**4. Pre-treatment technologies for biomass**

The most important difficulties encountered in the production of bioethanol are the pretreatment of biomass. The objectives of an effective pre-treatment are obtaining sugars directly or later by hydrolysis, preventing lost or degradation of obtained sugars, limiting the toxic materials which inhibit the ethanol production, reducing energy requirement for process and minimizing the production cost. There are four pre-treatment techniques including physical, chemical, physicochemical and biological pre-treatments that are applied to biomass [1]. Pretreatment process is the step that forms the significant part of the cost of ethanol production. Although there is no technique that can be considered as the best option, researches and developments are carried on to reduce cost and improve performance [3].

### **4.1. Physical pre-treatments**

### *4.1.1. Mechanical comminution*

Chipping, grinding and milling are the most used techniques for mechanical comminution. Comminutions improve the efficiency of the process for the next steps by reducing the polymerization degree and increase the specific surface by reducing cellulose cristallinity. Energy that is need in the process depends on the initial and final dimensions of particles, moisture content and structure of the raw material [1,47]. In order to assist enzymatic hydrol‐ ysis of lignocellulosic materials various milling techniques can be used. For instance, pretreatment of rice straw with wet disk milling gave higher hydrolysis yields than usual dry milling [48].

### *4.1.2. Pyrolysis*

Pyrolysis is an endothermic process which is a reaction needs low energy input and treats biomass over the temperature of 300°C and degrades cellulose to char and gaseous products like CO and H2. When the char is washed with water or diluted acid, remaining solution contains sufficient amount of carbon source to support microbial growth for the production of bioethanol. Approximately 55% of biomass weight is lost in the washing step [1]. It is reported in a study that Fan et al. [49] have performed 80-85% conversion of cellulose to reducing sugars.

### *4.1.3. Microwave oven pre-treatment*

Microwave oven pre-treatment is a simple method with short reaction time, high heating efficiency and low energy input. Thermal and non-thermal effects which are generated by microwaves in a liquid medium are used in this technique. The heat generated in biomass results in a polar bond vibration. This causes an explosion between the particles and degra‐ dation of lignocellulosic structure. Asetic acid is released from lignocellulosic material and an acidic medium is occurred for hydrolysis [50]. Ooshima et al. [51] investigated the effect of microwave pre-treatment on rice straw and baggase and it was found that an improvement in total reducing sugar production. In recent years, microwave pre-treatments are carried out with various chemical reagents and their potential are investigated. In the studies of alkali microwave pre-treatment, NaOH provides higher reducing sugar yields on switchgrass and coastal bermudagrass in comparison with other alkaline reagents such as Na2CO3, Ca(OH)2 [52,53]. Also for pre-treatment of rice straw and its hulls, this technique made cellulose more accessible to enzymes.

### **4.2. Physicochemical pre-treatments**

### *4.2.1. Steam explosion method*

treatment process is the step that forms the significant part of the cost of ethanol production. Although there is no technique that can be considered as the best option, researches and

Chipping, grinding and milling are the most used techniques for mechanical comminution. Comminutions improve the efficiency of the process for the next steps by reducing the polymerization degree and increase the specific surface by reducing cellulose cristallinity. Energy that is need in the process depends on the initial and final dimensions of particles, moisture content and structure of the raw material [1,47]. In order to assist enzymatic hydrol‐ ysis of lignocellulosic materials various milling techniques can be used. For instance, pretreatment of rice straw with wet disk milling gave higher hydrolysis yields than usual dry

Pyrolysis is an endothermic process which is a reaction needs low energy input and treats biomass over the temperature of 300°C and degrades cellulose to char and gaseous products like CO and H2. When the char is washed with water or diluted acid, remaining solution contains sufficient amount of carbon source to support microbial growth for the production of bioethanol. Approximately 55% of biomass weight is lost in the washing step [1]. It is reported in a study that Fan et al. [49] have performed 80-85% conversion of cellulose to

Microwave oven pre-treatment is a simple method with short reaction time, high heating efficiency and low energy input. Thermal and non-thermal effects which are generated by microwaves in a liquid medium are used in this technique. The heat generated in biomass results in a polar bond vibration. This causes an explosion between the particles and degra‐ dation of lignocellulosic structure. Asetic acid is released from lignocellulosic material and an acidic medium is occurred for hydrolysis [50]. Ooshima et al. [51] investigated the effect of microwave pre-treatment on rice straw and baggase and it was found that an improvement in total reducing sugar production. In recent years, microwave pre-treatments are carried out with various chemical reagents and their potential are investigated. In the studies of alkali microwave pre-treatment, NaOH provides higher reducing sugar yields on switchgrass and coastal bermudagrass in comparison with other alkaline reagents such as Na2CO3, Ca(OH)2 [52,53]. Also for pre-treatment of rice straw and its hulls, this technique made cellulose more

developments are carried on to reduce cost and improve performance [3].

**4.1. Physical pre-treatments**

146 Biofuels - Status and Perspective

*4.1.1. Mechanical comminution*

milling [48].

*4.1.2. Pyrolysis*

reducing sugars.

accessible to enzymes.

*4.1.3. Microwave oven pre-treatment*

Steam explosion method is a technique that provides accessibility on the biomass for degra‐ dation of cellulose. This method comprise the heating of biomass under high pressure steam (20–50 bar, 160-270 °C) for a few minutes, then reaction is stopped when the pressure condi‐ tions arrive to the atmospheric conditions. Diffusion of the steam into the lignocellulosic matrix leads to the dispersion of fibers. No catalyst is used during the applied method. Levulinic acid, xylitol and alcohols are obtained after the degradation of biomass [54,55]. Many types of biomass such as poplar [56], eucalyptus [57], olive residues [58], corn stover [59], wheat straw [60], sugarcane bagasse [61], grasses [62] have been pre-treated with steam explosion method efficiently.

### *4.2.2. Liquid hot water method*

Liquid hot water method treats biomass by using water which is kept in a liquid state under high pressure and temperature for 15 minutes without adding any chemical or catalyst. Instead of steam explosion method, this technique does not need rapid pressure drop or expansion. Pressure is used to prevent evaporation and to stabilize the water in this method [60]. Although it provides the release of hemisellulosic sugars as oligomers, it causes the formation of little amounts of undesirable components which inhibit microbial growth such as carboxylic acid, furfural [63]. Since there is no need for chemicals, it is an environmental and economic method [64]. It is reported that liquid hot water method improves the enzymatic hydrolysis by removing 80% of hemicelluloses when it is pre-treated corn stover, sugarcane bagasse and wheat straw [65].

### *4.2.3. Ammonia Ffiber Explosion (AFEX)*

Ammonia fiber explosion (AFEX) is a method that liquid ammonia and steam explosion are carried out together. In this method, biomass which has 15-30% moisture content is treated with liquid ammonia at a loading ratio of 1–2 kg NH3/kg dry biomass. To acquire appropriate temperature, pressure over 12 atm is required. Whereas being an easy method and have short reaction time, it is not effective on raw materials that contain high lignin content [54]. Ammonia has effects such as shredding biomass fibers, partially decrystallization of cellulose and destroying carbohydrate attachments [65]. Although sugars are not released directly with this method, it enhances polymers (hemicellulose and cellulose) to be attacked enzymatically. Thus, low amount of enzyme is enough for enzymatic hydrolysis after AFEX. In order to improve the process economically, ammonia must be recover after the pre-treatment. Ammo‐ nia loading, temperature, high pressure, moisture content of biomass, and residence time are the basic parameters which effect AFEX process. Up to 90% cellulose and hemicelluloses conversions can be acquired with this technique [3].

### *4.2.4. CO2 explosion*

CO2 explosion is similar to AFEX method. However this method has low process cost due to need low temperature. Also formation of inhibitors in the steam explosion is not occurred in this technique. In addition to that, its conversion yields are very high compared to steam explosion [50,66].

### *4.2.5. Wet oxidation*

Wet oxidation method is based on the treatment of biomass with water and air or oxygen as a catalyst over the temperature of 120 °C. Although solubility of hemicellulose and lignin are increased with this method, free hemicelluloses molecules do not hydrolyze. Whereas sugar monomers are formed in steam explosion and dilute acid pre-treatment, sugar which released in wet oxidation method are oligomers [67,68]. In a study performed by Pederson [69] et al. 40% glucose yield was obtained for wet oxidation of wheat straw.

### **4.3. Chemical pre-treatments**

Chemical pre-treatments include dilute acid, alkaline, ammonia, organic solvent pre-treat‐ ments and methods that use other chemicals. These processes are easy to perform and also good conversion yields are achieved in a short time [1].

### *4.3.1. Acid pre-treatment*

Acid pre-treatments are methods that acid is used as catalyst to make cellulose more accessible to the enzymes. These processes are divided into two groups as using concentrated acid or diluted acid. Using concentrated acid is less preferable than dilute acid because of forming high amount of inhibiting components and causing corrosion in the equipments [68]. Generally sulphuric acid, hydrochloric acid, nitric acid and phosphoric acid are used in these pretreatments. Dilute acid are applied at moderate temperatures to convert lignocellulosic structures to soluble sugars [54]. Nowadays biomass is pre-treated with dilute sulphuric mostly to hydrolyze hemicelluloses and facilitate enzymatic hydrolysis [70]. Dilute sulphuric acid hydrolyzes biomass to hemicelluloses, and then hydrolyzes to xylose and other sugar and break xylose down to furfural. Furfural which is a toxic component in ethanol production process, is recovered by distillation [54]. Miranda et al. have investigated the effect of acid pretreatments with the concentrations between 0.05-10 N, and have obtained the highest sugar yield under the condition of 2 N acid pre-treatment. In their experiments, 2 N to 10 N acid pretreatments, it is reported that a decrease have been observed in sugar yields [71]. Larsson et al. also mentioned that in an experiment about acid pre-treatment of soft wood, a decrease in ethanol yields have been observed with an increasing acid concentration. In addition to this, it is indicated that formic acid which is a toxic molecule, is presented in the media and inhibits the fermentation [72].

### *4.3.2. Alkaline pre-treatment*

These processes are carried out at low temperature and pressure compared to other techniques. Unlike acid pre-treatments, lignin can be removed without major effects on the other compo‐ nents. However there are limitations such as transformation of some alkaline to unrecoverable salts. In addition to that, solubility of hemicelluloses and cellulose are less in this pre-treatment compared to solubility in acid pre-treatment [73]. Alkaline pre-treatment reduces the lignin and hemicelluloses content of biomass and improves the surface area and helps water molecules for breaking bonds between hemicelluloses and lignin [54]. The most used catalysts in this method are sodium hydroxide, potassium hydroxide, calcium hydroxide and ammonia [74]. Effects of alkaline pre-treatments are varies according to biomass. In an alkaline pretreatment of coastal bermudagrass, reducing sugar yields are decrease with an increasing alkaline concentration [75]. However, Wang et al. reported that under the conditions of increasing alkaline concentrations, glucose yields were increased [76]. Like dilute acid pretreatments, dilute alkaline pre-treatments also can form inhibitory by-products such as furfural, hydroxymethylfurfural and formic acid [77]

### *4.3.3. Organosolv pre-treatment*

this technique. In addition to that, its conversion yields are very high compared to steam

Wet oxidation method is based on the treatment of biomass with water and air or oxygen as a catalyst over the temperature of 120 °C. Although solubility of hemicellulose and lignin are increased with this method, free hemicelluloses molecules do not hydrolyze. Whereas sugar monomers are formed in steam explosion and dilute acid pre-treatment, sugar which released in wet oxidation method are oligomers [67,68]. In a study performed by Pederson [69] et al.

Chemical pre-treatments include dilute acid, alkaline, ammonia, organic solvent pre-treat‐ ments and methods that use other chemicals. These processes are easy to perform and also

Acid pre-treatments are methods that acid is used as catalyst to make cellulose more accessible to the enzymes. These processes are divided into two groups as using concentrated acid or diluted acid. Using concentrated acid is less preferable than dilute acid because of forming high amount of inhibiting components and causing corrosion in the equipments [68]. Generally sulphuric acid, hydrochloric acid, nitric acid and phosphoric acid are used in these pretreatments. Dilute acid are applied at moderate temperatures to convert lignocellulosic structures to soluble sugars [54]. Nowadays biomass is pre-treated with dilute sulphuric mostly to hydrolyze hemicelluloses and facilitate enzymatic hydrolysis [70]. Dilute sulphuric acid hydrolyzes biomass to hemicelluloses, and then hydrolyzes to xylose and other sugar and break xylose down to furfural. Furfural which is a toxic component in ethanol production process, is recovered by distillation [54]. Miranda et al. have investigated the effect of acid pretreatments with the concentrations between 0.05-10 N, and have obtained the highest sugar yield under the condition of 2 N acid pre-treatment. In their experiments, 2 N to 10 N acid pretreatments, it is reported that a decrease have been observed in sugar yields [71]. Larsson et al. also mentioned that in an experiment about acid pre-treatment of soft wood, a decrease in ethanol yields have been observed with an increasing acid concentration. In addition to this, it is indicated that formic acid which is a toxic molecule, is presented in the media and inhibits

These processes are carried out at low temperature and pressure compared to other techniques. Unlike acid pre-treatments, lignin can be removed without major effects on the other compo‐ nents. However there are limitations such as transformation of some alkaline to unrecoverable salts. In addition to that, solubility of hemicelluloses and cellulose are less in this pre-treatment compared to solubility in acid pre-treatment [73]. Alkaline pre-treatment reduces the lignin and hemicelluloses content of biomass and improves the surface area and helps water

40% glucose yield was obtained for wet oxidation of wheat straw.

good conversion yields are achieved in a short time [1].

explosion [50,66].

148 Biofuels - Status and Perspective

*4.2.5. Wet oxidation*

**4.3. Chemical pre-treatments**

*4.3.1. Acid pre-treatment*

the fermentation [72].

*4.3.2. Alkaline pre-treatment*

Organosolv pre-treatment is a process that uses organic solvents such as methanol, ethanol, acetone, ethylene glycol. Catalysts are also can be added to the process along with solvents. Hydrochloric acid, sulphuric acid, sodium hydroxide and ammonia are the catalysts used in the process. Besides bonds of lignin and hemicellulose can be broken, pure and high quality lignin can be obtained as a by-product [78]. Removal of lignin improves the surface area and provides accessibility of enzymes to cellulose. After the pre-treatment, cellulosic fibers, solid lignin and liquid solution of hemicellulose sugars are obtained. This method has some disadvantages like oxidation, volatilization and creating high risk in process at high pressure. Also solvents must be recovered due to formation of significant amounts of furfural and soluble phenols and to reduce operation cost [50,67].

### **4.4. Biological pre-treatments**

Compared to the above methods applied to the production of bioethanol, using fungi in pretreatments is considered environmentally friendly because of not using chemicals, less energy input, not required reactors that resistant to corrosion and pressure, and minimum inhibitor formation [79]. Fungi which are used in biological pre-treatments are generally brown, white and soft mold. These fungi can be degrade lignin, hemicelluloses and cellulose partially. Despite of its advantages, long process time, large production are and need of control contin‐ uously for growth of microorganisms ensue as disadvantages for commercial productions[50].

Enzymatic hydrolysis is the step of hydrolysis of cellulose by specific cellulase enzymes. Obtained products after hydrolysis are reducing sugars that include glucose. Cost of the enzymatic hydrolysis are less than acid or alkaline hydrolysis due to reaction is carried out under mild conditions (4.8 pH, temperature of 45-50 °C) [50]. Cellulase enzymes that are used in hydrolysis can be produced by bacteria and fungi. These microorganisms can be aerobic, anaerobic, mesofilic or thermophilic. Bacteria which produce cellulase can be exemplify as *Clostridium, Cellulomonas, Bacillus, Thermomonospora, Ruminococcus, Bacteriodes, Erwinia, Acetovibrio, Microbispora* and *Streptomyces. Trichoderma, Penicillium, Fusarium, Phanerochaete, Humicola* and *Schizophillum sp.* are identified as cellulase produced fungi among the fungi [1]. Although there are anaerobic bacteria which produce cellulase with high specific activity, these bacteria are not suitable for commercial productions. Cellulase enzymes consist of mixture of endoglucanase, exoglucanase and b-glucosidase. While endoglucanase attacks the regions where cellulose fibers have low crystallinity, exoglucanase removes the cellulose units from released chains with the effect of endoglucanase and then degrades the molecule. B-glucosi‐ dase hydrolyzes the cellulose units and enables the formation of glucose [64]. Enzymatic hydrolysis can be affected by certain factors which are enzyme-related and substrate-related factors. Substrate-related factors have a directly influence on enzymatic hydrolysis. These factors are connected to each other and effect the enzymatic conversion. These factors can be defined as *degree of polymerization and crystallinity of cellulose, accessibility of the substrate, lignin and hemicelluloses content and pore size*.

Hydrolysis rates of biomass depend on the degree of polymerization and crystallinity of cellulose. Degree of polymerization is related to crystallinity. Cellulase enzymes can hydrolyze the crystalline structure of cellulose. Endoglucanase enzymes decrease polymerization degree of cellulosic component by cutting the internal sites of cellulose chains in the enzymatic hydrolysis [80]. Accessibility of the substrate is another main factor effect hydrolysis rate. The rate of hydrolysis increases with increasing substrate accessibility because of being surface area more available for enzymatic attack [80]. Lignin and hemicellulose are complex structures to hydrolyze in lignocellulosic materials. Due to have a role like cement, lignin acts as physical barrier and prevents the digestible parts of cellulose to hydrolyze and it becomes very difficult for enzymes to access cellulose. For this reason, they reduce the efficiency of hydrolysis. Removal of hemicellulose enhances the pore size and provides accessibility to cellulose for enzymes in order to perform hydrolysis efficiently [81,82]. Pore size of the substrate is one of the limiting factors in enzymatic hydrolysis process. In many lignocellulosic material, external area of the biomass is smaller than internal area and this situation causes cellulase enzymes to entrap in the pores of the material. In order to increase hydrolysis rate, porosity of the biomass should be increased [83].
