Thermochemical Delignification

Biological Delignification

In nature, lignin is degraded by an array of micro-organisms. Separation of lignin from the lignocellulosic biomass followed by depolymerization and mineralization is accomplished through synergistic interactions between bacteria and fungi, the latter being the major key players (Lee et al., 2019; Atiwesh et al., 2021). Typically, brown rot, white rot, and soft rot fungi are regarded as the main mycobionts involved in delignification, while white rot fungi being more effective. Few white rot fungi, apart from delignification, are able to degrade other components of lignocellulosic biomass as well, while others are selective delignifiers (Janusz et al., 2017). For example, Heterobasidium annosum (Maijala et al., 2003), Phlebia spp. (Arora and Sharma, 2009), Physisporinus rivulosus (Hilden et al., 2007), Irpex lacteus (Duan et al., 2018), Dichomitus squalens (Marinović et al., 2018), Phanerochaete chrysosporium, Pleurotus ostreatus (Kerem et al., 1992), Ceriporiopsis subvermispora (Honda et al., 2019), and Trametes versicolor (Bari et al., 2019). In comparison to fungal studies, research on potential of bacterial lignin deconstruction is scarce. Some of the lignin degrading bacteria mostly belong to the actinomycetes, firmicutes, and proteobacteria, mainly encompassing Sterptomyces, Rhodococcus, Pseudomonas, and Bacillus strains (Bugg et al., 2011; Wilhelm et al., 2019).

Fungal assemblages have been observed to depolymerize the lignin into monomers/low-molecular weight aromatics which are subsequently assimilated by bacteria for carbon and energy (Bugg and Winfield, 1998). Although bacteria are reported to play little role in direct delignification, mineralization is predominantly governed by them (Kamimura et al., 2017). Recently, bacterial systems have gained essence in lignin valorization due to their inherent processes, which are capable of channelizing multiple aromatic streams into a uniform compounds (Linger et al., 2014). Therefore, challenges associated with lignin heterogeneity and undesirable outcome can potentially be overcomed. Moreover, enzymatic hydrolysis yields monomers and subsequently mineralize them which prevents the re-polymerization of products. For example, in a bioconversion kinetic study, Pseudomonas putida KT2440 was incubated with two types of lignins—Kraft lignin and synthetic dehydrogenopolymer (DHP), and the phenolic monomers (dihydroferulic acid) formed transitorily were metabolized within 24 h (Rouches et al., 2021). Lignin degrading ability of such microbes are attributed to their potential to produce extracellular enzymes, called “ligninases”, which mainly include laccases, peroxidases, oxidoreductases, dye-decolorizing peroxidases, and versatile peroxidases.

Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are N-glycosylated extracellular multi-copper oxidases containing histidine-rich copper-binding domains (Messerschmidt and Huber, 1990). To date, laccases are reported from plants, fungi, and bacteria, however, most of the laccases characterized pertaining to their delignification ability are of fungal origin (Chauhan et al., 2017), with scarce incidences in bacteria. Although fungal laccases are commonly used owing to their higher redox potential, prokaryotic laccases which exhibit extreme thermostability, pH stability, and rapid proliferation make them ideal for industrial applications (Janusz et al., 2020). Unlike peroxidases, laccases do not produce toxic peroxide intermediates and function without the cofactors, like, NAD(P)H (Santhanam et al., 2011). Due to comparative low redox potential than other ligninases, laccases act only upon the phenolic compounds, however, their activity can be extended to non-phenolic compounds as well by the addition of mediators, such as, vanillin, p-coumaric acid, acetosyringone, syringaldehyde, 3-hydroxyanthranilic acid (HAA), 1-hydroxybenzotriazole (HBT), 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Kontro et al., 2020). Apart from enhancing the substrate specificity and oxidative capacity of laccases, mediators also prevent re-polymerization (Grelier and Koumbe, 2016; Huber et al., 2016). Together with mediators, these enzymes are able to perform Cα-Cβ, β ether and Cα oxidative cleavage. Besides mediators, some other enzymes are also used to enhance the lignin degradation by enzymatic hydrolysis viz., aryl-alcohol oxidase, catechol 2, 3-dioxygenase, feruloyl esterase, lipases, and quinone reductases (Kumar and Chandra, 2020).

Lignin peroxidases (1,2-bis(3,4-dimethoxyphenyl)propane-l,3-diol:hydrogen-peroxide oxidoreductases, EC 1.11.1.14) (LiP) are a group of heme-containing glycoproteins which use hydrogen peroxide (H₂O₂) for their activity to oxidize both phenolic and non-phenolic lignin constituents (Chowdhary et al., 2019). They exhibit high redox potential, less substrate specificity, besides maintaining their activity at low pH (Falade et al., 2017). These peroxidases preferably oxidize methoxylated aromatic ring without a free phenolic group. LiPs are used commercially to mineralize a variety of recalcitrant aromatic compounds, like, three- and four-ring polyaromatic hydrocarbons, polychlorinated biphenyls, and natural dyes (Falade et al., 2017). They mostly work by transforming the lignin degradation fragments released by the activity of manganese peroxidase.

Manganese peroxidases [Mn (ll): hydrogen-peroxide oxidoreductases, EC 1.11.1.13] (MnP) are glycosylated, heme-containing extracellular enzymes which require Mn²⁺ to oxidize lignin (Xu et al., 2017). On account of the lower redox potential of MnP-Mn complex in comparison to lignin peroxidases, it can oxidize both phenolic and non-phenolic substrates via lipid peroxidation reaction (de Eugenio et al., 2021). They are produced by the majority of fungi and acts by oxidising Mn²⁺ to Mn³⁺ which further oxidizes the phenolics into pheoxy radicals. These radicals, upon further reaction can result in the release of CO₂ or cleave the alkyl-alkyl or/and alkyl-phenyl bonds to form monomers/low molecular weight intermediates, such as, quinones and hydroxyl quinines (Kumar and Chandra, 2020).

Dye decolourizing peroxidases (Reactive-Blue-5: hydrogen-peroxide oxidoreductase, (EC 1.11.1.19) include those peroxidases (DyP) which decolorize anthraquinone derived dyes. The ability of delignification in such enzymes is due to the structural similarity of these dyes with that of lignin (Catucci et al., 2020). Therefore, these microbes can be detected through their dye-decolourizing capability. Two types of dye decolourizing peroxidases- DypA and DypB were identified based on bioinformatics analysis. DypB was characterized from a soil bacterium Rhodococcus jostii RHA1 which was able to degrade the Kraft lignin via C(α)-C(β) bond cleavage (Ahmad et al., 2011). Similarly, BaDyP, a dye decolourizing peroxidase from Bacillus amyloliquefaciens was found to decolourize dyes and degrade guaiacylglycerol-β-guaiacyl ether (GGE), a phenolic β-ether lignin model (Yang et al., 2019).

Versatile peroxidase (VP) reactive-black-5:hydrogen-peroxide oxidoreductase, EC 1.11.1.16) are the monomeric glycoproteins (40–50 kDa) with two conserved calcium binding sites and four conserved disulfide bridges, and are capable of acting without redox mediators (Perez-Boada et al., 2005). These constitute a unique type of lignin oxidizing peroxidases which possess the combined catalytic potential of manganese peroxidases and lignin peroxidases, the property that makes them the hybrid enzymes. However, direct cleavage of high reduction potential substrates and independent oxidation of Mn²⁺ ions makes them different from LiP and MnP (Camarero et al., 1999). Also, unlike the other oxidases, they work without the redox mediators (Moreira et al., 2007).

Delignifiers are widely distributed in nature and can be found in soil, water, air, coal, gut of rumens and termites, and inside the plants as endophytes (Xiong et al., 2014; Suman et al., 2016; Wang et al., 2016; Li et al., 2017; Zhou et al., 2017).

Endophytes as Potent Delignifiers

Endophytes are regarded as the fascinating microbes due to their asymptomatic existence inside the plants assisted by numerous secondary metabolites and enzymes. Most of the extracellular enzymes produced are being correlated to their penetration/entry inside the host plant (Abdalla et al., 2020). However, the interesting fact lies in their stable colonization in planta, where genes coding for these enzymes might be temporarily shut off, by the time any kind of stress prevails or host dies (Promputtha et al., 2007; Nelson et al., 2020). After host’s death, as a survival strategy, many endophytes switch their lifestyle to saprophytism and invade as the primary colonizers to feed on the dead host, thereby initiating decomposition of wood (lignocellulose) via the production of various enzymes (Song et al., 2017) (Figure 3A). Certain endophytes may join the degradation process after sometime but function upto the last stage of decomposition (Promputtha et al., 2010). This capability makes them competent for degrading numerous recalcitrant aromatic compounds including lignin into monomers, ultimately adding basic nutrients to soil. Hence, endophytes play an important role in bio-geochemical cycling.

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FIGURE 3

Endophytes as lignin decomposers. (A) Schematic overview of natural endophytic delignification. (B) Pictorial view of in vitro investigation of endophytic delignification. Where I, Inhibitor; LG, ligninase gene; ABTS, [2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid].

Exploration of endophytic microbes as agents of delignification is an emerging field. Monotospora sp. was the first endophytic fungus isolated from Cynodon dactylon that was reported to produce laccase (Wang et al., 2006). Most of the studies have focussed on the isolation of endophytes from healthy plant tissues followed by their investigation for ligninase producing ability or degradation of certain lignin-based substrates (Figure 3B). Both endophytic bacteria and fungi are known to produce ligninases in artificial cultures with ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and guaiacol (2-Methoxyphenol) as the frequently used substrates (Table 2). For example, endophytic fungi Pringsheimia smilacis, Neofusicoccum luteum, Neofusicoccum austral, Hormonema sp., and Ulocladium sp., isolated from the xylem of Eucalyptus spp. were found to degrade lignin-based model compound ABTS, the potency was associated with laccase production (Fillat et al., 2016). Similarly, Colletotrichum gloeosporioides, an endophyte of Piper betle produced laccase after growing in the medium containing Guaiacol (Sidhu et al., 2014). Trichoderma asperelloides LBKURCC2, a palm endophyte degraded both ABTS and guaiacol (Pisacha et al., 2020). Eight endophytic fungi isolated from Brunfelsia uniflora showed laccase activity (Marsola et al., 2022). Furthermore, five dark septate endophytic fungi derived from roots of P. merkusii also showed lignolytic activity (Akhir et al., 2022). Recently, it was observed that fungal endophytes of the grass species, Festuca sinensis, Stipa purpurea, and Achnatherum inebrians plays an important role in the litter decomposition of their hosts and nutrient trasition of ecosystem (Song et al., 2022).

On the other hand, as discussed earlier, reports on delignifying endophytic bacteria are comparatively lagging. An endophytic bacterium, Pantoea sp. Sd-1 isolated from the rice seeds however, was found to degrade both ABTS and guaiacol (Shi et al., 2015). Bacterial endophytes viz., Bacillus sp., Klebsiella sp., Pseudomonas sp., Clavobacter sp., Micrococcus sp., Xanthomonas sp., Enterobacter sp., Serratia sp., and Escherichia coli associated with Musa acuminata and Hevea brasiliensis also showed the ability to metabolize guaiacol (Wins et al., 2019). As reported, microbial degradation of ABTS is mainly associated with the oxidative catalysis by laccase, however, guaiacol is oxidized to catechol by cytochrome P450 monooxygenases (García-Hidalgo et al., 2019).

Certain endophytes are capable of producing multiple lignin degrading enzymes. For instance, marine fungi Alternaria alternata, A. solani, Gonatorrhodiella parasitica, Monascus ruber, Trematosphaeria mangrovei, and Ulocladium isolated from Paduia sp. Pterocladia sp. Cystoseira sp. Sargassum sp., and Corallina sp. Ulva exhibited laccase (Lac), lignin-peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (Atalla et al., 2010). Similarly, Myrothecium verrucaria produced all the above-mentioned ligninases (Su et al., 2018). The laccase procured also displayed various degrees of decolourization of several dyes in the presence of ABTS redox mediator. An endophytic fungus Fusarium proliferatum strain NRRL 31071 isolated from wheat showed extracellular laccase and aryl alcohol oxidase activities (Anderson et al., 2005). Likewise, Fusarium sambucinum from a mangrove plant Cananeia sp. showed lignin peroxidase and manganese peroxidase activity (Martinho et al., 2019). Also, basidiomycetous fungi endophytic to a macroalga Ecklonia radiata produced laccase and lignin peroxidase under oxic conditons (Perkins et al., 2021). Recently, Colletotrichum acutatum, Nigrospora sphaerica, Exserohilum rostratum, Diaporthe phaseolorum, and Pestalotiopsis arceuthobii isolated as endophytes from Taxillus chinensis showed manganese peroxidase and lignin peroxidase activity (Song et al., 2022). Also, an endophytic fungus Diaporthe phaeolorum recovered from the leaves of Dillenia indica exhibited Lac, LiP, and MnP activities (Kumar and Prasher, 2021). Upon investigating the effect of various carbon and nitrogen sources on the growth and production of ligninolytic enzymes, maximum laccase (44.5 U/ml), lignin peroxidase (3.5 U/ml), and manganese (6.88 U/ml) activity was found in pectin, sucrose, and glucose, respectively. Therefore, such endophytes can simultaneously break diverse bonds in lignin biopolymer and release different depolymerization products. As far as physical/chemical depolymerisation is concerned, different conditions needs to be maintained (high temperature, pH, irradiation), and many chemicals are to be supplemented in order to breakdown multiple kinds of linkages. Therefore, employing an endophyte with multiple ligninase ability can perform this function in an eco-friendly way with less energy consumption.

Being comparatively unexplored for delignification ability, endophytes can act as the promising tools for unearthing novel ligninases. As for example, an endophytic bacterium Pantoea sp. Sd-1 isolated from rice seeds showed outstanding ability of degrading lignin and rice straw (Xiong et al., 2014). This effect increased upon addition of glucose and 0·5% peptone to the rice straw or Kraft lignin containing medium. Upon genomic analysis of Sd-1, four putative laccases—Lac1, Lac2, Lac 3, and Lac4 were identified (Shi et al., 2015). However, only Lac4 possessed the complete signature sequence for laccase activity and resembles known bacterial multi-copper oxidases upto 64%. Apart from its acid-stable nature, recombinant Lac4 was capable of oxidizing both the phenolic and non-phenolic compounds, decolourize numerous synthetic dyes, and degrade ABTS and guaiacol at acidic pH (Shi et al., 2015). Similarly, a novel laccase enzyme, Colletotrichum gloeosporioides gr. laccase was recovered from an endophytic fungus, Colletotrichum gloeosporioides colonizing Piper betle (Sidhu et al., 2014). Also, a novel endophytic laccase (LACB3) isolated from Phomopsis liquidambari was cloned and investigated for its potential to promote peanut growth. Upon application in soil, laccase enhanced peanut biomass by 12% while downgraded the respective contents of coumaric acid, vanillic acid, and 4-hydroxybenzoic acid by 21%, 27%, and 40% (Wang et al., 2014). Recently, an extensive exploration revealed a novel laccase producing endophytic fungus Chaetomium globosum from Hibiscus manihot L. which selectively degraded lignin in larch sawdust into p-Hydroxyphenyl and Guaiacyl units (Dou et al., 2022). This indicates that discovery of novel microbial strains with the recognition of their enzyme systems for lignin degradation is imperative for the effective conversion of lignin into fuel and chemicals.

Despite being an eco-friendly, economical, and sustainable method of delignification, the slower rate of reaction halts the applicability of enzymatic lignin depolymerization at industrial scale. Although, white and brown rot fungi (Basidiomycetes) are regarded as the most influential and industrially competent enzymatic delignifiers, endophytic fungi showed more efficiency in saccharification of wood (and delignification) compared to that of Trametes sp. I-62, a white rot fungus, as exemplified in Eucalyptus (Fillat et al., 2016). It indicates that endophytes can surpass the enzymatic potential of basidiomycetes in depolymerizing lignin. This ability of certain endophytes, if not all, may be accompanied by their existence as primary colonizers or their competition on the decaying host with the potent basidiomycetes. Hence, explorations and extensive studies to unravel the delignifying endophytes are pivotal to unearth the more effective lignin degraders which can overcome the limitations of microbial delignification to be employed industrially.

Endophytic Dye-Decolourization: An Activity in Correlation With Lignin Deconstruction

On account of slow degradability and frequent binding property of lignocellulose to cationic molecules, complex chelating compounds get accumulated in the environment (Chandra et al., 2012). Therefore, industrial waste water from pulp industry potentially contributes to the environmental pollution (Sadh et al., 2018; Singh and Chandra, 2019). Several endophytic explorations are being made with respect to their dye-decolourizing potential, which find their applications in bioremediation and waste water pre-treatment. Interestingly, this ability is in positive correlation with their delignification potential owing to the structural similarity of aromatic dyes to that of lignin biopolymer. Various endophytes found to decolourize aromatic dyes and the enzymes involved are mentioned in Table 3. Although diverse methods are used in degradation of textile effluent dyes, endophytes can essentially augment the biodegradation in a sustainable and eco-friendly manner (Goud et al., 2020). A novel bacterial endophyte Exiguibacterium profundum strain N4 was isolated from a plant, Amaranthus spinosus growing on a textile dye effluents-contaminated site in Rajasthan, India (Shilpa and Shikha, 2015). It efficiently decolourized 901 ppm of Reactive Black 5 (RB5), an azo dye upto 84.78% after incubation at 30°C for 12 h. HPLC, GC-MS, and UV-Vis spectroscopy analysis confirmed biodegradation of the dye. Addition of glucose (10 g/L) and beef extract (2 g/L) significantly enhanced the decolourizing ability of the endophyte. Interestingly, another bacterium Proteus mirabilis isolated from the root nodules of Cactus decolourized and degraded RB5 under static conditions (Bibi et al., 2020). Upon elevation of temperature to 37°C and incubation time to 72 h, the activity was maximized upto 90%.

Dye decolourization by endophytes pertains to their potential of synthesizing laccases and peroxidases. An endophytic fungus Marasmius cladophyllus isolated from senduduk plant, Melastoma malabathricum was initially found to decolourize synthetic dyes, such as, Rubidium bromide (RBBR) (Ngieng et al., 2013). Upon detailed analysis, decolourizing ability was found to be associated with biodegradation of these dyes via the production of laccase and lignin peroxidase enzymes in the medium containing RBBR (Sing et al., 2017). Dye decolourization in the subsequent generations was also faster even without the addition of a mediator. Similarly, a dye decolourizing laccase was produced by a novel endophyte Myrothecium verrucaria MD-R-16 colonizing Cajanus cajan (Sun et al., 2017). The laccase decolourized dyes, such as, Congo red, Methyl orange, Methyl red, and Crystal violet in the presence of ABTS as mediator. Apart from its higher production, laccase was found to be acid-stable and thermostable upto 55°C. As stated by Davis and Moon (2020), lignin valorization will be facilitated by novel microbes capable of utilizing lignin or allied aromatic compounds. Therefore, endophytes can act as the promising tools for exploring potent ligninases possessing the ability of degrading lignin and other waste products, thereby valorizing lignin and performing phytoremediation, simultaneously. In a recent research, fungal dye-decolourizing peroxidases (DyPs) were studied for their gene sequences. Upon phylogenetic analysis of the DyPs gene sequences available in public domains, seven fungal clades were distinguished on the basis of sequence similarity network (Adamo et al., 2022). Sequences pertaining to one of the clades showed divergence from others having highest number of N-glycosylation sites, N-terminal sequence peptides for secretion, lower isoelectric points and hydropathy indices (Adamo et al., 2022). Interestingly, the putative proteins from the distinct clade were lacking from brown rot and ectomycorrhizal fungi which have lost the ability of enzymatic delignification. This study did not highlight whether the distinct clade occupied endophytic fungi nor did it identify these fungi in other clades. However, it showed the diversification of fungi based on DyPs genes. Hence, in-depth explorations regarding endophytic genes encoding dye-decolourizing enzymes are required to unearth the potential genes alongwith their properties and variations which could further be used in improving the enzyme secretion of these microbes using modern techniques.

Endophytes: A Promising Source of Stable Ligninases

To be compatible with industrial applications, stability or tolerance of enzymes to varied temperatures, pH, and high adaptability is the requisite. Extremophilic potential of ligninolytic enzymes is regulated by diverse factors, such as, specific genes, hydrophobic interactions, ion pairs, disulfide bridges, salt bridges, and hydrogen bonding between amino acids (Pace et al., 2014). Endophytes are well known to produce thermotolerant and acido-tolerant enzymes. For instance, endophytic Periconia sp. BCC2871 was found to produce thermotolerant β-glucosidase, BGL I (Harnpicharnchai et al., 2009). After cloning the complete gene encoding BGL1 into Pichia pastoris KM71, the recombinant enzyme exhibited similar characteristics to that of its native counterpart. Besides showing optimum activity at 70°C with pH of 5–6, engineered BGL1 retained its activity after several cycles of incubation. More so, it efficiently degraded rice straw into simple sugars indicating its potential for application in biomass conversion. Likewise, an acidotolerant and thermotolerant laccase was produced by endophytic Phomopsis liquidambari (Wang et al., 2014). The subsequently cloned and expressed enzyme showed remarkable features to be utilized at industrial scale. A novel acid-stable laccase Lac4 identified from the genome of an endophytic bacterium Pantoea ananatis Sd-1 was found to degrade lignin and decolourize various dyes (Shi et al., 2015). The enzyme was able to act on both phenolic and non-phenolic compounds under acidic conditions (pH 2.7–4.5) at a temperature range of 30°C–50°C. Same featured laccase was produced by another novel fungus Myrothecium verrucaria MD-R-16 endophytic to Cajanus cajan (Sun et al., 2017). The optimal conditions for laccase production included incubation at 30°C and pH 6.22 for 5 days, however, the enzyme also showed relative stability at pH 4.5–6.5 and temperature range of 35°C–55°C.

Although endophytes from all kinds of habitats can be exploited for their potency to synthesize stable enzymes, those from extreme environments have attracted attention due to their ability to produce higher amounts of valuable metabolites and enzymes (Singh and Dubey, 2018). This ability is attributed to the higher exposures to diverse array of stress factors, such as, heat, cold, salinity, high UV radiations, and numerous other oligotrophic conditions. All the organisms well adapted to such hostile environments have been observed to synthesize a plethora of stable enzymes (Sarmiento et al., 2015; Monsalves et al., 2020). However, of these microbes, endophytes being primary colonizers on their dead hosts could be the potent initiators of delignification. Similar to the endophytes recovered from contaminated sites which produce contamination tolerant enzymes (Lumactud et al., 2016; Goud et al., 2020), harsh habitats are the best sites to explore heat-stable, cold-stable, and pH stable enzymes (Martínez et al., 2018).

Interestingly, ligninolytic microbes from extremophilic environments, conventional proteins were found in completely denatured state (Chandra et al., 2017). For instance, a cold-adapted bacterium Paraburkholderia aromaticivorans AR20-38 isolated from the soil of Alpine coniferous forest in Italy (altitude of 1,724–1,737 masl) efficiently degraded the lignin monomers at low temperature (Magesin et al., 2021). Similarly, many endophytes explored from such habitats exhibited potent lignin deconstruction properties. For instance, from mangrove (high salinity, low pH) Brazilian plants, 19 endophytic fungi exhibited ligninoytic activities (Marinho et al., 2019; Marinho et al., 2020). Out of these, Fusarium sambucinum, Diaporthe sp., Fusarium sp., and Hypocrea lixii showed exceptional laccase, lignin peroxidase, and managnsese peroxidase activities. Conclusively, endophytes colonizing the plants growing in harsh environmental conditions could be the formidable source of lignin depolymerizing enzymes, therefore, explorations are required to exploit their maximum potential.

Mechanism of Enzymatic Delignification

Various microbes, including fungi and bacteria degrade lignin in nature which is accomplished by the complex synergy of diverse enzymes. Difficulty in delignification mainly occurs due to the presence of heterogeneous C-C linkages in phenylpropane building blocks (Castro-Sowinski et al., 2002). Fungal laccases and peroxidases are reported to oxidize lignin using oxygen (O₂) and hydrogen peroxide (H₂O₂), respectively, which assist in the formation of free hydroxyl (OH¯) ions. These ions which are mainly produced by three pathways—cellobiose dehydrogenase catalyzed reactions, quinone redox cycling, and Fenton reactions catalyzed by glycopeptides (Dashtban et al., 2010), attack the plant cell wall components including lignin governing cleavage of various bonds. It has been observed that post cleavage fragments of lignin are more susceptible to attack by diverse lignin modifying enzymes (Kantharaj et al., 2017). Thus diverse fungi are more efficient in pre-treatment of lignin to their intermediates or low molecular weight heterogeneous compounds (5/6-carbon chains) (Kumar and Chandra, 2020). Intriguingly, several bacteria capable of delignification have evolved pathways to funnel these heterogeneous oligomers into common products through the process called biological funnelling. It occurs via upper pathways which functions as “biological funnels” for channelizing the lignin-derived heterogeneous aromatic compounds to common central intermediates, mainly protocatechuate or catechol (Eltis and Singh, 2018). Subsequently, these intermediates experience ring cleavage and are further converted into central carbon metabolism via β-ketoadipate pathway (Linger et al., 2014) (Figure 4).

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FIGURE 4

Pathways involving the role of fungi and bacteria in enzymatic degradation of recalcitrant lignin. Where Lac, Laccase; LiP, Lignin peroxidase; MnP, Manganese peroxidase; VP, Versatile peroxidase; TCA, Tricarboxylic acid.

Biological funnelling is the property that makes microbial delignification the most important method for valorizing heterogeneous lignin into common desired products. For example, demonstration on Pseudomonas putida KT2440 revealed its role in the conversion of both aromatic model compounds and heterogeneous lignin-derived streams into polyhydroxyalkanoates (mcl-PHAs) (Linger et al., 2014). These are the intracellular energy-rich, carbon storage compounds utilized in carbon-deficient conditions by numerous microorganisms. PHA’s find their applicability in pharmaceutical and biomedical industries, packaging materials, energy and chemicals (Tsang et al., 2019). Similarly, Pseudomonas putida KT2440 funnelled various lignin-derived species into cis-cis muconic acid which was subsequently hydrogenated to adipic acid, a dicarboxylic acid used in confectionery, fats, flavouring agents, and in the synthesis of nylon 6.6 (Vardon et al., 2015).

Interestingly, these microbes not only funnel the variable lignin based constituents into common products but the lignin from variable biomass sources also. For instance, an engineered bacterium Novosphingobium aromaticivorans DSM12444 modulated by a targeted gene deletion, used its native funnelling pathways for the conversion of guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) aromatic units into 2-pyrone-4,6-dicarboxylic acid (PDC), a powerful polyester precursor (Perez et al., 2018; Perez et al., 2019). Surprisingly, this microbe could also funnel the heterogeneous mixture of aromatic monomers formed by the chemical depolymerization of poplar lignin into PDC. Currently, biological funnelling of other heterogeneous and toxic compounds has also gained essence (Borchert et al., 2022).

Endophytic Catalysis of Lignin to Value-Added Products

Endophytes have gained essence on account of their fabulous biotechnological relevance. However, their role as primary colonizers on dead plants has attracted attention due to their lignin deconstruction and bioremediation potential. They are one of the potent sustainable delignifiers capable of producing numerous essential products from the renewable underutilized resource—lignin (Figures 5, 6A–E). Exploration of various endophytic microbes for this potential will not only provide an additional source for the conversion of lignin into such valuable products, but may also unveil novel pathways of conversion with more feasibility in their industrial applications. The most valuable outcomes of delignification are briefly highlighted as follows:

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FIGURE 5

Schematic representation of the process involved in endophytic lignin valorization.

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FIGURE 6

Overview of various pathways involved in selective degradation of lignin into desired valuable products. (A) Key route to synthesize microbial lipids, with ergosterol as an example via TCA cycle. (B) Vannilin biosynthesis through lignin degradation to monomeric units. (C) Vannilin biosynthesis through degradation of ferulic acid. (D) Highlights of the multiple pathways in formation of polyhydroxyalkanoates where acetyl CoA acts as the main player. (E) Key reactions involved in cis-cis muconic acid formation. Where TCA, Tricarboxylic acid; β-KAP, β-ketoadipate pathway.

Microbial Lipids (Single Cell Oils)

Several oleaginous microorganisms are capable of producing lipids or microbial oils exhibiting applications in diverse fields including biodiesel production. Biodiesel is a non-toxic, renewable, and biodegradable fuel which comprises of fatty acid methyl esters. Similar to petroleum diesel, it is used to fuel compression-ignition engines. Currently, its industrial production is mainly dependent on the vegetable oils which can put a par on edible oils resulting in price elevation. Therefore, it is imperative to turn towards the alternative sources for biodiesel production. Interestingly, microbial lipids have attracted a lot of attention in the recent years as an efficient feedstock for biodiesel production. However, their production faces a number of challenges, such as, high costs for lipid extraction, cultivation of microbes, and continuous flux of carbon source (Santek et al., 2018). Intriguingly, utilizing lignocellulosic biomass as a renewable carbon source has effectively reduced the production cost of microbial lipids. Numerous bacteria and fungi are known to produce microbial lipids from lignin. For instance, oleaginous Rhodococcus opacus DSM 1069 efficiently used the Kraft lignin to accumulate lipids, mainly palmitic (46.9%) and stearic (42.7%) acids, with the maximum yield of 0.067 mg ml⁻¹ after incubation of 36 h (Wei et al., 2015).

Endophytes have great potential to be utilized for the sustainable production of microbial lipids from ligocellulosic biomass. Several studies have unravelled the oleaginous endophytes from diverse plant sources (Zhang et al., 2014; Wani et al., 2017; Das et al., 2022). Both endophytic bacteria and fungi are reported for their oil producing ability (Table 4). Mucor circinelloides Q531 isolated from mulberry stem and leaves outstandingly converted mulberry branches into lipids (Qiao et al., 2018). The maximum lipid content of 28.8 ± 2.85% was procured with the yield of 42.43 ± 4.01 mg per gram dry substrate (gds). Further, GC-MS analysis revealed palmitic acid (C16:0, 18.42%), palmitoleic acid (C16:1, 5.56%), stearic acid (C18:0, 5.87%), oleic acid (C18:1, 33.89%), linoleic acid (C18:2, 14.45%) and γ-linolenic acid (C18:3 n6, 22.53%) as the major fermentation products.

Endophytes are largely cherished for their potential to mimic their host plants in the production of certain essential metabolites, thereby acting as efficient alternatives to conserve these plants in nature. In view of this ability, explorations of oleaginous/biodiesel plants (plants that are traditionally used to produce biodiesel) for the recovery of oleaginous endophytes have gained essence (Strobel et al., 2001; Peng and Chen, 2007; Strobel et al., 2008; Dey et al., 2011). For example, biodiesel plants, such as, Jatropha curcas, Pongamia pinnata, Sapindus mukorossi, Mesua ferrea, Terminalia bellerica, Cascabela thevetia, and Ricinus communis proved competent sources for unveiling oleaginous endophytic fungi (Paul et al., 2020). Among the procured oleaginous fungi, the lipid content of Lasiodiplodia exigua SPSRJ27, Phomopsis sp. SPSRJ28, Phomopsis sp. SPSRJL36, and Pestalotiopsis microspora SPSRJL35 was 20% more compared to their dry biomass. Similarly, an endophytic bacterium Bacillus subtilis HB1310 isolated from thin-skinned walnut effectively used cotton stalk hydrolysate to produce lipids (Zhang et al., 2014). In 48 h of culture time, the lipid content of 39.8% (w/w) with the maximum productivity of 2.3 g/L, and cell dry weight of 5.7 g/L was achieved. Earlier, yeast and microalgae were regarded as the most promising oleaginous agents using glucose or acetic acid as the carbon source (Meng et al., 2009). However, low lipid yields were witnessed with the application of agricultural and industrial wastes as substrate (Santala et al., 2011). In comparison, HB1310, surprisingly, utilized cotton stalk hydrolysate to produce the lipid content comparable to that of yeasts. To have better insights into the lignocellulose conversion efficiency of HB1310, whole genomic information to trace the metabolic pathways has recently been reported (Zhang et al., 2021), which indicated the involvement of Embden–Meyerhof–Parnas, pentose phosphate, and fatty acid synthesis pathways as the major key players in substrate utilization and lipid biosynthesis (Figure 6A). Moreover, higher expression of fatty acid synthesis genes was observed compared to that of other Bacillus strains. Tricarboxylic acid (TCA) cycle ruthlessly shared the carbon flux flowing from acetyl CoA node before 48 h and acetic acid fermentation pathway competed after 72 h for the flux distribution of lipid synthesis. Conclusively, endophytes share excellent potential in microbial lipid production from lignocellulosic biomass, and can be used to develop efficient biofuel minifactories. Besides, in view of the abovementioned potential of endophytes, it is pivotal to explore the maximum endophytic microbes for their efficacy in microbial lipid production using lignocelluloses.

Vanillin (4-Hydroxy-3-Methoxybenzaldehyde)

Vanillin is an outstanding compound which apart from its cosmetic (fragrance) properties act as the precursor for numerous pharmaceutical polymers (Brianna et al., 2016). It can be naturally procured from plants or artificially through chemical synthesis, however, utilizing lignin as the feedstock for vanillin synthesis by microbes confers an eco-friendly, clean and green technology. Vanillin production from delignification has been carried out since 1950 on an industrial scale (Maeda et al., 2018). Initially it was synthesized by aerobic oxidation of sodium lignosulfonate in the presence of alkali NaOH (Forss et al., 1986). However, microbial conversion of lignin to yield vanillin involves a sustainable way to valorize lignin.

Vanillin can either be produced by the lignin depolymerization (Figure 6B), or degradation of ferulic acid. Ferulic acid, a kind of hydroxycinnamic acid is an important organic acid in the cell wall of herbaceous plants, acts as a standard model compound for G-lignin. Few endophytes are known to deconstruct this compound into vanillin. For instance, a bacterium Enterobacter sp. Px6-4 colonizing the roots of Vanilla plant metabolized ferulic acid into 4-vinylguaiacol and subsequently into vanillin (Li et al., 2008) (Figure 6C). Initially, vanillin was known to be produced from Vanilla plants only, however, many endophytic fungi colonizing this plant are known to mimic their host in the production of vanillin. Khoyratty et al. (2022) recently reviewed the vanillin biosynthetic potential of fungal endophytes in Vanilla plants to have better understanding of vanillin biosynthesis, bioproduction and biotechnology.

Likewise, Colletotrichum gloeosporioides TMTM-13 (an endophytic fungus) isolated from Ostrya rehderiana was found with potential to degrade ferulic acid into vanillin, acetovanillone, vanillic acid, and dihydroconiferyl alcohol (Zhang et al., 2019). Similarly, endophyte Phomopsis liquidambari used ferulic acid as the sole carbon source and within 48 h, more than 97% of ferulic acid added to mineral salt medium and soil was decomposed (Xie and Dai, 2015). The pathway involved decarboxylation of ferulic acid into 4-vinyl guaiacol followed by oxidation into vanillin and vanillic acid. These compounds upon subsequent demethylation yielded protocatechuic acid which was further degraded via β-ketoadipate pathway. Identification and quantification of metabolites was accomplished adopting GC-MS and HPLC-MS analyses (Xie and Dai, 2015). Interestingly, substrate and product concentrations affected the activities and expression levels of fdcB3 (ferulic acid decarboxylase), lacB3 (laccase), and pcaB3 (protocatechuate 3, 4-dioxygenase), however, transcription of all the three genes was induced in P. liquidambari. With the advent of multi-omics technology, more accessibility of metabolic pathways involved in microbial delignification is being achieved (Zhu et al., 2017; Moraes et al., 2018; Zhu et al., 2018).

Polyhydroxyalakanoates

Polyhydroxyalakanoates (PHA’s) are bioplastics with their promising role in pharmaceutical industry as anticancerous agents, drug carriers, and memory enhancers. Numerous microbes are being reported to produce PHA’s under stressful conditions of nitrogen, potassium, oxygen, phosphorus, and magnesium (Ray and kalia, 2017). Although PHA’s share similar properties with synthetic plastics, their biodegradable, biocompatible, and non-toxic behaviour makes them unique. Diverse microbes are known to produce PHA’s as the degradation product of lignin. For instance, Pseudomonas strains have marvellous contributions in lignin valorization via bioconversion of recalcitrant lignin into desirable bioplastics which paves their way for proper place in future biorefinaries. Using lignin as the sole carbon source, Psuedomonas putida NX-1 successfully produced PHAs (Xu et al., 2020). Moreover, the physical properties of PHAs liberated from lignin were similar to that synthesized from glucose thereby making lignin-derived aromatics as the efficient alternatives for PHA synthesis. As already mentioned, microbial PHA synthesis is positively correlated to diverse nutrient deficiencies, the biofunneling capacity of Pseudomonas strains to yield mcl-polyhydroxyalkanoate (mcl-PHA) was enhanced after subjected to nitrogen and oxygen limitations (Ramírez-Morales et al., 2021). Recently, conversion of lignin into PHA’s by Psuedomonas putida KT2440 was enhanced by the addition of glycerol to the lignin derivatives, such as, vanillin, vanillic acid and benzoic acid (Xu et al., 2021).

In a soil bacterium β-proteobacterium Pandoraea sp. ISTKB, lignin degradation and PHA production was witnessed, where Kraft lignin was efficiently degraded into bioplastic PHA’s (Kumar et al., 2018). The genomic and proteomic analysis revealed that various enzymes, such as, laccases, peroxidases, Dyp-type peroxidase, etherases, aldehyde oxidase, dehydrogenases, glycolate oxidase, GMC oxidoreductase, quinone oxidoreductase, dioxygenases, monooxygenases, glutathione-dependent, reductases, and methyltransferases were expressed during the process (Kumar et al., 2018). However, after detailed investigation, three enzyme systems viz., laccases, peroxidases, and Fenton-reaction enzymes came into limelight for their catalytic lignin biodegradation and gene clusters comprising bktB, phaR, phaB, phaA, and phaC genes to be involved in PHA synthesis (Liu et al., 2019) (Figure 6D).

Many endophytes have also shown their potential for PHA synthesis. Interestingly, the highest yield of PHA’s has been observed using non-conventional carbon sources (lignin-based substances). For instance, an endophytic bacterium Bacillus cereus HAL 03 colonizing leaves of Helianthus annuus was found to procude a homopolymer of 3(hydroxybutyric acid)/P(3HB), the most common polyhydroxyalkanoate. Identity of the compound was confirmed by the Fourier-transform infrared and proton nuclear magnetic resonance spectroscopic analysis (Das et al., 2016). Using sucrose (2%) and yeast extract (0.2%) as the carbon sources, P(3HB) production by the bacterial isolate reached 50.46 % and 53.19%, respectively, of its dry cell weight (CDW), while molasses as the carbon source could further scaele-uo the yield upto 54.05% of its CDW. Similarly, another strain Bacillus cereus RCL 02 isolated from the leaves of Ricinus communis was found to produce poly(3-hydroxybutyrate) [P(3HB)] (Das et al., 2017). Scanning electron microscopy revealed the release of these granules as a function of autolysis by the bacterium. The bacterial isolate produced 68%, 72.2%, and 81% P(3HB) of its CDW when grown in glucose with mineral salts, glucose with yeast extract, and metal stress (1.5 mM manganese), respectively. However, the polyester production was further enhanced to 83.6% CDW after using refined sugarcane molasses as the sole source of carbon. Bacteria, such as, Azospirillum, Burkholderia, and Herbaspirillum isolated as nitrogen fixing endophytes from Asparagus officinalis were also found to accumulate PHA’s in their cytoplasm (Altamirano et al., 2021).

Muconic Acid (C₆H₆O₄)

It is a diacrboxylic acid with three stereoisomeric forms—cis-cis muconic acid, trans-trans muconic acid and cis-trans muconic acid which differ in spatial arrangement of atoms/functional groups around the double bonds. cis-cis muconic acid is mainly produced by microbial degradation of aromatics and find its applications in making polyurethane, polyethylene terephthalate (PET), and nylon (Curran et al., 2013; Wang et al., 2020). Several microorganisms are reported to produce cis-cis muconic acid as an intermediate in lignin biodegradation (Wang et al., 2020). However, recently, metabolically engineered microbes for the selective conversion of lignin into cis-cis muconic acid has gained essence (Pyne et al., 2018; Choi et al., 2020; Aravind et al., 2021; Wirth and Nikel, 2021). Most of the microbes convert lignin into cis-cis muconic acid by using glucose as the additional growth substrate (Becker et al., 2018). However, engineered Pseudomonas putida KT2440 strain converted vanillic acid (guaiacol-based lignin model) and 4-hydroxybenzoic acid (p-hydroxyphenyl-based lignin model) into muconic acid (Sonoki et al., 2018). More interestingly, while KT2440 could yield 20% of cis-cis muconate, Sphingobium sp. SYK-6 yielded 45% after using syringic acid as the lignin-based model compound.

As far as endophytic delignification to yield muconic acid is concerned, very few reports are published so far. For instance, during an investigation, endophytic Phomopsis liquidambari was evaluated for its potential to degrade 4-hydroxybenzoic acid (4-HBA) as it comprises one of the phenolic allelochemicals produced after foliage decomposition. Intriguingly, the endophyte used 4-HBA as the sole carbon source thereby led to its degradation into cis-cis muconic acid (Chen et al., 2011). Unravelling the metabolic pathways following high performance liquid chromatography–mass spectrometry (HPLC–MS) and gas chromatography–mass spectrometry (GC–MS), 4-HBA first undergo hydroxylation to form 3,4-dihydroxybenzoic acid and then catechol followed by oxidation to cis-cis muconic acid in the tricarboxylic acid cycle (TCA) cycle. Upon feeding with ferulic acid as the sole carbon source, P. liquidambari produced vanillin and vanillic acid, as already mentioned under “Vanillin” sub-section. However, these intermadiates undergo demethylation yielded protocatechuic acid which was further degraded via β-ketoadipate pathway to form β-carboxy-cis, cis muconic acid (Xie and Dai, 2015) (Figure 6E). Research is essentially lagging regarding the application of endophytes in lignin depolymerization to yield such dicarboxylic acids. Therefore, various intermediates produced after lignin depolymerization/pre-treatment can be funnelled to produce muconic acid.

Optimization in Endophytic Delignification

The authors are highly thankful to the Head, Department of Botany, University of Jammu (India) and SAP-DRS-II for providing laboratory facilities. Thanks is also due to Council of Scientific and Industrial Research (CSIR) for granting fellowship to AM.