Ammonia infusion into biomass is a relatively low-cost pretreatment that is effective in removing lignin. The two most common ammonia-based pretreatments are ammonia recycle percolation (ARP) and ammonia fiber explosion (AFEX).

In ARP, ammonia interacts directly with lignin and solubilizes it. Hyun Kim and Lee (2005) reported 75%–85% removal of lignin along with 50%–60% removal of hemicellulose while retaining 92% of cellulose (Hyun Kim and Lee, 2005). This significant delignification not only opened up the structure of the biomass but also allowed the use of relatively low enzyme loadings since enzyme deactivation by lignin was substantially reduced (Hyun Kim and Lee, 2005).

AFEX exploits the fact that ammonia can be easily liquefied at moderate pressure, allowing it to readily diffuse into the biomass. When system pressure is subsequently reduced, the infused liquid ammonia instantly boils. The rapid expansion of the volatilized ammonia causes the biomass to explode, breaking the biomass into fibers. Furthermore, lignin tends to condense into droplets on the surface of the fibers and cellulose crystallinity is reduced (Mosier et al., 2005). The pretreated biomass is susceptible to enzymatic hydrolysis at modest enzyme loadings. Teymouri et al. (2005) demonstrated that ammonia pretreatment of corn stover for 5 minutes at 90°C yields almost 100% glucose and 80% xylose upon enzymatic hydrolysis. The advantage of using ammonia over other chemicals is that it is easily recovered and recycled (Kim et al., 2016). However, amide-containing by-products such as acetamide, phenolic amides, pyrazines, and imidazole are formed (Zhao et al., 2020).

Organic solvents are a well-known media for effective pretreatment of biomass. Solvent-based pretreatment typically implements either of the two types of solvents: polar protic or polar aprotic solvents. Each of these solvent-based pretreatment technologies is discussed below.

Biomass pretreatment using primarily polar protic solvents such as primary alcohols and formic acid is a well-established technology known as the organosolv process. Originally developed for the paper pulping industry to generate clean cellulose (Johansson et al., 1987), it was adopted as a pretreatment technique for enzymatic hydrolysis (Nitsos et al., 2018). In the organosolv process, ethanol is commonly used to solubilize the lignin and remove it from the biomass at moderate temperatures (Zhao et al., 2009). The use of ethanol as a solvent can make the pretreatment process more sustainable and more cost-effective (Silveira et al., 2015). Although not a protic solvent, acetone is also used widely for the organosolv process (Huijgen et al., 2010). An acid catalyst is often added to break apart the hemicellulose and liberate the lignin (Zhao et al., 2009). The organic solvent can be diluted with water causing the relatively unmodified lignin to precipitate from the solution, thereby facilitating its recovery (Nitsos et al., 2018). Because ethanol, methanol, and acetone can be obtained from biomass conversion technologies, these are considered green solvents. The organosolv process has the added benefit of producing a relatively pure lignin as a by-product (Sun et al., 2016).

The high rates of lignin removal make the organosolv process an attractive pretreatment for woody biomass, which is more resistance to enzymatic hydrolysis due to its high lignin content (Zhao et al., 2009). de la Torre et al. (2013) reached 87% lignin removal from wheat straw after 30 min using ethanol as the solvent. Nitsos et al. (2018) could achieve cellulose conversion yields of 72%–97% from softwood biomass. However, due to the high temperatures used, much of the hemicellulose and some of the cellulose are converted into biological inhibitors (Nitsos et al., 2018). Similar to ammonia pretreatment, organic solvents can be recovered and recycled.

Enzymatic hydrolysis of biomass polysaccharides can be significantly enhanced through thermochemical pretreatment with polar aprotic solvents. This type of pretreatment uses mild acid-catalyzed reaction conditions and water as the co-solvent for promoting solubilization and hydrolysis of hemicellulose, recovering xylose as a water-soluble product. Lignin is highly soluble in most polar aprotic solvents (Sameni et al., 2016). As a result, biomass so pretreated is rendered into a largely delignified, cellulose-rich feedstock (Nguyen et al., 2015). These compositional changes contribute to the opening of the polymeric matrix of biomass, and thus the biomass becomes substantially more susceptible to enzymatic hydrolysis (Nguyen et al., 2015; Shuai et al., 2016).

Nguyen et al. (2015) have demonstrated enhanced production of glucose, xylose, and arabinose from enzymatic hydrolysis of corn stover after pretreatment in a mixture of THF and water at 150°C with sulfuric acid serving as a catalyst. Significantly, the overall sugar yields as a result of this pretreatment reached approximately 95% even at the lowest enzyme loading of 2 mg g⁻¹ (glucan basis) after 14 days, whereas the yield from corn stover that had been pretreated with dilute acid, a commercial benchmark for cellulosic ethanol production, was only approximately 70% under the same enzymatic hydrolysis conditions. The high sugar recovery at such a low enzyme loading was probably the result of substantial lignin removal, a conclusion supported by compositional analysis, fractal kinetic modeling, and scanning electron microscopy images (Nguyen et al., 2015).

Shuai et al. (2016) describe a mild pretreatment using biomass-derived polar aprotic solvent, γ-valerolactone (GVL), for the production of concentrated sugar solutions from hardwood biomass. This process, using 80% GVL and 20% water as co-solvents with 75 mM sulfuric acid at 120°C, could achieve 80% delignification while retaining 96%–99% of the original cellulose in the pretreated biomass. The pretreated substrate upon enzymatic hydrolysis yielded 99% and 100% of the total glucose and xylose, respectively, which was recovered at 99.5% using a liquid carbon dioxide extraction method. This GVL pretreatment process attains a threefold higher enzyme digestibility than other organic solvents, such as THF and ethanol, and 20-fold higher than pure water as the solvent.

Alkaline pretreatment uses a base, such as sodium hydroxide, to cleave lignin–carbohydrate linkages. It also solubilizes hemicellulose by saponifying ester bonds in acetyl and uronic acid moieties (Whistler, 1993). Alkaline treatments have been used for many years by the pulp and paper industry to extract lignin from wood while leaving the cellulose fibers nearly unaltered. A strong base will partially solubilize lignin in addition to hemicellulose. Weaker bases, such as calcium hydroxide, are less effective, resulting in lower lignin removal rates (Kim et al., 2016). Using sodium hydroxide at 160°C, Karp et al. (2015) could achieve 80% cellulose and xylan conversion from switchgrass. They could remove up to 70% of the lignin while retaining most of the polysaccharides, even when low concentrations of sodium hydroxide were used (Karp et al., 2015). Since alkaline pretreatment does not break glycosidic bonds in hemicellulose, enzymatic hydrolysis must include xylanases as well as cellulases to recover pentose sugars (Chen et al., 2013).

Dilute acid pretreatments generally use mineral acids, often sulfuric acid due to its low cost. The goal is to preferentially hydrolyze hemicellulose, which is more accessible to acid molecules than the tightly bundled microfibrils of cellulose (de Oliveira Santos et al., 2018). This method has the advantage of selectively removing xylose while making the cellulose fibers more accessible to cellulase during subsequent enzymatic hydrolysis (Taherzadeh and Karimi, 2007). Hsu et al. (2010) converted 89% of the hemicellulose in rice straw to xylose after dilute acid pretreatment with sulfuric acid at 160°C and 180°C, eliminating hemicellulases from the enzyme cocktail employed for subsequent enzymatic hydrolysis. Lloyd and Wyman (2005) reported cellulose conversion of near 100% after enzymatic hydrolysis of dilute acid–pretreated corn stover with a 5-minute pretreatment at 180°C and pH of 1.25.

Dilute acid pretreatments can have the unintended consequence of converting some of the released sugars into fermentation inhibitors, such as furfural and hydroxymethylfurfural (Rasmussen et al., 2014). Additionally, enzymes can bind to residual lignin, increasing enzyme loadings for effective hydrolysis of cellulose. While non-productive binding of enzymes to lignin can occur with all kinds of treatments, it can be particularly problematic with dilute acid pretreatments, with the extent depending upon the severity of the dilute acid pretreatment (Kellock et al., 2019). Despite this prominent disadvantage, the relatively low cost of dilute acid pretreatment has made it the standard pretreatment for enhancing enzymatic hydrolysis (Humbird et al., 2011).

Using only hot water, it is possible to liberate acetyl groups from hemicellulose, which further react with water to form acetic acid. This produced acid can hydrolyze hemicellulose in a manner similar to dilute acid pretreatment, which is described as autohydrolysis. Because autohydrolysis depends upon the presence of acetyl groups in biomass, this simple and inexpensive pretreatment is restricted to biomass with high acetyl content such as hardwoods (Singh et al., 2015).

By pretreating hardwoods with hot compressed water, Ko et al. (2015) could achieve cellulose conversion yields of 70%, although this required high enzyme loadings. Unfortunately, lignin in the pretreated biomass bound and deactivated the enzymes, with the result that low enzyme loadings were ineffective in converting cellulose to sugars (Ko et al., 2015).

If the hot, liquid water in the biomass is rapidly depressurized, the resulting steam explosion will, like AFEX, shatter the biomass into fibers (Taherzadeh and Karimi, 2007). However, steam explosion, unlike AFEX, promotes conversion of acetyl groups into acetic acid. As in autohydrolysis, the acid hydrolyzes hemicellulose, further opening the structure of the biomass to enzymatic hydrolysis. Unfortunately, the elevated temperature promotes degradation of the sugars into enzyme inhibitors.

Ionic liquids (ILs) have drawn increasing attention as a pretreatment for enzymatic saccharification due to their high polysaccharide solubilization capability (van Osch et al., 2017) and tunable physiochemical properties (Hayes et al., 2015; van Osch et al., 2017). Ionic liquids consist of an organic cation and an inorganic or organic anion that promote disruption of cellulose crystallinity and, therefore, significantly increase sugar yields during enzymatic hydrolysis, even at temperatures below 100°C (Zhao et al., 2010; Tadesse and Luque, 2011).

In a typical pretreatment, cellulose is substantially dissolved in IL followed by the addition of an anti-solvent such as water to precipitate the cellulose, which can be enzymatically hydrolyzed in the absence of hemicellulose and lignin (Zhao et al., 2010). During IL pretreatment, a part of the hemicellulose and lignin are depolymerized and dissolved, which can end up in the regenerated biomass (Arora et al., 2010). Most ILs are inhibitory toward enzymatic activity, so their thorough removal is required prior to enzymatic hydrolysis.

Ionic liquid pretreatment can significantly delignify biomass (greater than 80% removal) (Sun et al., 2014; Verdía et al., 2014), which increases accessibility of enzymes to cellulose in pretreated biomass. Additionally, this ability is tunable. For example, imidazolium acetate-based ILs have greater delignification efficiency (86%) and can produce up to 96% of glucose yields by enzymatic hydrolysis post-treatment (Sun et al., 2014).

This approach separates pretreatment, enzyme production, hydrolysis, and fermentation into separate unit operations. Biomass is pretreated to solubilize hemicellulose and improve subsequent cellulose hydrolysis. The solubilized hemicellulose can be separated from the cellulose/lignin pulp for separate saccharification and fermentation or processed together. Sufficient water is added to the cellulose–lignin pulp to form a pumpable slurry, which is transferred into a saccharification tank along with enzymes that are either purchased or produced in a separate enzyme production facility (Humbird et al., 2011). After hydrolysis is complete, the resulting sugar solution(s) are pumped into fermentation tank(s) where appropriate yeasts or bacteria are introduced to metabolize the sugars to desired products. If the sugars from cellulose and hemicellulose are separately fermented, the processes can be optimized but at the cost of mechanical complexity (Brethauer and Studer, 2015).

Mechanical complexity can be significantly decreased by combining saccharification and fermentation in the same reactor. Pretreated biomass is combined with both enzymes and fermentation microorganisms to simultaneously produce and consume sugars (Brethauer and Studer, 2015). Not only does SSF simplify the process but the suppression of sugar concentration in the reactor also mitigates enzyme inhibition (Wright et al., 1988).

In typical SSF processes, the biomass, enzymes, and yeast are mixed together at the beginning of the process. Adding these components incrementally allows for a more consistent sugar level, the process that is known as fed-batch SSF. Fed-batch SSF improves yeast activity and product yields by overcoming substrate inhibition and increasing solids loading, which exacerbates mass transfer limitations (Hemansi and Saini, 2023). Nguyen et al. (2017) achieved an ethanol yield of 90.5% from pretreated corn stover using fed-batch SSF at a solids loading of 20 wt%. Cellulosic emulsions have also been applied to SSF to increase mass transfer between enzymes and cellulose fibers (Hoffman et al., 2021).

Another challenge is the incompatibility of temperature and pH for hydrolysis and fermentation steps (Afedzi and Parakulsuksatid, 2023). Attempting to optimize temperature for saccharification and fermentation processes has proven to show lower fermentation yields (Sarkar et al., 2012). Thermotolerant bacteria and yeasts are being explored for their ability to withstand the optimal temperatures of both fermentation and hydrolysis (Choudhary et al., 2016; de Barros et al., 2017; Wu et al., 2021; Ndubuisi et al., 2023; Panda and Maiti, 2024). A co-culture of high temperature-resistant Saccharomyces cerevisiae and xylose-utilizing S. cerevisiae could convert corn stover to 59.8 g L⁻¹ ethanol at 42°C (Zhu et al., 2020). A new approach shows cyclic temperature shifting with a thermotolerant yeast species to improve ethanol production from rice straw by up to 5.1-fold (Panda and Maiti, 2024).

Although mechanically simpler than SHF, SSF still requires purchase or separate manufacturing of enzymes. The capital and operating costs of SSF can be reduced if enzymes are produced on-site while also reducing the overall environmental impact (Nogueira et al., 2022).

The goal of CBP is to accomplish enzyme production, enzymatic hydrolysis, and fermentation using a single microorganism. The fact that this microorganism must produce a variety of enzymes and metabolize both pentose and hexose sugars into the desired product makes it the most complex bioconversion proposed for processing lignocellulosic biomass (Brethauer and Studer, 2015). The consolidation of these operations into a single reactor greatly reduces the capital and operating costs of a biorefinery (Dempfle et al., 2021). The difficulty of CBP lies in developing a microorganism with the desired metabolic pathways that is sufficiently robust to survive and produce high titers of the desired products under potentially harsh operating conditions (Lynd et al., 2016). Thus, thermophilic microorganisms have historically been the preferred microorganisms for CBP (Govil et al., 2022). The thermophile C. thermocellum converts pretreated empty fruit bunch from oil palm into glucose, acetic acid, and ethanol without the addition of external enzymes (Triwahyuni et al., 2022). Other naturally cellulolytic microorganisms have been explored, but they lack the ability to produce target compounds in desired quantities (Singh et al., 2018; Dutta and Suresh Kumar, 2023). Thus, research has pivoted toward either using a microbial consortium (Shahab et al., 2018) or genetic engineering non-cellulolytic microorganisms known to produce commercially relevant compounds (Yan and Fong, 2017; Banner et al., 2021). The non-conventional yeast Yarrowia lipolytica was genetically engineered to enhance cellulolytic function and achieved a 50% conversion rate of pretreated corn stover (Xu et al., 2018). Many studies emphasize the potential of yeast S. cerevisiae as a CBP candidate due to its heightened fermentation properties and ability to undergo genetic manipulation (Banner et al., 2021; Minnaar et al., 2024). Adding an enzyme cocktail of laccase, cellulase, and xylase to aid in lignin depolymerization enhanced ethanol production of S. cerevisiae (Rajak and Banerjee, 2020). Industrial scale implementation of CBP requires advanced research to identify robust microorganisms and widen the product range. Currently, no single microorganism has been identified nor engineered to enzymatically deconstruct lignocellulose, hydrolyze to sugars, and ferment to desired products (Minnaar et al., 2024).

A simple example of one-pot processing is the anaerobic digestion of mixed streams of lignocellulosic biomass and manure (Neshat et al., 2017). Raw feedstock without pretreatment is added into a reactor where enzymes released from a consortium of bacteria and fungi hydrolyze polysaccharides, fats, and proteins into simple sugars, fatty acids, and amino acids, respectively. Acetogens convert these intermediate products into carboxylic acids, which are subsequently converted by methanogens into methane, all within the same reactor. Even the separation of gaseous products from the reactants and solid products occurs in the same reactor. It might be argued that nature perfected this one-pot integrated process, but it is a useful model for envisioning engineered systems developed along the same principle of all reactions occurring in a single vessel.

Attempting to mimic the natural bioreactor process used by bovine animals has led to the development of co-treatment. This process, developed by the BioEnergy Science Center at the Oak Ridge National Laboratory, does not employ chemical pretreatment. Instead, biomass is deconstructed by continual or periodic milling, during or after partial enzymatic digestion (Paye et al., 2016; Balch et al., 2017). The combination of enzymes and milling creates a synergistic effect that achieves sugar yields from switchgrass up to 88% (Balch et al., 2017). As co-treatment is a relatively new technology, further development is required to improve microorganism survivability and optimize the in situ milling process. Eliminating toxic chemicals and harsh processing conditions makes co-treatment with CBP a very promising approach to liberate sugars from biomass (Lynd et al., 2017).

The Joint BioEnergy Institute at the Sandia National Laboratories has recently developed another approach to a single-pot process, made possible through the discovery of biocompatible ionic liquids such as cholinium lysinate ([Ch][Lys]) that readily solubilize cellulose (Liszka et al., 2016; Xu, F. et al., 2016). Because these so-called “bionic liquids” are non-toxic to enzymes and microorganisms, the solubilized carbohydrate does not have to be recovered from the solvent to perform enzymatic hydrolysis and fermentation. Its current manifestation still requires separate addition of enzymes although the microorganisms developed for CBP, in principle, could be used in this one-pot integrated process to truly combine all the steps required to convert lignocellulosic biomass in a single reactor.

To assure fast and efficient conversion of polysaccharides in biomass to fermentable sugars, enzyme cocktails must be tailored to the specific biomass feedstock. In addition to cellulases, hemicellulases can be added if the substrate includes significant hemicellulose (Dien and Bothast, 2003). Due to the variety and quantity of enzymes that must be produced, enzyme cocktails are expensive and represent a large operating cost for sugar production.

To reduce enzyme costs, on-site manufacture and recycling have been contemplated for commercial operations. Onsite enzyme production eliminates transportation costs and offers cost savings through integration with other processes in the plant (Humbird et al., 2011). Recycling has proven difficult to accomplish as processes used to concentrate the enzymes can also destroy them. One of the most promising methods recycles the solid residue from saccharification, which contains significant amounts of enzymes adsorbed onto the solid residue (Humbird et al., 2011).

Bio-oil is the liquid product from pyrolysis of biomass. Most of this liquid is condensed from vapors released during pyrolysis (Lindstrom et al., 2019b; Tiarks et al., 2019). Organic vapors must be rapidly removed from the pyrolyzer and cooled to prevent secondary decomposition reactions, reducing the yield of bio-oil such as in the decomposition of volatilized sugars (Shin et al., 2001; Evans and Nimlos, 2002; Zhang et al., 2012; Haverly et al., 2015). The liquid from pyrolysis of lignocellulosic biomass includes hundreds of compounds (Chaala et al., 2004; Patwardhan et al., 2011b; 2011a; 2009), which complicates its analysis and the separation of products (Carpenter et al., 2014). It is well known that phenolic compounds derived from lignin are chiefly responsible for the high viscosity (Bridgwater, 2003) of bio-oil, while phenolic compounds and carboxylic acids both contribute to its high corrosivity (Bridgwater, 2003; Keiser et al., 2014; Haverly et al., 2015). Less well known, bio-oil can also contain significant quantities of sugar, depending on how the biomass is prepared and processed.

Most sugars have vanishingly small vapor pressures (Oja and Suuberg, 1999; Westerhof et al., 2011), and their discovery in bio-oil was somewhat surprising, encouraging the idea that thermal ejection was responsible for their presence. However, studies on sugars and anhydrosugars, a class of carbohydrates that lack one or more water molecules when compared to standard carbohydrates, which are found in bio-oil, revealed that they had significant vapor pressures at pyrolysis temperatures (Oja and Suuberg, 1999). Heating these carbohydrates as pure compounds revealed that they can be readily volatilized (Bai et al., 2013). Table 2 depicts the most common sugar products from biomass fast pyrolysis. Sugar yield depends upon the type of polysaccharide, reaction conditions, and whether secondary reactions that degrade sugars proceed to a significant extent.

The yields of these sugars are also highly dependent on the presence of naturally occurring alkali and alkaline earth metals (AAEMs) in the biomass, which catalyze pyranose and furanose ring fragmentation in polysaccharides (Patwardhan et al., 2010; Kuzhiyil et al., 2012; Zhou et al., 2016). Secondary interactions with biochar, which contains AAEM, catalyze secondary reactions of levoglucosan decomposition and degrade pyrolytic vapors, leading to decreased sugar yields during pyrolysis (Plouffe et al., 2022). Biomass pretreatment to remove or passivate AAEM is therefore required to obtain high sugar yields from biomass.

During biomass pyrolysis, AAEM cations interact with polar moieties and functional groups in carbohydrates, leading to greatly reduced sugar yields compared to pure polysaccharides (Mayes et al., 2015). Sugar yields comparable to pure polysaccharides can be achieved for lignocellulose by either liquid extraction of soluble AAEMs or passivating the catalytic activity of AAEMs in situ prior to pyrolysis (Golova and Krylova, 1960; Kilzer and Broido, 1965; Broido, 1966; Patwardhan et al., 2010; Kuzhiyil et al., 2012; Bai et al., 2014; Dalluge et al., 2014; Wang et al., 2014; Oudenhoven et al., 2016a; 2016b). Regardless of the pretreatment method, AAEMs must be dealt with thoroughly as each metal atom is estimated to catalyze up to 1,000 fragmentations of pyranose or furanose rings (Zhou et al., 2016).

Both AAEM extraction and passivation improve sugar yields from fast pyrolysis of lignocellulosic biomass, but the application of these pretreatments differs substantially. Extraction involves using water to wash the AAEMs from the biomass. Inclusion of acids, such as carboxylic acid produced during pyrolysis, improves the extraction (Oudenhoven et al., 2016b). This technique increases sugar yields compared to no pretreatment (Oudenhoven et al., 2016b) but has significant drawbacks. Washing requires substantial water usage, which in turn is expensive to dry prior to pyrolysis and to treat prior to discharge (Oudenhoven et al., 2016b). For these economic reasons, this review focuses primarily on passivation.

Passivation predominantly uses mineral acids to convert AAEM cations in biomass into thermally stable salts (Kuzhiyil et al., 2012). When using acids, AAEMs likely bind to the conjugate base, forming inorganic salts. For example, if sulfuric acid is used, the conjugate base would be sulfate or hydrogen sulfate (bisulfate), which bonds with AAEMs forming salts such as calcium sulfate and sodium hydrogen sulfate (Kuzhiyil et al., 2012). AAEMs are less catalytically active when bonded as thermally stable salts. Although the protons released into the biomass in this manner do not appear to catalyze pyranose and furanose ring fragmentation, excessive acid can diminish sugar yields. Accordingly, there appears to be an optimal acid pretreatment level to passivate AAEMs (Figure 7) (Kuzhiyil et al., 2012).

Optimal infusion of mineral acid into lignocellulosic biomass can effectively passivate all catalytic activity of AAEMs, achieving sugar yields in (batch) micro-pyrolyzers equal to those from pyrolysis of pure polysaccharides, up to approximately 60 wt% (Patwardhan et al., 2010; Kuzhiyil et al., 2012; Wang et al., 2014). Enhancements from pretreating biomass are typically the greatest for biomass containing high AAEM content (Kuzhiyil et al., 2012; Dalluge et al., 2014). Continuous pyrolysis of pretreated biomass has also demonstrated enhanced sugar yields although to date they have only reached approximately 30 wt% monosaccharides on a polysaccharide basis (Kuzhiyil et al., 2012; Dalluge et al., 2014; Kim et al., 2014; David et al., 2018; 2017). Possible explanations for this discrepancy are subsequently discussed.

Kuzhiyil et al. (2012) developed a stoichiometric relationship between the AAEM content of biomass and amount of acid required for AAEM passivation. Polyprotic acids, such as sulfuric acid, can donate multiple protons, thereby generating more negative electric charges in the form of polyanionic conjugate bases, enhancing the AAEM passivation potential. Conversely, monoprotic acids, such as hydrochloric acid, can produce only monoanionic conjugate bases and are therefore less efficient on a molar basis. In addition to the number of protons available to donate, low acid dissociation constants (pKa) are very important. The conjugate base—not the proton—inhibits the AAEMs, so a stronger acid leads to more available conjugate base anions for a given amount of acid. In moderation, the proton itself appears unimportant and does not catalyze sugar destruction but is not wholly desirable because excessive acid addition produces lower sugar yields (Kuzhiyil et al., 2012). Theoretically, other chemicals could be used as long as they release appropriate anions and the corresponding cation does not catalytically degrade carbohydrates during pyrolysis. Additionally, these salts must be thermally stable. Kuzhiyil et al. (2012) claim that the greater thermal stability of potassium hydrogen sulfate compared to potassium dihydrogen phosphate explains the superior performance of sulfuric over phosphoric acid. This same explanation applies to carboxylic acids as they are not thermally stable and have higher dissociation constants and therefore yield less sugar (Kuzhiyil et al., 2012).

Maintaining the optimal level of acid pretreatment is essential to maximizing sugar yields, as illustrated in Figure 7. Adding too little or too much acid drastically reduces sugar yield, so pretreatment levels must be carefully monitored. Aside from this issue, acid pretreatment has another large operational challenge: agglomeration.

Pyrolysis of biomass without pretreatment usually yields a fine char, which can be elutriated and captured in a gas cyclone. Acid-pretreated biomass, on the other hand, results in large masses of agglomerated char and bed material. Agglomerates are not necessarily an impediment to proper reactor operation. For example, auger pyrolyzers readily grind agglomerated char as it is formed (Dalluge et al., 2014). On the other hand, agglomeration of char in a fluidized bed pyrolyzer forces operation at reduced biomass feed rates to maintain stable bed operation, which in turn reduces rates of sugar production. Understanding the mechanism by which agglomerates form is important to advancing the production of pyrolytic sugars.

The fact that biomass from which AAEM has been substantially extracted by water washing demonstrates that agglomeration arises from the loss of AAEM rather than the addition of protons to the biomass. The surface of agglomerated char from pyrolysis of washed biomass (Oudenhoven et al., 2016a) and acid-pretreated biomass (Rollag et al., 2020) suggests the formation of a melt phase prior to dehydration to char. This melt incorporates particles of bed media (sand), creating large agglomerates (Oudenhoven et al., 2016a). Rollag et al. (2020) have recently discovered that AAEM catalyzes depolymerization of both polysaccharides and lignin. It appears that AAEM increases the rate at which lignin depolymerizes and devolatilizes (Rollag et al., 2020) without substantially altering the final product's composition (Dalluge et al., 2017).

Tiarks et al. (2019) used optical and laser visualization techniques to explore the pyrolysis of lignocellulosic biomass and its components, which helps understand agglomeration of lignin. Upon pyrolysis on a heated metal strip, technical lignin powder produced large agglomerates, which was not observed for pyrolysis of wood powders. However, by mixing the lignin powder with nonreactive inorganic filler, simulating the dispersion strands of lignin polymers among the polysaccharides in biomass, agglomeration was eliminated. This suggests that char agglomeration occurs if dispersed strands of lignin have time to melt and coalesce before they can depolymerize into volatile molecules.

Since most biomass naturally contain sufficient AAEMs to catalyze lignin depolymerization, devolatilization proceeds more quickly than melt diffusion, with the result that lignin dehydrates in place to produce fine char particles that retain the cell wall structure. Removal or passivation of AAEMs slows depolymerization and devolatilization, giving time for melted lignin to diffuse to particle surfaces where it acts as an adhesive to bind together both pyrolyzing biomass particles and bed media. Untreated corn stover rind before and after pyrolysis have largely intact plant cell wall structures (Rollag et al., 2020). On the other hand, acid pretreated corn stover rind pyrolyzed for the same time duration has clearly melted (Rollag et al., 2020). Removing the catalytic activity of the AAEM promotes the melting and diffusion of lignin to the biomass surface, where it serves as an adhesive to bind particles together and subsequently dehydrate into agglomerated char (Rollag et al., 2020). Small additions of calcium hydroxide to a fluidized bed pyrolyzer has been shown to reduce although not eliminate char agglomeration of lignin (Zhou et al., 2015), and of course the addition of this alkaline earth metal when pyrolyzing whole biomass would be detrimental to sugar yields.

After discovering the role of AAEMs in depolymerizing lignin, Rollag et al. (2020) sought a biomass pretreatment that both passivated AAEMs and selectively catalyzed lignin depolymerization. Ferrous sulfate proved particularly effective, although magnesium sulfate was also successful (Rollag et al., 2020). The sulfate anion passivates AAEMs in the biomass, while the ferrous cation catalyzes lignin depolymerization without interfering with sugar yields. This new pretreatment intensifies volumetric sugar productivity (g L⁻¹ h⁻¹) by simultaneously increasing sugar yields and maintaining high biomass throughput. Rollag et al. (2020) also showed that the pyrolyzer could run autothermally, which further intensifies the process.

Adding small amounts of oxygen to a pyrolyzer in the form of air at low equivalence ratios, typically anathema for pyrolysis, can greatly increase biomass throughput for a reactor of given dimensions. Polin, Carr, et al. (2019) and Polin, Peterson, et al. (2019) increased the biomass throughput for an 8.9-cm-diameter reactor by threefold to fourfold through autothermal operation. Furthermore, intensification increases with reactor size, scaling linearly with reactor diameter (Brown, 2020). These methods may also reduce agglomeration by oxidizing char agglomerates (Kim et al., 2014). While the addition of oxygen to a pyrolyzer releases CO and CO₂, life cycle analyses of pyrolysis and autothermal pyrolysis demonstrate net carbon removal (Ganguly et al., 2022). Ganguly et al. (2022) demonstrated that for a 250-ton-per-day autothermal pyrolysis facility–processing corn stover, red oak, or yellow pine, the amount of carbon removed from the atmosphere was greater than the amount of carbon emitted. This is primarily due to the products of biochar and phenolic oil (for bio-asphalt application) that sequester biogenic carbon (Ganguly et al., 2022).

Pyrolysis has the potential to dramatically increase volumetric sugar productivity compared to enzymatic hydrolysis (Rollag et al., 2020). Under conventional (non-oxidative) pyrolysis of untreated corn stover, volumetric sugar productivity was 62 g L⁻¹ h⁻¹ compared to only 5.4 g L⁻¹ h⁻¹ for enzymatic hydrolysis of corn stover. However, the combination of ferrous sulfate pretreatment, which increased sugar yields on a biomass basis from less than 1% to over 12%, and autothermal pyrolysis, which increased biomass throughput by a factor of 2.5, increased volumetric sugar productivity to 2041 g L⁻¹ h⁻¹ (Rollag et al., 2020). An optimum reactor temperature of 450°C maximizes sugar production from autothermal pyrolysis of ferrous sulfate–pretreated corn stover (Peterson et al., 2023).

Figure 8 demonstrates that fast pyrolysis of untreated biomass yields less bio-oil while producing more gas and char than pretreated biomass. Furthermore, the concentration of sugar in bio-oil is higher when pyrolyzing pretreated biomass.

Fast pyrolysis rapidly converts polysaccharides into monosaccharides and, to a lesser extent, disaccharides; however, the elevated temperature of pyrolysis also promotes secondary reactions that degrade sugar monomers. For example, fast pyrolysis of cellulose even under ideal laboratory conditions rarely yields more than 60 wt% sugar, which stoichiometrically should be 100 wt% (Patwardhan et al., 2009). Competition among three condensed phase reactions may prevent higher yields than presently achieved: cracking (the primary depolymerization reaction), end-chain levoglucosan production, and degradation reactions (Lindstrom et al., 2019a). Cracking reactions convert polysaccharides to anhydro-oligosaccharides that are subsequently cracked into smaller anhydro-oligosaccharides (Radlein et al., 1987; Mayes and Broadbelt, 2012; Vinu and Broadbelt, 2012; Gong et al., 2014; Burnham et al., 2015; Lindstrom et al., 2019a). Levoglucosan can form directly from anhydro-oligosaccharides and readily volatilizes (Mayes and Broadbelt, 2012; Vinu and Broadbelt, 2012; Burnham et al., 2015; Lindstrom et al., 2019a). Secondary reactions limit sugar production by decomposing levoglucosan (Lindstrom et al., 2019a).

As discussed earlier, AAEMs promote pyranose and furanose ring fragmentation. The ion-dipole forces between the AAEM atoms and carbohydrates alter the reaction rates (Mayes et al., 2015), leading to reduced sugar yields. It is worth reiterating that preventing these reactions, by extracting or passivating the AAEM, only increases sugar yields to the levels observed for pure polysaccharides, which is much below the expected stoichiometric yields. Although some authors have claimed higher yields (Maduskar et al., 2018), these results have not been reliability reproduced by others.

The maximum sugar yield achieved in continuous pyrolysis systems is almost 50% lower than that achieved in microscale benchtop reactors even when using the same pretreated biomass for the experiments (Kuzhiyil et al., 2012; Dalluge et al., 2014). The lower yields from continuous pyrolysis are not fully understood but may be due to longer vapor residence times during which secondary reactions can decompose sugars. Indeed, Ronsse et al. (2012) demonstrated that alkali-rich char catalyzes vapor-phase decomposition of levoglucosan at pyrolysis temperatures.

The conventional approach to recovering condensable products of pyrolysis involves the rapid cooling of pyrolysis vapors in a spray of non-polar liquid (Venderbosch, 2019). This yields an emulsion of lignin-derived phenolic compounds in an aqueous phase in which most of the carbohydrate-derived compounds are dissolved. Although the phenolic compounds (often referred to as “pyrolytic lignin”) in this bio-oil can be precipitated by adding water, separating sugar from the aqueous phase is more challenging. Even the volatile components cannot be separated by distillation because bio-oil is extremely reactive. To combat these problems, condensable products of pyrolysis are increasingly being recovered in stages by exploiting differences in vapor pressure for the various components, as illustrated in Figure 9. High-boiling-point anhydrosugars such as levoglucosan and sugars such as xylose will condense at the highest temperatures, followed by phenolic compounds such as guaiacol and o-creosol, and ultimately lighter compounds such as furfural and acetic acids, as the temperature is lowered.

One scheme for selective recovery of the components of bio-oil is illustrated in Figure 10 (Pollard et al., 2012). Products of pyrolysis entrained in the gas flow leaving the pyrolysis reactor pass through gas cyclones for the removal of particulate matter (biochar), and then through a temperature-controlled condenser that cools the gas stream to 102°C, which is low enough to condense sugars, anhydrosugars, and most of the phenolic compounds in the stream. This so-called stage fraction 1 (SF 1) could, in principle, be operated at higher temperatures to encourage phenolic compounds to be collected downstream of SF 1, but success in this separation depends upon the relative vapor pressures of the sugars and phenolic compounds and the thermal stability of the sugars at higher quench temperatures (Dalluge et al., 2019). High-boiling-point aerosols produced during this cooling process must be separately recovered in a temperature-controlled electrostatic precipitator (ESP), designated as SF 2. The composition of liquids recovered from SF 1 and 2 have similar composition (Pollard et al., 2012), thus they are often combined and referred to as the “heavy ends” of bio-oil. A relatively simple water extraction can separate the water-insoluble phenolic compounds from the water-soluble sugars (Stanford et al., 2018). The sugar-rich aqueous solution (syrup) contains a small number of phenolic compounds that are well-known fermentation inhibitors (Rover et al., 2014) that must be removed before the syrup can be used as substrate in bioconversions. Filtration with resin columns has proved effective in this respect (Stanford et al., 2018). Stage fractions operating at progressively lower boiling points can collect other fractions of bio-oil.

Hydrolysis of cotton linen in concentrated sulfuric acid is one of the earliest examples of biomass liquefaction in an aqueous solvent to produce sugars (Braconnot, 1819). Later, dilute sulfuric acid was used to produce fermentable sugars from lignocellulosic biomass. Dilute acid hydrolysis can achieve 50%–83% yields of sugar, whereas concentrated acid produces 78%–99% yields of sugar (Bergius, 1937; Saeman, 1945; Fagan et al., 1971; Selke and Hawley, 1982; Franzidis et al., 1983; Harris et al., 1985; Malester et al., 1992; Lenihan et al., 2010; Ioelovich, 2012; Liu et al., 2012; Moe et al., 2012; Wijaya et al., 2014). Concentrated acid is typically operated at low temperatures for its strong catalytic effect promotes depolymerization of biomass polysaccharides selectively over sugar dehydration reactions. However, the use of acid for saccharification of biomass has not been highly industrialized due to the hazards of handling mineral acids, complicated recovery issues, and corrosive effects on reactors (Wright and Power, 1986; Taherzadeh and Karimi, 2007).

Cellulose hydrolysis has been studied extensively in supercritical water in the absence of any catalysts (Adschiri et al., 1993; Saka and Ueno, 1999; Sasaki et al., 2000; Ehara and Saka, 2005; Cantero et al., 2015). The high solubilization capability of supercritical water allows it to easily hydrolyze cellulose to glucose and other oligosaccharides, achieving up to 48%–54% yields (Adschiri et al., 1993; Saka and Ueno, 1999; Sasaki et al., 2000). Nevertheless, hot, pressurized water is highly corrosive requiring expensive corrosion-resistant materials of construction. Since cellulose decomposition occurs quickly in supercritical water, maximum sugar yields require precise control over reaction time (0.05–10 s) (Sasaki et al., 2000). On the other hand, using subcritical water (usually between 200°C and 300°C) encourages dehydration products of glucose, namely, 5-HMF, due to the increased catalytic activity of water caused by autodissociation into hydronium and hydroxide ions (American Society of Mechanical Engineers, 1993; Sasaki et al., 1998; Ehara and Saka, 2005; Bandura and Lvov, 2006). Furthermore, numerous studies show that 5-HMF and furfural, produced from acid-catalyzed dehydration of monosaccharides in water, can condense into undesirable water-insoluble polymeric material known as humins (Van Zandvoort et al., 2013; Tsilomelekis et al., 2016).

The use of polar protic solvents for production of cellulosic sugars is a comparatively less explored pathway. Ishikawa and Saka (2001) demonstrated the deconstruction of cellulose using supercritical methanol to produce methylated glucosides as the major products. Other alcohols, such as ethanol and ethylene glycol, can also be used for producing alkylated cellulosic sugars (Yamada and Ono, 2001; Deng et al., 2011). Interestingly, alcoholysis of cellulose in the presence of strong acid catalyst can produce methyl and ethyl glucosides at yields of 57%–63% when compared to only 5%–10% in pure water (Deng et al., 2011; 2010; Dora et al., 2012). Furthermore, a remarkably high yield of 99.5% of n-decyl pentosides was observed when wheat bran hemicellulose was treated in n-decanol at 100°C while using sulfuric acid catalyst (Marinkovic and Estrine, 2010). However, these alkylated sugars are not particularly attractive for fermentation applications due to additional upgrading steps to utilize them. These sugars are more attractive as precursors for production of cosmetics, detergents, emulsifiers, and nonionic surfactants (Marinkovic and Estrine, 2010; Villandier and Corma, 2010).

Polar aprotic solvents do not directly participate in biomass conversion reactions but influence the energetics and thermodynamics of reaction equilibria. These solvents have unique properties that make them attractive alternatives to pure water for hydrolysis and depolymerization of polysaccharides and are therefore receiving increased attention.

Compared to water, several polar aprotic solvents, such as GVL, 1,4-dioxane, and THF, reduce the apparent activation energy of acid-catalyzed hydrolysis of cellobiose, the structural unit of cellulose. At the same time, they increase activation energies for secondary reactions of the monosaccharides (Mellmer et al., 2014). In fact, increasing the ratio of GVL to water for cellobiose acid hydrolysis facilitates higher reaction rates, with a particularly sharp increase above 80 wt% GVL in the solvent mixture.

The favorable reaction energetics in polar aprotic solvents makes them highly selective for the production of sugars from cellulosic biomass. In the absence of water, polar aprotic solvents such as 1,4-dioxane, sulfolane, and acetone readily deconstruct cellulose, yielding levoglucosan as the major carbohydrate product (Koll et al., 1991; Kawamoto et al., 2003; GuiRong et al., 2008). Furthermore, the type of polar aprotic solvent also governs the yield and distribution of solubilized carbohydrates from cellulose with and without catalysts (Ghosh et al., 2018; 2016). Ghosh et al. (2016) showed that the yield of levoglucosan correlates linearly with the Hansen polar solubility parameter of the solvent, indicating a higher degree of polar interaction between the solvent and cellulose polymer. This effect reduces the activation energy of cellulose depolymerization in solvents of higher polarity (Ghosh et al., 2016).

Luterbacher et al. (2014) obtained 69%–73% yield of C5 and C6 sugars in a semi-continuous reactor system using corn stover and maple wood in a solution of GVL, water, and dilute sulfuric acid at approximately 157°C–217°C. In this work, the solvent flowed through a fixed bed reactor pre-loaded with biomass as shown in Figure 12. The sugar was subsequently separated from the solvent mixture using liquid carbon dioxide (CO₂) or sodium chloride (NaCl) solution. The system operated at relatively modest pressures compared to hydrothermal liquefaction due to the relatively low vapor pressure of GVL.

Similarly, Bai et al. (2014) have produced a mixture of glucose, levoglucosan, and xylose at a total yield of 19.8 wt% (on a biomass, not sugar, basis) by liquefaction of acid-infused switchgrass in 1,4-dioxane and water at 300°C. Taking this process further, Questell-Santiago et al. (2018) added formaldehyde to the system to react with C₅ and C₆ monosaccharide products, protecting them from decomposing into furanic compounds. This stabilization strategy overturns the typical kinetic limits of polysaccharide depolymerization and produces high yields of C₆ (over 70%) and C₅ (over 90%) sugars at approximately 5 wt% concentrations.

Ionic liquids have been explored widely for converting cellulose and whole biomass at low temperatures to fermentable sugars. Chlorine- and acetate-based imidazolium ILs are known to be highly effective in decrystallizing and dissolving cellulose (Wasserscheid and Keim, 2000; Swatloski et al., 2002; Pinkert et al., 2009). These basic anions can easily disrupt the extensive network of hydrogen bonding in cellulose and thus require only a small amount of thermal energy to break down the crystalline matrix. Moreover, ILs such as 1-ethyl-3-methylimidazolium, promote cellulose hydrolysis to glucose (Li and Zhao, 2007). Rinaldi et al. (2008) reported that ILs are also capable of effectively depolymerizing cellulose in the presence of solid catalyst.

Binder and Raines (2010) demonstrated high yields of glucose from corn stover using a chloride-based IL with gradual introduction of water into the system during hydrolysis. More recently, the Joint BioEnergy Institute (JBEI) group at Sandia National Laboratories has developed a lysinate based on biocompatible ionic liquid or “bionic liquids” for saccharification of whole biomass (Liszka et al., 2016; Xu, F. et al., 2016). Among the bionic liquids, cholinium lysinate ([Ch][Lys]) has the potential for one-pot conversion of biomass.

Feeding solid biomass into high-pressure reactors is a significant challenge for scaling up of solvent liquefaction technologies, especially at high mass loadings. Higher feedstock-to-solvent ratios allow for a smaller reactor with the same throughput, reduced solvent use, and higher product concentrations. These effects in turn can lower capital and operating costs. Loading of wood chips in solvents greater than 10–12 wt% can clog pumping systems (Thigpen and Berry, 1982). This can be partially mitigated by wet milling and other pretreatments to reduce biomass particle size, thus increasing bulk density of the feedstock, but can be expensive (Elliott et al., 1989; Ghosh and Haverly, 2019).

Extruder systems have been explored for high-pressure biomass feeding at pilot scale reactors (White et al., 1987; Haverly et al., 2018). Despite stable performance at high pressures and temperatures, successful operation requires uniform feedstock size and careful attention to the feedstock-to-solvent ratio. Progressive cavity, piston, and plunger pumps have also been employed to feed biomass slurries into high pressure reactors (Elliott et al., 2015; 1989; Zöhrer et al., 2014; Mørup et al., 2015; Zhang et al., 2015). Generally, these pumps have limited scalability and can be very expensive. Furthermore, both piston and plunger pumps deliver pulsed or discontinuous flow, and pulseless flow requires operating at least two pumps in tandem, which further increases the capital cost.

The high cost of solvent is one of the greatest challenges to the commercialization of solvent liquefaction biorefineries. A 1,000-ton-per-day hydrothermal liquefaction plant operating at 75% solvent (water) recovery and recycle would require approximately $3.9 M in make-up solvent annually (Ghosh and Haverly, 2019). Wastewater treatment adds $19.8 M to the annual operating costs. Of course, solvent recovery costs are even higher for more costlier organic solvents.

Despite the significant economic challenges, solvent recovery and recycle studies are scarce. Separation of polar solvents, such as GVL, could be complicated and expensive due to the high miscibility of the solvent with the aqueous phase where sugars are recovered. For example, a separation subsystem of compressed liquid CO₂ extraction units for recovering and recycling GVL is necessary for economic feasibility of cellulosic ethanol production (Han et al., 2015). Unremoved products in a recycled solvent stream or other impurities may adversely impact the reaction chemistry. Furthermore, purification of recycle streams by distillation or flash separation may reduce sugar quality due to degradation during rapid heating. In some cases, it may be environmentally or economically attractive to employ a product of the liquefaction process, a goal of some of the earliest work in biomass liquefaction (Thigpen and Berry, 1982) and more recently a process explored by Chevron (Haverly et al., 2018).

When employed, pretreatment and solvent liquefaction can be the largest components of the total cost for solvent liquefaction. Techno-economic analysis of GVL- and THF-based cellulosic ethanol production shows that biomass pretreatment and liquefaction could be 37.9% of the total capital cost and 31.7% of the total installed equipment cost, respectively (Han et al., 2015; Li et al., 2018). The liquefaction cost contribution is mainly due to the use of high solvent-to-biomass ratios and suboptimal solvent liquefaction performance, whereas pretreatment cost drivers are the large amounts of solvent required and long reaction times. Pretreatment and liquefaction processes deserve further development to understand how they can contribute to reducing the cost of sugars from solvent liquefaction.

Organic solvents contribute to process economics either from the costs of recovering or replacing them. The effect of the biomass-to-solvent ratio on economic feasibility of biomass conversion for sugar production is shown in Figure 14 for GVL-based solvent liquefaction (Han et al., 2015). By increasing biomass feeding capacity per unit volume of solvent used, the capital and operating costs can be lowered significantly and the heating requirements for large volumes of solvent, solvent replacement, and recycling costs can also be reduced. Additionally, the sugar produced per unit volume of solvent processed increases, thereby improving the economics of solvent liquefaction.

Solvent liquefaction has the potential to reach approximately 70% of theoretical sugar yields from biomass polysaccharides (Luterbacher et al., 2014). This combination of relatively high sugar yields without expensive enzymes and high conversion rates makes solvent liquefaction attractive for biomass processing. Nevertheless, the process is currently not economically competitive with biochemical processing (Han et al., 2015; Li et al., 2018), reflecting the fact that these systems require corrosion-resistant materials of construction, pressure vessels and piping, heat transfer equipment for thermal management, and solvent recovery or replacement. Accurate yields of bio-oil from solvent liquefaction should be generated using laboratory-scale systems that mimic the high heat transfer rates of continuous systems (Lindstrom et al., 2020).