**4. Steam-explosion pretreatment**

Several works have reported about the optimal LHW pretreatment conditions in terms of temperature and residence time. For the pretreatment of corn stover the best conditions were reported to be 190 °C and 15 min, resulting in a 90% of cellulose conversion after en‐ zymatic hydrolysis [33], while for wheat straw the optimum treatment temperature was found to be 188 °C during 40 min, which resulted in 79.8% of cellulose conversion and re‐ leasing of 43.6% of hemicellulose derived sugars to the liquid fraction. Nonetheless, when response variables were analyzed separately, the best conditions for the recovery of hemi‐ cellulose-derived sugars from wheat straw, at up to 71.2%, were found at 184 °C during 24 min, whereas the optimal conditions for a cellulose conversion of 90.6% were found to

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

For sugarcane bagasse, top-performing LHW runs are favored by high temperatures (≥ 220 °C) and a short residence time (≤ 2 min) associated with low solids concentrations (≤ 5%), reaching 87% of simultaneous saccharification and fermentation (SSF) conversion, and 81% of xylan recovery. However, it is reported that the use of LHW using a solid concentration of more than 1% can significantly decrease the ethanol fermentation rate

The LHW pretreatment of *Eucalyptus* biomass was studied in two steps as follows: in the first step the pretreatment in which a temperature range from 180 to 200 °C was studied gave the highest total xylose recovery yield of 86.4% at 180 °C for 20 min. In the second step of the pretreatment, a temperature range from 180 to 240 °C was studied for intervals of time up to 60 min. The authors concluded that the efficiency of LHW for the cellulose con‐ version rate was more sensitive to temperature than residence time. The optimum reaction conditions for the second step of the pretreatment with minimal degradation of sugars were found to be 200 °C for 20 minutes, where the total sugar recovery from *Eucalyptus grandis* after 72 h of enzymatic saccharification reached 96.63%, which is superior to the yield from a

LHW pretreatment of olive tree biomass resulted in a 72% glucose yield from cellulose hy‐ drolysis after 72 h of saccharification using 2% of solids concentration during pretreatment, while for higher solids content the glucose yields were strongly affected reaching 70%, 60%, 57%, and 43% when using 5%, 10%, 20%, and 30% of solids, respectively [41]. A two-stage process which combines the LHW for hemicellulose removal and a treatment for delignifica‐ tion (e.g. ammonia pretreatment) has also been suggested for further improvement of enzy‐

The steam treatment is quite similar to LHW, with the major difference between the process‐ es being related to the contact of the liquid phase with the biomass. For LHW, the biomass is in direct contact with the liquid phase at the bottom of the reactor, which prevents the use of high solids content, while in the steam pretreatment, the biomass is at the top of the reactor and not in direct contact with the liquid phase, in a similar manner to that of an autoclaving process. Using steam pretreatment means that it is possible to use a higher solids content of 50% or more, whereas for the LHW, in most cases, the solids content is lower than 10%. Sim‐

single-step pretreatment with hot water or diluted acid [40].

be 214 °C during 2.7 min [35].

due to inhibition [36].

matic hydrolysis [42].

**3.2. Steam pretreatment**

Steam explosion is one of the most used methods for lignocellulosic biomass pretreatment. This process was initially developed in 1926 by the Masonite Corporation, Canada, for the production of fiberboard from wood [44]. From 1970–1980, the process was adapted to treat wood and agriculture residues aiming at improving the cellulose enzymatic hydrolysis and cattle feed production from lignocellulosic materials. A batch-type device was available from Iotech Corporation at pilot-plant scale in 1983 and a continuous device was available from Stake Technology, both from Canada, in the '80s.

In this process, the biomass is subjected to pressurized steam at temperatures ranging from 160 to 260 °C (corresponding to 0.69–4.83 MPa) for a few seconds to 20 minutes, followed by a rapid decompression of the reactor, by opening up an outlet, sharply reducing the temper‐ ature and interrupting the reactions. This approach combines chemical and mechanical forces in order to solubilize the hemicellulose fraction and render the cellulose more accessi‐ ble to the enzymatic hydrolysis [1, 28, 45-47].

leaves, respectively. The production of toxic compounds from the dehydration of sugars

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Many patents [51-53] have been granted for steam-explosion, and these processes have been widely tested in pilot- and demonstration-scale ethanol plants, and are considered to be close to commercialization [47]. Iogen Corporation (Ottawa, Canada) was the first company to commercialize cellulosic ethanol; at full capacity, the demonstration plant was designed to process approximately 20–30 tons per day of wheat, barley, and oat straw, and to produce

In Brazil, the pretreatment of lignocellulosic materials by steam explosion was initially developed at the Foundation of Industrial Technology in 1981. Studies which were per‐ formed on bench scale (a 1.6-L reactor) with sugarcane bagasse, *Eucalyptus*, and elephant grass, produced a wealth of data regarding the degree of hydrolysis and solubilization of the hemicellulose fraction, and the susceptibility of the treated material to enzymatic sac‐ charification in the presence or absence of impregnating agents (SO2 and H2SO4). These studies [55, 56] showed that the enzymatic saccharification enhancement due to steam ex‐ plosion was more prominent for *Eucalyptus*, though steam exploded bagasse, and steam exploded grass were more accessible to attack by enzymes in comparison to treated *Eu‐ calyptus*. In contrast to *Eucalyptus*, treatment conditions for achieving higher saccharifica‐ tion yields for bagasse and elephant grass were similar to those for optimal hemicellulose recovery in the process liquid stream. The use of the optimum time and temperature conditions for sugarcane bagasse (200 °C for 5–7 min) resulted in a 63% C-5 sugar recovery as soluble oligomers and an increase in glucose yield after enzymatic hy‐ drolysis at standard conditions from 13% (untreated bagasse) to 56%. The use of 0.25% sulfuric acid as the impregnant agent promoted a sharp decrease in the optimal pretreat‐ ment time (1 min) and allowed a higher recovery of the hemicellulose fraction (90%) as monomers. Saccharification yields were at the same levels as those observed for the treatment without acid addition. For *Eucalyptus*, the best pretreatment conditions for C-5 sugar recovery (61%) were 200 °C for 3.5–5 min, while those for achieving higher enzy‐ matic hydrolysis yields were 200 °C for 9 min, in which saccharification yields increased from almost zero (untreated wood) to 29.9%. Moreover, a study evaluated the effects of the explosion step in the steam-explosion process by comparing sugarcane bagasse pre‐ treatment by steam explosion at 200 °C with that at the same temperature but without the explosion process [56]. Although both pretreatment processes showed almost identi‐ cal chemical effects (hemicellulose autohydrolysis and cellulose and hemicellulose recov‐ ery), steam-exploded bagasse was 14.4% more accessible to attack by cellulases. Engineering studies were also carried out in order to expand the reactor scale to 0.2 and

, both of which were tested and operated successfully [57]. These results allowed for the development of a joint project with the Sugar and Alcohol Plant, Iracema (Iracemáp‐ olis, SP), for the installation and operation of an unit with the capacity to produce treat‐ ed bagasse enough to feed 150 cows/day. These studies have contributed to the implementation of the steam-explosion process of sugarcane bagasse for the production

(mainly furfural and hydroxymethylfurfural) was less than 1%.

approximately 5000–6000 liters of cellulosic ethanol per day [54].

2 m3

of cattle feed, currently operating in Brazil.

In the first step, called autohydrolysis, the water at high temperature causes the release of organic acids (mainly acetic acid) from the hemicellulose biomass moiety, followed by a chain reaction where the hemicellulose is partially solubilized and hydrolyzed [46]. The me‐ chanical effects are caused by the rapid decompression, which results in the disruption of the cell wall fibers and, by extension, particle size reduction, and increased porosity [48]. In association with the partial hemicellulose hydrolysis, lignin is degraded to some extent, and a small portion is removed from the material [49].

At the end of the process, an insoluble solid fraction and a liquid fraction are obtained. The solid fraction contains primarily cellulose and the partially modified lignin. The liq‐ uid fraction—whose pH is in the range of 3.5 to 4.0 depending on how many of the acid chains are released [47]—contains soluble carbohydrates derived from hemicellulose in the form of oligomers and monomers in a proportion that is subject to the process conditions [11, 50]. A balance between the temperature and time of the pretreatment should be ach‐ ieved in order to minimize the formation of phenolic compounds, furfural, and hydroxy‐ methylfurfural from the degradation of lignin, five-carbon sugars (C5), and six-carbon sugars (C6), respectively. The formation of such compounds, as they may be inhibitory to the subsequent enzymatic hydrolysis and ethanol fermentation-production steps, should be minimized as much as possible.

Another approach for the steam-explosion process has been the use of impregnating agents such as SO2, CO2, and H2SO4, which can improve the effectiveness of the pretreatment, in‐ creasing the efficacy, and decreasing the residence time. In some materials, such as soft‐ wood, the intrinsic level of organic acids is not enough to promote the degradation of the hemicellulose backbone. Thus, the addition of a mineral acid can reduce the initial pH to be‐ low 2 and promote the hydrolysis. On the other hand, the use of acids as impregnating agents may require an additional step for pH adjustment. Moreover, such an approach may enhance partial carbohydrate and lignin degradation, resulting in an increase in the forma‐ tion of toxic compounds, which will affect enzymatic hydrolysis, and fermentation [50]. Steam-explosion technology without additives has been successfully performed for ethanol production from hardwoods and for a wide range of agricultural residues [1].

The use of CO2 was tested as an impregnating agent in the steam-explosion pretreatment of sugarcane bagasse and straw, and the pretreatment was assessed in terms of glucose yields after enzymatic hydrolysis [50]. For sugarcane bagasse, the highest glucose yield (86.6% of the theoretical level) was obtained after pretreatment at 205 °C for 15 min. For straw, the highest glucose yield (97.2% of the theoretical level) was obtained after pretreatment at 220 °C for 5 min. The reference pretreatment, using impregnation with SO2 and performed at 190 °C for 5 min, resulted in an overall glucose yield of 79.7% and 91.9% for bagasse and leaves, respectively. The production of toxic compounds from the dehydration of sugars (mainly furfural and hydroxymethylfurfural) was less than 1%.

In this process, the biomass is subjected to pressurized steam at temperatures ranging from 160 to 260 °C (corresponding to 0.69–4.83 MPa) for a few seconds to 20 minutes, followed by a rapid decompression of the reactor, by opening up an outlet, sharply reducing the temper‐ ature and interrupting the reactions. This approach combines chemical and mechanical forces in order to solubilize the hemicellulose fraction and render the cellulose more accessi‐

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

In the first step, called autohydrolysis, the water at high temperature causes the release of organic acids (mainly acetic acid) from the hemicellulose biomass moiety, followed by a chain reaction where the hemicellulose is partially solubilized and hydrolyzed [46]. The me‐ chanical effects are caused by the rapid decompression, which results in the disruption of the cell wall fibers and, by extension, particle size reduction, and increased porosity [48]. In association with the partial hemicellulose hydrolysis, lignin is degraded to some extent, and

At the end of the process, an insoluble solid fraction and a liquid fraction are obtained. The solid fraction contains primarily cellulose and the partially modified lignin. The liq‐ uid fraction—whose pH is in the range of 3.5 to 4.0 depending on how many of the acid chains are released [47]—contains soluble carbohydrates derived from hemicellulose in the form of oligomers and monomers in a proportion that is subject to the process conditions [11, 50]. A balance between the temperature and time of the pretreatment should be ach‐ ieved in order to minimize the formation of phenolic compounds, furfural, and hydroxy‐ methylfurfural from the degradation of lignin, five-carbon sugars (C5), and six-carbon sugars (C6), respectively. The formation of such compounds, as they may be inhibitory to the subsequent enzymatic hydrolysis and ethanol fermentation-production steps, should

Another approach for the steam-explosion process has been the use of impregnating agents such as SO2, CO2, and H2SO4, which can improve the effectiveness of the pretreatment, in‐ creasing the efficacy, and decreasing the residence time. In some materials, such as soft‐ wood, the intrinsic level of organic acids is not enough to promote the degradation of the hemicellulose backbone. Thus, the addition of a mineral acid can reduce the initial pH to be‐ low 2 and promote the hydrolysis. On the other hand, the use of acids as impregnating agents may require an additional step for pH adjustment. Moreover, such an approach may enhance partial carbohydrate and lignin degradation, resulting in an increase in the forma‐ tion of toxic compounds, which will affect enzymatic hydrolysis, and fermentation [50]. Steam-explosion technology without additives has been successfully performed for ethanol

The use of CO2 was tested as an impregnating agent in the steam-explosion pretreatment of sugarcane bagasse and straw, and the pretreatment was assessed in terms of glucose yields after enzymatic hydrolysis [50]. For sugarcane bagasse, the highest glucose yield (86.6% of the theoretical level) was obtained after pretreatment at 205 °C for 15 min. For straw, the highest glucose yield (97.2% of the theoretical level) was obtained after pretreatment at 220 °C for 5 min. The reference pretreatment, using impregnation with SO2 and performed at 190 °C for 5 min, resulted in an overall glucose yield of 79.7% and 91.9% for bagasse and

production from hardwoods and for a wide range of agricultural residues [1].

ble to the enzymatic hydrolysis [1, 28, 45-47].

a small portion is removed from the material [49].

be minimized as much as possible.

Many patents [51-53] have been granted for steam-explosion, and these processes have been widely tested in pilot- and demonstration-scale ethanol plants, and are considered to be close to commercialization [47]. Iogen Corporation (Ottawa, Canada) was the first company to commercialize cellulosic ethanol; at full capacity, the demonstration plant was designed to process approximately 20–30 tons per day of wheat, barley, and oat straw, and to produce approximately 5000–6000 liters of cellulosic ethanol per day [54].

In Brazil, the pretreatment of lignocellulosic materials by steam explosion was initially developed at the Foundation of Industrial Technology in 1981. Studies which were per‐ formed on bench scale (a 1.6-L reactor) with sugarcane bagasse, *Eucalyptus*, and elephant grass, produced a wealth of data regarding the degree of hydrolysis and solubilization of the hemicellulose fraction, and the susceptibility of the treated material to enzymatic sac‐ charification in the presence or absence of impregnating agents (SO2 and H2SO4). These studies [55, 56] showed that the enzymatic saccharification enhancement due to steam ex‐ plosion was more prominent for *Eucalyptus*, though steam exploded bagasse, and steam exploded grass were more accessible to attack by enzymes in comparison to treated *Eu‐ calyptus*. In contrast to *Eucalyptus*, treatment conditions for achieving higher saccharifica‐ tion yields for bagasse and elephant grass were similar to those for optimal hemicellulose recovery in the process liquid stream. The use of the optimum time and temperature conditions for sugarcane bagasse (200 °C for 5–7 min) resulted in a 63% C-5 sugar recovery as soluble oligomers and an increase in glucose yield after enzymatic hy‐ drolysis at standard conditions from 13% (untreated bagasse) to 56%. The use of 0.25% sulfuric acid as the impregnant agent promoted a sharp decrease in the optimal pretreat‐ ment time (1 min) and allowed a higher recovery of the hemicellulose fraction (90%) as monomers. Saccharification yields were at the same levels as those observed for the treatment without acid addition. For *Eucalyptus*, the best pretreatment conditions for C-5 sugar recovery (61%) were 200 °C for 3.5–5 min, while those for achieving higher enzy‐ matic hydrolysis yields were 200 °C for 9 min, in which saccharification yields increased from almost zero (untreated wood) to 29.9%. Moreover, a study evaluated the effects of the explosion step in the steam-explosion process by comparing sugarcane bagasse pre‐ treatment by steam explosion at 200 °C with that at the same temperature but without the explosion process [56]. Although both pretreatment processes showed almost identi‐ cal chemical effects (hemicellulose autohydrolysis and cellulose and hemicellulose recov‐ ery), steam-exploded bagasse was 14.4% more accessible to attack by cellulases. Engineering studies were also carried out in order to expand the reactor scale to 0.2 and 2 m3 , both of which were tested and operated successfully [57]. These results allowed for the development of a joint project with the Sugar and Alcohol Plant, Iracema (Iracemáp‐ olis, SP), for the installation and operation of an unit with the capacity to produce treat‐ ed bagasse enough to feed 150 cows/day. These studies have contributed to the implementation of the steam-explosion process of sugarcane bagasse for the production of cattle feed, currently operating in Brazil.

#### **4.1. Advantages and disadvantages of steam-explosion pretreatment**

The main advantages of steam explosion relate to the possibility of using coarse particles, thus avoiding a biomass-size conditioning step, the non-requirement for exogenous acid addition (except for softwoods, which have a low acetyl group content in the hemicellulo‐ sic portion), a high recovery of sugars, and the feasibility for industrial implementation. Moreover, the soluble stream rich in carbohydrates derived from hemicellulose in the form of oligomers and monomers may be easily removed and used as feedstock for the production of higher added-value products such as enzymes and xylitol [58]. Other attrac‐ tive features include less hazardous process chemicals and conditions, the potential for significantly lower environmental impact, and lower capital investment [59]. The fact that the steam-explosion process does not require previous grinding of the raw biomass is an important feature, considering that the energy required to reduce the particle size before the pretreatment (pre-grinding) can represent up to one-third of the total energy required in the process [60].

mass enzymatic saccharification, which is solely achieved by using a milling process that al‐ ters the biomass structure at a nanoscopic level. Ball milling and wet disk milling (WDM)

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The ball milling process uses mechanical shear stress and impaction to produce pow‐ dered material [62]. This process can be done in the wet or dry state. A combination of chemicals, such as acids, bases and organic solvents, can also be applied depending on the main treatment purpose. In general, the process uses a rotary drum and balls of dif‐ ferent sizes made from different materials (tungsten, ceramic or stainless steel). The ef‐ fect of ball milling on the biomass particle size, structure and crystalline degree depends on the rotation speed, operation time and ball size. Ball milling treatment can be consid‐ ered a kind of ultra-fine grinding and fibrous materials can present between 10-20 µm in terms of particle size [63]. Highly crystalline cellulose has a strong interchain hydrogenbonding network that confers a high resistance to enzymatic hydrolysis, whereas amor‐

For biomass pretreatment after the material is fed into the rotary drum equipped with balls, drum rotation around a horizontal axis causes a reduction on the material particle size [65]. The ball milling process can drastically alter the complex heterogeneous network structure of wood cell walls, and with a long pretreatment time, cellulose crystallinity can be sig‐ nificantly reduced, which increases the ratio of amorphous cellulose, thus improving the sac‐ charification yields [65]. However, it was found that nanofibrillation of woody biomass by ball milling in the wet state can improve the saccharification yield without a significant de‐ crease in cellulose crystallinity [65]. They found the crystallinity index on the ball-milled bi‐ omass to be ca. 41% in comparison to 68% for the raw material. Thus, the decrease in particle size to a powder-like material and the increase of surface area seemed to be the main factors

Results obtained with the pretreatment of sugarcane biomass have shown that ball mill‐ ing treatment of bagasse for 60 min and sugarcane straw for 90 min results in glucose and xylose yields of 78.7% and 72.1% for bagasse and 77.6% and 56.8% for straw, re‐ spectively. In both cases, the enhancement in cellulose digestibility was related to the reduction of cellulose crystallinity to nearly an amorphous level [66]. In another study, the 20 min pretreatment of *Eucalyptus* using a planetary ball milling process was insuffi‐ cient for improving enzymatic digestibility, even though the crystallinity index de‐ creased from 59.7% to 7.6%. When a prolonged milling time of 120 min was used, the enzymatic digestibility of both glucan and xylan increased, while the degree of crystal‐ linity of the material was almost the same as that milled for 20 min. Additionally, the digestibility of glucan and xylan and their total yield were 76.7%, 63.9% and 74%, re‐ spectively, even at a substrate concentration of 20% and an enzyme dosage of 4 FPU/g of substrate, indicating that ball milling is extremely efficient to enhance biomass reac‐

are the most common biomass milling pretreatments.

phous cellulose is readily digestible [64].

that promoted the hydrolysis of treated wood.

tivity to enzymes [39].

*5.1.1. Ball milling*

The main drawbacks related to steam-explosion pretreatment are the enzyme and yeast in‐ hibitors generated during the pretreatment, which include furfural and hydroxymethyl fur‐ fural; the formation of weak acids, mostly acetic, formic, and levulinic acids, the two latter acids being derived from furfural's and hydroxymethyl furfural's further degradation; and the wide range of phenolic compounds produced due to lignin breakdown. Several detoxifi‐ cation methods have been developed in order to reduce the inhibitory effect, which repre‐ sent additional costs in the overall process. Other limitations of this method include the incomplete disruption of the lignin–carbohydrate matrix [11].
