A review on organosolv pretreatment of softwood with a focus on enzymatic hydrolysis of cellulose

A clean and efficient fractionation of biomass into cellulose, hemicellulose and lignin streams is a key step in biofuel and biorefinery industries. Lignin-free cellulose and hemicellulose streams can be enzymatically hydrolysed to sugars for fermentation to different biofuels, high value biochemicals and biopolymers. Towards this objective, organosolv pretreatment is the promising strategy to separate lignin from carbohydrates. Scientific information on the organosolv pretreatment using lignocellulosic biomass in general and various organic wastes is available with a focus on lignin separation and its use. Amongst different lignocellulosic biomass, softwood is a challenging feedstock due to recalcitrance towards enzymatic hydrolysis of cellulose. The aim of this review is to describe technical and research efforts on various organosolv processes developed specifically for softwood as a feedstock and how it influences enzymatic hydrolysis of cellulose. Process severity factor, selection of the solvent and choice of a catalyst in organosolv process are discussed. The differences in conventional pulping versus organosolv pretreatment and physico-chemical changes that occur in organosolv fractionated cellulose, lignin and hemicellulose are explained. Pilot and demonstration scale organosolv treatment plants and the challenges they face going towards commercialisation, as well as a path to the future growth and advancement in softwood organosolv processes, are discussed.


Introduction
Non-food lignocellulosic biomass such as crop residues, energy crops and forest waste are the focus of global research in the production of second-generation biofuels and renewable biochemicals [1][2][3]. Commercial forestry based on softwood is widely spread in temperate and subtropical areas including Chile, Spain, South Africa, New Zealand and Australia [4]. In New Zealand, well-established sustainable softwood plantations are grown on 1.7 million hectares, which is about 7% of total New Zealand land area [5]. This provides an assured supply of fast-growing softwood for the pulp and paper industry and timber processing for furniture and housing. A considerable portion of waste is generated during the harvesting of the trees (such as twigs, branches, offcuts and stumps) and timber processing (such as wood chips and sawdust) that could be used as a feedstock for the production of biofuels and biochemicals [4]. One of the most promising approaches involve using enzymes to hydrolyse carbohydrate polymers to monomeric sugars and then fermenting these sugars to the various bio-based products [6].
Depending on the quality of the isolated lignin, it can be directly used for supplying energy or chemically/biochemically processed to get value-added products [7][8][9].
A typical composition of softwood on a weight basis is as follows: cellulose 42-45%, hemicelluloses 21-24%, lignin 25-30%, extractives 1-10% and less than 1% inorganic compounds. Softwood cellulose is a linear polymer of d-glucose units with a high degree of polymerisation (DP ≈ 10,000), high ordered structure and partially crystalline in nature. It is intertwined with hemicellulose, which is a short and branched structure consisting of partially acetylated glucomannans and galactoglucomannans with xylose and arabinose present in smaller amounts. Softwood lignin is mainly made from coniferyl alcohol as a precursor. It is a guaiacyl-and p-hydroxyphenyl-type lignin having lower methoxy content, being highly condensed, having crystalline and being resistant to deconstruction. Extractives are organic compounds including terpenes, aliphatic acids, alcohols, alkaloid, phenolics, gums, resins, flavonoids and essential oils which can have influence on the properties and processing quality of the softwood [10][11][12][13].
Different components of the softwood can be used as a feedstock in the production of biofuels and biochemicals, provided clean and efficient fractionation of each component into separate streams is achieved. Many pretreatments such as physical, biological, physico-chemical, chemical and combinations have been developed [12,14] for fractionating softwood. However, not many of them can efficiently fractionate cellulose, hemicellulose and lignin as a separate processable and value-added stream. An effective pretreatment that can remove lignin and hemicelluloses encasing the cellulose microfibrils, and make the cellulose more accessible to the enzymes, is critical to the sugar yields during enzymatic hydrolysis [6,15]. Towards this the most promising pretreatment is the organosolv process originally developed to separate lignin in high purity from carbohydrates [16]. This pretreatment was proposed as early as 1931 by Kleinert and v. Tayenthal for the delignification of pulp where lignin is extracted using organic solvents or aqueous solutions [17,18]. Main advantages of organosolv pretreatment over other pretreatments are high yield of enzymatic hydrolysable cellulosic pulp and generation of pure lignin; both are potential feedstocks for biorefinery. The other advantage is easy recovery and reuse of organic solvents at the end of organosolv process [8,9,19]. Organosolv pretreatment have some disadvantages such as high cost of organic solvents and high capital investment in the equipment due to highly volatile nature of many organic solvents [20].
There are recent reviews available on organosolv pretreatment of lignocellulosic biomass in general [21][22][23][24][25][26][27]; however, only one review by Nitsos et al. (2018) focus on organosolv pretreatment of softwood. Here, we aim to fill this gap in describing different organosolv pretreatment strategies specifically for softwood as a feedstock, with an emphasis on improving enzymatic hydrolysis of cellulose into a glucose stream. The specific objectives of this review are to (1) discuss the basic organosolv process and give guidelines on selection of a particular solvent and catalyst with a focus on softwood as a feedstock; (2) describe the effect of different organosolv treatments on the enzymatic hydrolysis of softwood; (3) explain the physico-chemical changes in cellulose, lignin and hemicellulose after organosolv treatment; and (4) discuss pilot and demonstration scale plants and the challenges faced towards commercialisation of organosolv pretreatment of softwood. Discussion on individual organosolv steps, reaction mechanism and factors influencing organosolv process is out of scope as this information is widely available in the literature.

Organosolv pretreatment
A representative organosolv process is schematically shown in Fig. 1. Typically, biomass is treated with organic solvents such as aqueous ethanol, acetone, acetic acid, methanol, butanol or glycerol with or without catalyst at high temperatures from 100 to 250 °C, for durations ranging from 30 to 60 min [7,8,19,24,[28][29][30][31][32][33][34]. The cellulose-rich pulp is separated by solid-liquid separation using filtration or centrifugation. In spent liquor, both lignin and hemicellulose fractions are dissolved. The lignin is precipitated from the liquor by dilution with water with partially hydrolysed hemicellulose, furans and soluble lignin which remained dissolved in the liquor. Generally, organic solvent is recovered and recycled from the spent liquor.

Severity factor
The severity of the organosolv process is measured as severity factor (S 0 ) which is a combination of pretreatment reaction time and temperature. This factor is expressed in Eq. 1 originally proposed by Overend et al. (1987) [17].
where t is the residence time (min) and T is the pretreatment temperature (°C). Equation 1 is modified to include pH term when organosolv process is performed in the presence of acid/base catalyst such as H 2 SO 4 or NaOH. This is expressed by combined severity (CS) factor which is a function of time, temperature and pH (Eq. 2) [23].
where T ref is 100 °C, t is the residence time (min) and T is the pretreatment temperature (°C). The relationship between pretreatment severity and enzymatic hydrolysis of cellulose depends on the type of reactor used and the scale of the process. Pretreatment studies conducted in different reactor configurations are often difficult to compare even when carried out at similar severity values [26,35]. The optimal severity of the organosolv process varies with the type and nature of softwood biomass, solvent and catalyst used in the organosolv process. The severity factor needs to be empirically selected to get good cellulose recovery and improve enzymatic digestibility. A high severity organosolv pretreatment is applied for delignification and removing hemicellulose; however, this results in significant degradation of cellulose and sugars creating enzyme or fermentation inhibitors [27].

Different organic solvents
The most common solvent used is ethanol, due to its low price, good solubility of lignin, lack of toxicity, miscibility with water and ease of recovery [25,[36][37][38]. Ethanol in organosolv pretreatment primarily affects the process in two ways: (1) enhancing the impregnation of the softwood by transferring the catalyst or reagent to the lignin, (2) transporting the soluble lignin fragments from the cellular matrix to the bulk liquor solution [39].
Ethanol-organosolv treatment is more efficient for hardwood and herbaceous biomass compared to softwood. Other solvents such as butanol, acetone, ethylene glycol and glycerol were tried for fractionation of softwood. Delignification of southern yellow pine was investigated at 175 °C with a 50% water-ethanol and water-butanol system for 80 min. The lignin removal of 16% for the ethanol and 28% for the butanol system was reported [40]. The butanol/sulphur dioxide (SO 2 ) pretreatment for the mountain beetle-killed lodgepole pine resulted in 82% and 100% cellulose hydrolysis in 12 h and 72 h respectively. The high severity in butanol pretreatments resulted in the substrate with less hemicellulose, smaller fibre length, increased ratio of large versus small pores, lower DP of cellulose and increased swelling. All these factors contribute to increase in enzymatic hydrolysis of the cellulose with minimum sugar degradation [41]. The use of acetone (50% v/v) in organosolv pretreatment of Pinus radiata D. Don completely solubilised hemicellulose and resulted in cellulose hydrolysis of 38-72% depending on the process severity [28]. The cost of acetone prohibits wide use of this process. The organosolv process can be performed at atmospheric pressure when high-boilingpoint (BP) alcohols such as ethylene glycol (BP = 197 °C) and glycerol (BP = 290 °C) employed as solvents. Different softwoods such as pine, spruce, cedar and Douglas fir were fractionated using dimethyl carbonate-ethylene glycol at 140 °C, 40 min leading to 98% extraction of monophenolic compounds from lignin and enzymatic cellulose hydrolysis resulted in 85% glucose yield [42]. Biodiesel side-product glycerol mixed with sodium salts of laurate (20%) was used as mixed solvent system in organosolv delignification of Japanese cedar and spruce at 250 °C for 1 h. This resulted in reduction of lignin by almost 27% in Japanese cedar and 18% in spruce. Enzymatic hydrolysis of delignified Japanese cedar (9.3% lignin) gave 0.55-0.67 g glucose/g cellulose after 72 h. Also, lignin dissolution in glycerol resulted in improved calorific value of recycled glycerol from 20 to 25 MJ/kg [43]. Alkaline pulping of Pinus sylvestris using glycerol (wood:glycerol = 1:13.3 w/w) as a solvent showed enzymatic hydrolysis of the pulp depends on the pulp composition, and pulp characteristics such as drying of the pulp reduced cellulose conversion [44]. The high cost and high energy consumption for the recovery of ethylene glycol and glycerol limited their use in organosolv processes.

Different solubility parameters
The ability of single solvents to dissolve or swell lignin increases as the cohesive energy density or hydrogen-bonding capacity of the solvents increases and associated solubility parameter (Hildebrand parameter, δ1 value), approach an estimated value of around 22.5 (J/cm 3 ) -1/2 which is the approximate solubility parameter of extracted wood lignin (δ2 value) [45]. If the δ1 value of the solvent is too dissimilar from the δ2 value of the lignin, the solvent cannot solubilise lignin or only can solubilise a small fraction of lignin. For example, water has strong hydrogen bonding ability, but its δ1 value is much higher (47.3), so that lignin is hardly solubilised in water. For polar molecules and mixture of solvents, Hansen and Björkman [46] proposed three different intermolecular interactions such as dispersive (δ D ), polar (δ p ) and hydrogen bonding (δ H ) which will affect solubility of lignin in a solvent or mixture of solvents. However, Hansen solubility parameter alone cannot explain lignin dissolution since the values are used to measure the intermolecular affinity between solvent and lignin but do not account for their intramolecular affinities [47,48]. The Hildebrand (δ1) and Hansen values of different organic solvents commonly used in the organosolv pretreatment of softwood are listed in Table 1, showing ethanol as a good solvent, generally for lignocellulosic biomass. The solubility of lignin in aqueous ethanol is significantly affected by ethanol concentration [49]. The optimal ethanol concentration in organosolv pretreatment is usually selected in the range of 50 to 60 wt% at a softwood loading of 10 to 25 wt% [10,25].

Organosolv process for pulping versus biomass fractionation for biorefinery
Traditional paper manufacturing processes such as Kraft and sulphite use hazardous chemicals resulting in wastewater disposal problem; a fraction of cellulose is lost in the liquid stream, and impure and degraded lignin collected in black liquor. Moreover, the cellulose pulp obtained from Kraft and sulphite processes did not respond well to the enzymatic hydrolysis, and recovery of pure lignin from black liquor is cost and energy intensive. To overcome these problems, organosolv process was developed which provided a clean fractionation of lignin and cellulose, both of which can be used as feedstocks in the biorefinery [11]. In the organosolv process, comparative mild chemicals are used which can be recycled and reused creating less hazardous waste. There are distinct differences when applying the organosolv process for paper manufacturing versus to achieve high enzymatic cellulose digestibility. These differences are as follows: (1) Pulping for paper manufacturing requires a high degree of delignification whereas for cellulose digestibility, partial delignification could be sufficient; (2) the pulping process for paper manufacturing is optimised for obtaining a high pulp yield and preserving a good fibre quality, while for cellulose digestibility the optimisation is generally approached by increasing cellulose accessibility with a compromise on fibre quality allowing some degradation of cellulose structure [50].

Different organosolv pretreatments applied to softwood
The basic organosolv pretreatment process involves only ethanol as a solvent without adding any catalyst. This leads to a relatively low delignification yield and lower enzymatic hydrolysis of cellulose to glucose. Therefore, other co-solvents/catalysts are added along with ethanol in the modified organosolv pretreatment. A catalyst in the form of dilute mineral acid such as sulphuric (H 2 SO 4 ) or hydrochloric (HCl) (0.1 to 2.5 wt%) is often used to accelerate the process and reduce pretreatment time [2,33,34,41,51]. Different modifications to the basic organosolv pretreatment are outlined in the following sub-sections:

Acid-catalysed ethanol organosolv pretreatment
Softwoods have low concentrations of acetyl groups, which when combined with an inherent recalcitrant nature do not allow self-catalysed reactions to improve the enzymatic digestibility of the organosolv pulp. Hence, addition of an acid catalyst supplements the low natural acidity of the softwood feedstock in the ethanol organosolv process. The glucosidic bonds of hemicelluloses and celluloses are susceptible to the acid catalyst. Acid-catalysed organosolv pretreatment is usually performed at a lower temperature, traditionally below 200 °C compared to autocatalysed ethanol pretreatment. The acid-catalysed pretreatment is conducted using an inorganic acid such as sulphuric, phosphoric or nitric acid. The most preferred acid is H 2 SO 4 at a concentration of 0.1 to 2.5 wt% with residence time and solids loading varying from 30 min to 1 h and 5 to 15% w/v, respectively [10]. The acid-catalysed ethanol-organosolv process was optimised for ethanol-water content and H 2 SO 4 concentration using different softwoods. The optimum system (60:40 ethanol-water, 175 °C, 0.25% H 2 SO 4 ) achieved cellulose yield and purity of 82% ± 3% and 71% ± 3%, respectively [52]. However, strong inorganic acids have more corrosive effects than organic acids. Thus, some organic acids, such as formic and acetic acid, are chosen as catalysts for use at high process severity. A complete enzymatic hydrolysis of Norway spruce cellulose was achieved using formic acid as a catalyst (maintain pH ~ 3.5 in aqueous phase) under high severity ethanol organosolv process at 63 wt% ethanol, 170-240 °C, 90 min [53]. The acid-catalysed organosolv processes are effective in solubilising hemicellulose but not so effective in removing lignin. After acid-organosolv treatment, the cellulosic pulp requires a wash with dilute alkali or ammonia to remove acetylated or formyl functional groups on cellulose. The enzymatic hydrolysis efficiency of cellulose is compromised if these ester groups are not removed. This was demonstrated in Douglas fir treated with 95% acetic acid and subsequent enzymatic hydrolysis of the acetylated substrate resulted in a meagre 10% cellulose conversion versus after complete deacetylation of the substrate resulted in enzymatic cellulose hydrolysis of 60% [54].

Alkaline-catalysed ethanol organosolv pretreatment
Alkaline catalysis is a selective delignification strategy which also lowers the DP and crystallinity of cellulose. The auto-hydrolysis and dissolution of cellulose and hemicellulose is reduced [14] and swelling of the biomass was improved. In one-step alkaline-organosolv treatment of pine sawdust, using 3% sodium hydroxide, 45% ethanol and 150-165 °C for 120 min resulted in 91% removal of lignin without loss of cellulose. Subsequent enzymatic hydrolysis of the cellulose gave almost 90% conversion [55]. Several alkaline catalysts are used in the ethanol organosolv, including inorganic bases such as sodium hydroxide, potassium hydroxide, calcium hydroxide and some organic amines such as ethyl-, propyl-, butyl-amines and ethylenediamine [27]. Generally, sodium hydroxide is used for low lignin containing substrates such as herbaceous and hardwood biomass whereas calcium hydroxide though expensive is applied to high lignin containing substrates such as softwood. Alkaline catalyst efficiency is dependent on the temperature and retention time of the process. This can range from room temperature to 150-250 °C and from 30 min to weeks, respectively [10]. The inorganic base-catalysed process usually generates significant wastewater that requires neutralisation, leading to additional disposal costs.

A two-stage combinative organosolv pretreatment
A two-stage combinative organosolv pretreatment of softwood is an important advancement because of higher efficiency, higher delignification rates and lower production of fermentation inhibitors. The first stage involves a pre-soaking or pre-hydrolysis of biomass with steam or hot water to partly hydrolyse and extract hemicelluloses mainly in the oligomeric form. This reduces the formation of furfural (F) and hydroxymethyl furfural (HMF) in the subsequent organosolv stage. Breaking lignin-hemicellulose bonds in the pre-extraction step enables increased lignin removal in the subsequent organosolv liquor [51,56]. A care should be taken to prevent repolymerisation and redeposition of highly condensed and insoluble lignin on the substrate in the pre-extraction step. This redeposited condensed lignin will impair the extraction of residual lignin in the subsequent organosolv step. It was observed that addition of carbonium ion scavengers such as 2-naphthol, cresol and hydroquinone can reduce repolymerisation of lignin. One of the initial examples of two-stage organosolv pretreatment was performed on Pinus sp. when first step hot water autohydrolysis (150-180 °C, 30-60 min) resulted in hemicellulose extraction in autohydrolysate liquor followed by second step acid-catalysed organosolv (50-75% v/v ethanol, 1 wt% H 2 SO 4 , 180-185 °C, 60-75 min) leads to delignification. Overall, this improved the fractionation of components with increased enzymatic hydrolysis of the cellulose fraction [4,10,51]. In another study, pre-extraction of hemicelluloses before a mild thermo-mechanical pretreatment increased enzymatic hydrolysis of the Pinus radiata [15].

Other organosolv pretreatments
Ethanol organosolv pretreatment using inorganic salts as catalysts can be environmentally friendly as the salts are less corrosive to the corresponding acids and can be recycled easily. The wastewater streams generated from inorganic salts do not require addition of extra lime for neutralisation. Inorganic salts such as iron (III) chloride and magnesium chloride can eliminate inhibition of lignin by metal complexation. However, added cost and the possibility of enzyme inhibition by the inorganic salts can prevent the use of such catalyst in the organosolv process. It was reported in ethanol-organosolv pretreatment of Pitch pine carried out in the presence of three separate catalysts such as 1% H 2 SO 4 , 1% magnesium chloride and 2% sodium hydroxide resulted in different percent of enzymatic hydrolysis of cellulose such as 55-60%, 60% and 80% respectively [51]. A combination of biological pretreatment followed by ethanol organosolv, known as bioorganosolv, used to treat Pinus radiata wood chips with white rot Ceriporiopsis subvermispora resulted in a pulp with 89 to 92% cellulose and low lignin content (6.1 to 8.1%). This combinatorial pretreatment leads to complete enzymatic hydrolysis of cellulose compared to only 55% hydrolysis in the case of ethanol organosolv substrate [57]. Pinus radiata chips were incubated with the brown rot fungus Gloeophyllum trabeum for 4 to 12 weeks followed by ethanol-organosolv treatment at 60% v/v ethanol, 200 °C for 60 min. Enzymatic hydrolysis of cellulose gave 55-70% glucose for brown rot + ethanol-organosolv combined pretreatment while without brown rot pretreatment gave cellulose conversion between 30 and 40%. The bioorganosolv process requires a very long incubation time and significant carbohydrate losses when brown-rot fungus such as Gloeophyllum trabeum is used resulted in 9 to 21 wt% loss of glucan [58].
A hybrid organosolv-steam explosion method efficiently fractionates and reduced size of spruce biomass and yields pretreated solids with high cellulose content (72% w/w), owing to efficient hemicellulose (90.2%) and lignin (79.4%) removal at steam explosion and organosolv stages respectively. At the end of the hybrid pretreatment, enzymatic hydrolysis gave almost 61% glucose yield compared to when only steam explosion was used as a control giving 50% glucose yield [59].
Oxiorganosolv process is a recent modification to the basic organosolv process using oxygen gas to depolymerise and remove lignin. This method can remove up to 97% of the lignin using various organic solvents such as acetone, ethanol and tetrahydrofuran. This process generates a minimum amount of sugar degradation products with 100% cellulose recovery in the pulp and almost 60% enzymatic cellulose conversion to glucose. This process has potential to explore further, provided safety and cost of the process is regulated [60]. A microwave-assisted ethanol-organosolv pretreatment showed enzymatic hydrolysis of cellulose up to 80% at 1.5% substrate consistency; however, scale up of the enzymatic hydrolysis step using higher substrate concentrations of 7.5% decreased enzymatic hydrolysis of cellulose to almost 60% [61]. Recently, ultrasonic and microwave (480 W)-assisted ethylene glycol (94 wt%) organosolv pretreatment performed at 150 °C on pine wood produced levoglucosan (55.9 wt%), hemicellulosic sugars (20 wt%) and phenols (14 wt%). This process exhibits no enzymes are required to get value-added products from softwood [52].
Another variation is mild (170 °C and 60 min) organosolv pulping in the presence of sub-/super-critical CO 2 , which resulted in increased delignification of industrial softwood sawdust [62]. However, removal of hemicellulose prior to organosolv pretreatment in the presence of sub-/super-critical CO 2 has a deleterious effect on delignification. This is opposite to the two-stage combinative organosolv process discussed in the Sect. 3.3, where pre-extraction of hemicelluloses has a positive effect on delignification.

Effect of different organosolv pretreatments on the enzymatic hydrolysis of softwood
There are two main groups of factors that affect the enzymatic hydrolysis of organosolv cellulose-enzyme-related and substrate-related factors [27]. Additionally, CS factor of organosolv process also affects enzymatic hydrolysis of cellulose. These factors are described below with examples from softwood.

Effect of enzyme-related factors
Enzyme-related factors mainly focus on the improvement in the enzyme activity, including improved enzyme adsorption on cellulose, increased enzyme thermal stability, enzyme reusability and synergism with accessory enzymes [24,63]. The enzymatic digestibility of organosolv Japanese cypress reached to 70% by changing the enzyme from acremonium cellulase to Accellerase1500. This increase was corroborated to the better binding of Accellerase enzyme to the organosolv-treated substrate [64]. It was observed during enzymatic hydrolysis of organosolv-treated Douglas fir and pine that supplementation of β-glucosidase improves cellulose hydrolysis by reducing end-product inhibition by cellobiose, while xylanase supplementation increases the accessibility of cellulose to cellulases [65]. Softwood lignin is a major hindrance in enzymatic hydrolysis due to its nonproductive adsorption of enzymes [66,67]. Ethanol organosolv pretreatment removes lignin and can depolymerise cellulose to form more short fibres and provide more binding sites for the enzymes [19]. Delignification of Norway spruce was increased from 20 to 65% with increased ethanol organosolv temperature from 175 to 235 °C. A complete enzymatic hydrolysis of cellulose was achieved at 65% delignification [53]. In addition, the physical redistribution of lignin in a softwood substrate also plays an important role in the improvement of enzymatic hydrolysis of cellulose.
Increasing the hydrophilic property of the substrate, such as introducing more acidic or sulphonic functionality on the lignin, can decrease the non-productive adsorption of cellulase on lignin [68].

Effect of substrate-related factors
The substrate-related factors are mainly concerned with the improvement of accessibility of enzymes to cellulose [69]. Del-Rio et al. [41] showed chemical composition of the substrate and substrate-related factors both have a significant effect on the enzymatic hydrolysis of organosolv-treated beetle-killed lodgepole pine. The same group [70] demonstrated that when organosolv-treated lodgepole pine was PFI-mill refined and fibre fractionated in the size range of 0.2 to 3.4 mm, the fibre size has little influence on enzymatic hydrolysis of cellulose. Hemicellulose can shield enzyme access to the cellulose [67,68]. As discussed in the twostage combinative organosolv pretreatment in Sect. 3.3, removing hemicellulose at the pre-hydrolysis stage can benefit subsequent organosolv process. Bouxin et al. [71] reported hydrolysis of hemicellulose and conversion of monosaccharides into ethyl glycosides in the acid-catalysed organosolv liquid fraction of Sitka spruce. Almost 12.5 wt% of ethyl glycoside was produced which is a valuable biorefinery product. In the literature, many substrate factors, such as the contents of hemicellulose and lignin [30,31,72], accessible surface area, porosity [41] and swelling of the fibres and crystallinity of cellulose [7,8], demonstrated to affect the enzymatic hydrolysis of cellulose after organosolv treatment. Ethanol-organosolv treatment on recycled wood (softwood + hardwood) which is a heterogeneous and dry material compared to the typical softwood used in the biorefinery leads to incomplete or severe organosolv treatment on the recycled wood. This reduced enzymatic hydrolysis of recycled wood compared to the typical softwood such as spruce [73]. It was noted that pulp drying after organosolv treatment also affects enzymatic hydrolysis of cellulose [44]. The effects of different ethanol organosolv pretreatments on the characteristics of substrate-related factors are shown in Table 2.
The results on % cellulose conversion by enzymatic hydrolysis of organosolv-pretreated softwood are compiled in Table 3. It is to be noted that enzyme loading and the amount of pretreated softwood biomass used in the enzymatic hydrolysis varied in the literature, thereby making direct comparison between different studies difficult.

Effect of pretreatment severity and temperature
In Fig. 2, the percent enzymatic hydrolysis of softwood cellulose data is plotted against severity factor (S 0 ) and pretreatment temperature as applied in different ethanol organosolv pretreatments in the literature. The pH of organosolv   [52,61] process in the presence of catalyst is not often reported in the literature and those reported are the starting pH, which gradually changes over the course of the organosolv reaction. Therefore, S 0 values in Fig. 2 are calculated from Eq. 1. This helps in comparison of different organosolv processes as shown in the Fig. 2. The most effective range of S 0 factor in the percent enzymatic hydrolysis of organosolv-treated softwood cellulose is between 3.5 and 4.8, which correlates with a pretreatment temperature of 165 to 205 °C. This pretreatment severity applied for softwood is higher compared to other lignocellulosic biomass such as hardwood, grasses and energy crops due to the recalcitrant nature of the softwood. It can be seen from Fig. 2 that process temperature can be a good indicator in understanding how pretreatment severity affects enzymatic hydrolysis of organosolv softwood cellulose. The acid-ethanol organosolv pretreatment gave consistently higher enzymatic hydrolysis yield, almost 100% conversion for Pinus contorta [41,69] and Norway spruce [53], compared to ethanol organosolv which reported only one 100% conversion for Pinus radiata [57] whereas alkaline and other organosolv processes gave a maximum of 95% [55] and 90% [41] cellulose conversion yields respectively. Generally, a spread of enzymatic cellulose conversion from less than 20% to almost 100% can be seen. From Fig. 2, the enzymatic conversion depends on the type of softwood, type of catalyst used and other process parameters.

Physico-chemical changes and applications of softwood fractionated components after organosolv pretreatment
The general physico-chemical changes associated with basic ethanol organosolv process are (1) hydrolysis of glycosidic bonds in hemicellulose and cellulose; (2) hydrolysis of the internal bonds in lignin as well as lignin-hemicellulose bonds, particularly ether and 4-O-methylglucuronic acid ester bonds to the α-carbons of the lignin units, giving smaller lignin fragments; (3) acid-catalysed degradation of pentose into F and hexose into HMF, of which both are enzyme and fermentation inhibitors; (4) condensation reactions between lignin fragments and the reactive aldehydes generated by the sugar degradation products which will produce pseudo-lignins; and (5) modification of the side-chain functional group, for example -OH by ethoxylation. The different degrees of physicochemical changes in the softwood due to organosolv pretreatment mainly depend on the CS of the pretreatment [24]. The physico-chemical changes and post-organosolv treatment application of individual softwood components are discussed below:

Cellulose
Under optimum pretreatment conditions, depolymerisation of the less-ordered (amorphous) fraction of cellulose leads to short cellulose fibres which could provide more binding sites and increased accessibility to the enzymes [19]. In the case of Loblolly pine, an increase in cellulose I β and decrease in cellulose I α + β and para-crystalline forms for organosolvtreated cellulose was reported. However, after enzymatic hydrolysis, the relative proportion of cellulose I β and cellulose I α + β was increased by almost three times with accompanied decrease in the para-crystalline form of cellulose [8].
The less-ordered amorphous cellulose is an ideal substrate for the enzymatic hydrolysis since cellulose hydrolysis mediated by fungal cellulase enzymes is typically 3 to 30 times faster for amorphous and para-crystalline forms of cellulose compared to long-range ordered crystalline cellulose [8,74]. Hence, the aim of any organosolv process is to increase the amorphous:crystalline ratio of cellulose. The cellulose-rich pulp is mainly used in enzymatic hydrolysis to produce glucose, which can then be a Hemicellulose to ethyl glucosides under optimal conditions b Ten-minute heating with ultrasonication followed by 10 min heating with microwave radiation c Yields of levoglucosan = 56%, hemicellulose sugars = 20% and phenol = 14%, respectively  [59] fermented to ethanol, acetone-butanol-ethanol (ABE) [7], biohydrogen, single cell oil, succinic acid and lactic acid. The other uses of cellulose-rich pulp are in the production of dissolving pulp, microcrystalline cellulose, carboxymethyl(ethyl)cellulose and nano-fibrillated cellulose [10,27,75].

Lignin
Delignification to reduce overall lignin content is a major attribute of the ethanol organosolv process. Lignin molecular weight and polydispersity index are influenced by pretreatment conditions. The fragmentation of lignin is mainly Fig. 2 Effect of organosolv pretreatment on enzymatic hydrolysis of different softwood substrates ascribed to the cleavage of ether linkages which are 40 to 65% of the total linkages in the softwood lignin. In acidicethanol organosolv process, easily hydrolysable α-ether linkages are most readily broken [68]. New phenolic -OH groups are produced by cleavage of the β-aryl ether linkage. However, the rate and reaction pathway of β-O-4 cleavage is dependent on the mineral acids used for catalysis as well as the combined pretreatment severity [76]. During the acid-catalysed process, pseudo-lignins may be formed by the copolymerisation of lignin-and carbohydrate-degraded products. This substance usually contains large amounts of unsaturated carbon with polyphenolic structure, and adheres to the surface of cellulose pulp, which is detrimental to further delignification and enzymatic hydrolysis of the organosolv substrates [22,77]. Generally, at the end of ethanol organosolv or organic acid-ethanol organosolv pretreatments, the total lignin content and aliphatic -OH functionality decreases. The amount of phenolic condensed lignin, phenolic -OH and carboxylic functionality increases compared to the original lignocellulosic biomass [78]. The carboxyl and sulfonyl groups introduced on the residual lignin in acid-catalysed ethanol organosolv-treated softwood substrate results in an increase in the anionic charge (therefore hydrophilicity) of the lignin present in the pulp that ultimately improves the enzymatic hydrolysis of cellulose [79]. Lai et al. [80] demonstrated correlation between the distribution of cellulase enzymes on organosolv lignin and the changes in cellulose hydrolysis yields. Further, Huang et al. [81] showed inhibitory or stimulatory effects of organosolv lignin as a function of both hydrophobic interactions and electrostatic repulsions with cellulase enzymes. Majority of the lignin-rich fraction is used in energy production and an additional small fraction has potential in value-added products such as manufacturing of resins [82,83], dispersants, binding agents [84], coating, plasticiser filler [85], stabilising agents [86], carbon fibres and nanoparticles [86]. In renewable bioenergy, the lignin-rich fraction can be used in the production of bio-oil, aromatics and syngas [87].

Hemicellulose
With increased concentration of acid in the ethanol organosolv process, hemicelluloses are hydrolysed easily to form oligomeric and monomeric sugars; however, as pretreatment severity increases further, enzyme and fermentation inhibitors such as F, HMF, levulinic acid and formic acid are formed. Acetic acid released is not a sugar degradation product but the product of the hydrolysis of the acetyl groups of hemicellulose [88]. The hemicellulose-rich stream can be used in the production of ethanol, methane, barrier films, hydrogels [89], prebiotics [90] and rhamnolipids.

Pilot and demonstration scale organosolv processes and challenges for commercialisation
Some organosolv processes studied or being developed towards pilot and demonstration scale include the alcoholbased Alcell™ (Lignol Innovations, Canada) and ECN (Netherlands), Organocell (Germany), American Science and Technology (AST) (USA), organic acid-based CIMV (France) and Chempolis (Finland), and glycerol-based Glycell™ (Leaf Resources, Australia) process [23]. However, the following organosolv processes were promising but did not progress to commercialisation owing to challenges including high process cost (e.g. solvent), energy consumption (e.g. solvent recovery) and subject to the fluctuation of fossil oil prices.

The Lignol process
This process was proposed for a biorefinery based on ethanol organosolv pulping of mixed softwoods. The cellulose fraction of the pulp was converted to glucose using enzymatic hydrolysis followed by subsequent fermentation to produce ethanol [32]. The lignin extracted is a suitable feedstock for production of lignin-based adhesives and other products due to its high purity, low molecular weight and abundance of reactive groups. Additional coproducts were derived from the hemicellulose sugars and furfural recovered from the water-soluble stream. Another advantage of the Lignol process is that the process has good economic fundamentals provided efficient engineering design is co-developed for the recovery of solvent and valuable co-products. Otherwise, high energy consumption for solvent recovery is a limitation of this process. Based on the Lignol process, the first end-to-end production of cellulosic ethanol from woodchips at fully integrated industrial scale biorefinery plant in Burnaby, British Columbia, was completed in 2009. Generally, wood biomass is treated under a cooking temperature of 180 to 195 °C for 30 to 90 min with an ethanol concentration of 35 wt to 70 wt% at a liquor to solids weight ratio from 4:1 to 10:1. The pH of the liquor might range from 2.0 to 3.8. The cellulose-rich pulp generates a high yield of glucose after enzymatic hydrolysis. The glucose is readily converted to ethanol or other sugar platform chemicals using appropriate fermentation technologies. The liquor after the organosolv step is processed to recover lignin, furfural, xylose and other extractives [91]. Lignol was acquired by Canadian Fibria Celulose SA and changed its name to Fibria Innovations Inc. which was running until the year 2018. It has now merged with Suzano pulp and paper, Brazil.

LignoFibre process
The LignoFibre process was developed at VTT, Finland. This process is based on cooking with organic solvents, such as acetic acid or ethanol, at elevated temperatures (130 to 150 °C) for 3 to 4 h. In this process, phosphinic acid (H 3 PO 2 ), an expensive catalyst, is used as a reducing agent in delignification by acidolysis. H 3 PO 2 also potentially protects lignin against typical condensation reactions via esterification under acidic conditions. However, at higher cooking temperature (> 150 °C) and longer time (5 h and above), degradation and condensation of dissolved carbohydrates and lignin with formation of pseudolignin is observed. LignoFibre process is found to be flexible towards various feedstocks such as hardwood and softwood, as well as various annual plants. This process yields high quality components, i.e. reactive and/or easily hydrolysed cellulose fibres, sulphur-free lignin and sugar compounds, which can be potentially used as bio-based materials in various applications [92]. Additional information on commercial use of this process is not available.

Alcell process
The Alcell process was originally called alcohol and pulping process [93]. It has three stages of extraction and uses an aqueous ethanol solution (40 to 60% v/v) to delignify wood in the temperature range 180 to 210 °C with pressure ranging from 2 to 3.5 MPa. The solvent was recovered by flash vaporisation, vapour condensation and vacuum stripping. In August 1983, a pilot plant for the Alcell process was started. This plant operation showed that the production costs were lower than those of Kraft or sulphite pulp [94]. Both lignin and hemicellulose sugars could be recovered in high yields as attractive by-products. The first demonstration scale plant using the Alcell process operated from 1989 to 1996 in New Brunswick, Canada. In 1993, more than 2,000 cooks were completed in the Alcell demonstration plant. Alcell process is economically viable at a smaller scale compared to the Kraft process to produce chemical pulp and has been considered an alternative to Kraft pulping [95]. The small scale makes it an attractive process especially for some limited wood resources.
In the Alcell process, there are no recovery boilers because no inorganic materials need to be recycled. The absence of recovery boilers means that the Alcell process can achieve a big saving in the capital cost. In addition, lignin free of sodium and sulphur but having more condensed phenolic units recovered from the solvent liquor can be used to produce value-added products. The condensation reactions are responsible for the deceleration of the delignification of softwoods. The residual condensed lignin in the pulp reduces enzymatic hydrolysis of cellulose. Alcell Inc. has been discontinued and there are no further plants running on this process; the reasons are unknown.

Future perspectives
Organosolv fractionation of softwood has been established at pilot and demonstration scale; however, developing commercial scale is hindered mainly by unfavourable process and energy cost and fluctuating prices of fractionated product streams. Therefore, overall cost reduction and energy balance of the organosolv process must be addressed with efficient separation of pure fractionated components. The organosolv pretreatment cost and energy requirement can be reduced by operating at low temperature and/or atmospheric pressures. This can be achieved by developing novel catalysts and/or combinatorial pretreatment processes using high-boiling-point organic solvents which are successfully demonstrated at the Laboratory scale discussed in Sect. 3.3. For the expected combinatorial organosolv pretreatment to have commercial success, it may be required to apply a presoaking or pre-extraction step in which hemicellulose sugars are extracted from the biomass and the remaining biomass used for the organosolv pretreatment for delignification. This two-stage combined pretreatment approach can result in a clean separation of cellulose, lignin and hemicellulose from softwood [51,56]. The resulting cellulose pulp can be enzymatically hydrolysed to sugars under mild and environment friendly operating conditions. However, fine tuning of the organosolv process is essential either from a viewpoint of different types of softwood used as a feedstock or focus on producing cellulose pulp with enhanced enzymatic hydrolysis. Further, investigation into the amount of residual lignin left in the cellulosic pulp, generation of degradation products/inhibitors or pure lignin recovery yields are vital components for a commercial organosolv process to be successful and viable.

Conclusions
Softwood biomass represents a significant lignocellulosic feedstock; however, its potential in the biorefinery has not been fulfilled due to its natural recalcitrance. There are different pretreatments developed for softwood to improve its value as a feedstock in biofuel and in the biorefinery industry. Ethanol-based organosolv pretreatment is an effective pretreatment to improve the enzymatic digestibility of softwood. Addition of catalysts such as acid or alkali could assist ethanol organosolv pretreatment to be performed at mild conditions. The organosolv process keeps most of the cellulose fraction in the pulp by selectively removing lignin and hemicellulose. The isolated lignin is of high purity with low sugar and inorganic ash content and with reactive functional groups that allow its use in a variety of applications. It is anticipated that lower amounts of residual lignin left in the pulp can reduce non-productive adsorption of enzymes and thereby allow expensive enzymes to be recycled and reused, influencing the cost of the softwood pretreatment process. Moreover, lower amounts of residual lignin and higher proportion of amorphous cellulose in the organosolv softwood pulp can result in the improved enzymatic hydrolysis to glucose; the latter can be fermented to various value-added platform biochemicals, biofuels and biopolymers.