Electron Paramagnetic Resonance in Lignocellulosic Biomass Pyrolysis Mechanism: Advancements, Applications, and Prospects

Abstract

Lignocellulosic biomass can be converted into high-value-added bio-based materials through pyrolysis; however, an unclear pyrolysis mechanism hinders its further application. Electron paramagnetic resonance (EPR) spectroscopy is the most common technology for detecting radicals, which are important intermediates of bond-breaking reactions and coupling reactions during pyrolysis. Hence, this article provides a dedicated review of recent applications, limitations, and prospects of EPR for lignocellulosic biomass pyrolysis. It starts with the advancements of EPR, including EPR spectroscopy principles, radical trapping methods, and spectrum analysis. This review establishes the radical-mediated reaction pathway spanning model compounds to native lignocellulosic biomass, via detecting and identifying the key radicals in the pyrolysis process and pyrolysis products. Furthermore, the effect of biomass pretreatment on the radical behavior during pyrolysis has been emphasized. By providing a comprehensive review of radical evolutionary patterns during biomass pyrolysis using EPR, we conclude with limitations and prospects, which may offer a new perspective on the mechanism of biomass pyrolysis and the optimization of pyrolysis conditions.

1. Introduction

Biomass, as the only renewable energy source capable of conversion into liquid hydrocarbon fuels via thermochemical processes, offers a sustainable route for producing liquid fuels and value-added chemicals [1]. Pyrolysis refers to the rapid heating of biomass to moderate temperatures (450–600 °C) under oxygen-free conditions, yielding liquid bio-oil, solid biochar, and non-condensable gas [2,3]. This transformation comprises a complex process involving multiple components, multi-stage reactions, and a variety of products [4]. Contemporary mechanistic studies utilize complementary approaches employing model compounds [5,6,7] and native biomass [8,9] to elucidate reaction pathways. Based on the analysis of experiments, reaction pathways were partly simulated by computational methods, and single-step formulations to complex kinetic models containing hundreds of reactions were established [10,11,12]. However, prevailing analytical methodologies dependent on gas/liquid chromatography [13,14] coupled with insufficient direct experimental validation of intermediate species evolution create fundamental limitations in confirming critical bond cleavage and radical recombination processes. These knowledge gaps persist in two key domains: (1) thermal decomposition mechanisms of biomass macromolecules and (2) transformation pathways of reactive radical intermediates, collectively impeding the development of a universal mechanistic framework for biomass pyrolysis.

Although biomass pyrolysis is a complex process, its chemical dynamics are fundamentally governed by the radical mechanism, and radical-induced reactions have been widely recognized [15,16]. During the pyrolysis process, radical reactions involve radical formation, transformation from one radical species to another, and the quenching stage. Bond-breaking reactions produce radicals, and participate in electron transfer to form new radicals or react with stable molecules to form new intermediates. Finally, the termination step involves dimerization, disproportionation, oxidation, or reduction of the radicals to form stable non-radical products [17]. These radical reactions are relevant to the final products generated from primary products. For instance, hydroquinone pyrolysis ultimately yields p-benzoquinone, necessitating experimental verification of the formation of the intermediate p-semiquinone radical and the loss of hydroxyl hydrogen (Figure 1) [18]. By tracking and detecting the key radical intermediates in the biomass pyrolysis process, critical mechanistic insights into the biomass pyrolysis process are provided, and a more complete reaction pathway of “reactants-intermediates-products” is constructed.

Recent advancements in analytical techniques have enabled direct detection of radical species in pyrolysis through electron paramagnetic resonance (EPR) spectroscopy [19,20,21,22]. Persistent radicals are easy to detect, being characterized by prolonged lifetimes and accumulative concentrations, but they only account for a minor fraction of the radicals in the pyrolysis process. Transient radicals, conversely, exhibit microsecond-scale lifetimes, exceeding the temporal resolution of conventional continuous-wave EPR [23], necessitating stabilization through radical trapping techniques. Recently, scholars have utilized in situ EPR to successfully track real-time concentration variations of transient radicals during pyrolysis [17,24,25]. And rapid freezing combined with spin trapping has proven effective for capturing highly reactive radical species.

As critical mechanistic indicators of intermediate transformations, radicals serve as pivotal evidence for elucidating lignocellulosic biomass pyrolysis pathways. Despite their diagnostic significance, a systematic synthesis of EPR advancements in biomass pyrolysis research remains absent. In this review, we start from typical monomer model compounds and dimer model compounds, and then expand to the three major components of biomass, namely cellulose, hemicellulose, and lignin, to elucidate the basic radical-induced reactions. As for native lignocellulosic biomass, we provide a detailed review of the changes in radicals during pyrolysis and the radicals in pyrolysis products, as well as the effects of pretreatment means on pyrolysis. Additionally, we make suggestions for the existing technological limitations and specific future research directions.

2. Electron Paramagnetic Resonance Spectroscopy

EPR serves as an electromagnetic spectroscopic technique for investigating paramagnetic substances. It can provide in situ and non-destructive information on the microscopic scale of unpaired electrons, orbitals, and hyperfine nuclear interactions [26]. The following section is about the operational principles of EPR spectroscopy, with particular emphasis on the methods for detecting and analyzing the radicals in the pyrolysis process.

2.1. Principle of EPR Detection

The electron possesses a magnetic moment with spin S = 1/2. When the external magnetic field (H) increases from 0, the electron’s energy level is split from the degenerate state into two energy levels due to the Zeeman effect. The difference in energy (ΔE) between the two electron spin levels is

$$∆\mathrm{E}=g_e{\mu }_B\mathrm{H}$$

(1)

where $g_e$ is the electron g-factor, and ${\mu }_B$ is the Bohr magneton.

Resonant absorption occurs if the microwave frequency v can meet the following equation:

$$hv=∆\mathrm{E}=g_e{\mu }_B\mathrm{H}$$

(2)

For field-swept EPR, the microwave frequency v is fixed at a constant value, and the microwave magnetic field is perpendicular to H. H is swept until the resonance condition is matched.

2.2. Radical Detection Methods

Organic radicals stabilized in bio-oil and biochar can be directly detected by EPR. However, the predominant radical population generated during pyrolysis demonstrates high reactivity, undergoing rapid chain reactions followed by annihilation. Their short lifetimes lead to difficulties in accumulating concentration and make them difficult to detect. Equipped with a variable-temperature device, EPR can realize in situ high-temperature and low-temperature detection. In situ high-temperature EPR configurations simulate the biomass pyrolysis conditions, thus enabling in situ detection of reactive radicals. However, due to the eruption of radicals and the carbon encapsulation, the EPR spectrum is a simple superposition of spectra of different radicals (Figure 2).

Cryogenic conditions significantly enhance radical detection sensitivity by prolonging transient radical lifetimes and reducing the EPR spectral linewidth [28]. Modern EPR systems implement two complementary cryogenic strategies: (1) in situ cooling via liquid nitrogen (−196 °C) or helium circulation systems, and (2) ex situ sample quenching using saturated dry ice/ethanol slurries (−78 °C). Researchers at Louisiana State University designed an isothermal flow reactor equipped with a cold finger called low-temperature matrix isolation (LTMI) EPR, schematically illustrated in Figure 3a. LTMI EPR affords the accumulation, detection, and characterization of trace quantities of radicals produced in gas-phase reactions [29]. For complex radical mixtures, by gradually raising the matrix temperature, some of the thermally labile radicals are annihilated, thereby simplifying spectral interpretation and possibly observing the types of remaining radicals [30], as shown in Figure 3b.

Spin trapping involves the addition of a spin trap to the reactive radicals, giving rise to a longer-lived radical adduct, which is more easily detected [31]. Commonly used spin traps are mainly 2-methyl-2-nitrosopropane dimer (MNP), N-tert-butyl-α-phenylnitrone (PBN), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).

2.3. Spectral Analysis of EPR

EPR spectroscopic analysis of pyrolyzed biomass radicals employs three principal spectral parameters—g-factor, linewidth, and peak intensity—as quantitative metrics for tracking radical speciation and concentration dynamics during thermal degradation processes [31].

The g-factor is used to determine the value of the magnetic field at the resonance position, for a given operating frequency. For a specific paramagnetic species, the g-value is fixed and particularly sensitive to the chemical environment [32], which is a quantity characteristic of the molecule where the unpaired electron is located, reflecting the nature of this orbital. In crystals or ordered solids, where the molecular arrangement is fixed, different magnetic interactions in different directions lead to anisotropy. In such cases, EPR spectra exhibit orientation-dependent features, such as anisotropy in the g-factor. The anisotropic g-factor can be represented by three principal g-values, g_xx, g_yy, g_zz, which apply to the orientation of the radical in the three principal directions of space with respect to the magnetic field. In isotropic conditions, the g-factor is represented by an average g-value [26]. For common organic radicals, the closer the unpaired electron is to an oxygen, the higher the g-value: oxygen-centered radicals, g > 2.004; carbon-centered radicals near an oxygen, g = 2.003–2.004; carbon-centered radicals, g < 2.003 [29].

The interaction between the unpaired electron and the nuclear magnetic moment of the atom involved in the unpaired electron orbital is called a hyperfine interaction. The hyperfine splitting is used to identify at what (kind of) nucleus the unpaired electron is localized (Figure 4). Nevertheless, the typical EPR spectra of biomass-derived matter are usually smooth curves without a hyperfine structure, as illustrated in Figure 1, because such matter contains multiple paramagnetic species.

The linewidth $(∆\mathrm{H})$ is defined as the half-width of the adsorption curve or the peak-to-peak distance of the derivative curve. Spin-exchange, spin–lattice relaxation, spin–spin relaxation, and hyperfine splitting are the factors affecting the linewidth and line shape [33]. As shown in Figure 5, an unresolved hyperfine structure gives rise to inhomogeneous broadening (“Gaussian” line shape), whereas lifetime broadening, due to a very short relaxation time, gives rise to homogeneous broadening (“Lorentzian” line shape) [34]. The spin concentration of radicals is proportional to the integrated intensity, which is calculated by double integration of the first derivative EPR line.

EPR, as a practical technique for exploring radicals, has been widely used to observe organic persistent radicals in bio-oil and biochar. For mixtures of multiple radicals, as shown in Figure 6, a numerical method has been proposed to resolve the EPR spectra as a superposition of one Gaussian line and three Lorentzian lines [35,36], which are a Gaussian curve (g = 2.00255–2.00285, ∆H = 9.1924–10.69973 G), representing simple aromatic radicals; a Lorentz 1 curve (g = 2.00357–2.00424, ∆H = 6.79062–8.14362 G), representing oxygen-containing radicals; a Lorentz 2 curve (g = 2.00241–2.00305, ∆H = 4.54199–6.98046 G), representing aliphatic hydrocarbon radicals and aromatic-structured radicals; and a Lorentz 3 curve (g = 2.00109–2.00193, ∆H = 3.5648–4.97517 G), representing oxygen-containing radicals of the σ and π types. Using these four radical types, a simple qualitative analysis of mixed radicals can be performed.

3. EPR for Model Compounds’ Pyrolysis

Due to the complexity associated with the structural heterogeneity of biomass, the tendency is to conduct experimental studies using simple model compounds, including the simplest monomer compounds, dimeric compounds, and three major components of lignocellulosic biomass, which may lead to a simpler and sharper EPR spectrum.

3.1. Monomer Model Compound

Due to the complex and irregular structure of lignin, the model compounds representing the building blocks of lignin, such as guaiacol, coniferyl alcohol (CFA), cinnamyl alcohol (CNA), and p-coumaryl alcohol, are significant in providing detailed mechanistic insights into the thermal decomposition of lignin. Lavrent Khachatryan’s team at Louisiana State University used LTMI-EPR to deeply explore the pyrolysis mechanism of simple model compounds, namely phenol [30], hydroquinone [37], and catechol [38], which are common intermediates in the pyrolysis of lignocellulosic biomass. The cyclopentadienyl radical (CPD), phenoxy radical, semiquinone radical, etc., were identified in the pyrolysis process (Figure 7). These important intermediates were consistent with the observation of cyclopentadiene, phenol, and benzoquinone, which further clarified the pyrolysis reaction pathway. The LTMI EPR spectrum of hydroquinone was dominated by p-semiquinone radicals (g = 2.0044 and ∆H = 12G) from 350 to 725 °C, which was consistent with the formation of p-benzoquinone. At higher temperature, phenol decomposed to form phenoxy radicals (above 600 °C), which converted into CPD (six distinctive lines with g = 2.0043 and ∆H = 3 G) by eliminating CO, and CPD formed naphthalene via a homogeneous radical recombination reaction [18].

3.1.1. Guaiacol

Guaiacol, a principal aromatic compound derived from lignin pyrolysis, serves as a key model compound for investigating lignin thermal decomposition mechanisms [5]. Through integrated experimental analyses of gas/liquid-phase products and computational simulations, researchers have proposed mechanistic pathways governing guaiacol conversion to dominant products including catechol, phenol, 2-methylphenol, benzofuran, and salicylaldehyde [39,40,41]. However, there is a lack of direct evidence for the processes of methylation, proton transfer, decomposition, and combination of intermediates, leaving guaiacol pyrolysis mechanisms incompletely resolved.

Li et al. [27] performed in situ EPR experiments to observe radical changes in the guaiacol pyrolysis process, which showed a clear radical signal with a g-value of 2.00018 for the central peak. To gain further insights into free radicals generated by the pyrolysis of guaiacol, complex EPR profiles (Figure 8a) were obtained by using DMPO. The g-value was 2.00036, and by isolating the characteristic peaks, the methyl radical was clearly fitted (Figure 8b), which indicated that the pyrolysis of guaiacol was initiated by a demethylation reaction and generated a large amount of methyl.

Zhou et al. [42] categorized the pyrolysis of guaiacol at 600 °C into three stages based on the reaction’s progression, and detected radicals in each stage using two methods, namely pyrolysis-frozen trapping (PFT) and pyrolysis-spin trapping (PST). In the PFT method, the pyrolysis products were rapidly frozen and detected in the high-sensitivity resonance cavity at −196 °C, while PST incorporated DMPO spin-trap agents into liquid nitrogen-quenched intermediates for radical adduct formation. Through stage-resolved radical characterization, the authors reconstructed guaiacol’s thermal decomposition pathways via radical-mediated mechanisms, as schematically summarized in Figure 9.

In the initial pyrolysis stage, only a small portion of guaiacol was pyrolyzed, and the PST method detected the presence of methyl radicals, hydroxyl radicals, and methoxy radicals, which indicated that demethylation and demethoxylation occurred. In the middle pyrolysis stage, o-semiquinone radicals (4), phenoxy radicals (7), CPD (15) (Figure 10), and methyl radicals were detected, which were intermediates of catechol (5), phenol (6), and 2-methyl phenol (3). Semiquinone radicals undergo facile disproportionation to form quinones or catechol; however, an insufficient amount of the hydrogen radical limits complete conversion to catechol. o-Benzoquinone (10) was formed from the o-semiquinone radical, which verified that the semiquinone radical is an important precursor of coke. Under a hydrogen atmosphere, the content of o-semiquinone radicals was reduced, which greatly inhibited the formation of coke, thereby improving the bio-oil yield. 2-Methyl phenol was mainly produced by the methylation of phenol. At the completion of the pyrolysis of guaiacol, phenoxy radicals, CPD, phenyl radicals (11), hydroxyl radicals, and methyl radicals were observed, which were strongly associated with the formation of benzene (12). Phenoxy radicals originated via hydrogen abstraction or unimolecular decomposition, subsequently releasing CO to generate CPD. The interactions of methyl and CPD produced fulvene (16) through dehydrogenation, followed by aromatization to benzene. Enhanced methylation promoted toluene (13) and xylene (14) formation via radical-mediated alkylation.

3.1.2. Cinnamyl Alcohol

CNA is the fundamental model compound and principal primary product in lignin pyrolysis [43], providing critical insights into radical-mediated decomposition mechanisms. Khachatryan et al. [44] performed LTMI-EPR experiments to investigate CNA pyrolysis. In the pyrolysis temperature range of 400–800 °C, anisotropic EPR spectra (Figure 11a) revealed spectral superposition from multiple radical species. The g-value decreased from 2.0096 to 2.0045 in the annealing process, indicating that the pyrolyzed radicals were mainly oxygen-centered persistent radicals, while the carbon-centered radicals dominated at lower pyrolysis temperatures. The radical adduct of PBN presents six lines with hyperfine splitting constants (${\mathsf{\alpha }}^{\mathrm{N}}=13.8 \mathrm{G}$ and ${\mathsf{\alpha }}^{\mathrm{H}}=2.18 \mathrm{G}$, where the ${\mathsf{\alpha }}^{\mathrm{N}}$ value refers to the hyperfine interaction of the N atom, and the ${\mathsf{\alpha }}^{\mathrm{H}}$ value refers to the hyperfine interaction of the H atom) in Figure 11b, which further confirms the formation of oxygen-centered radicals.

Potential energy surface analysis of cinnamyl alcohol (CNA) pyrolysis revealed a critical bond dissociation pathway, identifying the C₆H₅CH₂(CHCHO)· radical with spin density partially located on the O atom (calculated g = 2.0051–2.0063) as the predominant intermediate in cinnamaldehyde formation. As the primary thermal degradation product of CNA, cinnamaldehyde formed through two distinct mechanisms, namely (1) β-H elimination from the C₆H₅CH₂(CHCHO)· radical or (2) intermolecular hydrogen abstraction between radical species, as illustrated in Figure 12.

3.1.3. p-Coumaryl Alcohol

Xu et al. [45] employed LTMI-EPR to probe intermediate radical formation during gas-phase pyrolysis of p-coumaryl alcohol. The radicals captured in the pyrolysis reaction at 700–1000 °C all exhibited an anisotropic character with high g-values and broad linewidths, ranging from 2.0088 to 2.0101 and 14.7 to 15.3 G, respectively, indicating that oxygen-centered radicals are prevalent in the gas-phase pyrolysis products. A small amount of CPD was observed in further microwave power saturation experiments, since CPD is easily detected in large-microwave-power regions. Combined analysis of the EPR profiles and DFT calculations suggested that other signals in the profiles were related to the presence of phenoxy groups, the acetylenyl phenoxy radical (g = 2.0059), and the allenyl phenoxy radical (g = 2.0055). Phenoxy radicals were formed by scission of the phenolic hydroxyl group, and subsequently degraded by elimination of CH₃OH or dehydration and transformed into the acetylenyl phenoxy radical and allenyl phenoxy radical.

3.1.4. Coniferyl Alcohol

Coniferyl alcohol (CFA) is one of the major structural units of lignin [46], and an important model compound in the pyrolysis of lignocellulosic biomass. Barekati-Goudarzi et al. [47] investigated the intermediate radicals using LTMI-EPR. In the pyrolysis temperature range of 400–700 °C, EPR detected anisotropic spectra with high g-values of 2.009 to 2.011 and high linewidths of 13.5 to 13.0 G (Figure 13), indicating that the radicals in the gas-phase products were mainly oxygen-centered radicals. The annealing experiments showed a large number of CH₃O₂ radicals, generated by methyl groups and traces of oxygen. The CH₃O₂ methylperoxy radical showed a characteristic shape of the EPR spectrum and a hyperfine structure of 4.5–5.3 G at g = 2.0028 and g_average = 2.0103 at the crossing of the baseline. Consequently, demethylation can be regarded as a key primary decomposition process of CFA pyrolysis.

3.2. Dimeric Model Compound

The linking bonds between lignin monomers primarily consist of α-O-4, β-O-4, 4-O-5, β-5, β-β, and biphenyl bonds, with ether bonds constituting approximately 60% of the total linkages [48]. Ether bond breaking is considered a key step in lignin depolymerization. Understanding the pyrolysis mechanisms of ether-bonded compounds is essential for elucidating the initial stages of lignocellulosic biomass pyrolysis. Britt et al. [49] conducted the flash vacuum pyrolysis of methoxy-substituted β-O-4 lignin model compounds and proposed a complex reaction pathway including free radical reactions. This work demonstrated that the pyrolysis of phenylethyl phenyl ether (PPE) conforms to a free radical chain mechanism [50]. While empirical studies have confirmed radical species formation during pyrolysis [51], quantum mechanical calculations indicated predominant concerted retro-ene reactions at temperatures below 1000 °C [52]. EPR is a promising method for clarifying radical-mediated ether bond cleavage in dimeric model compounds.

3.2.1. Dimeric Model Compound Containing α-O-4 Bond

The α-O-4 bond is the second most abundant linkage in lignin after β-O-4, exhibiting the highest propensity for primary cleavage during pyrolysis to generate radical species. Kim et al. [53] used EPR to detect the pyrolysis products of benzyl phenyl ether (BPE) and two other dimeric model compounds containing α-O-4 bonds. Samples were subjected to pyrolysis at 500 °C, with reactive radicals stabilized by immediate trapping in 1,4-dioxane maintained under liquid nitrogen conditions. As shown in Figure 14, all three dimeric compounds exhibited similar anisotropic profiles with g-values of 2.0035 to 2.0036. Additionally, the radical concentrations in the methoxy substituted dimers were obviously higher than in BPE. Structural analysis combined with reaction pathway evaluation suggested these spectral features originated from carbon-centered benzyl radicals and oxygen-centered phenoxy radicals. These critical transient species, arising from homolytic C-O bond cleavage, provided direct experimental evidence supporting radical-mediated reactions in α-O-4 dimer pyrolysis.

3.2.2. Dimeric Model Compound Containing β-O-4 Bond

2-Phenylethyl phenyl ether (PPE) is the main lignin model compound of the β-O-4 linkage bond type. The pyrolysis mechanism of PPE conforms to an intermolecular interaction mechanism with the help of EPR. Lignin-derived radicals achieve stabilization through hydrogen abstraction from available donors, including native lignin structures and pyrolytic byproducts. The intermolecular interaction reactions have a much lower energy barrier than R-H homolysis, suggesting that it is more advantageous to stabilize radicals by intermolecular interaction reactions during lignin pyrolysis. Furthermore, radical propagation through chain-transfer processes accelerates lignin depolymerization, generating both new radical species and stable degradation products. For lignin model compounds (PPE, 2-methoxyphenol, and 2,6-dimethoxyphenol), hydrogen donation mediates radical termination, yielding characteristic products such as phenol, ethylbenzene, catechol, and 3-methoxycatechol. At the same time, the presence of radicals can lower the decomposition energy barriers of lignin and its derivatives through the corresponding intermediates. This synergistic mechanism between radical generation and intermolecular interactions enhances the overall efficiency of lignin thermolysis.

3.3. Cellulose

Cellulose is the most abundant polymer in nature, accounting for up to 50% of biomass [54]; therefore, it is often used as a representative of lignocellulosic biomass for probing pyrolytic product distributions under varying pyrolysis conditions. Extensive mechanistic investigations employing experimental and computational approaches have established the reaction kinetics framework [55]. Regarding the formation of cellulose pyrolysis products, radical, concerted, and ionic mechanisms have been proposed. In particular, the radical pathway requires precise elucidation through time-resolved sampling methodologies [56], which underscores the imperative for operando radical detection via in situ EPR coupled with tandem spectroscopic techniques.

3.3.1. Cellulose In Situ EPR Pyrolysis

Ma et al. [57] performed in situ EPR experiments of cellulose pyrolysis at a temperature of 300–500 °C for 10 min, followed by cooling for 5 min, with the EPR spectra recorded every 50 s. As shown in Figure 15, cellulose first decomposed into volatile and non-volatile persistent radicals, and the radical content increased dramatically. With the growth of the coke precursors, the radical concentration increased slowly. When the heating was stopped, the radical content changed significantly due to the structure shrinkage of coke and the breaking of weak bonds. This is consistent with the experimental results of Tao et al. [58], who observed that when the pyrolysis temperature reached 300 °C, numerous covalent bonds were broken and the EPR signal was significantly enhanced. Furthermore, the g-values of radicals produced during cellulose pyrolysis were less than 2.0030, which were assigned to carbon-centered radicals.

3.3.2. Radicals in Cellulose Pyrolysis Products

Radical characterization during cellulose pyrolysis presents inherent analytical challenges due to spectral superposition from multiple radicals. Persistent radicals in bio-oil and biochar exhibit a steady state linearly correlated with transient radical concentrations [17], enabling indirect monitoring of radical-mediated reactions through spin density quantification. Kim et al. [19] determined the radicals in cellulose-derived tars generated at 600 °C. The acquired spectra displayed a broad, featureless EPR signature (g = 2.0031, ΔH = 5.01 G) devoid of characteristic hyperfine splitting, indicative of either radical multiplicity or strong matrix interaction effects. This spectral pattern aligns with stabilized glucose-derived radical structures, providing critical experimental validation for a homolytic glycosidic bond cleavage mechanism during cellulose thermolysis.

Liang et al. [59] observed radicals in biochar generated from cellulose at different pyrolysis stages, and the g-values at the active pyrolysis stage (325–350 °C) were 2.0041–2.0042, which were assigned to oxygen-centered radicals. As the pyrolysis temperature increased, the radicals shifted to carbon-centered oxygen-containing radicals, the value of the g-factor gradually decreased, and the concentration of radicals increased, thus positively indicating the existence of radical reactions during cellulose pyrolysis. Bi et al. [60] detected the radicals in coke generated from cellulose pyrolysis from 300 °C to 500 °C using EPR and observed a similar phenomenon. The experimental results indicated that as the pyrolysis temperature increased, the polycyclic aromatic structure underwent condensation, which enhanced the spin–lattice interactions and prolonged the relaxation time, resulting in a narrowing of the linewidth of the spectra. By further deconvolution of the EPR spectra, it was found that at a lower pyrolysis temperature, oxygen-containing radicals dominated, and as the temperature increased, some of the ether and quinone oxygenated compounds transformed into simple aromatic ring compounds, which subsequently generated complex heavy aromatic compounds by polymerization reactions, leading to a decrease in the g-value from 2.0032 to 2.0030.

3.4. Hemicellulose

Hemicellulose is the most unstable component of biomass, comprising branched heteropolysaccharides dominated by pentoses and hexoses [61]. The prevailing reaction mechanism during its pyrolysis—whether radical, concerted, or ionic—remains contentious due to the limited experimental evidence characterizing transient intermediates. While computational studies suggest concerted pathways exhibit lower activation barriers and better align with carbohydrate pyrolysis product distributions [62], EPR analyses have detected persistent radicals in both pyrolytic vapors and biochar, confirming radical participation in the depolymerization process [63,64].

The existing studies mainly focused on the radicals in the solid products produced by xylan pyrolysis. Liang et al. [63] used xylan as a model compound, and the signals of radicals began to appear in the solid products during the active pyrolysis stage of xylan (200–350 °C), and were enhanced with the increase in the pyrolysis temperature to generate carbon-centered radicals. The g-values of the radicals decreased from 2.0035 to 2.0029 in the temperature range of 300–550 °C, indicating that the increase in the pyrolysis temperature promoted the breakage of oxygen-containing groups in the side chain of xylan. At the same time, the more intense degree of pyrolysis reaction promoted xylan to undergo the main-chain depolymerization and cyclization reaction to produce simple aromatic radicals and phenyl π radicals [60].

3.5. Lignin

Lignin, a heterogeneous aromatic biopolymer, exhibits an intricate and highly disordered three-dimensional structure composed of crosslinked phenylpropanoid units. At higher pyrolysis temperatures (e.g., >400 °C), a radical mechanism predominates [65], involving the formation of radicals formed by homolytic breakage between phenolic substituents [66], some of which undergo condensation reactions leading to the formation of coke. However, the lack of reliable mechanistic evidence has led to the radical mechanism still being debated.

3.5.1. Lignin In Situ EPR Pyrolysis

Klason lignin extracted from poplar (hardwood) and pine (softwood) has been used as a raw material for in situ high-temperature pyrolysis. The quantity of radicals during pyrolysis depends on two basic processes: the number of broken bonds to generate radicals, and the ability of the radicals to undergo quenching reactions such as recombination. Since hardwood contains more β-O-4 bonds, which are more susceptible to breaking than the 5-5 linkages, it produces a higher content of radicals. In addition, softwood contains mainly G-type units, and the 5-position is more prone to recombination reactions, resulting in a lower stability of radicals [24].

In addition, the changes in the radicals during the pyrolysis of different types of biomass feedstocks are different. For instance, the number of radicals in hardwood and softwood increased rapidly within 500 s at the beginning of the pyrolysis, and the growth rate of the number of radicals slowed down after the lignin was pyrolyzed completely. When the pyrolysis temperature exceeded 550 °C, the concentration of radicals produced by the pyrolysis of hardwood decreased, and the radical quenching reaction dominated, conjectured to be related to the disproportionation reaction of two semiquinone radicals to produce hydroquinone or quinone (Figure 16), whereas the radicals produced by the pyrolysis of softwood continued to increase.

3.5.2. Radicals in Lignin Pyrolysis Products

Zhu et al. [15] systematically investigated radical evolution during the staged pyrolysis (50–500 °C) of dealkylated lignin, elucidating key mechanistic pathways (Figure 17). A large number of C-O-C (α-O-4 and β-O-4 bonds) and C-C bonds were broken to generate G-type monomers in the temperature interval of 250–350 °C, while the concentration of radicals in the biochar increased dramatically with the temperature to generate semiquinone radicals, such as o-methoxy-substituted phenoxy radicals. The generation of catechol radicals (g = 2.00490–2.00443, ∆H = 7.72–11.67 G), which are precursors of phenolic compounds, was detected at 300–350 °C. This finding was similar to the results of lignin LTMI-EPR experiments, which detected phenoxy radicals and substituted phenoxy radicals [67]. Phenolic products detected in GC-MS accounted for more than 40% [68], which further proved that demethylation and demethoxylation reactions generate phenolic compounds such as catechol and phenol for G-type compounds. Further cleavage of lignin occurred at 350–400 °C, where C_aromatic-OCH₃ bond scission produced carbon-centered radicals. Above 450 °C, phenolic compounds converted to benzene derivatives through dehydroxylation reactions, and radical species became more complex, with σ-type oxygen-containing radicals dominating. When the temperature reached 500 °C, oxygen-centered radicals escaped in gaseous form due to accelerated molecular diffusion and reaction rates [60]. Simple carbon-centered aromatic radicals, such as phenyl radicals, predominated in the char. Carbon-centered aromatic radicals underwent polycondensation reactions to produce aromatic radicals with one to five rings. At pyrolysis temperatures below 600 °C, the concentration of radicals in the char continued to increase with increasing temperature. However, the radical content in the bio-oil was low, since the carrier gas carried the reactive intermediates from the pyrolysis vapor out in time. When the pyrolysis temperature exceeded 600 °C, lignin pyrolysis intensified, and the collision and binding of radical intermediates were enhanced, leading to radical quenching and stabilization to form the final products. In addition, the graphitization of char intensified, leading to the weakening of its ability to stabilize radicals, and thus the spin concentration of the char radicals gradually decreased. At the same time, the secondary reactions in the gas phase became violent, favoring the fragmentation of vapor molecules, which not only decreased the bio-oil yield and increased the gas yield, but also led to a sharp increase in the spin concentration of detectable radicals in the bio-oil. The three inflection temperatures for bio-oil radicals, biochar radicals, and bio-oil yields were perfectly matched at 550–600 °C [69], which indicates that the radical content resulting from radical-mediated reactions is a key indicator for pyrolysis temperature optimization to obtain a high yield of bio-oil. Overall, the radical-mediated lignin pyrolysis process was demonstrated by observing the changes in active radicals during lignin pyrolysis and the types of persistent radicals in the pyrolysis products.

4. EPR for Lignocellulosic Biomass Pyrolysis

Biomass pyrolysis exhibits complex mechanistic behavior arising from synergistic interactions among its three primary components. The mechanistic complexity originates from massive radical generation via chemical bond dissociation, producing oxygen-centered and carbon-centered radicals that partition into gas-phase volatiles, condensed bio-oil, and solid biochar. These persistent radical species, detectable through EPR spectroscopy, enable sequential characterization of their cooperative transformation dynamics during thermal decomposition.

4.1. Radical Behavior of Three Components

The three lignocellulosic constituents—cellulose, hemicellulose, and lignin—exhibit distinct pyrolytic behaviors due to their contrasting molecular structures. Lignin, characterized by branched aryl-ether networks, undergoes preferential β-O-4 bond cleavage during initial pyrolysis, generating persistent radicals with elevated spin densities [19]. The pyrolysis process in lignin contains a large number of phenols, of which the guaiacyl unit is the main section for generating radical signals [70], leading to slightly higher g-values of 2.0030–2.0040 for radicals in the pyrolysis products than in cellulose and hemicellulose, which are oxygen- and carbon-centered mixed radicals [58].

The cross-component interactions among cellulose, hemicellulose, and lignin fundamentally govern pyrolytic reaction networks through radical-mediated pathways. Synergistic effects between cellulose and hemicellulose during co-pyrolysis preferentially convert aromatic/aliphatic radicals into oxygenated species at 300–650 °C, as evidenced by EPR analyses of biochar. When the pyrolysis temperature exceeded 650 °C, the hydrogen radicals provided by hemicellulose were consumed to generate small and stable structures, resulting in a lower hydrogen yield compared to the pyrolysis of individual components [64]. Cellulose–lignin interactions exhibited distinct temperature-dependent behavior: Lignin initiated pyrolysis at lower temperatures (200–350 °C), generating stabilized phenoxyl radicals [61] that became encapsulated within nascent char. Subsequent cellulose pyrolysis (400–500 °C) released volatile oxygen-centered radicals, which underwent coupling reactions with lignin-derived aromatic intermediates, accelerating polycyclic condensation through Diels–Alder cyclization [57].

4.2. Radicals in Lignocellulosic Biomass Pyrolysis Products

The native lignin in biomass feedstock inherently contains stabilized radicals (g = 2.003, $∆H$= 6 G) with a spin density of 10¹⁷ spins/g order of magnitude [57], which are formed by polymerization of the natural lignin in wood through acid or fungal attack [71]. Biomass pyrolysis generates not only a significantly higher quantity of radical species but also greater diversity compared to monomeric systems, rendering direct spectroscopic characterization particularly problematic.

Biomass pyrolysis is mechanistically categorized into two distinct stages: primary and secondary reactions. Primary reactions usually contain depolymerization, cracking, and carbonization reactions [72]. The depolymerization and cleavage reactions generate a large number of radical intermediates, such as the breaking of the C_aromaticO-CH₃ bond in lignin to generate o-methoxy-substituted phenoxy radicals and σ-type oxygen-containing radicals [73]. Short-lived methyl radicals can react with other radicals, such as hydrogen radicals, to form methane. Some of the longer-lived persistent radicals are encapsulated on the nascent char and stabilized by steric hindrance of the macromolecules [74]. The secondary reactions mainly contain secondary cracking and secondary polymerization reactions. Secondary cracking reactions refer to the pyrolysis vapor component further undergoing a depolymerization reaction or a cleavage reaction, forming smaller molecular substances, which continue to be cracked to generate the non-condensable gas [75]. The reactive radicals generated during this process are partially annihilated as the carrier gas is blown away, and some of the longer-lived radicals may remain in the generated pyrolysis products. Secondary polymerization reactions refer to the conversion of volatile pyrolysis vapor into products of higher molecular weight, involving polymerization and recombination reactions of radicals, such as secondary condensation of primary G-type guaiacol and furan to form coke, where EPR signals attributed to the one- to five-ring carbon-centered aromatic radicals were detected [73].

4.2.1. Radicals in Bio-Oil

When the biomass pyrolysis temperature is low (<450 °C), a large number of transient radicals are generated by the breaking of covalent bonds as the temperature rises, but these radical reactions occur mainly in the solid phase, while the gas phase (including condensable bio-oil and non-condensable gas) is instantaneously moved out of the pyrolysis zone by an inert carrier gas. As the pyrolysis vapors condense, the gas-phase radicals are quenched so rapidly that only a few radicals within the bio-oil are eventually detected. However, as the pyrolysis temperature exceeds the critical point (600 °C), the secondary reactions in the gas phase become violent, and these disordered vapor molecules are cleaved in large quantities, leading to the production of extremely abundant radicals.

The presence of stable radicals in bio-oil with g-values of 2.0026 to 2.0033 and ∆H of 3.2 to 5.2 G suggested that they are predominantly carbon-centered radicals, with a spin concentration of about 10¹⁶ spins/g order of magnitude [20]. Similar to the EPR signals obtained from isolated lignin, no hyperfine structure was observed from bio-oil, suggesting that the EPR spectra are simple averages of highly complex radical species. During the aging process of the bio-oil, there was a slight decrease in the radical signal, indicating that slight free radical annihilation occurred. However, the g-value and linewidth remained constant, suggesting that radicals in bio-oil can be stabilized under heating aging and the addition of radical scavengers, and do not have a serious effect on the aging of the bio-oil [76]. Highly reactive functional groups such as vinyl, which were used in lignin biogenic oils, play an important role in the re-polymerization process [19].

The high oxygen content (35–50 wt%) of crude bio-oil necessitates upgrading for fuel applications, as it confers a low calorific value (<20 MJ/kg), high acidity (pH 2–3), and instability during storage [77]. During the heating process of bio-oil refining, the active components in the bio-oil will polymerize to form solid carbon deposits through radical-mediated reactions [78]. The coking process occurs very rapidly and is difficult to observe, and the evolution of radicals during the reaction process can be observed using in situ EPR [79]. As illustrated in Figure 18, in the early stage of bio-oil pyrolysis, the small-molecule radicals generated by side-chain cleavage react rapidly to generate gas. The content of stabilized radicals enters an induction period, at the end of which a large number of stabilized radicals are rapidly produced. After a sufficiently vigorous reaction, the content of stabilized radicals increases slowly with the reaction time [80]. At the same time, light oxygen-containing components gradually polymerize into heavy components to generate coke precursors. Some oxygen-containing components form oxygen-centered radicals, and σ- and π-type oxygen-containing radicals are trapped together with aromatic radicals in the nascent char [81]. Finally, they are stabilized depending on the steric hindrance of the macromolecular structure. Therefore, the content of stabilized radicals is roughly proportional to the char yield. When the heating is stopped, the carbon structure shrinks due to cooling stresses, leading to the breakage of some oxygen-containing heterocycles or oxygen bridge bonds and the generation of some oxygen-centered radicals. These radicals will be gradually annihilated, and a small portion will be converted to oxygen-containing stable radicals on the branched chain. In this process, the aromatic cluster supports the structure of the biochar and the stabilized radicals remain unchanged [25].

Semiquinone radicals identified during biomass pyrolysis implicate o-quinone methide as a critical intermediate in lignin-derived coke formation [82]. In addition, coking is the biggest problem for in situ EPR in detecting reactive radicals. Through hydrogenation reactions, semiquinone radicals are susceptible to being converted to catechol, while inhibiting the conversion of semiquinones to quinones [42]. Therefore, introducing an external hydrogen source to increase the hydrogen concentration promotes the active radicals to combine with hydrogen radicals to form small molecules, thus blocking the reaction path of polymerization into large molecules. After introducing hydrogen as a source during biomass pyrolysis, named hydropyrolysis, the results showed that H₂ can effectively reduce the relative radical concentration [83]. 9,10-Dihydrophenanthrene (DHP) is a typical hydrogen-donating solvent, and the radicals generated by pyrolysis can be coupled or stabilized by obtaining hydrogen radicals from the solvent, which is converted to the corresponding aromatic hydrocarbon (phenanthrene) [84]. Zhou et al. [85] compared the effects of H₂, CH₄, and CH₃OH on the pyrolysis of poplar wood. The concentrations of all radicals were decreased with the addition of the hydrogen source. The addition of methanol caused the highest decrease, with a 48% reduction in concentration, promoting a reduction in coke yield on the catalyst surface. Thus, the added hydrogen radicals can react with the coke to enhance its aromaticity.

Bio-oil upgrading is usually centered on deoxygenation, which infers catalytic hydrogenation, and the selection of suitable catalysts for catalytic cracking is vital [86,87]. A suitable catalyst is the key to generating high-yield hydrocarbons, and the ideal catalyst must have good hydrodeoxygenation activity. Oxygen vacancies (OVs) on the catalyst surface have a crucial influence on the catalytic performance, and EPR is a powerful technique for detecting the strength of OVs in solid catalysts [82,88]. The results of experimental studies have shown the presence of symmetric peaks near g = 2.006, which is a typical diagnostic signal for OVs [89]. OVs, as the main sites for the adsorption of reactants, optimize the adsorption energies of the reactants on the catalyst surface, lower the reaction energy barriers, and synergistically act with the nearby active metal sites to improve catalyst deoxygenation activity [90].

When using catalysts for the in situ hydrodeoxygenation of biomass pyrolysis oils for quality enhancement, the effectiveness of the catalytic reaction can be evaluated by the total radical concentration of the pyrolysis oil products, with lower radical concentrations indicating more effective catalysis [1,42]. Changes in radical concentration are associated with chemical bond-breaking reactions and radical recombination reactions. Zeolite-based catalysts stabilize the transition state of bond dissociation reactions through close contact of the surface with the broken bonds, thus slowing down the reaction rate of radical recombination [91], and reducing the generation of macromolecular char. The results of a biomass pressurized hydropyrolysis vapor upgrading system demonstrated that the catalysts screened by EPR characterization exhibited high conversion efficiency and resistance to carbon accumulation in biomass hydropyrolysis, with C4+ hydrocarbon yields as high as 23.81% [85].

4.2.2. Radicals in Biochar

During biomass pyrolysis, the changes in radicals in biochar can be divided into three stages. In the first stage, the concentration of radicals in biochar increases rapidly due to homolytic cleavage of C-C and C-O bonds. In the second stage, the content of stabilized radicals increases slowly in the nascent char at a constant pyrolysis temperature [74]. This is because the nascent char gradually polymerizes small aromatic rings into larger ones through radical condensation reactions, so that more stable radicals can be retained in the biochar [92]. The secondary reaction of biochar is relatively slow and therefore the rate of increase of stabilized radicals is lower than in the first stage. Finally, in the cooling stage, the cooling stress can induce radical reactions [93,94], and the content of radicals increases slightly and then gradually decreases and remains stable [95]. These stabilized radicals are called environmentally persistent free radicals (EPFRs), which are defined as surface-stabilized metal–radical complexes [96,97,98]. Due to the spectral overlap caused by the complex composition of radicals, all pyrolyzed biochars show similar overall spectral characteristics. A typical EPR spectrum is a single symmetric resonance line. The g-values vary slightly, commonly in the range of 2.0028–2.0041, consisting of either oxygen-centered or carbon-centered radicals [99]. The relative predominance of O- or C-centered radicals in the radical mixture depends mainly on the pyrolysis temperature, residence time, and structure of the precursor [100]. EPFRs are considered to be a special type of oxygen-centered radical, such as hydroxyl, hydroperoxy, and superoxide anion radicals, as well as non-radical derivatives of O₂, such as alkoxy, peroxy, semiquinone radicals, and hydroperoxide [101,102,103]. DFT calculations suggest that stable radicals are most likely to be hydroxylated polycyclic aromatic hydrocarbon structures, such as phenoxy radicals [21]. The maximum spin concentration of all biogenic carbons is 10¹⁹–10²⁰ spins/g in the temperature range of 500–600 °C [22].

4.2.3. Influencing Factors of EPFRs

Understanding the presence of radicals and oxygen heteroatoms is essential for understanding and predicting the evolution of biochar. EPFRs depend on the physicochemical properties of the biochar, including its conductivity, pH, charge density, calcium carbonate content, volatile and non-volatile organic molecules, and elemental composition, which are related to the feedstock type and pyrolysis conditions [104].

The pyrolysis temperature is a key factor affecting biochar radicals. The g-value decreases with increasing temperature, indicating a shift in the type of EPFRs from oxygen-centered and carbon-centered radicals to carbon-centered radicals [105]. The increasing temperature leads to the breaking of C-O, C=O, and O-H bonds, and subsequently these oxygenated functional groups convert into gaseous form, thus reducing the oxygen-containing radicals in biochar [106]. Oxygenated persistent radicals in biochar originate from C=O functional groups in quinones, aldehydes, or ketones. If KBH₄ treatment is added, some of the carbonyl groups will reduce, resulting in the reduction of radicals [58]. The increase in pyrolysis temperature promotes the formation of aromatic C=C bonds, which corresponds to the increase in carbon-centered radicals [107,108]. Thus, a higher pyrolysis temperature leads to the decomposition of oxygen-centered radicals into carbon-centered radicals.

A moderate pyrolysis temperature (400–600 °C) favors the formation of EPFRs, and the volatiles released in this temperature range are transferred into tar and radicals in the char [109]. In contrast, an excessive temperature (700 °C) leads to continued decomposition of char and a combination of highly reactive radicals and other molecules to generate small-molecule gas, which causes a significant decrease in the intensity of the EPR signal. In general, the free radical concentration shows a tendency to first increase and then decrease with increasing temperature.

The increase in pyrolysis temperature causes an increase in the degree of aromatization of the biochar, and the large polycyclic aromatic structure enhances the spin–lattice interaction, prolongs the relaxation time, and narrows the linewidth. Ma et al. [57] further found a positive correlation between the concentration of the persistent radicals and the area ratio of the aromatic structure bands (the area ratio of the D band to the sum of the V_r, V_l, and G_r bands, which represents the ratio of large and small aromatic rings in the coke, reflecting the degree of condensation of the aromatic structure), and the correlation coefficient R² was about 0.94. This is mainly because the persistent radicals are active sites in the coke, and their existence depends on the steric hindrance of the macromolecules. Both the steric hindrance and conjugation effect of aromatic structures contribute to the stability of EPFRs. This steric hindrance effect also occurs in pyrolyzed carbon from biomass pellets. Under the same reaction conditions, the concentration of EPFRs in pyrolysis char from biomass pellets was consistently higher than that from powdered biomass, due to the fact that the generated EPFRs were more readily captured by the stabilized chemical environment of the pellet char. Furthermore, due to the higher mass transfer resistance, it is difficult for radicals to react with other radicals in the neighboring region [110].

The effect of the residence time on persistent radicals in biochar is similar to that of the pyrolysis temperature. At a shorter residence time, EPFRs can be formed by side-chain breaks and gradually accumulate on the surface of pyrolyzed biochar [93]; excessively long residence times lead to radical recombination and the formation of nonparamagnetic materials, resulting in a decrease in radical content [106]. Increasing the residence time caused a decrease in the linewidth of the persistent radicals, a decrease in the g-value, and a shift in the type of EPFRs from a mixture of oxygen-centered and carbon-centered radicals to carbon-centered radicals [111].

In addition to pyrolysis conditions, the elemental composition of the biomass feedstock, ash content, and three-component content affect the concentration of EPFRs [112,113]. The pyrolysis of the three components in biomass produces radicals with different behaviors, with lignin contributing to the persistent radical signal over a wide temperature range, from feedstock (100%) to 600 °C (55%), and cellulose contributing 12–23% of the EPFR signal over the 360–600 °C temperature range, whereas hemicellulose contributes 5% [114]. In addition to this, biomass contains inorganic compounds that affect the generation of polycyclic aromatic compounds during pyrolysis, causing a decrease in the radical spin concentration [115,116]. Thus, biomass feedstocks have an important influence on persistent radical signaling. The persistent radicals produced by different biomass feedstocks are shown in

Table 1. Concentration, g-factor, and linewidth of EPFRs in biochar samples.

Biomass Type	Pyrolysis Temperature (°C)	g-Factor	Linewidth (G)	EPFR Concentration (1018 spins/g)	EPFR Type	References
Pine needles	400	2.0037	6.8	15.2 ± 0.03	Oxygenated carbon-centered	[117]
Pine needles	550	2.0028	4.5	13.7 ± 0.06	Carbon-centered	[105]
Wheat straw	400	2.0030	5.0	16.5 ± 0.09	Oxygenated carbon-centered	[105]
Wheat straw	500	2.0029	4.8	28.6 ± 0.12	Carbon-centered	[105]
Maize straw	400	2.0031	6.2	6.25 ± 0.12	Oxygenated carbon-centered	[105]
Maize straw	500	2.0029	5.2	30.2 ± 0.09	Carbon-centered	[105]
Rice husk	300	2.0041	2.77	6.9 ± 0.1	Oxygen-centered	[118]
Rice husk	700	2.0036	0.16	1.8 ± 0.1	Oxygenated carbon-centered	[118]
Corn straw	500	2.0030	3.2	1.9 ± 0.03	Oxygenated carbon-centered	[106]
Peanut husk	500	2.0032	4.8	2.2 ± 0.02	Oxygenated carbon-centered	[106]
Cotton stalk	500	2.0032	4.1	2.2 ± 0.02	Oxygenated carbon-centered	[106]

.

The metallic constituents in biomass ash, particularly transition metals (e.g., Fe, Mn, Al) and alkali/alkaline earth metals (e.g., K, Mg), exert a significant influence on EPFR concentrations through distinct interfacial mechanisms. Transition metals facilitate EPFR stabilization via a three-stage process: phenolic precursors initially adsorb onto metal oxide surfaces through physical interactions, followed by chemisorption establishing metal–oxygen–carbon bonds, ultimately enabling electron transfer from transition metal d-orbitals to organic molecules that stabilize unpaired electrons [58,119]. This electron delocalization mechanism creates positive correlations between EPFR concentrations and the transition metal content (Al, Fe, Mn). Concurrently, alkali and alkaline earth metals have a catalytic effect on biomass pyrolysis [120,121], thus affecting the intensity of radicals in biochar. K and Mg contents have a catalytic effect on the concentration of radicals [106,122], where the interaction between Mg and oxygen-containing functional groups is capable of weakening the strength of intramolecular bonds, and K is capable of causing the formation of reactive aliphatic radicals by promoting the breakage of the C-C bond in the aryl ring at elevated temperatures, which are capable of further generating stabilized polyaromatic compounds through polymerization/crosslinking reactions [123,124]. Similarly, Ma et al. [74] found that alkali and alkaline earth metals at elevated temperatures (>450 °C) can promote coupling reactions of radicals, leading to a decrease in the content of stabilized radicals.

5. EPR for Pretreatment of Biomass Pyrolysis

The inherent heterogeneity of lignocellulosic biomass in composition and structure necessitates tailored pretreatment protocols to enhance compatibility with pyrolysis reactors and improve product uniformity. Common pretreatment methods include drying and crushing, acid treatment, torrefaction, and hydrothermal treatment [125,126,127]. Systematic evaluation combining EPR analysis of pretreated feedstocks with radical speciation in pyrolysis products enables quantitative assessment of pretreatment effects on radical generation/annihilation kinetics. This methodology facilitates mechanistic correlations between pretreatment parameters and radical-mediated reaction pathways, ultimately guiding the development of energy-efficient pretreatment protocols optimized for targeted product distributions.

5.1. Effect of Lignin Extraction Method on Pyrolysis

Lignin isolated from biomass contains substantial stabilized organic radicals [128,129], and most of its species are semiquinone radicals stabilized in the polyphenolic lignin matrix [130,131]. The extraction method of lignin affects the chemical structure of lignin, which causes changes in the electronic structure of the radicals. Moreover, it was found that the plant source has less influence on the concentration and g-value of radicals in lignin than the extraction method. The extraction methods involve different temperature and pH environments, which cause lignin to undergo bond breaking or radicals to undergo protonation or deprotonation processes, leading to changes in the concentration and species of radicals.

Klason lignin, dioxane lignin, and organosolv lignin obtained from three different extraction methods showed different changes in the concentrations and g-values of the radicals. The approximately 10-fold increase in radical concentration in organic solvent-extracted lignin was attributed to the fracture of the homolytic bond at 200 °C. In addition, the g-value of the semiquinone radicals decreased with decreasing pH, attributed to the conversion of the radical anion to the radical cation via the neutral species, as shown in Figure 19 [132]. When the environmental pH reaches alkaline conditions, hydroquinone and quinones are converted to semiquinone radicals and semiquinone anions, and the content of radicals increases significantly [133,134].

However, lignin tends to be extracted in a way that does not significantly alter its chemical structure, and the stabilized radicals have a minor influence on the pyrolytic behavior of lignin [17,135]. For technical hot-water-extracted lignin, kraft lignin, and soda lignin obtained by different extraction methods, the behavioral trends, such as temperature-induced changes in yield distributions and changes in radicals, were similar. There are subtle differences in the concentrations of persistent radicals [69], which may be related to the radical changes due to the lignin extraction method. Since the distribution of pyrolysis products is similar, it is still not possible to determine the effect of radical species changes on the intermediates of the pyrolysis process.

5.2. Effect of Pre-Oxidation Method on Pyrolysis

Shielding the benzyl hydroxyl group effectively inhibits the condensation of lignin during the conversion process, and has been successfully used for the hydrodepolymerization of lignin [136]. Fan et al. [16] carried out the hydroxyl shielding of lignin using pre-oxidative treatments, and the experimental results showed that pre-oxidation weakened the thermal stability of lignin. Nevertheless, an increase in char yield and a decrease in pyrolysis oil yield were observed. Characterization of the radicals in the liquid-phase products and solid-phase products by EPR revealed a large number of oxygen-containing radicals, which was attributed to the disruption of the carbonyl groups produced by pre-oxidation and the 1,3-dioxane side-chain structure. These oxygen-containing radicals exacerbated the condensation of pyrolysis intermediates, resulting in the formation of char and coke, while decreasing the yield of the liquid compounds. Consequently, the provision of hydrogen radicals to react with oxygen-containing radicals and promote the hydrodeoxygenation reaction is an effective method for inhibiting the formation of char.

5.3. Effect of Torrefaction Method on Pyrolysis

The higher oxygen content (~40%) in bio-oil limits its direct application as fuel [137]. Torrefaction treatment effectively reduces the oxygen content of the biomass feedstock [138,139,140]. For beech trunks roasted at 200–300 °C, torrefaction changed the chemical environment of the H atoms [141], and the lignin demethoxylation reaction occurred, leading to an exponential increase in radicals. Meanwhile, the linewidth of the EPR spectral lines decreased linearly with increasing torrefaction temperature. The carbon content increased, while the hydrogen and oxygen contents decreased, correlating with the trend of a linear decrease in the g-value of the radicals. In addition, torrefaction at 300 °C may destroy cell walls, causing a significant increase in the concentration of radicals [142]. Therefore, roasted biomass pyrolysis oils have a lower oxygen content, higher effective hydrogen-to-carbon ratios [143], and more stable chemical compositions, which are expected to reduce the formation of coke during the pyrolysis oil refining process [144], and contribute to the pyrolysis production of high-quality bio-oil.

5.4. Effect of Plasma Method on Pyrolysis

Non-thermal plasma technology is an efficient pretreatment method for increasing the roughness of cellulose by ablating the biomass surface [145], which can improve the hydrolysis [146,147] and catalytic upgrading of bio-oil [148]. Plasma treatment was found to significantly change the surface structure of cellulose by EPR spectroscopic examination. As exhibited in Figure 20, the plasma-irradiated glucose units at the C1 position formed alkoxy radicals by hydrogen abstraction, and hydroxyalkyl radicals at the C2 and C3 positions dehydrated to further generate acyl alkyl radicals [149]. Moreover, the low-temperature plasma method was able to significantly promote cellulose pyrolysis to produce high yields of levoglucosan; however, the changes in cellulose microstructure, crystallinity, solubility, thermal stability, and functional groups could not account for the elevated levoglucosan yields. The radical profile of plasma-treated cellulose was examined by EPR and showed a significant positive correlation between the concentration of radicals in cellulose and levoglucosan yield, which was attributed to the homolytic cleavage of glycosidic bonds by the plasma treatment, resulting in the formation of persistent radicals, which promote chain-negative growth reactions and levoglucosan formation [150]. Plasma-induced chain excitation and radical formation reduce the formation of interchain hydrogen bonds, which reduces the energy demand during subsequent pyrolysis and promotes biomass depolymerization.

6. Current Limitations and Future Prospects

Extensive investigations employing electron paramagnetic resonance (EPR) have established radical-mediated pathways as central to lignocellulosic pyrolysis mechanisms. Many studies have detected persistent radicals in bio-oil and biochar, and further discussed the relationship between the quantity and type of radicals and pyrolysis conditions, which has provided a radical view of depolymerization kinetics and product selectivity. In particular, the generation and influencing factors of EPFRs in biochar have been comprehensively explored, and the rule of radicals’ evolution varying across different pyrolysis conditions has been established. For transient radicals, spin trapping and cryogenic EPR have made progress in detecting some longer-lifetime radicals. In situ EPR has also observed some obvious characteristics of reactive radicals, such as their explosion and quenching. However, the types of radicals that can be identified in the pyrolysis process are very limited, which hinders further investigation into radical-mediated reactions.

The generation of radicals is an instantaneous process with massive radical outbursts. It is challenging to capture and distinguish these complex mixtures in time. In addition, different types of radicals are generated in an inconsistent time and space. The existing X-band EPR detection accuracy is insufficient to differentiate these radicals and their reactions, so it is necessary to introduce a high-frequency EPR instrument to resolve the three principal values of the g-factor, g_xx, g_yy and g_zz, and to deeply analyze the complete fine-structure spectrum. Due to the lack of standard spectra of radicals in different solvents, it is not completely accurate to judge the specific structure of EPR spectra. The development of standardized radical reference libraries through collaborative initiatives is urgently needed to enable cross-study comparisons. Using AI-driven spectral deconvolution algorithms to analyze the obtained spectra is a practical solution. Now, there are related research and explorations in the market, which is effective for a single-radical spectrum. Nevertheless, the current system is still immature, and further optimization is still needed for spectra of multiple radicals.

Hydropyrolysis is an important development direction of biomass pyrolysis technology, which introduces the hydrogen source to reduce coke and upgrade the quality of bio-oil. But the mechanism of the radical-mediated reaction between hydrogen radicals and reactive radicals remains unclear. The hydrogen radicals play a pivotal role in determining the distribution of products. By tracking the hydrogen radicals and their addition reaction pathways through EPR combined with isotopic labeling, it is possible to gain a deeper understanding of the principle of hydrogenation, and to regulate the effect of the hydrogen source on the distribution and quality of the biomass products.

The influencing factors of EPFRs have been comprehensively investigated, while the radical evolutionary pattern of bio-oil has not yet been systematically theorized. The yield and quality of bio-oil can be improved by optimizing the pyrolysis conditions. Existing studies mainly focus on temperature effects, while systematic investigation of pressure modulation, residence time optimization, and carrier gas composition remains underdeveloped. Particular emphasis should be placed on bridging the molecular-to-macroscopic divide by correlating radical signatures (g-values, hyperfine constants) with bulk fuel properties (oxygen content, efficiency of hydrogenation and aging).

7. Conclusions

Pyrolysis is a vital technique for converting biomass into sustainable fuel. However, the mechanism of pyrolysis remains unclear and lacks direct evidence of intermediate product generation and transformation. EPR, a powerful tool for detecting intermediate radicals, has been successfully applied to the analysis of the pyrolysis process and pyrolysis products. This review comprehensively elaborated on the application of EPR spectroscopy in exploring the radical-mediated reactions in lignocellulosic biomass pyrolysis, covering radical detection methods, transient and persistent radicals of model compounds and biomass pyrolysis, and the effects of pretreatment on the radical behavior of pyrolysis. The application of EPR in model compounds’ pyrolysis has helped to identify a variety of radicals and clarify their changing rules and reaction paths. Furthermore, the persistent radicals in bio-oil and biochar were emphasized, focused on their formation and impacts.

However, EPR for biomass pyrolysis research still encounters problems, such as its resolution being below the free radical lifetime, its difficulty in spectral analysis, and coke wrapping. At present, by comprehensively investigating the relationship between “pyrolysis conditions—chemical bond breaking—product properties”, EPR has proved its unique advantages in studying the changing laws of radicals and exploring the regulation mechanisms. In the future, high-frequency EPR and pulse EPR combined with intelligent analytical methods will be used to systematically study the evolution of reactive radicals and further clarify the pyrolysis mechanism, so as to expand the application of EPR in hydropyrolysis, catalytic hydrodeoxygenation, and other aspects.