can be reduced by increasing temperature [54–57]. Crystallinity index increases in lime pretreatment because of the removal of lignin and hemicellulose. Structural features resulting from lime pretreatment affect the hydrolysis of pretreated biomass. Correlation of three structural factors, viz., lignin, acetyl content and crystallinity, and enzymatic digestibility, was reported by Chang and Holtzapple [58]. He concluded that (1) regardless of crystallinity and acetyl content, in order to obtain high digestibility, extensive delignification is enough. (2) Parallel barriers to hydrolysis are removed by delignification and deacetylation. (3) Crystallinity does not affect ultimate sugar yield; however, it plays some role in initial hydrolysis. It is evident from these points that lignin content should be reduced to 10% and all acetyl groups should be removed by an effective pretreatment process. Thus in exposing cellulose to enzymes, alkaline pretreatment plays an important role. By increasing enzyme access to cellulose and hemicellulose and eliminating nonproductive adsorption sites, lignin removal can play its role in increasing effectiveness of enzyme.

## **3.3 Organosolv**

Aqueous organic solvents like methanol, acetone, ethanol, and ethylene glycol are used in this method with specific conditions of temperature and pressure. Organosolv pretreatment is usually performed in the presence of salt catalyst, acid, and base. The biomass type and catalyst involved decide the temperature of pretreatment, and it can go up to 200°C. Lignin is a valuable product, and to extract lignin this process is used mainly. Cellulose fibers are exposed when lignin is removed, which leads to more hydrolysis. During organosolv pretreatment, fractions and syrup of cellulose and hemicellulose, respectively, are also produced. There are certain variable factors like catalyst type, temperature, and concentration of solvent and reaction time which affects the characteristics of pretreated biomass like crystallinity, fiber length, and degree of polymerization. Inhibitor formation is triggered by long reaction, high temperature, and acid concentrations [59, 60]. In a study by Park et al. [61], effect of different catalyst was checked for the production of ethanol and among sulfuric acid, sodium hydroxide, and magnesium sulfate, and sulfuric acid was found to be most effective in ethanol production, but for enhancing digestibility the use of sodium hydroxide is proven to be effective. Sulfuric acid is a good catalyst, but its toxicity and inhibitory nature make it less favorite. Organosolv is not a cost-effective pretreatment process because of the high cost of catalysts, but it can be made cost-effective by recovering and recycling of solvents. Solvent removal is important because its presence effects fermentation, microorganism growth, and enzymatic hydrolysis. There is added risk of handling such harsh organic solvents. Acid helps in hydrolysis and depolymerization of lignin. Upon cooling lignin is dissolved in phenol, and in the aqueous phase, sugars are present. Formasolv involving formic acid, H2O, and hydrochloric acid is a type of organosolv

**25**

*Different Pretreatment Methods of Lignocellulosic Biomass for Use in Biofuel Production*

lignin extraction were reported by Panagiotopoulos et al. [65].

ture, cations and anions, and time of pretreatment.

in which lignin is soluble and at low temperature process can be carried out. For pretreatment with ethanosolv cellulose, hemicellulose and pure lignin can be recovered, but high pressure and temperatures are required when ethanosolv is used, and less toxic nature of ethanol as compared to other organosolv makes it favorite for pretreatment. Ethanosolv when used in pretreatment effects the enzymatic hydrolysis, so to prevent this low ethanol, water is used [62]. Recovery of ethanol and water reduces the overall cost of the pretreatment. For sugarcane bagasse Mesa et al. [63] used ethanosolv at 195°C for 60 min, and results showed formation of 29.1% sugars from 30% ethanol. Alcohol-based organosolv pretreatment is combined with ball milling by Hideno et al. [24] to pretreat Japanese cypress and observed a synergistic effect on digestibility. 50.1, 41.7, and 48.1% yield of organosolv pulping was obtained from ethylene glycol-water, acetic acid-water, and ethanol-water in a study done by Ichwan and Son [64]. Poplar wood chips were first treated with stream and then with organosolv to separate cellulose, lignin, and hemicellulose. About 88% hydrolysis of cellulose to glucose, 98% recovery of cellulose, and 66% increase in

For the pretreatment of lignocellulose, scientist took a great interest in using ionic liquids, for decades. Ionic liquids containing cations or anions are a new class of solvents with high thermal stability and polarity, less melting point, and negligible vapor pressure [66, 67]. Normally large organic cations and small inorganic anions compose ionic liquids. Factors like degree of anion charge delocalization and cation structure significantly effect physical, biological, and chemical ionic liquid properties. Interactions between ionic liquids and biomass get affected by tempera-

Ionic liquids actually compete for hydrogen bonding with lignocellulosic components, and in this competition disruption of network occurs. 1-Ethyl-3 methylimidazolium diethyl phosphate-acetate, 1-butyl-3-methylimidazoliumacetate, cholinium amino acids, cholinium acetate, 1-ethyl-3-methylimidazolium diethyl phosphate-acetate, 1-allyl-3-methylimidazolium chloride, and chloride are ionic liquids used for the treatment of rice husk, water hyacinth, rice straw, kenaf powder, poplar wood, wheat straw, and pine. Among other ionic liquids are imidazolium salts which are most commonly used [42]. 1-Butyl-3-methylimidazolium chloride is used for pretreatment by Dadi et al. [68] who observed a twofold increase in yield and rate of hydrolysis. For the pretreatment of rice straw, Liu and Chen [69] used 1-butyl-3-methylimidazolium chloride also known as (Bmim-Cl) and observed significant enhancement in the process of hydrolysis due to modifications in the structure of wheat straw by Bmim-Cl. Bmim-Cl played role in the reduction of polymerization and crystallinity. A twofold increase in hydrolysis yield from sugarcane bagasse was observed in a study by Kuo and Lee [70] as compared to untreated bagasse. 1-Ethyl-3-methylimidazolium-acetate is used in a study by Li et al. [71] for the pretreatment of switch grass in order to remove lignin at a temperature of 160°C for 3 hours. Results showed 62.9% lignin removal enhanced enzymatic digestibility, and reduced cellulose crystallinity was reported by Tan et al. [72] on palm tree pretreatment with 1-butyl-3-methylimidazolium chloride. Slight changes in composition of biomass occurred after ionic liquid pretreatments although significant changes were observed in the structure of biomass. Ionic liquid pretreatment is less preferred over other techniques because of high thermal and chemical stability, less dangerous conditions for processing, low vapor pressure of solvents, and retaining liquid state at wide range of temperature. Ionic liquids can be recycled easily and are non-derivatizing. Disadvantage of using ionic liquid pretreatment is that

*DOI: http://dx.doi.org/10.5772/intechopen.84995*

**3.4 Ionic liquids**

#### *Different Pretreatment Methods of Lignocellulosic Biomass for Use in Biofuel Production DOI: http://dx.doi.org/10.5772/intechopen.84995*

in which lignin is soluble and at low temperature process can be carried out. For pretreatment with ethanosolv cellulose, hemicellulose and pure lignin can be recovered, but high pressure and temperatures are required when ethanosolv is used, and less toxic nature of ethanol as compared to other organosolv makes it favorite for pretreatment. Ethanosolv when used in pretreatment effects the enzymatic hydrolysis, so to prevent this low ethanol, water is used [62]. Recovery of ethanol and water reduces the overall cost of the pretreatment. For sugarcane bagasse Mesa et al. [63] used ethanosolv at 195°C for 60 min, and results showed formation of 29.1% sugars from 30% ethanol. Alcohol-based organosolv pretreatment is combined with ball milling by Hideno et al. [24] to pretreat Japanese cypress and observed a synergistic effect on digestibility. 50.1, 41.7, and 48.1% yield of organosolv pulping was obtained from ethylene glycol-water, acetic acid-water, and ethanol-water in a study done by Ichwan and Son [64]. Poplar wood chips were first treated with stream and then with organosolv to separate cellulose, lignin, and hemicellulose. About 88% hydrolysis of cellulose to glucose, 98% recovery of cellulose, and 66% increase in lignin extraction were reported by Panagiotopoulos et al. [65].

## **3.4 Ionic liquids**

*Biomass for Bioenergy - Recent Trends and Future Challenges*

treatment. Ammonium, sodium, calcium, and potassium hydroxides are used for alkaline pretreatment, but among these sodium hydroxide is the most commonly used alkaline pretreatment agent, whereas calcium hydroxide is the cheapest yet effective among all other alkali agents for pretreatment. By neutralizing calcium with carbon dioxide, calcium can be recovered easily in form of insoluble calcium carbonate. Using lime kiln technology, calcium hydroxide can be regenerated. Apparatus required for alkali pretreatment is basically temperature controller, a tank, CO2 scrubber, water jacket, manifold for water and air, pump, tray, frame, temperature sensor, and heating element. The first step of pretreatment consists of making lime slurry with water. The next step is spraying of this slurried lime on biomass; after spray, store the biomass for hours or, in some case, days. Contact time can be reduced by increasing temperature [54–57]. Crystallinity index increases in lime pretreatment because of the removal of lignin and hemicellulose. Structural features resulting from lime pretreatment affect the hydrolysis of pretreated biomass. Correlation of three structural factors, viz., lignin, acetyl content and crystallinity, and enzymatic digestibility, was reported by Chang and Holtzapple [58]. He concluded that (1) regardless of crystallinity and acetyl content, in order to obtain high digestibility, extensive delignification is enough. (2) Parallel barriers to hydrolysis are removed by delignification and deacetylation. (3) Crystallinity does not affect ultimate sugar yield; however, it plays some role in initial hydrolysis. It is evident from these points that lignin content should be reduced to 10% and all acetyl groups should be removed by an effective pretreatment process. Thus in exposing cellulose to enzymes, alkaline pretreatment plays an important role. By increasing enzyme access to cellulose and hemicellulose and eliminating nonproductive adsorption sites, lignin removal can play its role in increasing effectiveness

Aqueous organic solvents like methanol, acetone, ethanol, and ethylene glycol are used in this method with specific conditions of temperature and pressure. Organosolv pretreatment is usually performed in the presence of salt catalyst, acid, and base. The biomass type and catalyst involved decide the temperature of pretreatment, and it can go up to 200°C. Lignin is a valuable product, and to extract lignin this process is used mainly. Cellulose fibers are exposed when lignin is removed, which leads to more hydrolysis. During organosolv pretreatment, fractions and syrup of cellulose and hemicellulose, respectively, are also produced. There are certain variable factors like catalyst type, temperature, and concentration of solvent and reaction time which affects the characteristics of pretreated biomass like crystallinity, fiber length, and degree of polymerization. Inhibitor formation is triggered by long reaction, high temperature, and acid concentrations [59, 60]. In a study by Park et al. [61], effect of different catalyst was checked for the production of ethanol and among sulfuric acid, sodium hydroxide, and magnesium sulfate, and sulfuric acid was found to be most effective in ethanol production, but for enhancing digestibility the use of sodium hydroxide is proven to be effective. Sulfuric acid is a good catalyst, but its toxicity and inhibitory nature make it less favorite. Organosolv is not a cost-effective pretreatment process because of the high cost of catalysts, but it can be made cost-effective by recovering and recycling of solvents. Solvent removal is important because its presence effects fermentation, microorganism growth, and enzymatic hydrolysis. There is added risk of handling such harsh organic solvents. Acid helps in hydrolysis and depolymerization of lignin. Upon cooling lignin is dissolved in phenol, and in the aqueous phase, sugars are present. Formasolv involving formic acid, H2O, and hydrochloric acid is a type of organosolv

**24**

of enzyme.

**3.3 Organosolv**

For the pretreatment of lignocellulose, scientist took a great interest in using ionic liquids, for decades. Ionic liquids containing cations or anions are a new class of solvents with high thermal stability and polarity, less melting point, and negligible vapor pressure [66, 67]. Normally large organic cations and small inorganic anions compose ionic liquids. Factors like degree of anion charge delocalization and cation structure significantly effect physical, biological, and chemical ionic liquid properties. Interactions between ionic liquids and biomass get affected by temperature, cations and anions, and time of pretreatment.

Ionic liquids actually compete for hydrogen bonding with lignocellulosic components, and in this competition disruption of network occurs. 1-Ethyl-3 methylimidazolium diethyl phosphate-acetate, 1-butyl-3-methylimidazoliumacetate, cholinium amino acids, cholinium acetate, 1-ethyl-3-methylimidazolium diethyl phosphate-acetate, 1-allyl-3-methylimidazolium chloride, and chloride are ionic liquids used for the treatment of rice husk, water hyacinth, rice straw, kenaf powder, poplar wood, wheat straw, and pine. Among other ionic liquids are imidazolium salts which are most commonly used [42]. 1-Butyl-3-methylimidazolium chloride is used for pretreatment by Dadi et al. [68] who observed a twofold increase in yield and rate of hydrolysis. For the pretreatment of rice straw, Liu and Chen [69] used 1-butyl-3-methylimidazolium chloride also known as (Bmim-Cl) and observed significant enhancement in the process of hydrolysis due to modifications in the structure of wheat straw by Bmim-Cl. Bmim-Cl played role in the reduction of polymerization and crystallinity. A twofold increase in hydrolysis yield from sugarcane bagasse was observed in a study by Kuo and Lee [70] as compared to untreated bagasse. 1-Ethyl-3-methylimidazolium-acetate is used in a study by Li et al. [71] for the pretreatment of switch grass in order to remove lignin at a temperature of 160°C for 3 hours. Results showed 62.9% lignin removal enhanced enzymatic digestibility, and reduced cellulose crystallinity was reported by Tan et al. [72] on palm tree pretreatment with 1-butyl-3-methylimidazolium chloride. Slight changes in composition of biomass occurred after ionic liquid pretreatments although significant changes were observed in the structure of biomass. Ionic liquid pretreatment is less preferred over other techniques because of high thermal and chemical stability, less dangerous conditions for processing, low vapor pressure of solvents, and retaining liquid state at wide range of temperature. Ionic liquids can be recycled easily and are non-derivatizing. Disadvantage of using ionic liquid pretreatment is that

noncompatibility of cellulase and ionic liquids results in the unfolding and inactivation of cellulase. At less viscosity cellulose solubilizes at low temperature; that's why while using ionic liquids, viscosity is an important factor to be considered regarding the energy consumption of the whole process. High temperatures trigger more side reactions and negative side effects like reducing ionic liquid stability [73].

#### **3.5 Ozonolysis**

Ozone pretreatment is a great option for lignin content reduction in lignocellulosic biomass. In vitro digestibility of biomass is enhanced by the application of ozone pretreatment. Inhibitors are not formed in this pretreatment which is a great advantage because other chemical pretreatments produce toxic residues. In ozone pretreatment, ozone acts as an oxidant in order to break down lignin. Ozone gas is soluble in water and being a powerful oxidant, by breaking down lignin, releases less molecular weight, soluble compounds. Wheat straw, bagasse, cotton straw, green hay, poplar sawdust, peanut, and pine can be pretreated with ozone in order to degrade lignin and hemicellulose; however, only slight changes occur in hemicellulose, whereas almost no changes occur in cellulose. Ozonolysis apparatus consists of ozone catalytic destroyer, iodine trap used for testing efficiency of catalyst, oxygen cylinder, ozone generator, three-way valve, ozone UV spectrophotometer, pressure regulation valve, process gas humidifier, vent, and automatic gas flow control valve [40, 41, 74–76]. Moisture content hugely effects oxidization of lignin via ozone pretreatment as lignin oxidation decreases with increase in the moisture content of biomass. Ozone mass transfer is limited at less water concentration, which ultimately effects its reactivity with biomass. Longer residence time of ozone is caused by the blockage of pores by water film [77]. During ozonolysis, pH of water decreases because of the formation of organic acids. Alkaline media trigger delignification because it removes lignins that are bonded to carbohydrates [78, 79].

Biomass delignification is associated with the production of inhibitory compounds. Certain aromatic and polyaromatic compounds are produced as a result of delignification [80]. Structural changes in lignin are observed by Bule et al. [81] in a study; different lignin subunits showed aromatic opening and degradation of β-O-4 moieties in NMR analysis. How do aromatic structures of control- and ozone-pretreated samples differ? A spectrum showed a decrease in aromatic carbon signal concentration. Changes were observed in methoxy groups that suggest the breakdown of ester-linked structure. Different reactor designs are used for the ozone pretreatment of biomass, for example, batch reactor, Drechsel trap reactor, fixed bed reactor, rotatory bed reactor, and multilayer fixed bed reactor. Plug flow reactors are used by most researchers [82]. Heiske et al. [83] compared the characteristics of single layered and multiple layered bed reactors in order to improve the wheat straw conversion to methane. Straw with 16.2% lignin concentration was obtained from single layered reactor, whereas in multiple layered reactor, lignin concentration decreased up to 7.2% at the bottom layer. Due to wax degradation in ozone-pretreated wheat straw, production of fatty acid compounds is observed by Kádár et al. [84]. About 49% lignin degradation was observed when corn stover was pretreated with ozonolysis in a study by Williams [85].

### **4. Physicochemical pretreatment**

#### **4.1 Ammonia fiber expansion (AFEX)**

AFEX technique belongs to the category of physicochemical pretreatment methods. In this low temperature process, concentrated ammonia (0.3–2 kg ammonia/

**27**

nia is used.

**4.2 Steam explosion**

*Different Pretreatment Methods of Lignocellulosic Biomass for Use in Biofuel Production*

with high lignin content, AFEX pretreatment is not a suitable choice.

Ammonia recycle percolation (ARP) is another method that uses ammonia. Aqueous ammonia (10–15 wt %) is used in this method. With a fluid velocity of 1 cm/min and temperature of 150–170°C and residence time of 14 minutes, aqueous ammonia passes through biomass in this pretreatment, and ammonia is recovered afterwards. Under these conditions, ammonia reacts with lignin and causes the breakdown of lignin breakdown linkages. Liquid ammonia is used in AFEX technique whereas in ammonia recycle percolation method/technique, aqueous ammo-

In this method, high-pressure saturated steam is used to treat lignocellulosic biomass, and then suddenly pressure is reduced, due to which lignocellulosic biomass undergoes explosive decompression. Initiation temperature of steam explosion 160–260°C and 0.69–4.83 MPa pressure is provided for few seconds to minutes, and then lignocellulosic biomass is exposed and retained at atmospheric pressure for a period of time; this triggers hydrolysis of hemicellulose and at the end explosive decompression, terminated the whole process. Cellulose hydrolysis potential increases due to the cellulose degradation and lignin transformation caused by high temperature. During the steam explosion pretreatment, acid and other acids formed, which played their role in the hydrolysis of hemicellulose. Fragmentation of lignocellulosic material occurs due to turbulent material flow and rapid flashing of material to atmospheric pressure [86–88]. In steam explosion pretreatment, the use of sulfuric acid or carbon dioxide decreases time, temperature, and formation of inhibitory products and increases hydrolysis efficiency that ultimately leads to complete removal of hemicellulose. Steam explosion pretreatment is not that effective for pretreating soft woods; however, acid catalyst addition during the process is a prerequisite to make the substrate accessible to hydrolytic enzymes. By using steam, targeted temperature can be achieved to process the biomass without the need of excessive dilution. Sudden release of pressure quenches the whole process at the end and also lowers the temperature. Particulate structure of biomass gets opened by rapid thermal expansion which is used to terminate the reaction. Steam explosion gets affected by certain factors like moisture content, residence time, chip size, and temperature. By two ways optimal hydrolysis and solubilization of hemicellulose can be achieved; either use high temperature and short residence

kg of dry weight) is used as a catalyst. Ammonia is added to biomass in a reactor of high pressure; after 5–45 min of cooking, pressure is released rapidly. Normally temperature around 90°C is used in this process. Ammonia can be recovered and reused because of its volatility. The principle of AFEX is similar to steam explosion. Apparatus for AFEX includes reactor, thermocouple well, pressure gauge, pressure relief valve, needle valve, sample cylinder, temperature monitor, and vent. Rate of fermentation is seen to be improved by AFEX pretreatment of various grasses and herbaceous crops. For treatment of alfalfa, wheat chaff and wheat straw AFEX technology is used. Hemicellulose and lignin cannot be removed by using AFEX technology; hence, small amount of material is solubilized only. Degradation of hemicellulose into oligomeric sugars and deacetylation occur during AFEX pretreatment which is the reason of hemicellulose insolubility. After AFEX pretreatment of Bermuda grass and bagasse, 90% hydrolysis of cellulose and hemicellulose was achieved. Effectiveness of AFEX pretreatment decreases with increase in the lignin content of biomass, for example, newspaper, woods, nutshells, and aspen chips. In case of AFEX pretreatment for newspaper and aspen chips, maximum hydrolysis yield was 40% and 50%, respectively. So for the treatment of biomass

*DOI: http://dx.doi.org/10.5772/intechopen.84995*

#### *Different Pretreatment Methods of Lignocellulosic Biomass for Use in Biofuel Production DOI: http://dx.doi.org/10.5772/intechopen.84995*

kg of dry weight) is used as a catalyst. Ammonia is added to biomass in a reactor of high pressure; after 5–45 min of cooking, pressure is released rapidly. Normally temperature around 90°C is used in this process. Ammonia can be recovered and reused because of its volatility. The principle of AFEX is similar to steam explosion. Apparatus for AFEX includes reactor, thermocouple well, pressure gauge, pressure relief valve, needle valve, sample cylinder, temperature monitor, and vent. Rate of fermentation is seen to be improved by AFEX pretreatment of various grasses and herbaceous crops. For treatment of alfalfa, wheat chaff and wheat straw AFEX technology is used. Hemicellulose and lignin cannot be removed by using AFEX technology; hence, small amount of material is solubilized only. Degradation of hemicellulose into oligomeric sugars and deacetylation occur during AFEX pretreatment which is the reason of hemicellulose insolubility. After AFEX pretreatment of Bermuda grass and bagasse, 90% hydrolysis of cellulose and hemicellulose was achieved. Effectiveness of AFEX pretreatment decreases with increase in the lignin content of biomass, for example, newspaper, woods, nutshells, and aspen chips. In case of AFEX pretreatment for newspaper and aspen chips, maximum hydrolysis yield was 40% and 50%, respectively. So for the treatment of biomass with high lignin content, AFEX pretreatment is not a suitable choice.

Ammonia recycle percolation (ARP) is another method that uses ammonia. Aqueous ammonia (10–15 wt %) is used in this method. With a fluid velocity of 1 cm/min and temperature of 150–170°C and residence time of 14 minutes, aqueous ammonia passes through biomass in this pretreatment, and ammonia is recovered afterwards. Under these conditions, ammonia reacts with lignin and causes the breakdown of lignin breakdown linkages. Liquid ammonia is used in AFEX technique whereas in ammonia recycle percolation method/technique, aqueous ammonia is used.

## **4.2 Steam explosion**

*Biomass for Bioenergy - Recent Trends and Future Challenges*

**3.5 Ozonolysis**

noncompatibility of cellulase and ionic liquids results in the unfolding and inactivation of cellulase. At less viscosity cellulose solubilizes at low temperature; that's why while using ionic liquids, viscosity is an important factor to be considered regarding the energy consumption of the whole process. High temperatures trigger more side

Ozone pretreatment is a great option for lignin content reduction in lignocellulosic biomass. In vitro digestibility of biomass is enhanced by the application of ozone pretreatment. Inhibitors are not formed in this pretreatment which is a great advantage because other chemical pretreatments produce toxic residues. In ozone pretreatment, ozone acts as an oxidant in order to break down lignin. Ozone gas is soluble in water and being a powerful oxidant, by breaking down lignin, releases less molecular weight, soluble compounds. Wheat straw, bagasse, cotton straw, green hay, poplar sawdust, peanut, and pine can be pretreated with ozone in order to degrade lignin and hemicellulose; however, only slight changes occur in hemicellulose, whereas almost no changes occur in cellulose. Ozonolysis apparatus consists of ozone catalytic destroyer, iodine trap used for testing efficiency of catalyst, oxygen cylinder, ozone generator, three-way valve, ozone UV spectrophotometer, pressure regulation valve, process gas humidifier, vent, and automatic gas flow control valve [40, 41, 74–76]. Moisture content hugely effects oxidization of lignin via ozone pretreatment as lignin oxidation decreases with increase in the moisture content of biomass. Ozone mass transfer is limited at less water concentration, which ultimately effects its reactivity with biomass. Longer residence time of ozone is caused by the blockage of pores by water film [77]. During ozonolysis, pH of water decreases because of the formation of organic acids. Alkaline media trigger delignification because it removes lignins that are bonded to carbohydrates [78, 79]. Biomass delignification is associated with the production of inhibitory compounds. Certain aromatic and polyaromatic compounds are produced as a result of delignification [80]. Structural changes in lignin are observed by Bule et al. [81] in a study; different lignin subunits showed aromatic opening and degradation of β-O-4 moieties in NMR analysis. How do aromatic structures of control- and ozone-pretreated samples differ? A spectrum showed a decrease in aromatic carbon signal concentration. Changes were observed in methoxy groups that suggest the breakdown of ester-linked structure. Different reactor designs are used for the ozone pretreatment of biomass, for example, batch reactor, Drechsel trap reactor, fixed bed reactor, rotatory bed reactor, and multilayer fixed bed reactor. Plug flow reactors are used by most researchers [82]. Heiske et al. [83] compared the characteristics of single layered and multiple layered bed reactors in order to improve the wheat straw conversion to methane. Straw with 16.2% lignin concentration was obtained from single layered reactor, whereas in multiple layered reactor, lignin concentration decreased up to 7.2% at the bottom layer. Due to wax degradation in ozone-pretreated wheat straw, production of fatty acid compounds is observed by Kádár et al. [84]. About 49% lignin degradation was observed when corn stover was pretreated with ozonolysis in a study by Williams [85].

reactions and negative side effects like reducing ionic liquid stability [73].

**26**

**4. Physicochemical pretreatment**

**4.1 Ammonia fiber expansion (AFEX)**

AFEX technique belongs to the category of physicochemical pretreatment methods. In this low temperature process, concentrated ammonia (0.3–2 kg ammonia/

In this method, high-pressure saturated steam is used to treat lignocellulosic biomass, and then suddenly pressure is reduced, due to which lignocellulosic biomass undergoes explosive decompression. Initiation temperature of steam explosion 160–260°C and 0.69–4.83 MPa pressure is provided for few seconds to minutes, and then lignocellulosic biomass is exposed and retained at atmospheric pressure for a period of time; this triggers hydrolysis of hemicellulose and at the end explosive decompression, terminated the whole process. Cellulose hydrolysis potential increases due to the cellulose degradation and lignin transformation caused by high temperature. During the steam explosion pretreatment, acid and other acids formed, which played their role in the hydrolysis of hemicellulose. Fragmentation of lignocellulosic material occurs due to turbulent material flow and rapid flashing of material to atmospheric pressure [86–88]. In steam explosion pretreatment, the use of sulfuric acid or carbon dioxide decreases time, temperature, and formation of inhibitory products and increases hydrolysis efficiency that ultimately leads to complete removal of hemicellulose. Steam explosion pretreatment is not that effective for pretreating soft woods; however, acid catalyst addition during the process is a prerequisite to make the substrate accessible to hydrolytic enzymes. By using steam, targeted temperature can be achieved to process the biomass without the need of excessive dilution. Sudden release of pressure quenches the whole process at the end and also lowers the temperature. Particulate structure of biomass gets opened by rapid thermal expansion which is used to terminate the reaction. Steam explosion gets affected by certain factors like moisture content, residence time, chip size, and temperature. By two ways optimal hydrolysis and solubilization of hemicellulose can be achieved; either use high temperature and short residence

time or low temperature and high residence time. Low energy requirement is a great advantage of steam explosion pretreatment, whereas in mechanical pretreatment 70% more energy is required as compared to steam explosion pretreatment in order to obtain the same, reduced particle size. So far steam explosion pretreatment with addition of a catalyst is tested and came closest to scaling up at commercial level due to its cost-effectiveness. In Canada, at Iogen demonstration plant, steam explosion pretreatment is used at a pilot scale. For hardwood and agriculture residues, steam explosion pretreatment is a very effective pretreatment process.

#### **4.3 Carbon dioxide explosion**

Supercritical carbon dioxide explosion treatment falls in the category of physiochemical pretreatment. Scientists had tried to develop a process cheaper than ammonia fiber explosion and a process which would operate at temperature lower than stream explosion temperature. In this process, supercritical carbon dioxide is used that behaves like a solvent. Supercritical fluids are compressed at room temperature above its critical point. When carbon dioxide is dissolved in water, carbonic acid is formed which causes less corrosiveness due to its special features. During the process, carbon dioxide molecules enter into small pores of lignocellulosic biomass due to its small size. Carbon dioxide pretreatment is operated at low temperature which helped in prevention of sugar decomposition by acid. Cellulosic structure is disrupted when carbon dioxide pressure is released which ultimately increased the accessibility of the substrate to the cellulolytic enzymes for the process of hydrolysis [11, 40, 41, 43]. Dale and Moreira [89] used carbon dioxide pretreatment for alfalfa and observed 75% theoretical release of glucose. Zheng et al. [90] performed experiments to show comparison among ammonia explosion, steam pretreatment, and carbon dioxide pretreatment of recycled paper and sugarcane bagasse. The results showed that carbon dioxide explosion pretreatment is cost-effective than AFEX.

#### **4.4 Liquid hot water (LHW)**

Hot compressed water is another terminology used for this method of treatment. High temperature (160–220°C) and pressure (up to 5 MPa) are used in this type of pretreatment in order to maintain the liquid state of water. However, chemicals and catalysts are not used in liquid hot water pretreatment method [42]. In this method, water in liquid form remains in contact with lignocellulosic biomass for about 15 min. In this treatment pressure is used to prevent its evaporation, and sudden decompression or expansion in this pretreatment process is not needed. This method has proved to be very effective on sugarcane bagasse, wheat and rye straw, corncobs, and corn stover. Different terms like solvolysis, aqueous fractionation, aquasolv, and hydrothermolysis are used by different researchers to describe this pretreatment method [42, 60, 91]. Based on biomass flow direction and water flow direction into reactor, liquid hot water pretreatment can be performed in three different ways. The first method is co-current pretreatment, which is carried out by heating biomass slurry and water at high temperature, holding it for a controlled residence time at pretreatment conditions, and finally applying cool environment. The second method involves the countercurrent pretreatment that engages pumping of hot water against biomass at controlled conditions. The third method is the flow-through pretreatment, which can be carried out by the flow of hot water through lignocellulosic biomass which acts like a stationary bed.

To investigate the effect of liquid hot water pretreatment, a study was conducted by Abdullah et al. [92] that determined the different hydrolysis rates of cellulose

**29**

*Different Pretreatment Methods of Lignocellulosic Biomass for Use in Biofuel Production*

and hemicellulose. Two steps of optimization of various conditions were considered. The first step was performed at less severity for hydrolyzing hemicellulose, whereas the second step was performed at high severity for cellulose depolymerization. Disadvantage of liquid hot water pretreatment is high energy consumption requirement for downstream process because of the involvement of large amount of water. However, the advantage of this process is that chemicals and catalysts are not

In this pretreatment method, oxygen/air and water or hydrogen peroxide is used to treat biomass at high temperatures (>120°C) for half an hour at 0.5–2 MPa pressure [11, 93]. This pretreatment method is also used for the treatment of waste water and soil remediation. This method has proven to be very effective for pretreatment of lignin enriched biomass. Certain factors like reaction time, oxygen pressure, and temperature effect the efficiency of wet oxidation pretreatment process. Water acts like acid at high temperature, so it induces hydrolysis reaction as hydrogen ion concentration increases with increase in temperature which ultimately decreases the pH value. Pentose monomers are formed as a result of hemicellulose breakdown in wet oxidation pretreatment, and oxidation of lignin occurs, but cellulose remains least affected. There are certain reports on the addition of alkaline peroxide or sodium carbonate. The addition of these chemical agents help in bringing down temperature reaction and reduce the formation of inhibitory compounds. Efforts to improve the degradation of hemicellulose at high temperature lead to the formation of inhibitory compounds like furfural and furfuraldehydes. However, amount of the production of inhibitors in wet oxidation pretreatment is certainly less than that of liquid hot water pretreatment or steam explosion method. There is extremely less possibility of using this process at industrial scale because of two reasons. One is the combustible nature of oxygen, and the other is the high cost of

SPORL stands for sulfite pretreatment to overcome recalcitrance of lignocellulose, and this technique is used for pretreatment of lignocellulosic biomass [95]. SPORL is performed in two steps. The first step involves treatment of biomass with magnesium or calcium sulfite for the removal of lignin and hemicellulose fractions. The second step involves the reduction in size of pretreated biomass via mechanical disk miler. Effect of SPORL pretreatment was studied by Zhu et al. [22, 23] on spruce chips by employing conditions like temperature 180°C, half an hour time duration, 8–10% bisulfite, and 1.8–3.7% sulfuric acid. By employing these conditions, more than 90% substrate was converted to cellulose when cellulase of 14.6 FPU and 22.5 CBU β-glucosidase was used in hydrolysis. Low-yield inhibitors like hydroxymethyl furfural (HMF) (0.5%) and furfural (0.1%) were produced during this process. These percentages are far less as compared to acid-catalyzed steam pretreatment of spruce. In another study, SPORL-pretreated Popular NE222, beetle-killed lodgepole pine, and Douglas fir were purified. Low contents of sulfur and molecular

SPORL pretreatment on switch grass with temperature ranging between 163 and 197°C, 3–37 min time duration, 0.8–4.2% sulfuric acid dose, and 0.6–7.4% sodium sulfite dose was performed by Zhang et al. [97]. The results with enhanced digestibility by the removal of hemicellulose due to sulfonation and decreased hydrophobicity of lignin were obtained. SPORL yielded 77.3% substrate as compared to 68.1%

mass were obtained with high phenolic derivative production [96].

*DOI: http://dx.doi.org/10.5772/intechopen.84995*

required and no inhibitor is formed [60].

hydrogen peroxide used in the process [94].

**4.6 SPORL treatment**

**4.5 Wet oxidation**

*Different Pretreatment Methods of Lignocellulosic Biomass for Use in Biofuel Production DOI: http://dx.doi.org/10.5772/intechopen.84995*

and hemicellulose. Two steps of optimization of various conditions were considered. The first step was performed at less severity for hydrolyzing hemicellulose, whereas the second step was performed at high severity for cellulose depolymerization. Disadvantage of liquid hot water pretreatment is high energy consumption requirement for downstream process because of the involvement of large amount of water. However, the advantage of this process is that chemicals and catalysts are not required and no inhibitor is formed [60].

### **4.5 Wet oxidation**

*Biomass for Bioenergy - Recent Trends and Future Challenges*

**4.3 Carbon dioxide explosion**

ment is cost-effective than AFEX.

**4.4 Liquid hot water (LHW)**

explosion pretreatment is a very effective pretreatment process.

time or low temperature and high residence time. Low energy requirement is a great advantage of steam explosion pretreatment, whereas in mechanical pretreatment 70% more energy is required as compared to steam explosion pretreatment in order to obtain the same, reduced particle size. So far steam explosion pretreatment with addition of a catalyst is tested and came closest to scaling up at commercial level due to its cost-effectiveness. In Canada, at Iogen demonstration plant, steam explosion pretreatment is used at a pilot scale. For hardwood and agriculture residues, steam

Supercritical carbon dioxide explosion treatment falls in the category of physiochemical pretreatment. Scientists had tried to develop a process cheaper than ammonia fiber explosion and a process which would operate at temperature lower than stream explosion temperature. In this process, supercritical carbon dioxide is used that behaves like a solvent. Supercritical fluids are compressed at room temperature above its critical point. When carbon dioxide is dissolved in water, carbonic acid is formed which causes less corrosiveness due to its special features. During the process, carbon dioxide molecules enter into small pores of lignocellulosic biomass due to its small size. Carbon dioxide pretreatment is operated at low temperature which helped in prevention of sugar decomposition by acid. Cellulosic structure is disrupted when carbon dioxide pressure is released which ultimately increased the accessibility of the substrate to the cellulolytic enzymes for the process of hydrolysis [11, 40, 41, 43]. Dale and Moreira [89] used carbon dioxide pretreatment for alfalfa and observed 75% theoretical release of glucose. Zheng et al. [90] performed experiments to show comparison among ammonia explosion, steam pretreatment, and carbon dioxide pretreatment of recycled paper and sugarcane bagasse. The results showed that carbon dioxide explosion pretreat-

Hot compressed water is another terminology used for this method of treatment. High temperature (160–220°C) and pressure (up to 5 MPa) are used in this type of pretreatment in order to maintain the liquid state of water. However, chemicals and catalysts are not used in liquid hot water pretreatment method [42]. In this method, water in liquid form remains in contact with lignocellulosic biomass for about 15 min. In this treatment pressure is used to prevent its evaporation, and sudden decompression or expansion in this pretreatment process is not needed. This method has proved to be very effective on sugarcane bagasse, wheat and rye straw, corncobs, and corn stover. Different terms like solvolysis, aqueous fractionation, aquasolv, and hydrothermolysis are used by different researchers to describe this pretreatment method [42, 60, 91]. Based on biomass flow direction and water flow direction into reactor, liquid hot water pretreatment can be performed in three different ways. The first method is co-current pretreatment, which is carried out by heating biomass slurry and water at high temperature, holding it for a controlled residence time at pretreatment conditions, and finally applying cool environment. The second method involves the countercurrent pretreatment that engages pumping of hot water against biomass at controlled conditions. The third method is the flow-through pretreatment, which can be carried out by the flow of hot water

through lignocellulosic biomass which acts like a stationary bed.

To investigate the effect of liquid hot water pretreatment, a study was conducted by Abdullah et al. [92] that determined the different hydrolysis rates of cellulose

**28**

In this pretreatment method, oxygen/air and water or hydrogen peroxide is used to treat biomass at high temperatures (>120°C) for half an hour at 0.5–2 MPa pressure [11, 93]. This pretreatment method is also used for the treatment of waste water and soil remediation. This method has proven to be very effective for pretreatment of lignin enriched biomass. Certain factors like reaction time, oxygen pressure, and temperature effect the efficiency of wet oxidation pretreatment process. Water acts like acid at high temperature, so it induces hydrolysis reaction as hydrogen ion concentration increases with increase in temperature which ultimately decreases the pH value. Pentose monomers are formed as a result of hemicellulose breakdown in wet oxidation pretreatment, and oxidation of lignin occurs, but cellulose remains least affected. There are certain reports on the addition of alkaline peroxide or sodium carbonate. The addition of these chemical agents help in bringing down temperature reaction and reduce the formation of inhibitory compounds. Efforts to improve the degradation of hemicellulose at high temperature lead to the formation of inhibitory compounds like furfural and furfuraldehydes. However, amount of the production of inhibitors in wet oxidation pretreatment is certainly less than that of liquid hot water pretreatment or steam explosion method. There is extremely less possibility of using this process at industrial scale because of two reasons. One is the combustible nature of oxygen, and the other is the high cost of hydrogen peroxide used in the process [94].

#### **4.6 SPORL treatment**

SPORL stands for sulfite pretreatment to overcome recalcitrance of lignocellulose, and this technique is used for pretreatment of lignocellulosic biomass [95]. SPORL is performed in two steps. The first step involves treatment of biomass with magnesium or calcium sulfite for the removal of lignin and hemicellulose fractions. The second step involves the reduction in size of pretreated biomass via mechanical disk miler. Effect of SPORL pretreatment was studied by Zhu et al. [22, 23] on spruce chips by employing conditions like temperature 180°C, half an hour time duration, 8–10% bisulfite, and 1.8–3.7% sulfuric acid. By employing these conditions, more than 90% substrate was converted to cellulose when cellulase of 14.6 FPU and 22.5 CBU β-glucosidase was used in hydrolysis. Low-yield inhibitors like hydroxymethyl furfural (HMF) (0.5%) and furfural (0.1%) were produced during this process. These percentages are far less as compared to acid-catalyzed steam pretreatment of spruce. In another study, SPORL-pretreated Popular NE222, beetle-killed lodgepole pine, and Douglas fir were purified. Low contents of sulfur and molecular mass were obtained with high phenolic derivative production [96].

SPORL pretreatment on switch grass with temperature ranging between 163 and 197°C, 3–37 min time duration, 0.8–4.2% sulfuric acid dose, and 0.6–7.4% sodium sulfite dose was performed by Zhang et al. [97]. The results with enhanced digestibility by the removal of hemicellulose due to sulfonation and decreased hydrophobicity of lignin were obtained. SPORL yielded 77.3% substrate as compared to 68.1% for dilute acid treatment and 66.6% through alkali pretreatment. When sodium sulfite, sodium hydroxide, and sodium sulfide were used in SPORL pretreatment of switch grass, an improved digestibility of switch grass was achieved. When SPORL treatment was applied with optimized conditions, 97% lignin and 93% hemicellulose were removed from water hyacinth, and 90% hemicellulose and 75% lignin were achieved for rice husk [98].
