**2. Diluted acid pretreatment**

Different pretreatment methods produce different effects on the biomass in terms of its struc‐ ture and composition [2] (Figure 1). For example, the hydrothermal and acidic pretreatments conceptually remove mainly the biomass hemicellulose fraction and alkaline pretreatments remove lignin, whereas the product of a milling-based pretreatment retains its initial biomass composition. As such, the choice of pretreatment as well as its operational conditions deter‐ mines the composition of the resulting biomass hexose and pentose syrups. Furthermore, cel‐ lulose crystallinity is not significantly reduced by pretreatments based on steam, or hydrothermal, or acidic procedures, whereas ionic liquid-based techniques can shift crystal‐ line cellulose into amorphous cellulose, substantially increasing the enzymatic hydrolysis rates and yields. The activity profile of the enzyme blend and the enzyme load for an effective saccharification may also vary according to the pretreatment. Indeed, a low hemicellulase load can be used for a xylan-free biomass and a lower cellulase load will be needed for the hy‐

48 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

drolysis of a low crystalline and highly amorphous pretreated biomass material.

**Figure 1.** Flow diagram for biomass ethanol production showing different pretreatments options and the composi‐

As the pretreatment choice will also be affected by the type of biomass, the envisaged biore‐ finery model will need to consider the main types of biomass that will be used for the biore‐ finery operation so as to select an appropriate, and versatile pretreatment method [3]. To date, sugarcane and woody biomass, depending on the geographic location, are strong can‐ didates as the main renewable resources to be fed into a biorefinery. However, due to major differences regarding their physical properties and chemical composition, the relevant pre‐ treatments to be used in each case are expected to be selective and customized. Moreover, a necessary conditioning step for wood size reduction, prior to the pretreatment, may not be necessary for sugarcane bagasse, affecting the pretreatment energy consumption and costs.

tion of the solid pretreated material. SSF: simultaneous saccharification and fermentation

The use of mineral acids for biomass processing has a historical record dating back to 1819, when concentrated acid was used for wood saccharification aiming at ethanol production [8]. Nevertheless, different technologies using mineral acids have been developed over the last two centuries for converting plant biomass into monosaccharides [9, 10]. The use of acid for biomass pretreatment is conducted with diluted sulfuric or hydrochloric acid (1 to 5%) at 150 °C and pressures up to 10 atm [11]. The efficiency of hemicellulose removal in acid pre‐ treatments is approximately 90%, with sugar losses by degradation at around 1% [12].

The diluted acid pretreatment allows for the deconstruction of the lignocellulosic material structure and the release of sugar monomers, mostly derived from the hemicellulose. If acid pretreatment is carried out under mild conditions of acid concentration and temperature, the hemicellulose fraction can be extracted without significantly affecting the cellulose and lignin biomass content. Unlike cellulose, the hemicellulose is amorphous and branched, be‐ ing more accessible to hydrolysis agents. This structure allows for the diffusion of acids, which accelerate the hydrolytic process. Therefore, in diluted acid pretreatment, the hemi‐ cellulose is preferably removed and hydrolyzed.

The process conditions are crucial in preventing undesirable reactions, which could promote a decrease in monosaccharide yields by the formation of sugar-derived toxic compounds. Temperatures lower than 150 °C reduce sugar degradation, but can result in the decrease of sugar extraction, while temperatures above 160 °C favor the unwanted hydrolysis of the cel‐ lulosic fraction, and the formation of toxic compounds [13, 14].

The inductive effect describes the fact that different substituents on the ring promote changes in the electron density of the ring oxygen. Electrophilic substituents such as carbon‐ yl and carboxyl groups reduce the protonation and inhibit the C–O fission, thus having a

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**Figure 2.** A simplified illustration for the mechanism of hemicellulose acid hydrolysis (adapted from [16]).

glycosidic bond between A and B is activated by the same effect [16].

**Figure 3.** The inductive effect of the carboxyl group on acid hydrolysis (adapted from [16]).

Figure 3 shows the inductive effect caused by the presence of glucuronic acid in the glycosi‐ dic chain. The carboxyl group induces different electron densities on the oxygen atom of the glycosidic bonds between A–B and B–C. The nucleophilicity is higher in the oxygen between B and C and this reduces the capacity for protonation. Thus, the bond is stabilized, while the

stabilizing effect on the glycosidic bond.

The mechanism of the acid hydrolysis reaction of lignocellulosic materials is described by the following steps (Figure 2) [15]:


With respect to the material to be treated, some intrinsic characteristics also have an influ‐ ence during the pretreatment such as the sample phase, the structure and physical accessi‐ bility (in the case of heterogeneous hydrolysis), conformation effects, and, finally, the structure and substituents of the sugar ring [16].

The theoretical and fundamental relations among molecular structure, molecular conforma‐ tion, and the inter-unities bonds of polysaccharides have been evaluated for numerous mod‐ el experiments. The hydrolytic behavior of glycosidic bonds is also substantially influenced by the conformation of the sugar unit and the inductive effect in these molecules caused by certain substituents in the chain. The half-chair conformation occurring intermittently dur‐ ing the hydrolytic attack is caused by a small rotation of the substituents around the links between carbon atoms 2 and 3, and between carbon atoms 4 and 5, respectively. Generally, the hydrolysis is supported if the axial substituents change to an equatorial position. As the rate of hydrolysis increases with the number of axial groups, the β-anomers are hydrolyzed faster than the corresponding α-forms, with the exception of L-arabinose [16].

Other effects of conformation can accelerate hydrolysis; for example, reducing end bounds are easily hydrolyzed when compared to non-reducing end bounds in polysaccharide chains. C5 substituent's can also hinder hydrolysis reactions [16].

Furanosidic ring structures are hydrolyzed faster than the pyranosidic rings due to the dif‐ ference in structural angular tension between furanosidic and pyranosidic rings. For exam‐ ple, in woods, α-D-galactofuranosides are hydrolyzed approximately 100 times faster than α-D-galactopyranosides [16].

The inductive effect describes the fact that different substituents on the ring promote changes in the electron density of the ring oxygen. Electrophilic substituents such as carbon‐ yl and carboxyl groups reduce the protonation and inhibit the C–O fission, thus having a stabilizing effect on the glycosidic bond.

The process conditions are crucial in preventing undesirable reactions, which could promote a decrease in monosaccharide yields by the formation of sugar-derived toxic compounds. Temperatures lower than 150 °C reduce sugar degradation, but can result in the decrease of sugar extraction, while temperatures above 160 °C favor the unwanted hydrolysis of the cel‐

The mechanism of the acid hydrolysis reaction of lignocellulosic materials is described by

**3.** The breakage of the ether bond and the generation of a carbocation as an intermediate;

**5.** The regeneration of protons and the cogeneration of sugar monomers, oligomers, or

**6.** The distribution of products in the liquid phase (if permitted by their shape and size);

With respect to the material to be treated, some intrinsic characteristics also have an influ‐ ence during the pretreatment such as the sample phase, the structure and physical accessi‐ bility (in the case of heterogeneous hydrolysis), conformation effects, and, finally, the

The theoretical and fundamental relations among molecular structure, molecular conforma‐ tion, and the inter-unities bonds of polysaccharides have been evaluated for numerous mod‐ el experiments. The hydrolytic behavior of glycosidic bonds is also substantially influenced by the conformation of the sugar unit and the inductive effect in these molecules caused by certain substituents in the chain. The half-chair conformation occurring intermittently dur‐ ing the hydrolytic attack is caused by a small rotation of the substituents around the links between carbon atoms 2 and 3, and between carbon atoms 4 and 5, respectively. Generally, the hydrolysis is supported if the axial substituents change to an equatorial position. As the rate of hydrolysis increases with the number of axial groups, the β-anomers are hydrolyzed

Other effects of conformation can accelerate hydrolysis; for example, reducing end bounds are easily hydrolyzed when compared to non-reducing end bounds in polysaccharide

Furanosidic ring structures are hydrolyzed faster than the pyranosidic rings due to the dif‐ ference in structural angular tension between furanosidic and pyranosidic rings. For exam‐ ple, in woods, α-D-galactofuranosides are hydrolyzed approximately 100 times faster than

faster than the corresponding α-forms, with the exception of L-arabinose [16].

chains. C5 substituent's can also hinder hydrolysis reactions [16].

α-D-galactopyranosides [16].

lulosic fraction, and the formation of toxic compounds [13, 14].

**1.** The diffusion of protons through the wet lignocellulosic matrix;

polymers, depending on the ether connection that is broken;

**2.** The protonation of the ether–oxygen link between the sugar monomers;

50 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

the following steps (Figure 2) [15]:

and

**4.** The solvation of the carbocation with water;

**7.** The restart of the process from step 2.

structure and substituents of the sugar ring [16].

**Figure 2.** A simplified illustration for the mechanism of hemicellulose acid hydrolysis (adapted from [16]).

Figure 3 shows the inductive effect caused by the presence of glucuronic acid in the glycosi‐ dic chain. The carboxyl group induces different electron densities on the oxygen atom of the glycosidic bonds between A–B and B–C. The nucleophilicity is higher in the oxygen between B and C and this reduces the capacity for protonation. Thus, the bond is stabilized, while the glycosidic bond between A and B is activated by the same effect [16].

**Figure 3.** The inductive effect of the carboxyl group on acid hydrolysis (adapted from [16]).

The main problems associated with acid hydrolysis relate to the formation of toxic com‐ pounds from biomass degradation and from equipment corrosion. Such toxic products en‐ tail inhibition in cell metabolism when biomass hydrolyzates are used for bioconversion. Steps to remove these inhibitory compounds have been employed to improve the yields in bioconversion processes.

**3. Hydrothermal pretreatments**

acid in the liquid fraction.

flow-through process [30].

**3.1. Liquid hot water (LHW) pretreatments**

The liquid hot water (LHW) treatments are also called hot compressed water treatments, hy‐ drothermolysis [22, 23], aqueous or steam/aqueous fractionation [24], uncatalyzed solvolysis [25, 26], and aquasolv [27]. LHW is based on the use of pressure to keep water in the liquid state at elevated temperatures (160–240 °C). This process changes the biomass native struc‐ ture by the removal of its hemicellulose content alongside transformations of the lignin structure, which make the cellulose more accessible to the further enzymatic hydrolysis step [1, 28]. Differently from steam-explosion treatment, LHW does not use rapid decompression and does not employ catalysts or chemicals. Nevertheless, as with the acid treatment, LHW depolymerizes hemicelluloses to the liquid fraction. In this case, sugars are removed mostly as oligosaccharides, and the formation of the inhibitors furfural and 5-hydroxymethyfurfu‐ ral (HMF) is at a slightly lower level [28], depending on the process conditions. To avoid the formation of inhibitors, the pH should be kept at between 4 and 7 during the pretreatment, because at this pH, hemicellulosic sugars are retained in oligomeric form, and monomer for‐ mation is minimized. The removal of hemicellulose also results in the formation of acetic

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LHW pretreatment, whose most important parameters are the biomass moisture content, the operation temperature, and the residence time [29], is usually done in a pressure tank reac‐ tor where two streams can be obtained after filtration of the biomass slurry: a solid, cellu‐ lose-enriched fraction and a liquid fraction rich in hemicellulose-derived sugars. The solid phase is therefore constituted by cellulose and lignin along with residual hemicellulose. There are three types of rector design for LHW pretreatment. For co-current reactors, the bi‐ omass liquid slurry passes through heat exchangers where it is heated to the appropriate temperature (140–180 °C) and kept for 10–15 minutes as the slurry passes through an insu‐ lated plug-flow snake-coil, followed by the slurry-cooling concomitant to heat recovery via the countercurrent heat exchange with the incoming slurry. Flow-through technologies pass hot water at 180–220 °C and approximately 350–400 psig. The resulting pretreated biomass has enhanced digestibility and a significant portion of the lignin is also removed. In counter‐ current pretreatment, the biomass slurry is passed in one direction while water is passed in another in a jacketed pretreatment reactor. Temperatures, back pressures, and residence times are similar. In the flow-through pretreatment reactor, water or acid is passed over a stationary bed, and removes some of the biomass components including lignin. Although LHW can result in the partial depolymerization and solubilization of lignin, the re-conden‐ sation of lignin-derived, soluble compounds is also observed. Flow-through systems have been reported to be more efficient in terms of hemicellulose and lignin removal in compari‐ son to batch systems for some types of biomass via the addition of external acid during the

There have been many studies on the use of LHW for the pretreatment of corn fiber [28, 30-33], wheat straw [34, 35], and sugarcane bagasse [36, 37]. Studies on woody biomass from

*Eucalyptus* [38-40], and olive tree biomass [41] have also been reported.

Table 1 presents different conditions of acid pretreatment for different lignocellulosic mate‐ rials for enzymatic hydrolysis, as well as the cellulose conversion efficiency for hardwood, softwood, and sugarcane bagasse and straw. Historically, acid pretreatment has been the main choice for wood pretreatment [16].


**Table 1.** Examples of sugarcane and woody biomass pretreated with diluted acid.

#### **2.1. Advantages and disadvantages of acid pretreatment**

Pretreatment with diluted sulfuric acid has been reported as one of the most widely used processes due to its high efficiency [14]. This pretreatment removes and hydrolyzes up to 90% of the hemicellulose fraction, rendering the cellulose fraction more accessible to hydro‐ lytic enzymes. However, it presents important drawbacks related to the need for a neutrali‐ zation step that generates salt and biomass sugar degradation with the formation of inhibitors for the subsequent fermentation step such as furfural from xylose degradation. The removal of inhibitors from the biomass sugar syrups adds cost to the process and gener‐ ates a waste stream. Additionally, mineral acids are corrosive to the equipment, calling for the use of more sturdy materials alongside higher maintenance costs. Acid recovery is also costly. The availability of the biomass acid pretreatment and the knowledge that has been built up on this subject highlights its important and costly drawbacks. In addition, the envi‐ ronmental problems caused by its waste streams have called for the need for other options for the pretreatment of lignocellulosic materials.
