Transforming Lignin Biomass to Value: Interplay Between Ligninolytic Enzymes and Lignocellulose Depolymerization

Lignin is the main constituent of lignocellulosic biomasses, which have a significant untapped ability to replace ecologically unfavorable and non-renewable fossil fuels. The lignin is broken down by ligninolytic bacteria, which also use a peripheral pathway to transform heterogeneous lignin derivatives into central intermediates like protocatechuate or catechol. By undergoing ring cleavage through the -ketoadipate pathway, these intermediates become metabolites by producing acetyl-CoA for internal product biosynthesis, including the creation of triacylglycerols and polyhydroxyalkanoates. Expanding our understanding of ligninolytic microbial communities, strains, and enzymes through bioprospecting can help us better understand the metabolism of aromatics. The most viable idea for sustainable development is the valorization of lignin into biopolymers as well as other high-value goods. This process is now being used to generate a variety of biopolymers, including polyesters, epoxies, phenol resins, poly (lactic acids), poly hydroxyl alkanoates, and polyurethanes. Furthermore, lignin recalcitrance remained a possible barrier to efficient lignin valorization, prompting several efforts to design high-efficiency bioprocesses to produce specific polymer types as well as other important bioproducts.


Introduction
The lignin molecule is well-known as the 2nd most abundant infinite organically grown polymer (acts as the most active material) which allows plants to create hard chemical structures and protects hemicellulose and cellulose from hydrolysis in lignocellulosic biomass [1]. Lignocellulosic biomass, which is abundant regionally, demands minimal capital expenditure for bioconversion, minimizes greenhouse gas emissions, and generates job chances for rural habitats. Lignin (15-20%), cellulose (40-50%), and hemicelluloses (25-35%) make up about two-thirds of lignocellulosic [2]. Lignin is among the most prevalent biomolecules accessible for biorefining as a raw material. It is, nevertheless, among the most underutilized plant elements in biorefineries, where it is now largely employed to generate process electricity and steam [3]. The primary cause is lignin's intrinsic obstinacy and structural variability, which makes the availability of important aromatic side chains difficult. Nonetheless, lignin valorization, or the transformation of lignin into fine chemicals, minerals, or fuels is regarded as critical for the effective realization of low-cost lignocellulosic biorefineries [4].
Speedy expansion and the need to feed an expanding population resulted in a massive volume of lignocellulosic biomass being produced. According to some estimates, each year approximately 709,200,000 metric tons of waste from wheat, 731 million tons of residues from rice crop [5], one hundred and forty billion metric tons of biomass trash, five thousand million metric tons of crop remains [6] are generated. The most common source of lignin is lignocellulosic biomass (Fig. 1). Softwood and grasses are largely composed of G (guaiacyl) unit of lignin with a concentration of > 95% and 35-80% respectively while hardwood is mainly composed of S (syringyl) unit of lignin with a concentration of 45-75% [7]. Total dry biomass can be produced on forest and agricultural sites. Despite this, pulp and paper companies create 30% of lignocellulosic material in form of lignin. Consequently, the sugar industry produces lignocellulosic waste, which contains between 27 and 30% lignin by dry weight. As per the current situation, biorefineries primarily use hemicellulose and cellulose lignocellulosic biomass components to make important goods such as levulinic acid, pyrones, and furfuryl alcohol, ethanol, furfural, and butanol, leaving lignin as a residual waste [8]. Pulp and paper sectors solely create 50-70 million tonnes of lignin every year, and the developing biofuel industries are expected to produce 62 million tonnes per year. According to some estimates, this might reach 225 million tonnes per year by 2030 [9]. In comparison to crude oil, one of the most plentiful and cheapest biomasses is lignocellulosic biomass.
Globally, large amounts of agro-industrial byproducts are produced each year, and they usually include 15-25% lignin [1]. In 2017, rice husks, barley straw, soybean hulls, wheat straw, maize stoves, and sugarcane bagasse, contributed nearly 3.9 Gt of biomass. As a result, certain new chemical pathways are suitable for producing great premium lignin molecules [3]. One of the most efficient bio-based refinery procedures now contains four essential distributions: enzymatic hydrolysis, material collection and stockpiling, pre-treatment, and fermenting of liberated sugar into biofuels [1]. Furthermore, lignin seems to be a significant renewable feedstock with a substantial quantity of cyclic monomers [10]. Hydrolytic enzymes are critical in the generation of biomass-derived sugars, bio-based products, and biochemicals by breaking down complex biomass into macromolecules and subsequently simple sugars [2]. Furthermore, developments in combining computational designing, analytical chemistry, and genetic engineering modeling aspects for lignin preprocessing have made lignin depolymerization and transformation simpler [11]. The aromaticity and side chains on the C-backbone of lignin have also been established to make it a more useful property than the other current biomass traits [1]. In a nutshell, 2 primary process concerns that define the importance and utility of lignin biorefineries are the convenience of taking out lignin through a decline in cross-linkages with other polymeric materials in biomass and depolymerization routes of intrinsically compound lignin structure.

Ligninocellulose Depolymerization Techniques
The biorefinery approach is focused on using biomass feedstocks to generate a wide variety of industrially relevant bioproducts, like chemicals and fuels while reducing dependency on fossil fuels. Former publications distinguished biorefineries depending on the processing amenability, feedstock, and bio-based outputs. Using a fixed bioprocessing capability, dried distillers' grain, ethanol and carbon dioxide, are created from dry grains in the 1st method. Grain is used to create CO 2 , high-fructose syrup, ethanol, corn oil, dried distillers' grain, and starch in the 2nd technique, which provides process flexibility. The use of integrated processing technologies and a wide range of feedstocks is an extra Fig. 1 Various sources of lignin and composition of hardwood, softwood, and grasses sophisticated idea in biorefinery, which is at a halt in the early stages of research and development. Biorefinery entails the biosynthesis of classic and novel fuels and chemicals by incorporating specifically created biomass conversion methods and technology. A primary goal of the biorefinery is to create innovative bioproducts that are not available from fossil sources, besides emulating petro-refinery. Nevertheless, to build effective bioconversion techniques that create optimal products, a thorough understanding of biomass chemistries is required [12].
Biorefineries use processed wood, cellulose, and hemicellulose to make a variety of goods. The majority of integrated biorefinery ideas have four primary sections: feedstock gathering and storage, pretreatment of yeast biomass, hydrolysis of enzymes, and fuel transformation. The majority of biologically based biorefinery designs depend on cellulose biomass, and roughly 60% more lignin is produced than is necessary. SDGs' synergistic deployment and ligninolytic armory's enzyme-assisted biological transformation perspective will serve as a prospective edge for producing numerous goods from a variety of bio-based resources. To depolymerize lignin, some fungal and bacterial species are created having powerful metabolic systems. These bacteriarestricted strains frequently lack desirable industrial features. With today's microbes, for example, yield recovery and productivity are key challenges [13].
Nowadays, biorefineries are concentrating on generating fuels from feedstocks having high sugar content. Lignin valorization includes a specific effective method known as the hybrid biorefinery route, which has the advantages of increased lignin depolymerization effectiveness via certain chemical approaches and good selectivity of focused elevated value-added bio compounds or chemical products via green microbial methods [14]. Lignin has a market value of roughly $300 million [3]. Lignin is predicted to become the primary sustainable source for biofuel and chemical industries because of its economic performance. Furthermore, lignin can be used as an environmentally benign substitute for a variety of thermoplastics, petrochemicals, rubber additives, and medicines [15]. There are several roadblocks to the complete development and deployment of lignin-based biorefineries caused by a lack of new processing methods. Consequently, commercialized biochemical synthesis from lignin-based feedstocks must turn into a complicated technique.
Generally, lignin-originated compounds are a combination of lignin products created during depolymerization; nevertheless, extraction of active compounds is time-intensive, and solitary-derived yields are commercially low. Compounds such as alcohol, carboxyl, alkyl, ester and carbonyl, molecules are used as polymers, solvents, renewable fuels, and aromatic organic chemicals during the depolymerization of lignin. There are profitable avenues for converting lignin into worthy chemicals and fuels, which is one of the most significant issues related to lignin valorization. HDO (Hydrodeoxygenation) of lignin depolymerization intermediates is generally a critical and key improving step in prime focus modifications that influence the phenolic core. Ligninderived hydro deoxygenation cyclohexane products, that are produced from methoxylated phenols via a synergistic act of rubidium, palladium, platinum (noble), or hydrogenation, proceeded by dehydration and demethylation may contribute as mid-range fuel additives [16]. Moreover, there is the possibility of certain harmful interactions among the generated intermediate compounds during the lignin conversion process. Even though lignin extraction and utilization technologies have been employed since the early 1900s, researchers are still looking for effective lignin valorization approaches. A reasonable association among diverse depolymerization demands the same lignin substrate and vice versa. When these prerequisites are met, depolymerization and fractionation processes could be evaluated across numerous explorations. In any instance, an additional challenge is a difference in analytical procedures that exist between various research, resulting in varying outcomes. As a result, identical evaluations that recall the recommended criteria and adopt a similar analytical method might give important attention to promote a suitable and reliable assessment of the various fractionations and depolymerization approaches. An optimum lignin valorization into synthetic goods necessitates the best usage of whole lignin from feedstock, which demands a mixture of an elevated return lignin confinement and depolymerization processes.
Cutting-edge technology allows us to utilize garbage while also developing a key global economy. Lignin (byproduct of the biorefining sectors, paper, and pulp) is gaining an extra notice than ever before. The aromaticity of lignin has significant market viability. Its use is both environmentally and commercially beneficial. Lignin has also been used as an unprocessed ingredient for artificial polymers like epoxy resin, polyester, polyurethane resin, and phenol-formaldehyde resin [17]. Lignin thus provides an exceptional environment for lignocellulosic biomass valorization and boosting lignin-based biorefineries. Lignin could be a font of a variety of cyclic molecules and chemicals. Chemically altered lignin could work as reinforcement material or filler in polymer blends and composites [18]. Aromatic and aliphatic acids, quinones, vanillic acid, aromatic aldehydes, cyclohexanol, DMSO, and -keto adipate were all produced when lignin was oxidized under varied oxidation circumstances [19]. Lignin depolymerization had been investigated utilizing mild oxidants (CuO) to create phenolic aldehydes in the presence of an alkaline media [3]. Aromatic hydrocarbons including catechol, phenol, resorcinol, cresols, substituted phenols, eugenol, benzene, and guaiacols can all be produced from lignin. Essential features of lignocellulosic biomass made it a suitable substrate for developing a range of products with huge biotechnological and industrial value [20]. Sugar extraction from lignocellulose is a crucial step in converting these biomasses into important products. To maximize resource recovery and offer a cost-effective solution, several pretreatment myths have been suggested. Chemical, physicochemical, physical, and biological pretreatment procedures are used [21]. Because sugar separation from lignocellulose is such an important step in the process, choosing appropriate solutions is critical.

Pretreatment Approaches for Converting Lignocellulose in Valuable Products
The important stage in the breakdown of essential parts of biomass and subsequent alteration to important products is lignocellulosic biomass pretreatment [22]. It is resistant to decomposition because of its chemical and structural features, and the appropriate digesting process is defined by biomass type and chemical makeup. The basic objective of pretreatment is to produce hemicellulose, cellulose, and lignin more available to be transformed into sugar. Enzymatic hydrolysis, pretreatment, and alteration to end products were the three essential phases in biomass conversion to important products (Table 1) [23]. Pretreatment is a critical phase in the biomass conversion process, and it is also the most energyintensive [23]. Several pretreatment technologies are currently offered, some of which are substantially competent and environmentally friendly. Pretreatment techniques are split up into three groups: chemical, physical, and biological. The pretreatment procedures are chosen based on several factors. For example, the chosen approach should not alter biomass size, other fractions such as hemicellulose must be preserved, the degraded product must be minimized, cost-effective ways that are proficient should be chosen, and techniques must use cost-effective pretreatment catalysts [24].

Catabolic Pathways Involved in Lignin Degradation
The following catabolic pathways are involved in the degradation of lignin-derived compounds (Fig. 2) [3].

Beta-Aryl Ether Degradation Pathways
In Sphingomonas paucimobilis SYK-6, the bacterial catabolism routes for a number of lignin constituents have been thoroughly investigated [33]. The NAD-dependent dehydrogenase LigD catalyzes the first oxidation of the alphahydroxyl group to the corresponding ketone. Pseudomonas acidovorans has also been shown to degrade this model b-aryl ether molecule [34]. Using Pseudomonas putida and Rhodococcus jostii RHA1, the ketone product was found to be a by-product of the breakdown of lignocellulose. It may have formed through a b-etherase cleavage reaction or a b-elimination reaction [35]. The ketone product is next converted to vanillic acid by oxidation of the g-hydroxyl group to the carboxylic acid and a further C-C cleavage reaction similar to fatty acid b-oxidation. The creation of the methyl ketone acetovanillone, which is a recognised lignin breakdown byproduct and acts as a mediator for fungal laccases, may be explained by the decarboxylation of the carboxylic acid intermediate [36]. The fungus ascomycete strain 2BW-1 has also been shown to contain a b-etherase enzyme [37].
In S. paucimobilis SYK-6, a tetrahydrofolate-dependent demethylase named LigM catalyzes the demethylation of vanillic acid to protocatechuic acid, producing 5-methyl tetrahydrofolate as a byproduct [38]. In contrast, a non-heme iron-dependent demethylase enzyme catalyzes the identical demethylation process in Pseudomonas and Acinetobacter [39,40]. Protocatechuic acid, the end result, is then Ammonia fiber expansion lignin Lipids Apex-lignin preceded by fermentation [29,30] Rhodococcus opacus (PD630) Corn fodder Lipids Pretreatment with chemicals is preceded by fermenting [31,32] used as a substrate for an oxidative ring cleavage, as will be discussed below. It is known that Phanerochaete chrysosporium in white-rot fungi metabolizes b-aryl ether model compounds mostly by Ca-Cb oxidative cleavage, producing vanillin as a byproduct [41]. The lignin peroxidase from P. chrysosporium is known to catalyze this oxidative cleavage process in vitro [42]. Vanillate dehydrogenase, which is produced by the bacteria S. paucimobilis SYK-6 and Pseudomonas sp. HR199 [4], converts vanillin into vanillic acid. 2-hydroxyacetaldehyde is a by-product of oxidative C-C breakage. Additionally, metabolites from the bacterial degradation of lignocellulose have been found to include oxalic acid [35].

Biphenyl Degradation Pathways
Through oxidation to 2,3-dihydroxybiphenyl and oxidative meta-cleavage, a number of soil bacteria may break down biphenyl and mildly chlorinated biphenyls [33]. One of the key elements of lignin is the biphenyl bond, which frequently exists between two guaiacyl units. An enzyme called LigX catalyzes the demethylation of one methoxy group [40]. Two decarboxylases, LigW, and LigW2 have been discovered in S. paucimobilis SYK-6. The catecholic product of LigX is then a substrate for oxidative meta-cleavage by the extradiol dioxygenase LigZ, and the ring fission product is then cleaved by C-C hydrolase LigY, resulting in 5-carboxyvanillic acid and 4-carboxy-2-hydroxypentadienoic [43].

Diarylpropane Degradation Pathways
In P. chysosporium, the C α -C β link is oxidatively broken, producing aromatic aldehyde compounds by lignin peroxidase. An enzyme activity that in bacteria catalyzes the removal of formaldehyde and water from a model molecule called diarylpropane has been discovered and isolated from Pseudomonas paucimobilis TMY1009 [44]. Although the gene encoding this enzyme has not yet been discovered, the metabolism of the reaction's end product, lignostilbene, has received considerable attention. From Pseudomonas paucimobilis TMY1009, lignostilbene dioxygenase isozymes have been isolated, characterized, and the associated genes have been found [40]. The larger family of carotenoid cleavage dioxygenases, which is in charge of the manufacture of apocarotenoid signalling molecules in plants, shares sequence similarities with this enzyme [45].

Degradation of Phenylcoumarane and Pinoresinol
The white-rot fungus (Phanerochaete chrysposporium) degraded the alkylated phenylcoumarane by first oxidizing the side chain, then oxidizing the heterocyclic ring to furan, and eventually cleaving the oxidative C α -C β bond [46]. Conversely, the Fusarium solani M-13-1 (filamentous fungus) broke down the phenolic phenylcoumarane through a direct C α -C β bond, yielding 5-acetylvanillone. Although in S. paucimobilis (bacteria) SYK-6 has been shown to break down phenylcoumarane compounds in lignin [3], the responsible genes have yet to be identified.
The breakdown of the (lignin component) pinoresinol has also been analyzed in Fusarium solani M-13-1. The benzylic group was oxidized, resulting in the cleavage of the C-O bond and the formation of a monocyclic ketone intermediate. A carboxylic acid product and the associated lactone were produced through further aryl-alkyl oxidation. In bacteria, S. paucimobilis SYK-6 has been shown in bacteria to degrade pinoresinol compounds of lignin, but the genes that are responsible are still to be identified. Catabolic pathways for both heterocyclic components of lignin appear to begin with a-hydroxylation. It is thus worth noting that a-hydroxylation of phenylpropane molecules using iron porphyrin bio-inspired models has a chemical precedent [47].

Bacterial Degradation of Ferulic Acid
Ferulic acid is another phenolic component of lignocellulose (released by esterase enzymes found in fungi and bacteria) that is connected to hemicellulose through ester linkages [48]. Ferulic acid esterase has already been extracted from Aspergillus niger, [49] and esterase enzymes that can produce ferulic acid from hemicelluloses have also been observed in Pseudomonas fluorescens [50] and Streptomyces avermitilis [51]. In bacteria, ferulic acid is degraded by 2 catabolic pathways. Feruloyl CoA synthetase enzyme FerA in S. Paucimobilis SYK-6 works by converting ferulic acid into feruloyl CoA, after which it is transformed into vanillin and acetyl CoA by a feruloyl CoA hydratase [4]. Some bacteria have a decarboxylase enzyme that converts hydroxycinnamic acids into 4-vinyl aromatic compounds (it was discovered in Bacillus sp. BP-7) [52].

Oxidative Cleavage of Protocatechuic Acid
Vanillin or its oxidation product vanillic acid is produced by many catabolic pathways, and vanillin or vanillic acid is then transformed by demethylation to protocatechuic acid. Numerous soil bacteria have the ability to break down protocatechuic acid through oxidative ring breakage while utilizing catechol dioxygenase enzymes that are not heme iron reliant. Intradiol cleavage between the two phenolic hydroxyl groups, catalyzed by iron(III)-dependent dioxygenases, and extradiol cleavage next to the phenolic hydroxy groups, catalyzed by iron(II)-dependent dioxygenases, are the two types of oxidative cleavage processes that are analyzed [33]. A protocatechuate 4,5-dioxygenase enzyme named LigAB has been discovered in Sphingomonas paucimobilis SYK-6. Pyruvate and oxaloacetate are then produced by hydratase LigJ and aldolase LigK. Using the trisubstituted gallic acid and 3-methyl-gallic acid, which are both cleaved by catechol dioxygenases DesB and DesZ, two iron (II)-dependent dioxygenases, S. paucimobilis SYK-6 may also break down compounds of syringyl lignin [53]. Therefore, the routes for the degradation of guaiacyl and syringyl lignin components merge upon subsequent ring cleavage. Other bacteria use protocatechuate 3,4-dioxygenase to cleave protocatechuic acid into cis,cis-muconic acid, which is then metabolized through the β-keto-adipate pathway or ortho-cleavage pathway [3].

Biological Approach for Lignin Breakdown
For efficient depolymerization of lignin, several pre-treatment procedures (catalytic and thermochemical) have been stated. Such approaches, on the other hand, necessitate stringent reaction circumstances, together with temp and pressure. Furthermore, such technologies are frequently associated with significant environmental concerns and necessitate a significant amount of energy to carry out activities [33]. Several microorganisms have well-organized metabolic systems for mortifying lignin to cyclic compounds and then bioconverting the resultant products into energy through a variety of reaction pathways [3].

Lignin Depolymerization from Enzymes Produced by Fungi
Fungi are common microorganisms associated with lignin breakdown and depolymerization. The most often used basidiomycetes white-rot fungi for ligninolytic enzyme synthesis and lignin depolymerization aspects are basidiomycetes white-rot fungi [54]. When compared to bacteria, fungi have a higher rate of depolymerization and catalytic conversion [55]. White-rot fungi have been intensively investigated for a variety of industrial applications, including the production of biomethane, degradation of textile dye, biomass de-lignification, and removal of the phenolic chemical from pollutants, because of their high depolymerization and degradation capacity of lignin [56,57]. Enzymes generated by white-rot fungus strains have been discovered to be extremely effective at degrading lignin [58]. Laccases, lignin peroxidases (LiPs), MnPs, and versatile peroxidases (VPs) are among the enzymes they create to depolymerize lignin. Pleurotus species manufacture various kinds of peroxidases to metabolize phenolic chemicals including methoxybenzene, and benzoic acid [53]. Pleurotus ostreatus and Trametes hirsute, two white-rot fungus strains, were investigated due to their capability to generate ligninolytic enzymes. The inclusion of fungal inoculates resulted in a twofold increase in fibrinolysis, as well as an increase in organic matter composability from 26.5 to 31.2%. The rate of lignin decomposition in tobacco stalks was improved by utilizing fungal ligninolytic enzymes, according to the study [59]. Coniophora puteana has been described as a laccase generator that may destroy cell walls in the presence of tannic acid [60]. Laccases and peroxidases work together to depolymerize the lignin polymer into aromatic compounds, which bacteria can then transform into high-valued commercial goods [61].
Linares et al. [62] used shock wave-induced acoustic cavitation to overexpress laccases and peroxidase enzymes in Phanerochaete chrysosporium to produce ligninolytic enzymes. Wheat bran and sugarcane bagasse were used to test the enzymes for potential delignification. When recombinant enzymes were used instead of native enzymes, outcomes showed a 25% increase in the depolymerization of lignin. Recombinant fungi were found to have 2.6-fold elevated activity of peroxidase and fourfold elevated activity of laccase than control fungi. Ligninolytic enzymes generated by Phanerochaete chrysosporium have been reported to depolymerize up to 99% of lignin in other research [63].
Improved fungal efficiency toward lignin depolymerization has been demonstrated in multiple routes, proteomic, and genomic studies. Lac I extracted from a strain of basidiomycetes (Phlebia brevispora) was reported. Gene was converted into Pichia pastoris for protein expression. Consequences showed that the enzyme created by lac I had a good solvent/salt tolerance, demonstrating that it might be used in a variety of biotechnological applications [64]. Jin et al. [65] stated the identification of the lac I gene from Ganoderma tsugae, which was found to have the ligninolytic capacity in knockout tests. The principal fungal enzymes concerned with the depolymerization of lignin are various peroxidases and laccases [66]. Penicillium chrysogenum and Aspergillus niger, two soft-rot fungal strains with the ability to degrade pine wood and sycamore, were found [67].

Lignin Depolymerization from Enzymes Produced by Bacteria
In comparison to fungus, bacterial strains are thought to be less efficient in ligninolytic. Only a few bacterial strains are capable of degrading lignin. Various bacteria have been shown to alter and depolymerize lignin into chemicals with remarkable efficiency. Rhodococcus jostii (RHA1 specie) had been widely explored for its lignin breakdown capabilities as well as lignin consumption and transformation to other value-added chemicals [68]. The metabolically re-routed mechanism was found in Rhodococcus jostii for breaking down lignin to produce aromatic dicarboxylic acids, which could be utilized to make bioplastics. Orthocleavage to -the keto-adipate pathway is extensively used in protocatechuic acid metabolism [69]. Two forms of DyPs were identified in Rhodococcus jostii (RAH1): DypA (periplasmic) and DypB (non-periplasmic). Peroxidases have substantially lower activity than other DyPs kinds, according to enzyme kinetics. When compared to pyrogallol or ABTS, DypA had a greatly enhanced (sixfold) decolorization capacity for Reactive Blue 4 dye. For ABTS and oxidized Mn(II), DypB had the highest misleading specificity. The work proposed a metabolic engineering technique to increase Rhodococcus jostii's lignocellulose breakdown capacity (RAH1) [4]. Pseudomonas spp. can assimilate a wide range of cyclic compounds and frequently present in bacteria separated for their capability to nurture on cyclic substrates. They efficiently grow in minimal medium and have an inclusive genetic toolbox for metabolic engineering [70]. As a result, it is thought to be an excellent model organism for investigating lignin biotransformation and generating new industrial strains.
Previous studies have shown that Pseudomonas putida has a high lignin degradation capacity and can break down lignin into low molecular weight components. By changing lignin-rich fractions, bioconversion leads to the buildup of polyhydroxyalkanoates (PHA), competent unprocessed material for creating bioplastics [71,72] using genome sequencing to extensively investigate lignin transforming enzymes in Pseudomonas putida strain NX-1. Peroxidases with dye decolorization capability were investigated, and they were found to be effective on dyes as well as ligninderived aromatic compounds. The P. putida KT2440 strain helped researchers better understand aromatic compound uptake [73,74].
Apart from the bacterial strains mentioned above, Amycolatopsis specie had been extensively reported for having elevated bioconversion of high-molecular-weight chemicals into compounds with low molecular weight and excellent lignin depolymerization capability when compared to other genera [75].To study the metabolism of aromatic chemicals in Amycolatopsis spp., a variety of metabolic engineering methods have been applied [76]. The vanillin dehydrogenase gene was extracted and cloned from the genome of Amycolatopsis (39,116 spp.). Vanillin production was improved after vanillin dehydrogenase was knocked out (up to 2.3 times). The bacteria lost their ability to use vanillin as a C-source for the production of energy after knocking out the vanillin dehydrogenase gene, so produced vanillin was competently stored in cells [77].
Depolymerizing lignin biomass could also produce valueadded cis-muconic acid. Genome engineering was used to study the Amycolatopsis species for biomass conversion and important compound manufacturing, including biofuels [78]. Using pre-engineered Amycolatopsis, lignin depolymerization found to be capable of producing cis-muconic acid (39,116 spp.). Because the cis-muconic acid hydrolyzing enzyme (catB gene) was knocked out, cis-muconic acid cannot be collected in the cell. Even though the gene knockdown reduced the growth of the cell, the modified microbe accumulated more than three times the amount of original microbes. Furthermore, several ligninolytic bacteria have previously been discovered and their metabolic routes have been described, and there is yet a wide variety of organisms to be discovered. As a result, there is a pressing need to create effective screening approaches that allow for the discovery of novel microbial activity [79].

Other Microbial Enzymes for Lignin Depolymerization
Various bacterial and fungal strains have been found to produce enzymes with the ability to degrade lignin. The use of in vitro enzymes in ligninolytic processes can alleviate some of the drawbacks, such as direct contact and culturing time between the substrate and the microbe [80]. The use of a single enzyme to digest biomass containing lignin has been described in several studies. Further inquiry and exploration of molecular mechanisms will require multi-enzymatic complicated systems [81]. Non-specific cleavage is used by the majority of ligninolytic enzymes to break down cyclic compounds. Laccases and peroxidases are the two most common groups of in vitro enzymes. By oxidative cleavage, both enzymatic families depolymerize lignin. Rather than cleaving links or substrates, these enzymes target lignin molecules at random. MnP and LiP are the two most common forms of peroxidases. Furthermore, due to their wide applicability, two other kinds, DyP and VP, have also gained attention [13].

Manganese Peroxidases (MnPs)
The MnP (E.C1.11.1.13) enzyme is a glycosylated protein that needs H2O2 (oxidant) to begin catalysis [33]. It is found all over the world. MnP manufacturing is gaining popularity as a result of its numerous uses in the industrial and biological industries [82,83]. It is a high-potential enzyme having a highly flexible nature that plays a key role in industrial bioprocesses. MnP enzyme is involved in the oxidation of non-phenolic and phenolic substances [84]. MnPs are extracellular enzymes that are generated by several white and brown-rot fungus taxa, as well as a few bacterial species. MnPs are regarded as the proficient biocatalysts for lignin depolymerization when conjugated with other enzymes such as laccases. MnP production was first discovered in Phanerochaete chrysosporium [85].
The ability to synthesize MnP in sufficient quantities is critical for developing an environmentally benign substitute to lower the chemical cost of producing industrial-scale biofuels. MnPs have a wide range of applications, including pollution bioremediation/biodegradation, bio-bleaching, biopulping, and more [86]. A variety of microbial taxa, including fungi, bacteria, and algae, have been found to produce MnP efficiently [87]. Extracellular MnP production has been reported from Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Phanerochaete sordida, Physisporinus rivulosus, Dichomeris squalene and Phlebia radiata. MnP production was reported by utilizing Phanerochaete chrysosporium BKM-1767 under a variety of nutritional and climatic circumstances. To boost the biodegradation efficiency of xenobiotics, more research into MnP synthesis with improved inorganic/organic ions tolerance is needed [88].

Lignin Peroxidases (LiPs)
LiP is a heme-containing monomeric protein that destroys lignin-containing biomass residues by oxidative degradation [89]. LiP was first studied in Phanerochaete chrysosporium's extracellular matrix [90]. Following that, LiP isozymes from different microbial species such as Trametes versicolor, Phanerochaete sordida, Phlebia radiata, and others have been discovered [91]. Approximately 96% of all species produced MnPs. Daedaleopsis septentrionalis and Cylindrobasidium evolve, two novels, LiPs-producing fungal strains, were found in 26% of the total strains tested. The automated screening was used to identify ligninolytic enzymes in this study, which resulted in enhanced test conditions. To begin and catalyze phenolic and non-phenolic chemicals, LiP requires H 2 O 2 [92]. Due to its larger redox potential than other enzymes in the same family that impart significant lignin disruption features, LiP is the most prevalent peroxidase type. Apart from depolymerizing lignin, LiP can also be used to generate bioplastics and other value-added chemicals due to its great delignification capacity [58,93,94]. LiPs have been shown to depolymerize and degrade lignin with great selectivity and yield when used alone or in conjunction with other ligninolytic enzymes [95].

Laccases
Laccase enzymes are oxidoreductases found in a variety of bacterial and fungal species. Species of brown-rot and whiterot fungus (with redox potentials up to 800 milli volts) have been reported to produce highly powerful and efficient laccases [66]. Laccases are blue copper phenoloxidases that utilize O 2 as an electron acceptor to oxidize phenolic molecules [96]. It is termed the "greenest" enzyme because it has a basic obligation of utilizing O 2 from the environment and converting it into the H 2 O as a by-product [97]. Laccases can be found all over the place in nature. They are involved in a variety of processes, including degradative and synthetic ones [66]. Their biological functions may differ from the synthesis and breakdown of lignin (in developed plants) and chitin (in arthropods) by diverse fungal and bacterial strains. More than sixty fungal strains with excellent laccase production capacity have been reported, according to a preliminary estimate. Laccase-producing fungal species include Dichomitus squalene, Lentinula edodes, Current maxima, Trametes versicolor, Pleurotus ostreatus, Phanerochaete chrysosporium, and Irpex lacteus [98][99][100].
Laccases have lower oxidative adaptability and require less catalysis. These enzymes catalyze a variety of chemical processes, such as aromatic compound oxyfunctionalization and ring cleavage, monomeric crosslinking, and polymer degradation [101]. Laccases are used in a variety of redox processes, including fiber alteration, ethanol generation, bioremediation, biofuel cell production, effluent treatment, and biosensors [102]. Furthermore, due to their ecologically friendly nature, fungal enzymes reduce pollution.

Ligninocellulose Depolymerization Products
The following valuable products are obtained from ligninocellulose depolymerization (Fig. 3).

Industrial Biocatalysts
The focus of current research is on developing sustainable methods for manufacturing enzymes to make overall bioprocess less costly [103,104]. In several industries, enzymes have piqued interest as potential biocatalysts. The use of lignocellulosic waste biomass reduces the cost of raw materials (60%), making the entire industrial bioprocess more cost-effective [81]. Several investigations had reported the production of industrially important enzymes such as cellulases, laccases, xylanases, proteases, lipases, peroxidases, pectinases, and -galactosidases from agricultural-industrial lignocellulosic materials by microbial fermentation. Using delicious peels of citrus fruit as a substrate for growth, Pectinases were manufactured for the fruit juice business from the Schizophyllum commune [105]. A lipase yield of 1038.86 U/gds was attained from Penicillium fellutanum Fig. 3 Lignin depolymerization products from a canola oilseed cake (solid-state culture) under optimal processing conditions [106]. Among a wide range of agricultural-industrial wastes tested, sesame, canola, cotton, and linseed oilseed cake, wheat and rice bran triggered the greatest lipolytic enzyme synthesis by P. notatum [107]. Laccase manufacture had produced by Myrothecium gramineum from cowpea pods. Xylanase was obtained from sugarcane straw under submerged fermentation conditions using Trichoderma reesei QM 9414 [108].

Biologically Active Compounds
Pharmaceutical and food sectors value biological active compounds such as phytoconstituents and phenolics, which have antioxidant and health-promoting properties. Phenolic chemicals, such as tannins, flavonoids, phenolic acids, and others, are abundant in vegetables and [109]. Agricultural waste is a rich basis of bioactive compounds which could be extracted via microbial fermentation [110]. From solid-state bioprocessing of Aspergillus niger HT4 by-products, 8 distinct phenolics were discovered with the possible antioxidant ability [111]. Microbial transformation of apricot pomace resulted in a 30% increase in neochlorogenic acid, chlorogenic acid, rutin, and quercetin 3-acetyl-glucoside titer with A. niger and a 78% enhancement with Rhizopus oligosporus in a variety of phenolic compounds titer unambiguously, quercetin 3-acetylgluco, rutin, and neochlorogenic acid [112].

Biofuels
Known negative effects of traditional fuels, microbesassisted biofuels manufactured from lignocellulosic substrates like rice straw, pods, molasses, bagasse, sweet potato, maize waste, and sugar beet waste have attracted enormous attention in the last 20 years. Biofuel production from edible food crops has been heavily criticized because the majority of the world's population is previously suffering from severe food shortages and hunger. Investigating non-edible feedstocks for biofuels promises to be a viable research field, and the use of lignocellulosic biomasses is an appealing count to industry. Agro-industrial leftovers include an elevated number of carbohydrates, lipids, and amino acids which could be used as substitute resources for biofuel production [113]. S. cerevisiae cultured in yeast extract, D-glucose, and Bacto peptone-based fermentation medium produced bioethanol from date palm waste [114]. Fermentative bioprocess produced 15% ethanol under ideal conditions.

Biodiesel
Trans-esterification of fats and oils from plants, animals, and microorganisms produces biodiesel, which is a variety of fatty acid alkyl esters with various chain lengths [115,116].
Because a major amount of the molecular structure of the feedstock is preserved in the ensuing bioproduct, feedstocks for biodiesel had a significant impact on product attributes. Enzyme-assisted production of biodiesel has several distinct advantages, including low energy requirements, efficient bioconversion of free fatty acid-containing oils, pure glycerol by-products, and easy product recovery and reprocessing [115,117]. In presence of endogenous lipolytic enzymes, triglycerides in certain microalgal cells are transformed to FFA in favorable settings [118]. Membrane lipids of these cells also produce a significant amount of polar lipids in glycolipids and phospholipids form. Microalgae lipids appear to be potential feedstocks for biodiesel production, although they are high in phospholipids and FFA.

Biogas
Another bioenergy-raised area that may be made from bioresources such as hydrogen and methane is biogas. Several biological methods for producing biohydrogen have been proposed; however, the type of synthesis chosen may have an impact on the yield. Biohydrogen has been produced using a variety of fermentative microbial strains (Clostridium, Enterobacter, and Bacillus) [119,120]. Species of Bacillus and Thermotogales were dominating bacteria yielding both H 2 and CH 4 in mesophilic fermentation settings of food waste [121]. In thermophilic circumstances, however, Desulfotomaculum geothermicum and Thermoanaerobacterium thermosaccharolytium were dominating strains, ensuring the formation of sole hydrogen.
Along with the growing population of human beings, the production of agricultural waste is expected to rise [122]. Agro-waste is primarily made up of bagasse, crop peelings, and animal manure, all of which contain a large number of carbohydrates that could be converted to fermentable sugars through the production of biogas. Many additional industrial waste materials had also been identified as a potential source for the synthesis of biogas, including effluent from oilseed mills, a slurry of rice, and cassava waste [123][124][125].

Biopolymers
Many polymeric compounds can be produced by fungi and bacteria from lignocellulosic wastes, including polysaccharides (endo and Exo), polyphosphates, and polyhydroxyalkanoates [126]. Bioconversion of lignocelluloses to commercially important biopolymers such as xanthan, chitosan, cellulose, pullulan, and others has been studied extensively. Paper mill wastewater and apple pomace are suitable substrates for Penicillium citrinumin and Gongronella butleri to synthesize chitosan in a cost-effective and environmentally friendly manner [127,128]. Pullulan was synthesized in a stirred tank bioreactor utilizing Aureobasidium pullulans P56 and pre-treated beet molasses in a study.

Biosurfactants
Biosurfactants are biomolecules with significant benefits over artificial complements in terms of selectivity, biodegradability, and toxicity. They are divided into many types, such as lipoproteins, lipopeptides, and glycolipids, and have a wide variety of dietary, medical, cleansing, bioremediation, and cosmetic applications. They could be used as a food preservative to improve the product's rheological properties; however, their high production costs limit their use on a broader scale [129]. Paneibacillus sp. D9 produced lipopeptide-type biosurfactant using sunflower oil and discarded coconut as substrates for fermentation, according to Jimoh and Lin [130]. Serratia nematodiphila has proven synthesis of a glycolipid biosurfactant with emulsifying and antibacterial properties utilizing rice straw [131].

Bioplastics
At present, bioplastic manufacturing sectors have huge costcompetitiveness difficulty when compared to petro-based plastics, that is much less expensive. These findings have piqued researchers' interest in using agro-based fermentative feedstocks to make cheaper bioplastics using fungal and bacterial strains. Sugar refinery waste was exploited to produce PHA by a Pseudomonas aeruginosa submerged fermentative bioprocess [132]. PHB bioplastics were produced and accumulated from Ralstonia eutropha fermentation using hemp hurds biomass [133], Bacillus sp. fermentation using sugarcane bagasse [134], Lactobacillus acidophilus fermentation using date molasses [135], and Cupriavidus necator fermentation using cashew apple juice [136].

Biocontrol Agents
By using microbial-assisted fermentative processes, agriculture lignocellulosic residues are being investigated to produce new biocontrol agents like bioinsecticides and biopesticides. A recent study used rice husk as a lignocellulosic substrate to produce bioinsecticide from Trichoderma harzianum and Beauveria bassiana in a packed bed bioreactor. Furthermore, cassava dregs and palm kernel cake blends were revealed to be promising substrates for generating bioinsecticides from Bacillus thuringiensis subsp. aizawai [137].

Polyurethane Products
Polyurethanes are exceptional polymeric compounds that are among the most flexible polymers for the purposes they are designed for. Due to its ability to replace fossil fuelbased rigid polyurethane foam (RPUF) components, the valorization of lignin in RPUF has been the goal of research.
However, the overall viability of RPUF is determined by a variety of factors, such as processability, cost-effectiveness, and performance retention throughout the foam's service life [138]. Furthermore, lignin can be consolidated as a polyol in polyurethane production and thus structurally changed before use [139]. Furthermore, the hydroxyl (-OH) functional lignin polymer can efficiently substitute polyols in the manufacture of polyurethane [140].

Vanillin
Since 1937, vanillin has been economically extracted from lignin, and in 2011, the global market for vanillin was predicted to be 16,000 metric tonnes with a value of $230 million. Around 20% of produced vanillin comes through the lignin valorization process, with the remaining 80% coming from crude petroleum [141]. Vanillin is one of the most widely used flavoring agents in the world [63], and it is made from lignin via an oxidative catalytic mechanism rather than a hydrogenolysis pathway [77].

The Techno-Economic Analysis of Lignin Valorization
Bioproducts made from biological lignin have not been produced at commercial, demonstration, or pilot scales. In order to reduce the technical risk for scale-up and boost cost competitiveness across the board in the next-generation biorefinery, techno-economic analysis is currently used to not only identify key cost drivers of lignin valorization but also to prioritize research directions and project the economic impacts of the coproduction integration of lignin-based products through process design. As depicted in Fig. 4, several lignin scenarios that may be utilized to create goods with additional value were found. After burning lignin-rich wastes, scenario 1 of the NREL 2011 design case [142] plans to use 23.9% of the total energy for processing steam or heat, 52.5% for electricity consumption, and 23.6% for revenuegenerating sales to the grid. In case 2, only 23.6% of the lignin leftovers would be turned into lignin-based coproducts, with the remainder being burned to provide electricity and steam for the plant and no extra electricity being sold to the grid. The manufacturing of lignin-based coproducts would get 76.1% of the lignin leftovers under scenario 3, while the remaining 23.9% would be burned to generate plant steam and heat [143].
In case three, no lignin-derived electricity would be produced, therefore plant electricity would have to be obtained from the grid. Additional operation units and processing steps in the integrated new biorefinery design will result in higher operating and total capital costs when the coproduction facility is envisioned as a bolt-on process into a lignocellulosic ethanol plant [142]. The results of the techno-economic study demonstrated that the economic viability of lignin valorization in biorefineries depended on high yields of lignin conversion to value-added products and low costs of product separation. Thus, the development of biorefineries that turn plant biomass into fuel ethanol will result in the production of significantly more lignin; nevertheless, in order to create a viable biorefinery, efforts must still be made to convert this lignin into coproducts of higher value.

Future Perspectives
Current lignin-based developments are still mostly bound by low fuel and energy consumption. The commercialization of lignin in the same way as an originator of chosen macromolecular materials and aromatic compounds will necessitate more exploration and improvement. Concerning the evaluation of natural lignin configuration, there are still obstacles in the treatment of lignin depolymerization, and the applications of end products. Even though such a zone is being seriously considered. However, major efforts in terms of powerful collaboration amongst many fields in science and engineering are required. There is also an issue of feedstock variation from one region to the next, as well as from one growing season to the next. Despite these challenges, lignin is able to be used as a substitute for fossil-based feedstock in a variety of applications. Furthermore, by establishing strong approaches that push for fossil fuel substitutes, the government can play a significant part in stimulating commercialization. Lignin valorization is expected to progress and thrive rapidly, according to a multidisciplinary and collaborative team. Achieving optimal lignin valorization in chemicals needs combining an efficient lignin separation and depolymerization activity. Likewise, the sustainability of lignin depolymerization is determined by the depolymerization technique and the chemical makeup of the lignin substrate. Because of multifaceted features of lignin, that can strongly affect the involved reaction pathways in lignin valorization, not only is progressive in cost-effective approaches at a standstill, but implicated processes also significantly distort the structural individuality of the lignin composition. As a result, the yield for current and future biorefineries, as well as certain high-value-added platform chemicals, has been constrained.

Conclusions
Pulp and paper mills, power plants, and biorefineries all use lignin biomass to generate energy and value-added chemicals. Traditional lignin valorization methods limit its use as a low-quality solid fuel in biorefinery businesses to generate power and heat. Its valorization could open up new frontiers in biofuel and value-added chemical synthesis because of its high aromatic characteristics and large accessibility of renewable feedstock. Furthermore, an integrated biorefinery with lignin conversion will allow for a cost-effective, longterm, and diverse business based on lignocellulosic biomass. Less reactivity, highly asymmetrical polymer formation, and robust hydrogen interactions during diverse industrial processes are the key obstacles to efficient lignin utilization. The subsequent product recovery is mostly governed by the fractionation procedure. The main concern is that the amount of lignin produced will much outnumber the existing global marketplace for lignin utilized in specialty stocks. To turn lignin into value-added goods, new ideas and technologies are required.
Acknowledgements The research leading to these results has received funding from the Norwegian Financial Mechanism 2014-2021 under the Project number 2020/37/K/ST8/03805. The authors are thankful to the University of Punjab, Pakistan for the scholastic support.

Conflict of Interest
The authors declare no competing interests.
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