Microbial lignin valorization through depolymerization to aromatics conversion

Lignin is the most abundant source of renewable aromatic biopolymers and its valorization presents significant value for biorefinery sustainability, which promotes the utilization of renewable resources. However, it is challenging to fully convert the structurally complex, heterogeneous, and recalcitrant lignin into high-value products. The in-depth research on the lignin degradation mechanism, microbial metabolic pathways, and rational design of new systems using synthetic biology have significantly accelerated the development of lignin valorization. This review summarizes the key enzymes involved in lignin depolymerization, the mechanisms of microbial lignin conversion, and the lignin valorization application with integrated systems and synthetic biology. Current challenges and future strategies to further study lignin biodegradation and the trends of lignin valorization are also discussed.     

Lignin valorization meets synthetic biology

Lignin, an abundant renewable resource in nature, is a highly heterogeneous biopolymer consisting of phenylpropanoid units. It is essential for sustainable utilization of biomass to convert lignin to value‐added products. However, there are technical obstacles for lignin valorization due to intrinsic heterogeneity. The emerging of synthetic biology technologies brings new opportunities for lignin breakdown and utilization. In this review, we discussed the applications of synthetic biology on lignin conversion, especially the production of value‐added products, such as aromatic chemicals, ring‐cleaved chemicals from lignin‐derived aromatics and bio‐active substances. Synthetic biology will offer new potential strategies for lignin valorization by optimizing lignin degradation enzymes, building novel artificial converting pathways, and improving the chassis of model microorganisms.     

生物炼制过程中木质素高值转化研究进展

木质素高值转化对于提升生物炼制经济性,促进社会经济绿色发展具有重要意义。然而,木质素结构复杂且不均一,其高值化利用仍存在技术壁垒,使得木质素应用尚未形成规模。文中首先综述了当前生物炼制过程中木质素高值转化面临的主要挑战。然后通过比较不同预处理技术对木质素分离、性质及其利用的主要影响,详细阐述了基于生物炼制理念发展的新型组合预处理技术。其次,针对木质素本征结构特性导致其利用效率低等问题,进一步详述了溶剂分级、膜分级、梯度沉淀分级等分级利用策略对克服木质素不均一性,改善其可加工性能的重要影响。再次,针对木质素利用策略,系统比较了木质素热化学转化和生物转化,结合生物质预处理及木质素分级,阐述了以生物炼制理念进行木质素高值转化的新策略。最后,总结了木质素利用过程中存在的挑战性问题,展望了木质素高效分离、分级及转化过程发展的新策略和新趋势。     

Biocatalysis in ionic liquids for lignin valorization: Opportunities and recent developments

Lignin holds tremendous potential as a renewable feedstock for upgrading to a number of high-value chemicals and products that are derived from the petroleum industry at present. Since lignin makes up a significant fraction of lignocellulosic biomass, co-utilization of lignin in addition to cellulose and hemicelluloses is vital to the economic viability of cellulosic biorefineries. The recalcitrant nature of lignin, originated from the molecule's compositional and structural heterogeneity, however, poses great challenges toward effective and selective lignin depolymerization and valorization. Ionic liquid (IL) is a powerful solvent that has demonstrated high efficiency in fractionating lignocellulosic biomass into sugar streams and a lignin stream of reduced molecular weight. Compared to thermochemical methods, biological lignin deconstruction takes place at mild temperature and pressure while product selectivity can be potentially improved via the specificity of biocatalysts (lignin degrading enzymes, LDEs). This review focuses on a lignin valorization strategy by harnessing the biomass fractionating capabilities of ILs and the substrate and product selectivity of LDEs. Recent advances in elucidating enzyme-IL interactions as well as strategies for improving enzyme activity in IL are discussed, with specific emphases on biocompatible ILs, thermostable and IL-tolerant enzymes, enzyme immobilization, and surface charge engineering. Also reviewed is the protein engineering toolsets (directed evolution and rational design) to improve the biocatalysts' activity, stability and product selectivity in IL systems. The alliance between IL and LDEs offers a great opportunity for developing a biocatalytic route for lignin valorization.     

The emerging role for bacteria in lignin degradation and bio-product formation

The microbial degradation of lignin has been well studied in white-rot and brown-rot fungi, but is much less well studied in bacteria. Recent published work suggests that a range of soil bacteria, often aromatic-degrading bacteria, are able to break down lignin. The enzymology of bacterial lignin breakdown is currently not well understood, but extracellular peroxidase and laccase enzymes appear to be involved. There are also reports of aromatic-degrading bacteria isolated from termite guts, though there are conflicting reports on the ability of termite gut micro-organisms to break down lignin. If biocatalytic routes for lignin breakdown could be developed, then lignin represents a potentially rich source of renewable aromatic chemicals.     

木质素的微生物解聚与高值转化

木质纤维素是地球上最丰富的可再生资源。我国每年产生约9亿吨农业秸秆,因得不到有效利用,不仅造成资源浪费,也产生了诸多严峻的环境问题。缺少木质素的高效降解和资源化利用技术是限制木质纤维素产业化的主要瓶颈之一。虽然木质素的降解与转化多年来一直都受到关注,但是由于木质素结构的复杂性及异质性,使其高效利用受限。近年来,微生物具有的“生物漏斗”式转化特性为木质素的高值转化和利用提供了新方向。本文就生物质利用研究以来,微生物在木质素解聚与转化方面的研究历程与最新进展进行了简要的回顾与总结,并初步讨论了目前木质素高值转化面临的机遇与挑战。     

Polymer properties of softwood organosolv lignins produced in two different reactor systems

Lignin, the second most abundant biopolymer on earth and with a predominantly aromatic structure, has the potential to be a raw material for valuable chemicals and other bio-based chemicals. In industry, lignin is underutilized by being used mostly as a fuel for producing thermal energy. Valorization of lignin requires knowledge of the structure and different linkages in the isolated lignin, making the study of structure of lignin important. In this article, lignin samples isolated from two types of reactors (autoclave reactor and displacement reactor) were analyzed by FT-IR, size exclusion chromatography, thermogravimetric analysis (TGA), and Py-GC-MS. The average molecular mass of the organosolv lignins isolated from the autoclave reactor decreased at higher severities, and FT-IR showed an increase in free phenolic content with increasing severity. Except for molecular mass and molecular mass dispersity, there were only minor differences between lignins isolated from the autoclave reactor and lignins isolated from the displacement reactor. Carbohydrate analysis, Py-GC–MS and TGA showed that the lignin isolated using either of the reactor systems is of high purity, suggesting that organosolv lignin is a good candidate for valorization.     

Microbial degradation of lignin

The transformations of lignin that occur during its biodegradation are complex and incompletely understood. Certain fungi of the white-rot group, and possibly other fungi and bacteria, completely decompose lignin to carbon dioxide and water. Other fungi and bacteria apparently degrade lignin incompletely. Differences in lignin-degrading abilities observed for different organisms may result from differences in the completeness of their ligninolytic enzyme systems. Not all lignin components may be attacked by a particular organism. Alternatively, different organisms may differ in their basic mechanisms of attack on lignin. The basic pathways of lignin degradation have been elucidated only for certain representatives of the white-and brown-rot fungi. Although it is known that each of the principal structural components of lignin is attacked by other fungi and bacteria, the biochemistry of that attack has not been elucidated. Work with low molecular weight lignin models has provided only limited information on possible pathways of lignin degradation by microorganisms. There is little evidence to suggest a correlation between abilities to degrade single-ring aromatic or lignin model compounds and the ability to degrade polymeric lignin. More evidence has come from analysis of spent culture media for lignin breakdown products and from comparative chemical analyses of sound lignins versus decayed lignin residues. Accumulated evidence with the most thoroughly studied white-rot fungi suggests that with these fungi lignin degradation proceeds by way of extracellular mixed-function oxygenases and dioxygenases, which catalyse demethylations, hydroxylations and ring-fission reactions within a largely intact polymer, concomitant with some release of low molecular weight lignin fragments. There are also apparent relationships between lignin, carbohydrate and nitrogen metabolism for some organisms, but the relationships may vary from one organism to another. Although research is now mostly at a basic level, industrial applications may result from lignin degradation research. Considerable potential exists for the development of bioconversions which might produce low molecular weight chemicals from waste lignins, and thereby reduce our dependence on petroleum as a source of these chemicals. Alternatively, such bioconversions might produce chemically altered forms of polymeric lignin that may be valuable industrially.     

Microbial utilization of lignin: available biotechnologies for its degradation and valorization

Lignocellulosic biomasses, either from non-edible plants or from agricultural residues, stock biomacromolecules that can be processed to produce both energy and bioproducts. Therefore, they become major candidates to replace petroleum as the main source of energy. However, to shift the fossil-based economy to a bio-based one, it is imperative to develop robust biotechnologies to efficiently convert lignocellulosic streams in power and platform chemicals. Although most of the biomass processing facilities use celluloses and hemicelluloses to produce bioethanol and paper, there is no consolidated bioprocess to produce valuable compounds out of lignin at industrial scale available currently. Usually, lignin is burned to provide heat or it remains as a by-product in different streams, thus arising environmental concerns. In this way, the biorefinery concept is not extended to completion. Due to Nature offers an arsenal of biotechnological tools through microorganisms to accomplish lignin valorization or degradation, an increasing number of projects dealing with these tasks have been described recently. In this review, outstanding reports over the last 6 years are described, comprising the microbial utilization of lignin to produce a variety of valuable compounds as well as to diminish its ecological impact. Furthermore, perspectives on these topics are given.     

Tailoring microbes to upgrade lignin

Lignin depolymerization generates a mixture of numerous compounds that are difficult to separate cost-effectively. To address this heterogeneity issue, microbes have been employed to ‘biologically funnel’ a broad range of compounds present in depolymerized lignin into common central metabolites that can be converted into a single desirable product. Because the composition of depolymerized lignin varies significantly with the type of biomass and the depolymerization method, microbes should be selected and engineered by considering this compositional variation. An ideal microbe must efficiently metabolize all relevant lignin-derived compounds regardless of the compositional variation of feedstocks, but discovering or developing such a perfect microbe is very challenging. Instead, developing multiple tailored microbes to tolerate a given mixture of lignin-derived compounds and to convert most of these into a target product is more practical. This review summarizes recent progress toward the development of such microbes for lignin valorization and offers future directions.     

Bacterial enzymes for lignin depolymerisation: new biocatalysts for generation of renewable chemicals from biomass

The conversion of polymeric lignin from plant biomass into renewable chemicals is an important unsolved problem in the biorefinery concept. This article summarises recent developments in the discovery of bacterial enzymes for lignin degradation, our current understanding of their molecular mechanism of action, and their use to convert lignin or lignocellulose into aromatic chemicals. The review also discusses the recent developments in screening of metagenomic libraries for new biocatalysts, and the use of protein engineering to enhance lignin degradation activity.     

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

Biodegradation of lignin by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Q18 and the characterization of a novel bacterial DyP-type peroxidase

Lignin valorization can be obtained through cleavage of selected bonds by microbial enzymes, in which lignin is segregated from cellulose and hemicellulose and abundant phenolic compounds can be provided. In this study, Pseudomonas sp. Q18, previously isolated from rotten wood in China, was used to degrade alkali lignin and raw lignocellulosic material. Gel-permeation chromatography, field-emission scanning electron microscope, and GC–MS were combined to investigate the degradation process. The GC–MS results revealed that the quantities of aromatic compounds with phenol ring from lignin increased significantly after incubation with Pseudomonas sp. Q18, which indicated the degradation of lignin. According to the lignin-derived metabolite analysis, it was proposed that a DyP-type peroxidase (PmDyP) might exist in strain Q18. Thereafter, the gene of PmDyP was cloned and expressed, after which the recombinant PmDyP was purified and the enzymatic kinetics of PmDyP were assayed. According to results, PmDyP showed promising characteristics for lignocellulosic biodegradation in biorefinery.     

Change in composition of hydrolyzed lignin during composting

Microbial assemblages were composed for composting hydrolysed lignin. Data on bioconversion of aromatic compounds with various types of substitution in the ring were used for this purpose. Composting of hydrolysed lignin reduced the contents of lignin, low-hydrolyzable polysaccharides, resins, and low-molecular-weight phenols and resulted in accumulation of humic acids. The resulting compost showed no phytotoxicity.     

Changes in the composition of hydrolyzed lignin during composting

Microbial assemblages were composed for composting hydrolyzed lignin. Data on bioconversion of aromatic compounds with various types of substitution in die ring were used for this purpose. Composting of hydrolyzed lignin reduced the contents of lignin, low-hydrolyzable polysaccharides, resins, and low-molecularweight phenols and resulted in accumulation of humic acids. The resulting compost showed no phytotoxicity.     

Multi-catalysis reactions: new prospects and challenges of biotechnology to valorize lignin

Considerable effort has been dedicated to the chemical depolymerization of lignin, a biopolymer constituting a possible renewable source for aromatic value-added chemicals. However, these efforts yielded limited success up until now. Efficient lignin conversion might necessitate novel catalysts enabling new types of reactions. The use of multiple catalysts, including a combination of biocatalysts, might be necessary. New perspectives for the combination of bio- and inorganic catalysts in one-pot reactions are emerging, thanks to green chemistry-driven advances in enzyme engineering and immobilization and new chemical catalyst design. Such combinations could offer several advantages, especially by reducing time and yield losses associated with the isolation and purification of the reaction products, but also represent a big challenge since the optimal reaction conditions of bio- and chemical catalysis reactions are often different. This mini-review gives an overview of bio- and inorganic catalysts having the potential to be used in combination for lignin depolymerization. We also discuss key aspects to consider when combining these catalysts in one-pot reactions.     

Biotransformation of lignin: Mechanisms,applications and future work

As one of the most abundant polymers in biosphere, lignin has attracted extensive attention as a kind of promising feedstock for biofuel and bio-based products. However, the utilization of lignin presents various challenges in that its complex composition and structure and high resistance to degradation. Lignin conversion through biological platform harnesses the catalytic power of microorganisms to decompose complex lignin molecules and obtain value-added products through biosynthesis. Given the heterogeneity of lignin, various microbial metabolic pathways are involved in lignin bioconversion processes, which has been characterized in extensive research work. With different types of lignin substrates (e.g., model compounds, technical lignin, and lignocellulosic biomass), several bacterial and fungal species have been proved to own lignin-degrading abilities and accumulate microbial products (e.g., lipid and polyhydroxyalkanoates), while the lignin conversion efficiencies are still relatively low. Genetic and metabolic strategies have been developed to enhance lignin biodegradation by reprogramming microbial metabolism, and diverse products, such as vanillin and dicarboxylic acids were also produced from lignin. This article aims at presenting a comprehensive review on lignin bioconversion including lignin degradation mechanisms, metabolic pathways, and applications for the production of value-added bioproducts. Advanced techniques on genetic and metabolic engineering are also covered in the recent development of biological platforms for lignin utilization. To conclude this article, the existing challenges for efficient lignin bioprocessing are analyzed and possible directions for future work are proposed.     

The structure of a prokaryotic feruloyl-CoA hydratase-lyase from a lignin-degrading consortium with high oligomerization stability under extreme pHs

In the context of increasing demand for renewable alternatives of fuels and chemicals, the valorization of lignin emerges as a value-adding strategy in biorefineries and an alternative to petroleum-derived molecules. One of the compounds derived from lignin is ferulic acid (FA), which can be converted into valuable molecules such as vanillin. In microorganisms, FA biotransformation into vanillin can occur via a two-step reaction catalyzed by the sequential activity of a feruloyl-CoA synthetase (FCS) and an feruloyl-CoA hydratase-lyase (FCHL), which could be exploited industrially. In this study, a prokaryotic FCHL derived from a lignin-degrading microbial consortium (named LM-FCHL) was cloned, successfully expressed in soluble form and purified. The crystal structure was solved and refined at 2.1 Å resolution. The LM-FCHL is a hexamer composed of a dimer of trimers, which showed to be quite stable under extreme pH conditions. Finally, small angle X-ray scattering corroborates the hexameric state in solution and indicates flexibility in the protein structure. The present study contributes to the field of lignin valorization to valuable molecules by establishing the biophysical and structural characterization for a novel FCHL member of unique characteristics.     

A new bacterial alcohol dehydrogenase active on degraded lignin and several low molecular weight aromatic compounds

A new intracellular bacterial dehydrogenase has been purified. It was active in the reversible reduction by NADH of conjugated carbonyl groups in partially degraded lignin. It was also active on various aromatic aldehydes such as vanillin, syringaldehyde and cinnamaldehyde, but had no effect on acetovanillone and lignin models carrying a conjugated ketone. It is proposed that this enzyme functions as a broadly specific lignin dehydrogenase at the level of aldehydic groups that are present in the lignin preparations.