Microbial degradation of lignin (Enzymes, Steps, Mechanisms)
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Lignin is a complex organic polymer that is found in the cell walls of most plants, especially in wood and bark. It is formed by the oxidative coupling of 4-hydroxyphenylpropanoids, which are derived from phenylalanine and tyrosine. Lignin is the most abundant aromatic biopolymer on Earth, accounting for about 30% of the organic carbon and 15-30% of the dry weight of lignocellulosic biomass .
Lignin plays a crucial role in the structure and function of plant cell walls. It provides mechanical strength, rigidity, and resistance to biotic and abiotic stresses, such as pathogens, insects, UV radiation, and water loss . Lignin also facilitates the transport of water and nutrients in the plant vascular system by making the cell walls more hydrophobic and less permeable. Lignin crosslinks with polysaccharides, such as cellulose and hemicellulose, to form a three-dimensional network that reinforces the cell wall matrix .
Lignin is also an important source of bioenergy and bioproducts. It can be converted into biofuels, such as ethanol and biodiesel, by biological or chemical processes. Lignin can also be used to produce various value-added chemicals, such as phenols, aromatics, and antioxidants . Lignin has many potential applications in various industries, such as paper, textile, pharmaceutical, cosmetic, and agricultural sectors.
However, lignin also poses some challenges for the utilization of lignocellulosic biomass. Lignin is a highly heterogeneous and recalcitrant polymer that is difficult to degrade and separate from polysaccharides . Lignin hinders the enzymatic hydrolysis of cellulose and hemicellulose into fermentable sugars, which reduces the efficiency and yield of biofuel production. Lignin also affects the quality and properties of paper and pulp products by causing coloration, brittleness, and reduced strength.
Therefore, understanding the biosynthesis, structure, and degradation of lignin is essential for improving the valorization of lignocellulosic biomass. In this blog post, we will discuss the following topics:
- The structure of lignin
- Microorganisms involved in lignin degradation
- Enzymes involved in the degradation of lignin
- Factors affecting lignin degradation
- The process of lignin degradation
- Mechanisms of microbial degradation of lignin
- Example of lignin degradation by Pseudomonas
Lignin is a complex organic polymer that forms key structural materials in the support tissues of most plants. It is the most abundant aromatic biopolymer on Earth, accounting for about 30% of the organic carbon and 20 to 35% of the dry mass of wood.
Lignin is composed of phenylpropane units linked together by various chemical bonds, such as ether and carbon-carbon bonds. The phenylpropane units are derived from three main precursors, called monolignols: p-coumaryl alcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S). These precursors differ in the number and position of methoxy groups attached to the phenyl ring.
The relative amount and ratio of the three monolignols vary depending on the plant source and environmental factors. Lignins are typically classified according to their syringyl/guaiacyl (S/G) ratio, which reflects the proportion of S and G units in the polymer. Lignins from softwoods (gymnosperms) have a high S/G ratio, as they are mainly composed of G units. Lignins from hardwoods (angiosperms) have a lower S/G ratio, as they contain both G and S units. Lignins from grasses (monocots) have a very low S/G ratio, as they also include H units.
Lignin is not a uniform or regular polymer, but rather a heterogeneous and irregular one. The monolignols are randomly coupled and cross-linked by different types of bonds, resulting in a three-dimensional network with a high molecular weight and a high degree of branching. The most common type of bond in lignin is the β-O-4 ether bond, which accounts for about 50% of all linkages in lignin. Other types of bonds include 5-O-4, α-O-4, β-β, β-5, β-1, 5-5, and 4-O-5 linkages.
The structure of lignin is one of the most challenging problems in natural polymer chemistry, as it is difficult to determine by conventional methods. Lignin does not have a single structural formula, but rather a range of possible structures that vary in composition, sequence, and configuration. The properties of lignin depend on its elemental composition, functional groups, molecular weight, degree of polymerization, branching pattern, and type and frequency of linkages.
Lignin has several important functions in plants. It provides structural support and rigidity to the cell wall, especially in vascular tissues such as xylem and sclerenchyma. It helps in water and nutrient transport by making the cell wall hydrophobic and preventing its collapse under negative pressure. It also protects the plant from chemical and biological attacks by acting as a barrier and an antioxidant. Lignin is believed to have played a role in the evolution of terrestrial plants by enabling them to withstand the compressive forces of gravity and to colonize dry habitats.
Lignin is a complex aromatic polymer that is resistant to degradation by most microorganisms. However, some fungi and bacteria have evolved the ability to degrade lignin by producing various enzymes and metabolites that can break down the lignin structure. The microorganisms involved in lignin degradation can be classified into three main groups: lignin-degrading fungi, lignin-degrading bacteria, and lignin-degrading actinomycetes.
Lignin-degrading fungi
Fungi are the most efficient and diverse lignin degraders in nature. They can degrade lignin by producing extracellular enzymes that catalyze oxidative reactions on the lignin molecules. The most common enzymes involved in fungal lignin degradation are lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase. These enzymes generate free radicals that can cleave various bonds in the lignin polymer, resulting in the formation of smaller fragments that can be further metabolized by the fungi or other microorganisms.
Among the fungi, white-rot fungi are the most effective lignin degraders, as they can degrade all the components of wood, including cellulose, hemicellulose, and lignin. White-rot fungi belong to the Basidiomycetes group and include species such as Phanerochaete chrysosporium, Pleurotus ostreatus, Ganoderma lucidum, and Trametes versicolor. Some white-rot fungi can also produce lignin-degrading auxiliary enzymes (LDA) that enhance the activity of LiP, MnP, and laccase by providing hydrogen peroxide or redox mediators.
Other groups of fungi that can degrade lignin are brown-rot fungi and soft-rot fungi. Brown-rot fungi belong to the Basidiomycetes group and include species such as Gloeophyllum trabeum, Postia placenta, and Serpula lacrymans. They degrade wood by preferentially attacking cellulose and hemicellulose, leaving behind a brownish residue of modified lignin. Brown-rot fungi produce hydroxyl radicals that depolymerize cellulose and hemicellulose, but they do not produce LiP or MnP. They may produce laccase or other phenol oxidases that modify the lignin structure.
Soft-rot fungi belong to the Ascomycetes group and include species such as Chaetomium globosum, Aspergillus niger, and Penicillium chrysogenum. They degrade wood by producing cellulases and hemicellulases that hydrolyze cellulose and hemicellulose, as well as esterases and lipases that cleave ester bonds in lignin. Soft-rot fungi do not produce LiP or MnP, but they may produce laccase or other phenol oxidases that modify the lignin structure.
Lignin-degrading bacteria
Bacteria are less efficient than fungi in degrading lignin, as they lack extracellular enzymes that can directly attack the lignin polymer. However, some bacteria can degrade lignin by producing intracellular enzymes that act on smaller fragments of lignin that are released by fungal or chemical degradation. The most common intracellular enzymes involved in bacterial lignin degradation are β-ketoadipate pathway enzymes, which convert aromatic compounds into central metabolites such as acetyl-CoA.
The most studied group of bacteria that can degrade lignin are sphingomonads, which belong to the Alphaproteobacteria class and include species such as Sphingobium sp., Sphingomonas sp., Novosphingobium sp., and Sphingopyxis sp. These bacteria can degrade a wide range of aromatic compounds derived from lignin, such as vanillin, syringaldehyde, ferulic acid, p-coumaric acid, guaiacol, and catechol. They can also produce extracellular enzymes such as laccase or peroxidase that modify the lignin structure.
Other groups of bacteria that can degrade lignin are actinobacteria, which belong to the Actinobacteria phylum and include species such as Streptomyces sp., Rhodococcus sp., Nocardia sp., and Mycobacterium sp. These bacteria can degrade a variety of aromatic compounds derived from lignin, such as benzoate, protocatechuate, 3-hydroxybenzoate, 4-hydroxybenzoate, gentisate, homogentisate, salicylate, phthalate, and anthranilate. They can also produce extracellular enzymes such as laccase or peroxidase that modify the lignin structure.
Lignin-degrading actinomycetes
Actinomycetes are a group of filamentous bacteria that share some characteristics with fungi. They belong to the Actinobacteria phylum and include species such as Streptomyces sp., Amycolatopsis sp., Thermobifida sp., Saccharopolyspora sp., Saccharomonospora sp., Thermomonospora sp., Micromonospora sp., Nocardiopsis sp., Actinosynnema sp., Frankia sp., Nocardia sp., Rhodococcus sp., Mycobacterium sp., Corynebacterium sp., Arthrobacter sp., Micrococcus sp., Cellulomonas sp., Brevibacterium sp., Propionibacterium sp., Actinomyces sp., Nocardioides sp., Gordonia sp., Dietzia sp., Tsukamurella sp., Kineococcus sp., Kocuria sp., Microbacterium sp., Agromyces sp., Curtobacterium sp., Leifsonia sp., Rathayibacter sp., Clavibacter sp., Dermabacter sp., Dermacoccus sp., Brachybacterium sp., Brevibacillus sp., Paenibacillus sp., and Bacillus sp. These bacteria can degrade a variety of aromatic compounds derived from lignin, such as vanillin, syringaldehyde, ferulic acid, p-coumaric acid, guaiacol, catechol, protocatechuate, 3-hydroxybenzoate, 4-hydroxybenzoate, gentisate, homogentisate, salicylate, phthalate, and anthranilate. They can also produce extracellular enzymes such as laccase, peroxidase, or dye-decolorizing peroxidase (DyP) that modify the lignin structure.
Lignin is a complex and recalcitrant biopolymer that requires oxidative enzymes to break its chemical bonds. The main enzymes involved in lignin degradation are laccase, lignin peroxidase, manganese peroxidase, and versatile peroxidase . These enzymes are produced by various microorganisms, especially fungi, that can utilize lignin as a source of carbon and energy. Here, we briefly describe the structure, function, and mechanism of these enzymes.
Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic and non-phenolic substrates with the reduction of molecular oxygen to water . Laccase has four copper atoms in its active site, which are classified into three types: T1, T2, and T3. The T1 copper is responsible for electron transfer from the substrate to the enzyme, while the T2 and T3 coppers form a trinuclear cluster that reduces oxygen to water. Laccase can oxidize a wide range of lignin-related compounds, such as methoxylated phenols, benzenediols, aromatic amines, and polyphenols . However, laccase alone cannot degrade non-phenolic lignin units, which constitute the majority of lignin structure. Therefore, laccase requires the presence of low molecular weight mediators, such as 2,2`-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT), or violuric acid (VA), that can act as electron shuttles between the enzyme and the lignin substrate . The mediators are oxidized by laccase to form radicals that can attack and cleave non-phenolic lignin bonds .
Lignin peroxidase is a heme-containing peroxidase that catalyzes the oxidation of lignin by hydrogen peroxide . Lignin peroxidase has a high redox potential and can directly oxidize non-phenolic lignin units without mediators . Lignin peroxidase has a single iron protoporphyrin IX (heme) prosthetic group in its active site, which cycles between two oxidation states: Fe(III) and Fe(IV). The catalytic cycle of lignin peroxidase involves two steps: (i) activation of hydrogen peroxide by Fe(III) to form compound I, a ferryl-oxo intermediate with a porphyrin radical cation; and (ii) oxidation of lignin by compound I to form compound II, a ferryl intermediate without a radical cation. Compound II is then reduced back to Fe(III) by another molecule of hydrogen peroxide. Lignin peroxidase can oxidize various types of lignin bonds, such as β-O-4, α-O-4, 5-5`, and β-5`, by abstracting hydrogen atoms from the benzylic or α-carbon positions to form carbon-centered radicals . These radicals can undergo further reactions, such as rearrangement, fragmentation, or condensation, leading to the depolymerization of lignin .
Manganese peroxidase is another heme-containing peroxidase that catalyzes the oxidation of lignin by hydrogen peroxide in the presence of manganese ions . Manganese peroxidase has a similar structure and catalytic cycle as lignin peroxidase, except that it uses Mn(II) as an electron donor instead of lignin. Manganese peroxidase oxidizes Mn(II) to Mn(III), which then complexes with organic acids, such as oxalate or malate, to form low molecular weight chelates . These chelates can diffuse into the lignocellulosic matrix and oxidize phenolic lignin units to form phenoxyl radicals . The phenoxyl radicals can then initiate the degradation of non-phenolic lignin units by radical coupling or Cα-Cβ bond cleavage .
Versatile peroxidase is a hybrid enzyme that combines the catalytic properties of both lignin peroxidase and manganese peroxidase . Versatile peroxidase has a heme prosthetic group and two manganese-binding sites in its active site. Versatile peroxidase can directly oxidize non-phenolic lignin units like lignin peroxidase, as well as oxidize Mn(II) to Mn(III) like manganese peroxidase . Versatile peroxidase can also use other substrates, such as veratryl alcohol or dyes, as electron donors or mediators for lignin oxidation .
These enzymes work synergistically to degrade lignin into smaller fragments that can be further metabolized by microorganisms. The degradation of lignin is influenced by various factors, such as moisture content, nitrogen availability, glucose concentration, and oxygen level.
Lignin degradation is a complex and dynamic process that involves various microorganisms, enzymes, and environmental factors. Some of the important factors that affect lignin degradation are:
- Moisture content: Degradation of lignin occurs most rapidly when the source is completely saturated, and free water is present. Increasing the amount of water has little effect on the degradation process until aeration becomes impaired.
- Added Nitrogen: The growth and biosynthesis of lignin-degrading enzymes require a particular concentration of nitrogen. It is observed that the rate of lignin degradation increases with the increase in nitrogen concentration but only to a certain degree. Besides, the addition of a very high concentration of nitrogen tends to have a detritus effect on the overall carbohydrate degradation, along with lignin degradation.
- Added glucose: The process of lignin degradation slows down with the addition of glucose as an excess of readily available energy source like glucose causes decreased consumption of lignin. When all the available energy sources are omitted, the microbial action on the lignin-rich compounds increases.
- Aeration: The rate of lignin and carbohydrate metabolism increases with the increase in oxygen content in the environment. This is due to the fact that most of the microorganisms involved in lignin degradation are aerobic microorganisms that thrive in a high oxygen atmosphere. Besides, efficient lignin degradation is also seen under a CO2 concentration of 30% and an O2 concentration of 10%. However, a pure O2 atmosphere might be toxic if any other form of energy like glucose or cellulose is present as it suppresses lignin degradation.
These factors can influence the activity, stability, and specificity of lignin-degrading enzymes, as well as the diversity and abundance of lignin-degrading microorganisms. Therefore, optimizing these factors can enhance the efficiency and yield of lignin degradation and valorization.
Lignin degradation is a complex and challenging process that involves the action of various microorganisms and enzymes. The process can be divided into two main steps: depolymerization and mineralization.
Depolymerization is the initial step that breaks down the large and heterogeneous lignin polymer into smaller and more soluble fragments. This step is mainly carried out by extracellular enzymes secreted by lignin-degrading microorganisms, such as fungi and bacteria. The most important enzymes involved in depolymerization are lignin peroxidase (LiP), manganese peroxidase (MnP), laccase (Lac), and versatile peroxidase (VP) .
These enzymes are oxidative in nature and catalyze the cleavage of various linkages in lignin, such as β-O-4, β-β, β-5, 5-5, and α-O-4 bonds. The enzymes act on both phenolic and non-phenolic subunits of lignin, generating free radicals that further react with each other or with oxygen to produce smaller molecules. The products of depolymerization include aromatic aldehydes, ketones, acids, alcohols, phenols, quinones, and other compounds .
Mineralization is the final step that converts the depolymerized lignin fragments into simple molecules that can be assimilated by the microorganisms or released into the environment. This step is mainly carried out by intracellular enzymes within the microbial cells. The enzymes involved in mineralization include various oxidases, dehydrogenases, demethylases, dioxygenases, hydrolases, and decarboxylases .
These enzymes catalyze a series of reactions that degrade the aromatic ring structure of lignin derivatives and introduce functional groups such as hydroxyl, carboxyl, keto, and sulfhydryl groups. The products of mineralization include simple organic acids, alcohols, aldehydes, ketones, carbon dioxide, water, and other compounds .
The process of lignin degradation can be affected by various factors such as moisture content, nitrogen availability, glucose concentration, oxygen level, pH, temperature, and microbial diversity . These factors can influence the production and activity of lignin-degrading enzymes as well as the metabolic pathways of lignin degradation.
Lignin degradation is an important process for the utilization of lignocellulosic biomass as a renewable source of energy and chemicals. Lignin degradation can also contribute to the carbon cycle and the bioremediation of environmental pollutants . However, lignin degradation is still not fully understood and requires further research to optimize its efficiency and applications.
Microbial degradation of lignin is a complex process that involves various enzymes, mediators, and metabolic pathways. Microorganisms can degrade lignin by two main mechanisms: extracellular depolymerization and intracellular mineralization.
Extracellular depolymerization: This mechanism involves the secretion of oxidative enzymes by microorganisms that attack the lignin polymer and break it into smaller fragments. The main enzymes involved in this process are lignin peroxidases (LiP), manganese peroxidases (MnP), and laccases. These enzymes catalyze the oxidation of phenolic and non-phenolic subunits of lignin, generating free radicals that can further react with each other or with other molecules. The products of this process are low molecular weight aromatic compounds, such as vanillin, syringaldehyde, and ferulic acid, that can be transported into the cells for further metabolism.
Intracellular mineralization: This mechanism involves the uptake and catabolism of lignin-derived compounds by microorganisms that possess specific metabolic pathways for their degradation. The main pathways involved in this process are the β-O-4 ether cleavage pathway, the biphenyl degradation pathway, and the protocatechuate 4,5-cleavage pathway. These pathways convert the aromatic compounds into central intermediates, such as protocatechuate, catechol, and vanillate, that can be further metabolized into tricarboxylic acid (TCA) cycle intermediates or other products. The end products of this process are CO2, H2O, and biomass.
The mechanisms of microbial degradation of lignin vary depending on the type of microorganism, the source of lignin, and the environmental conditions. Some microorganisms can degrade both phenolic and non-phenolic subunits of lignin, while others can only degrade phenolic subunits. Some microorganisms can use lignin as a sole carbon source, while others require additional nutrients or co-substrates. Some microorganisms can degrade lignin under aerobic conditions, while others can degrade it under anaerobic conditions.
Microbial degradation of lignin is an important process for the recycling of carbon and energy in nature. It also has potential applications for the production of biofuels, bioplastics, and other value-added products from lignocellulosic biomass. However, there are still many challenges and limitations for the efficient and sustainable utilization of lignin by microorganisms, such as low enzyme activity and stability, enzyme inhibition by lignin-derived compounds, low solubility and bioavailability of lignin, and complex regulation of lignin-degrading genes. Therefore, further research is needed to understand the molecular mechanisms, genetic diversity, and ecological roles of lignin-degrading microorganisms and to develop novel strategies for enhancing their performance and productivity.
Pseudomonas is a genus of bacteria that can metabolize various low molecular weight lignin-derived compounds found in lignocellulosic biomass . Some species of Pseudomonas, such as Pseudomonas putida KT2440, have been shown to produce lignin-degrading enzymes that can catalyze the oxidative cleavage of lignin . These enzymes include laccases, DyP-type peroxidases, multicopper oxidases, and other accessory enzymes . The degradation of alkali lignin by Pseudomonas putida KT2440 involves a series of extracellular and intracellular reactions, as described below.
Extracellular degradation
The extracellular degradation of lignin by Pseudomonas putida KT2440 is mainly mediated by two types of enzymes: oxidases and peroxidases/Mn2+-peroxidases . These enzymes are secreted by the bacteria when they grow on glucose as the only carbon source . The secretome of Pseudomonas putida KT2440 reaches the highest enzyme activity at 120 h of the fermentation time .
The oxidases, such as multicopper oxidases and laccases, preferentially attack the C-C bonds (β-β, β-5, and β-1) in lignin, resulting in the cleavage of aryl and biaryl compounds . The products of oxidase action are phenolic and methoxyphenolic acids that can be further degraded intracellularly .
The peroxidases/Mn2+-peroxidases, such as DyP-type peroxidases, require H2O2 or organic peroxides as co-substrates and Mn2+ as reducing substrates . These enzymes oxidize Mn2+ to Mn3+, which in turn oxidizes phenolic structures to phenoxyl radicals . The phenoxyl radicals can then react with each other or with other lignin fragments, leading to the cleavage of C-O bonds (β-O-4) in lignin . The products of peroxidase action are aromatic monomers and dimers that can be further degraded intracellularly .
The degradation rate of alkali lignin by the secretome of Pseudomonas putida KT2440 was found to be only 8.1% by oxidases, but increased to 14.5% with the activation of peroxidases/Mn2+-peroxidases . This indicates that both types of enzymes are important for lignin depolymerization.
Intracellular degradation
The intracellular degradation of lignin by Pseudomonas putida KT2440 involves a series of metabolic pathways that convert the phenolic and methoxyphenolic acids into vanillate, a central intermediate for further catabolism . Two examples of these pathways are:
β-O-4 ether degradation pathway: This pathway cleaves the β-O-4 ether bond that represents about 50% of all linkages in lignin . The pathway starts with LigD, a Cα-dehydrogenase that oxidizes the hydroxyl group at Cα position. Then, the β-etherase LigE or LigF cleaves the β-O-4 ether bond and generates vanillin and α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) as intermediates. Finally, GS-HPV is oxidized by LigG, a glutathione-S-transferase that cleaves glutathione and produces β-hydroxypropiovanillone, which can be further oxidized to vanillin .
Bi-phenyl degradation pathway: This pathway cleaves the bi-phenyl linkage that represents 10% of the total linkages in softwood lignin . The pathway starts with LigX, a 5,5`-dehydrodivanillate (DDVA) O-demethylase that demethylates one of the methoxy groups and converts it to a hydroxyl group. Then, LigZ, an OH-DDVA dioxygenase performs an oxidative meta-cleavage and produces a meta-cleavage compound. Next, LigY, a hydrolase for the meta-cleavage compound cleaves it into 4-carboxy-2-hydroxypentadienoic acid and 5-carboxyvanillic acid (5CVA). Finally, 5CVA is further metabolized into vanillate .
Vanillate can then enter the protocatechuate branch or the catechol branch of the β-ketoadipate pathway for further degradation into central metabolites such as acetyl-CoA and succinyl-CoA . These metabolites can then be used for energy production or biosynthesis by Pseudomonas putida KT2440.
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