**5. Soaking in aqueous ammonia (SAA)**

#### **5.1. Description**

**4. Ammonium fiber explosion (AFEX)**

In AFEX, liquid (anhydrous) ammonia at moderate-to-high temperatures (60–200°C) and pressures (6.5–45 bar) is mixed with moist biomass for about 5–30 min, followed by a sudden drop in pressure to atmospheric. Ammonia is usually fed at less than 2 kg/kg of dry biomass. AFEX leads to the removal of lignin and some hemicelluloses, in addition to the decrystallization of cellulose, partly due to the strong affinity of ammonia for such biomass components. According to Chundawat et al. [53], pretreatment causes morphological and physicochemical changes to cell walls of the material, by creating nanoscale network of interconnected tunnels within the cell wall structure through the cleaving of lignin-carbohydrate ester bonds, and the partial removal and subsequent deposition of extractives on cell wall surfaces, leading to enhanced enzymatic access to cellulose. Further, Maillard reactions between ammonia and

AFEX is generally affected by the moisture content and particle size of biomass, ammonia loading and process conditions including temperature and residence time. Higher temperatures cause more ammonia to flash causing greater disruption of the fibrous structure. Both glucan and xylan conversion (at fixed temperature and ammonia loading) was found to increase with moisture content of switchgrass [55]. In another study, particle size reduction increased the conversion of cellulose and xylan during pretreatment of corn stover [56].

AFEX has been widely applied to various class of lignocellulosic materials. Some results

AFEX is a dry-to-dry process since no liquid stream is produced, making it potentially less costly compared to steam explosion [63] and dilute acid methods [64]. The process is simple as it reduces requirements of post-pretreatment washing, stream separation and nutrient supplementation, and produces intermediates that are of value in developing advanced bioproducts. Reaction temperatures are moderate and energy requirements are low. Large solids (up to 5 cm) can be fractionated with good yields. Moreover, desired solid loadings are easily obtained, and high solid loadings are easier to implement due to low water demands. High glucose and xylose yields are both obtained under similar process conditions which simplify the optimization of process parameters. Moreover, except for some phenolic fragments of lignin and cell wall extractives that may form on the surface of pretreated solids, no enzymeinhibitors are produced [50]. AFEX give high sugar yields at low enzyme loadings of 1–10 FPU cellulase/g of dry biomass [1]. Klason lignin and carbohydrates are preserved and pretreated substrates possess high fermentability. Recently, process improvements bordering on ammonia loading and recovery, ammonia recycle concentration, and enzyme loadings have been developed and shown to reduce the cost of operation of AFEX-based biorefinery [65].

carbonyl-based aldehydic groups give rise to several intermediate products [54].

obtained from AFEX pretreatment of some biomass are given in **Table 3**.

**4.1. Description**

48 Fuel Ethanol Production from Sugarcane

**4.2. Applications**

**4.3. Positive attributes and drawbacks**

SAA involves treatment of biomass with aqueous ammonia (5–50%w/w) at low temperatures (25–90°C) under ambient pressure in a batch reactor. Pretreatment is undertaken for residence times ranging from about 1 h to 3 months. Pretreatment efficiency is depended on variables such as temperature, reaction time and ammonia concentration. Lignin dissolves in the aqueous solution without appreciable decrease in the carbohydrate content, and high levels of solubilization are observed with high temperatures and times. In addition, severe conditions also cause release of acetyl groups, hemicelluloses, extractives and ash into pretreatment liquor [66]. In other aqueous ammonia treatment, moderate temperatures (≥100°C) are used to achieve high delignification of biomass using pressure vessels [67]. Higher temperatures are compensated using lower reaction times.


**5.2. Applications**

Miscanthus 150°C/30 wt% NH<sup>3</sup>

NH<sup>3</sup>

News paper 4 wt% NH<sup>3</sup> + 2 wt% H2

Conditions: 70°C, 10 h, 20 wt% NH<sup>3</sup>

40°C, 3 h

, 1 h (not optimum)

Eth: ethanol yield after fermentation, SSF, SSCF, etc.

Oil palm trunk

Oil palm empty fruit bunch

a

in **Table 4**.

barley hull, SAA pretreatment (15w/w NH<sup>3</sup>

harsh pretreatment conditions and lower costs.

**5.3. Positive attributes and drawbacks**

Chen et al. [68] used aqueous ammonia to pretreat silvergrass, napiergrass and rice straw at room temperature, resulting in over 90% of cellulose recovery in 4 weeks. On destarched

DL: delignification; RT: room temperature; PBI: proton beam irradiation; X/H: percentage of xylan/hemicellulose retained in the solids after pretreatment; Glu: maximum theoretical glucose yield after enzymatic hydrolysis;

**Biomass Optimal pretreatment DL, % X/H, % Hydrolysis Yield, % Reference**

80°C, 8 h and 7 wt% NH<sup>3</sup> 40–50 50°C, 96 h, 60

60°C, 12 h, and 21 wt% NH<sup>3</sup> 40.9 60 FPU/g-

, 180°C/10 wt%

O2 ,

.

**Table 4.** Sugar and ethanol yields from selected SAA pretreated biomass.

saccharification yield using 15 FPU/g-glucan; and with the addition of a xylanase in simultaneous saccharification and co-fermentation (SSCF), a high ethanol yield of 89.4% of the maximum theoretical was obtained [69]. High ethanol concentration and yields from SAApretreated corn stover followed the use of a two-phase SSF involving pentose and hexose conversion with the help of *S. cerevisiae* and a recombinant bacterium, respectively [70]. Recently, the addition of surfactants such as Tween 80 and PEG 400 was found to improve sugar and ethanol yields [71]. In a similar study Raj and Krishnan [72] obtained high sugar yield by adding laccase and a mediator to enhance enzymatic hydrolysis of pretreated biomass. Nahar and Pryor [73] also found out that pelleting of samples before SAA application required less

Two-stage processes targeting separate removal of hemicelluloses and lignin have also been investigated. Kim et al. [74] employed acetic acid medium to remove hemicelluloses followed by aqueous ammonia at elevated temperatures. Results obtained from other studies are given

SAA retains most of the hemicelluloses in the solid, eliminating the need to separately process hemicellulose and cellulose sugars. It leads to efficient delignification, producing low levels of enzyme inhibitory compounds. The reactor configuration is simpler and less costly, while ammonia recovery is easier compared to AFEX [18]. It can be adapted to small-scale production. Further, neutralized salts from liquid hydrolysates could be used as nutrient source in fermentation.

, 75°C, 48 h) produced zero glucan loss and 83%

**Glu Eth**

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Emerging Physico-Chemical Methods for Biomass Pretreatment

95.4 78.3 [89]

41.4 65.6 [90]

53.4 [91]

90 [92]

FPU/g-glucan

glucan, 96 h

FPU/g-glucan

50°C, 72 h, 60 FPU/g-glucan

>65 39.3–77.1 50°C, 96 h, 20


DL: delignification; RT: room temperature; PBI: proton beam irradiation; X/H: percentage of xylan/hemicellulose retained in the solids after pretreatment; Glu: maximum theoretical glucose yield after enzymatic hydrolysis; Eth: ethanol yield after fermentation, SSF, SSCF, etc.

a Conditions: 70°C, 10 h, 20 wt% NH<sup>3</sup> .

**Table 4.** Sugar and ethanol yields from selected SAA pretreated biomass.

#### **5.2. Applications**

**Biomass Optimal pretreatment DL, % X/H, % Hydrolysis Yield, % Reference**

, 69°C, 10 h 60.6<sup>a</sup> 15 FPU/g-

, 65°C, 8 h 76–78 50°C, 72 h,

, 10–60 days, RT 56–74 85 50°C, 72 h,

, 60°C, 12 h 62 85 15

, 30°C, 4 weeks 55 15 FPU/g-

, 25°C, 2 wk 42 71 44–49 [75]

69.8 77 50°C, 15 FPU/g-

glucan, 30 CBU/g-glucan

glucan, 15 CBU/g-glucan

50°C, 60 FPU/gglucan, 10 CBU/g-glucan

15.57 FPU/gglucan, 30 CBU/g-glucan

15 FPU/gglucan, 30 CBU/g-glucan

FPU/g-glucan

glucan, 30 CBU/g-glucan

15 FPU/gglucan, 30 CBU/g-glucan

15 FPU/gglucan, 30 CBU/g-glucan

50°C, 72 h, 22–25 FPU/gglucan, 44–50 CBU/g-glucan, + xylanase

glucan, 30 CBU/g-glucan

, 69°C, 12 h >80 84 [70]

68 50°C, 24 h,

, UV 70 50°C, 24 h,

O2 65 15 FPU/g-

+ ZnO, UV 82

, 10 days, RT 40–50 50 72 [85]

Semi-aseptic 52–74

O2 77 74.3

Rice straw 27 wt% NH<sup>3</sup>

50 Fuel Ethanol Production from Sugarcane

Rice straw 21 wt% NH<sup>3</sup>

Corn fiber (destarched) 15 wt% NH<sup>3</sup>

60°C, 15 wt% NH<sup>3</sup> , 24 h

15 wt% NH<sup>3</sup>

15 wt% NH<sup>3</sup>

50 wt% NH<sup>3</sup>

15 wt%NH<sup>3</sup>

15 wt% NH<sup>3</sup>

12.5 wt% NH<sup>3</sup>

60°C, 24 h, O2

Switchgrass 29.5 wt% NH<sup>3</sup>

30 wt% NH<sup>3</sup>

, 5 days (pilot-scale)

15 wt% NH<sup>3</sup>

15 wt% NH<sup>3</sup>

120°C, 24 h

,

60°C, 8 h

,

,

Corn stover 29.5 wt% NH<sup>3</sup>

, 130°C, 325 psig, 20 min

No acid treatment

+ acid treatment

PBI: 3 kGy, 45 MeV

Hot water, 10 min

+ TiO2

Aseptic conditions

, 40°C/24 h, 60°C/8 h 40.8–

No H2

+ 5% H2

46.9

**Glu Eth**

71.1 83.1 [76]

83.2 [77]

90 [78]

85.4 [79]

86–89 73–77 [80]

85 77 [81]

86.5 73 [82]

96 [83]

85 [84]

>85 [87]

53.7 [88]

73 [86]

90.8

Chen et al. [68] used aqueous ammonia to pretreat silvergrass, napiergrass and rice straw at room temperature, resulting in over 90% of cellulose recovery in 4 weeks. On destarched barley hull, SAA pretreatment (15w/w NH<sup>3</sup> , 75°C, 48 h) produced zero glucan loss and 83% saccharification yield using 15 FPU/g-glucan; and with the addition of a xylanase in simultaneous saccharification and co-fermentation (SSCF), a high ethanol yield of 89.4% of the maximum theoretical was obtained [69]. High ethanol concentration and yields from SAApretreated corn stover followed the use of a two-phase SSF involving pentose and hexose conversion with the help of *S. cerevisiae* and a recombinant bacterium, respectively [70]. Recently, the addition of surfactants such as Tween 80 and PEG 400 was found to improve sugar and ethanol yields [71]. In a similar study Raj and Krishnan [72] obtained high sugar yield by adding laccase and a mediator to enhance enzymatic hydrolysis of pretreated biomass. Nahar and Pryor [73] also found out that pelleting of samples before SAA application required less harsh pretreatment conditions and lower costs.

Two-stage processes targeting separate removal of hemicelluloses and lignin have also been investigated. Kim et al. [74] employed acetic acid medium to remove hemicelluloses followed by aqueous ammonia at elevated temperatures. Results obtained from other studies are given in **Table 4**.

#### **5.3. Positive attributes and drawbacks**

SAA retains most of the hemicelluloses in the solid, eliminating the need to separately process hemicellulose and cellulose sugars. It leads to efficient delignification, producing low levels of enzyme inhibitory compounds. The reactor configuration is simpler and less costly, while ammonia recovery is easier compared to AFEX [18]. It can be adapted to small-scale production. Further, neutralized salts from liquid hydrolysates could be used as nutrient source in fermentation.

There are few disadvantages associated with SAA pretreatment. Since pretreated solids contain high fractions of hemicellulose, a high demand for C5 conversion enzymes is needed to produce xylose and other pentose monomers [18]. Post-treatment washing usually result in carbohydrate losses.

higher ethanol concentration and yield than substrates that did not receive any radiation. In another study, yields of 25.3, 21.2, and 46.5 g/100 g biomass, respectively, was obtained during radio frequency-assisted NaOH pretreatment (27.12 MHz, 0.20–0.25 g NaOH/g biomass; 90°C) of switchgrass at solids content of 20% [94]. In an investigation to ascertain the effects of microwave chemical pretreatment on sweet sorghum bagasse (12% moisture, 1–2 mm), lime was found to enhance lignin removal, with sugar yields reaching 23.2 g/100 g biomass (38% of theoretical yield) for lime concentration of 0.1 g/10 ml of water. Microwave has also been used in conjunction with eutectic solvent, with enhanced lignin and hemicellulose removal

Emerging Physico-Chemical Methods for Biomass Pretreatment

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Under electron beam application, Karthika et al. [105] obtained 79% sugar yield from the saccharification (30 FPU/g-biomass, 144 h) of a hybrid grass exposed to 250 kGy of radiation, while Bak et al. [106] realized 52.1% from rice straw when it was exposed to 80 kGy and saccharified using 60 FPU/g-glucan for 132 h. Prior removal of hemicellulose using dilute acid and alkaline before irradiation exposes cellulase to enzymatic action during hydrolysis, and culminates in higher sugar yields [107]. Electron beam has also been applied together with other physico-chemical methods such as SE with good results [108]. The main challenge regarding the use of electron beam pertains to its low energy and as such some interest are

The mode of heating is uniform, energy efficient and offers rapid processing of biomass. Pretreatment is performed at low temperatures and at shorter period. It has the potential to be used for effective isolation of hemicelluloses. Irradiation generates no/low levels of inhibitors

Irradiation-chemical methods do not come without disadvantages. Microwave-assisted pretreatment comes with the risk of causing extensive degradation of hemicelluloses and contamination of dissolved lignin at severe conditions, releasing toxic compounds that inhibit enzymatic hydrolysis. Hu and team [94] argue that practical issues with scaling-up is more of a challenge in microwave than in radio frequency which can be used on large quantities of biomass, and at relatively high solids loading (20–50%) with uniform temperature profile

Among the three main stages of cellulosic ethanol production, namely, pretreatment, hydrolysis and fermentation, pretreatment presents the most practical and economic challenges in the attempt to produce ethanol at industrial-scale due its influence on both upstream and downstream processes. Thus, emerging and promising pretreatment methods that rely on physico-chemical fractionation of biomass are discussed, with prominence given to process description, advantages, drawbacks, and innovations employed to counteract inherent

and by carefully controlling the chemical pretreatment, inhibitor levels are reduced.

and improved cellulose digestibility [104].

focusing on proton beam.

**6.3. Positive attributes and drawbacks**

when combined with chemical methods.

**7. Conclusion**
