**2.1 Milling**

*Biomass for Bioenergy - Recent Trends and Future Challenges*

Bioenergy products like bioethanol, biohydrogen, and biodiesel can be obtained

from lignocellulose biomass which is considerably large renewable bioresource and obtained from plants. The term "lignocellulosic biomass" is defined as lignin, cellulose, and hemicellulose that constitute the plant cell wall. Strong cross-linking associations are present between these components that cause hindrance in the breakdown of plant cell wall. Polysaccharides and lignin are cross-linked via ester and ether linkages [2–4]. Microfibrils that are formed by cellulose, hemicellulose,

Lignocellulose was first produced from food crop such as corn, oilseed, and sugarcane. But the use of edible feedstock for bioenergy products formation is being discouraged to prevent the rise in food competition. Thus, second-generation biofuels are obtained from plants wastes to avoid competition of land and water resources between energy crops and food crops. Currently, lignocellulose is being produced from wood residues, agricultural residues, food industry residue, grasses, domestic wastes, municipal solid wastes, and nonfood seeds [7–9]. The lignocellulose wastes (LCW) are largest renewable bioresource reservoir on earth that is being wasted as pre and postharvest agricultural wastes. Thus, many steps need to be adopted for use of these renewable resources for the production of bioenergy products. Recovery of many products like enzymes, methane, activated carbon, lipids, resins, methane, carbohydrates, surfactants, resins, organic acids, ethanol, amino acids, degradable plastic composites, biosorbents, biopesticides, and biopromoters can be achieved by utilizing LCW. The added benefits of using LCW besides recovery of different products are the removal of LCW waste from the environment. Also, utilization of LCW eliminates the use of food for bioethanol production. The US government has planned the production of 21 billion gallon of biofuels by 2022 [2, 5]. Biofuel production from lignocellulosic biomass reduces the emission of greenhouse gases. Pretreatment brings physical, biological, and chemical changes to biomass structure; therefore, it is very important to consider the type of pretreatment. In order to break down the hindrance caused by strong association within the cell wall, pretreatment is an important step which can increase the availability of lignocellulosic biomass for cellulase enzymes, their digestibility, and product yield. Before subjection to enzymatic hydrolysis, pretreatment of biomass can increase the rate of hydrolysis by 3–10-fold. Pretreatment of LCW is not an easy step as it seems after the installation of power generator; pretreatment is the second most costly process at industrial level. In crystalline cellulose, the disruption of hydrogen bonds, cross-linked matrix disruption, and increase in porosity as well as surface area of cellulose are the three tasks that are performed via a suitable pretreatment methods. The outcome of pretreatment also differs due to the difference in the ratio of cell wall components [10, 11]. The option to use dilute acid pretreatment method is more effective against poplar tree bark or corn as compared to the same method used for sweet gum bark or cornstalks. Few requirements of an effective, efficient, and economically suitable pretreatment process that including use of cheap chemicals, very less consumption of chemicals, prevention of hemicellulose and cellulose from denaturation, minimal energy requirement and consumption, cost-effective size reduction process, and reactive cellulosic fiber production are the factors that need to be considered for pretreatment. There are several methods of pretreatment that can be divided into four categories, namely, chemical,

and lignin help in the stability of plant cell wall structure [5, 6].

physical, biological, and physiochemical pretreatment [12–15].

Pore size and surface area of lignocellulosic biomass can be increased, whereas crystallinity and degree of polymerization of cellulose can be decreased with the

**16**

**2. Physical methods**

On the inherent ultrastructure of cellulose and degree of crystallinity, milling can be performed to render lignocelluloses more amenable to cellulases. Cellulases are enzyme that catalyze cellulose, but for the catalysis and best results, the substrate availability needs to be enhanced for optimized functioning of the enzymes. Before the subjection of the LCW to enzymatic hydrolysis, milling and size reduction of the lignocellulosic matter should be performed. Milling process has several types like ball milling, colloid milling, vibro-energy milling, hammer milling, and two-roll milling. For wet material, colloid mill, dissolver, and fibrillator are suitable, whereas for dry materials hammer mill, extruder, cryogenic mill, and roller mill are used. For both wet and dry material, ball milling can be used. For waster paper, hammer milling is the most suitable pretreatment option. Enzymatic degradation can be improved by milling as it reduces the degree of crystallinity and material size. Up to 0.2 mm reduction in particle size can be seen by milling and grinding. Reduction in particle size of biomass can be achieved up to a certain limit; beyond that limit reduction in particle size does not effect in the pretreatment procedure. Corn stover with small particle size, i.e., from 53 to 75 μm, is more productive as compared to large particle size corn stover ranging from 475 to 710 μm. The difference in particle size shows that productivity can significantly affect the pretreatment process. Ball milling causes a massive drop in crystallinity index from 4.9 to 74.2% which makes this process more suitable for saccharification of straw at mild hydrolytic conditions with more production of fermentable sugars [12, 16–18]. For better results of hydrolysis, milling can be used in combination with enzymatic hydrolysis. Mechanical action, mass transfer, and enzymatic hydrolysis can be achieved at the same time when two methods are combined. A number of ball beads in bill mill reactor play a crucial role in the α-cellulose hydrolysis, as less enzyme loading is required, and 100% rate of hydrolysis can be achieved in comparison to pretreatment of biomass that is carried without the use of milling procedure. Highest hydrolysis rate with high yield of reducing sugar was obtained when rice straw was put into fluidized bed opposed jet mill for fine grinding after cutting, steam explosion, and pulverization. For pretreatment of biomass, ball milling is an expensive option in terms of energy consumption, which is a huge disadvantage at industrial scale. Also, incapability of milling for removing lignin makes it a less suitable option as enzyme accessibility to the substrate is reduced in the presence of lignin. Reduction in crystallinity, degree of polymerization, and increase in surface area can be effected by the type of biomass, type of milling used for pretreatment, and duration of the milling process [19–21].

For improving digestibility and reducing crystallinity, vibratory ball milling is very effective. Low energy consumption has an important advantage of using wet disk milling which produces fibers that improve hydrolysis of cellulose, whereas hammer milling produces finer bundles. Due to this reason milling is not preferred when wet disk milling is available [22, 23]. Other study results of conventional ball and disk milling are compared. With the use of conventional ball milling, maximum yields of xylose and glucose were obtained, i.e., 54.3 and 89.4%, respectively [24]. Wet milling produces less yield, but it has the advantage of not producing inhibitors and very low energy consuming capability. An increase of 110% in enzymatic hydrolysis was achieved when wet milling was combined with alkaline pretreatment. Optimum parameters for wet milling pretreatment of corn stover were 10 mm diameter 20 steel balls, 1:10 solid-to-liquid ratio, 350 rpm/min speed, and 0.5 mm particle size [25] (**Figures 1** and **2**).

#### **Figure 1.** *Colloid milling (Pharmapproach.com).*

**Figure 2.** *Hammer milling (Solidswiki.com).*
