Creative biological lignin conversion routes toward lignin valorization

Lignin, the largest renewable aromatic resource, is a promising alternative feedstock for the sustainable production of various chemicals, fuels, and materials. Despite this potential, lignin is characterized by heterogeneous and macromolecular structures that must be addressed. In this review, we present biological lignin conversion routes (BLCRs) that offer opportunities for overcoming these challenges, making lignin valorization feasible. Funneling heterogeneous aromatics via a ‘biological funnel’ offers a high-specificity bioconversion route for aromatic platform chemicals. The inherent aromaticity of lignin drives atom-economic functionalization routes toward aromatic natural product generation. By harnessing the ligninolytic capacities of specific microbial systems, powerful aromatic ring-opening routes can be developed to generate various value-added products. Thus, BLCRs hold the promise to make lignin valorization feasible and enable a lignocellulose-based bioeconomy.     

Degradation of lignin and lignin derivatives by Acinetobacter sp.

An Acinetobacter sp. utilized 2-methoxy-4-formylphenoxyacetic acid, dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin as sole carbon source. It also degraded ¹⁴C-labelled DHP lignin and teakwood lignin. Vanillic acid, protocatechuic acid and catechol were separated from 2-methoxy-4-formylphenoxyacetic acid grown cultures. Both protocatechuic acid and catechol were formed from dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin. On the dimeric lignin model substances this Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase.     

环境微生物介导的木质素代谢及其资源化利用研究进展

木质素是一种丰富的芳烃生物大分子聚合物,其分解代谢与地球元素循环和生物资源利用密切相关。但由于木质素结构的复杂性和无规则性导致其难以降解,使得木质素降解的研究成为全球碳循环和生物质资源利用研究的难点。近年来,来自不同环境的微生物陆续被发现具有木质素降解能力,并解析出参与木质素分解代谢的多种氧化还原酶。然而对木质素详细的代谢过程仍不十分清楚,因此,探究木质素降解酶系、作用机理和代谢网络是研究微生物代谢木质素机理的关键。本文综述环境中参与木质素降解的微生物,重点解析其木质素解聚酶系组成、分泌机制和木质素的代谢途径,并在此基础上阐明近年来木质素生物转化的最新研究进展,以期为今后环境微生物代谢木质素机理及其资源化利用的研究提供参考。     

Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase

The present study maps the active site of lignin peroxidase in respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant lignin peroxidases. A nonphenolic tetrameric lignin model was synthesized that contains beta-O-4 linkages. The fungal and recombinant wild type lignin peroxidase both oxidized the tetrameric model forming four products. The four products were identified by mass spectral analyses and compared with synthetic standards. They were identified as tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. All four of these products were also formed from single turnover experiments. This indicates that lignin peroxidase is able to attack any of the C(alpha)-C(beta) linkages in the tetrameric compound and that the substrate-binding site is well exposed. Mutation of the recombinant lignin peroxidase (isozyme H8) in the heme access channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidation of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidation of the tetrameric model. These results are consistent with lignin peroxidase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators.     

The oxidation of veratryl alcohol, dimeric lignin models and lignin by lignin peroxidase: The redox cycle revisited

Abstract: The mechanism of oxidation of veratryl alcohol and β-0–4 dimeric lignin models is reviewed. Veratryl alcohol radicals are intermediates in both oxidation pathways. The possible role of the veratryl alcohol radical cation as a mediator is discussed. The lignin peroxidase (LIP) redox cycle is analyzed in terms of the Marcus theory of electron transfer. Reduction of both LiP-Compound I (LiP-I) and LiP-Compound II (LiP-II) by veratryl alcohol occurs in the endergonic region of the driving force. The reduction of LiP-II has a higher reorganization energy due to the change in spin state and the accompanying conformational change in the protein. It is suggested that a reversible nucleophilic addition of a carbohydrate residue located at the entrance of the active site channel plays a key role in the LiP redox cycle. Moreover. (polymeric) hydroxysubstituted benzyl radicals may reduce LiP-II via long-range electron transfer.     

The fate of lignin and lignin‐derived compounds in anaerobic environments

During ODP Leg 201 microbial communities in Eastern Equatorial Pacific Ocean and Peru Margin sediments were investigated. The sediment layers sampled extended down to 420 m below the sea floor, with estimated ages of up to 40 million years. Contamination-free anoxic slurries were inoculated into media containing different substrate combinations, all at micromolar concentration. These culture media were designed for a broad spectrum of physiological groups. A total of 162 pure cultures were isolated that could be grouped into 19 different phylotypes based on 16S rRNA gene analysis. The isolates belonged to the Alpha-, Gamma- and Deltaproteobacteria, the Firmicutes, Actinobacteria, and Bacteroidetes. The genera most frequently isolated were Bacillus (68 isolates) and Rhizobium (40 isolates). Comparison of strains with the same phylotypes by enterobacterial repetitive intergenic consensus (ERIC-PCR) analysis revealed the presence of several subgroups that did not correlate with medium, sediment depth or sampling site. The majority of the isolates, although obtained from anoxic environments and isolated under strictly anoxic conditions, turned out to be facultativly aerobic. Physiologically, the isolates were characterized as generalists, able to utilize a broad variety of electron donors with either oxygen, nitrate and in some cases manganese oxides as electron acceptors. The diversity inferred from physiological tests was even higher than that on the phylogenetic or genomic level. The outcome of the contamination tests, the isolation of close relatives of already known subsurface bacteria, the repeated finding of the same phylotype from different sites and the level of diversity present in the culture collection strongly suggest that indigenous deep-biosphere bacteria had been isolated.     

Rewriting the lignin roadmap

Considerable interest in lignin biosynthesis has been fueled by the many roles that lignin plays in development and in resistance to biotic and abiotic stress, as well as its importance to industry and agriculture. Although the pathway leading to the lignin polymer has been studied for decades, new insights into the enzymes of the pathway have required a complete re-evaluation of how we think lignin precursors are synthesized. Although free hydroxycinnamic acids have long been thought to be key intermediates, it has become apparent that many of the hydroxylation and methylation steps in the pathway occur instead at the level of hydroxycinnamic acid esters, and their corresponding aldehydes and alcohols.     

Bacteria and lignin degradation

Lignin is both the most abundant aromatic (phenolic) polymer and the second most abundant raw material. It is degraded and modified by bacteria in the natural world, and bacteria seem to play a leading role in decomposing lignin in aquatic ecosystems. Lignin-degrading bacteria approach the polymer by mechanisms such as tunneling, erosion, and cavitation. With the advantages of immense environmental adaptability and biochemical versatility, bacteria deserve to be studied for their ligninolytic potential.     

Bacterial decomposition of synthetic 14C-labeled lignin and lignin monomer derivatives.

Nocardia sp. which was isolated from soil is capable of degrading synthetic lignin and utilizing its monomer derivatives. Decomposition was monitored by measuring the 14CO2 evolved and O2 consumed, when the bacterium was grown on a medium containing specifically 14C-labeled ligning or monomer phenolic compounds as major carbon source. The time course of the 14CO2 release and O2 uptake indicates a significant depolymerization and utilization of lignin by the Nocardia sp.     

Bacteria and lignin degradation

Lignin is both the most abundant aromatic (phenolic) polymer and the second most abundant raw material.It is degraded and modified by bacteria in the natural world,and bacteria seem to play a leading role in decomposing lignin in aquatic ecosystems.Lignin-degrading bacteria approach the polymer by mechanisms such as tunneling,erosion,and cavitation.With the advantages of immense environmental adaptability and biochemical versatility,bacteria deserve to be studied for their ligninolytic potential.     

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.     

Fungal degradation of kraft lignin and lignin sulfonates prepared form synthetic 14C-lignins

Kraft lignins (KL), bleached kraft lignins (BKL), and lignin sulfonates (LS) were prepared from synthetic ¹⁴C-lignins labeled in the aromatic nuclei or in the propyl side chains. These and control lignins (CL) were incubated with the lignin-decomposing white-rot fungus, Phanerochaete chrysosporium Burds., in a defined culture medium containing cellulose as growth substrate. Decomposition was monitored by measuring the ¹⁴CO₂ evolved. Average percentages of the [ring-¹⁴C]- and [side chain-¹⁴C]-lignins, respectively, recovered as ¹⁴CO₂ at the cessation of ¹⁴CO₂ evolution were: KL, 41 and 31; BKL, 42 and 26; LS, 28 and 21; and CL, 26 and 24. Gel permeation chromatography of radiolabeled materials extracted from spent cultures showed that substantial degradation to nonvolatile products had occurred. The polymeric components in the extracts were further degraded in fresh cultures. These results indicate that industrial lignins are significantly bioalterable, and that under favorable conditions industrial lignins are substantially biodegradable.     

Degradation of lignin and lignin related compounds by protoplasts isolated from Fomes annosus

Protoplasts from a lignolytic fungus Fomes annosus were prepared through enzymatic hydrolysis of mycelium utilizing Novozym, a wall lytic enzyme preparation. Isolated protoplasts and living mycelium were compared in their ability to degrade ¹⁴C-labelled lignin related phenols and dehydropolymers of labelled coniferyl alcohol (synthetic lignin). The amounts of ¹⁴CO₂ released from O¹⁴CH₃-groups, ¹⁴C-2-side chains and ¹⁴C-rings by protoplasts was in the same range as those for intact mycelium. The methoxyl groups of synthetic lignin were more rapidly metabolized by protoplasts than by mycelium. When calculated in dpm of released ¹⁴CO₂ per mg protein the decomposition of ¹⁴C-labelled synthetic lignin and lignin-related monomers in a hyphae-free system of protoplasts was considerable higher than that obtained by the intact mycelium. The presence of intact hyphae is thus not necessary for lignin degradation to occur.Non-common-abbreviations used DHP Dehydropolymer of coniferyl alcohol - LS lignosulfonates prepared from DHP     

Influence of lignin peroxidase concentration and localisation in lignin biodegradation by Phanerochaete chrysosporium

Summary The lignin mineralization rate in cultures of Phanerochaete chrysosporium increases with lignin peroxidase concentration up to 20 nkat ml^–1. At higher concentrations the rate of lignin mineralization decreases with increasing lignin peroxidase concentration. The amount of mycelium is not a limiting factor for lignin mineralization at high exocellular lignin peroxidase in association with the mycelium as pellets and no free exocellular enzyme induce a lignin mineralization rate equivalent to cultures reconstituted with washed pellets supplemented with 15 nkat ml^–1 of exogenous free enzyme. These results show that although lignin degradation by lignin peroxidase seems to be facilitated when lignin peroxidase is localised on the surface of the mycelium, free exocellular lignin peroxidase can also efficiently enhance mineralization of lignin by P. chrysosporium.     

Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification

Lignocellulosic biomass is utilized as a renewable feedstock in various agro‐industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these ‘DP reducers’ is achieved by expressing a bacterial hydroxycinnamoyl‐CoA hydratase‐lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side‐chain of hydroxycinnamoyl‐CoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild‐type plants. However, cell wall characterization of extract‐free stems by thioacidolysis and by 2D‐NMR revealed an increased amount of unusual C₆C₁ lignin monomers most likely linked with lignin as end‐groups. Moreover the analysis of lignin isolated from these plants using size‐exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C₆C₁ monomers (‘DP reducers’) into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis.     

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.     

Antibiotic properties of lignin components

Inhibitory effects of compounds with guaiacyl and syringyl structure, representing the structure of native lignin, were studied on model cultures of bacteria, yeasts, yeast-like microorganisms and moulds. Isoeugenol exhibited the most pronounced inhibitory effect on growth of the studied microorganisms.