Search

Lignin degradation by biocatalysts is a key strategy to develop a plant-based sustainable carbon economy and thus alleviate global climate change. This process involves synergy between ligninases and auxiliary enzymes. However, auxiliary enzymes within secretomes, which are composed of thousands of enzymes, remain enigmatic, although several ligninolytic enzymes have been well characterized. Moreover, it is a challenge to understand synergistic lignin degradation via a diverse array of enzymes, especially in bacterial systems. In this study, the coexpression network of the periplasmic proteome uncovers potential accessory enzymes for B-type dye-decolorizing peroxidases (DypBs) in Pseudomonas putida A514. The catalytic network of the DypBs-based multienzyme complex is characterized. DypBs couple with quinone reductases and nitroreductase to participate in quinone redox cycling. They work with superoxide dismutase to induce Fenton reaction for lignin oxidation. A synthetic enzyme cocktail (SEC), recruiting 15 enzymes, was consequently designed with four functions. It overcomes the limitation of lignin repolymerization, exhibiting a capacity comparable to that of the native periplasmic secretome. Importantly, we reveal the synergistic mechanism of a SEC-A514 cell system, which incorporates the advantages of in vitro enzyme catalysis and in vivo microbial catabolism. Chemical analysis shows that this system significantly reduces the molecular weight of lignin, substantially extends the degradation spectra for lignin functional groups, and efficiently metabolizes lignin derivatives. As a result, 25% of lignin is utilized, and its average molecular weight is reduced by 27%. Our study advances the knowledge of bacterial lignin-degrading multienzymes and provides a viable lignin degradation strategy., Competing Interests: Competing interests statement:The authors declare no competing interest.

Biochemical methane potential tests using water hyacinth (WH), pretreated water hyacinth (PWH), and Hydrilla verticillata (HV) as substrates using sewage media were explored. This study replaced the freshwater required to prepare the slurry for AD of organic solid waste with domestic sewage. Cow dung was used as the inoculum. WH (241.5 mL CH 4 /g VS added ), PWH (200.5 mL CH 4 /g VS added ), and HV (212 mL CH 4 /g VS added ) produced significant amounts of methane in the sewage medium. 16S-rRNA analysis showed that, in sewage, ~ 85% of the microbes were hydrolytic bacteria, and 7% were methanogens. This abundant quantity of hydrolytic microbes from sewage accelerated lignin degradation, achieving 28.32% and 38.34% degradation for WH and HV, respectively, within 14 days. Field emission-scanning electron microscopy images visually confirmed the enhanced substrate degradation in the presence of sewage. The net energy produced from the AD of WH and HV was significant (4664 J/g VS added and 4109 J/g VS added ), but for PWH, it was negative, indicating that using sewage medium may be better than costly pretreatment techniques. This study demonstrated the potential of using sewage as an alternative to freshwater in AD, offering a sustainable solution for freshwater conservation and the possible utilisation of sewage for improved methane production, especially for substrates with lignin that are difficult to degrade., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: This study was strictly experimental and did not involve human or animal participants. Institutional ethics approval was not required. Consent to participate: This study does not involve any human subjects. Consent to publish: This study does not involve any human subjects., (© 2024. The Author(s).)

Lignin utilization is one of the key challenges in the valorziation of lignocellulose. Filamentous fungi are promising candidates for lignin degradation and mineralization. However, novel lignin-degrading species are underexplored and the mechanism of lignin degradation is not fully understood. Here we isolated and characterized a novel species, Myrothecium wuxin, capable of utilizing lignosulfonate as the sole carbon source. To understand the mechanism of lignin degradation, genomic, transcriptomic and metabolic analyses were performed. The genome was sequenced, and assembled to a size of 48.55 Mb, with a contig N50 size of 5.67Mb. A total of 14,221 protein-coding genes were predicted, including a high number of potential ligninolytic enzymes. Transcriptomic analysis revealed a pronounced effect of lignosulfonate on gene expression profiles. More than twenty intermediate aromatic metabolites were identified during lignosulfonate utilization. Through genomic annotation, the genes potentially involved in lignin degradation were identified, and more than nine metabolic pathways of lignin-derived aromatic intermediates were predicted, including the homogentisate pathway, benzoic acid pathway, as well as the tree-branched β-ketoadipate pathway. The genomic information will provide a valuable resource for lignin degradation, while the elucidated catabolic pathways and associated enzymes provide exciting biotechnological opportunities for lignin valorization and production of valuable chemicals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lignin is the third most abundant organic resource in nature. The utilization of white-rot fungi for wood degradation effectively circumvents environmental pollution associated with chemical treatments, facilitating the benign decomposition of lignin. Trametes gibbosa is a typical white-rot fungus with rapid growth and strong wood decomposition ability. The lignin content decreased from 23.62 mg/mL to 17.05 mg/mL, which decreased by 27 % in 30 days. The activity of manganese peroxidase increased steadily by 9.44 times. The activities of laccase and lignin peroxidase had the same trend of change and reached peaks of 49.88 U/L and 10.43 U/L on the 25th day, respectively. The change in H 2 O 2 content in vivo was opposite to its trend. For FTIR and GC-MS analysis, the fungi attacked the side chain structure of lignin phenyl propane polymer and benzene ring to crack into low molecular weight aromatic compounds. The side chains of low molecular weight aromatic compounds are oxidized, and long-chain carboxylic acids are formed. Additionally, the absorption peak in the vibration region of the benzene ring skeleton became complex, and the structure of the benzene rings changed. In the beginning, fungal growth was inhibited. Fungal autophagy was aggravated. The metal cation binding proteins of fungi were active, and the genes related to detoxification metabolism were upregulated. The newly produced compounds are related to xenobiotic metabolism. The degradation peak focused on the redox process, and the biological function was enriched in the regulation of macromolecular metabolism, lignin metabolism, and oxidoreductase activity acting on diphenols and related substances as donors. Notably, genes encoding key degradation enzymes, including lcc3, lcc4, phenol-2-monooxygenase, 3-hydroxybenzoate-6-hydroxylase, oxalate decarboxylase, and acetyl-CoA oxidase were significantly upregulated. On the 30th day, the N-glycan biosynthesis pathway was significantly enriched in glycan biosynthesis and metabolism. Weighted correlation network analysis was performed. A total of 1452 genes were clustered in the coral1 module, which were most related to lignin degradation. The genes were significantly enriched in oxidoreductase activity, peptidase activity, cell response to stimulation, signal transduction, lignin metabolism, and phenylpropane metabolism, while the rest were concentrated in glucose metabolism. In this study, the lignin degradation process and products were revealed by T. gibbosa. The molecular mechanism of lignin degradation in different stages was explored. The selection of an efficient utilization time of lignin will help to increase the degradation rate of lignin. This study provides a theoretical basis for the biofuel and biochemical production of lignin. SYNOPSIS: Trametes gibbosa degrades lignin in a pollution-free way, improving the utilization of carbon resources in an environmentally friendly spontaneous cycle. The products are the new way towards sustainable development and low-carbon technology., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Biofuel production from lignocellulose feedstocks is sustainable and environmentally friendly. However, the lignocellulosic pretreatment could produce fermentation inhibitors causing multiple stresses and low yield. Therefore, the engineering construction of highly resistant microorganisms is greatly significant. In this study, a composite functional chimeric cellulosome equipped with laccase, versatile peroxidase, and lytic polysaccharide monooxygenase was riveted on the surface of Saccharomyces cerevisiae to construct a novel yeast strain YI/LVP for synergistic lignin degradation and cellulosic ethanol production. The assembly of cellulosome was assayed by immunofluorescence microscopy and flow cytometry. During the whole process of fermentation, the maximum ethanol concentration and cellulose conversion of engineering strain YI/LVP reached 8.68 g/L and 83.41%, respectively. The results proved the availability of artificial chimeric cellulosome containing lignin-degradation enzymes for cellulosic ethanol production. The purpose of the study was to improve the inhibitor tolerance and fermentation performance of S. cerevisiae through the construction and optimization of a synergistic lignin-degrading enzyme system based on cellulosome., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Lignin, as an abundant organic carbon, plays a vital role in the global carbon cycle. However, our understanding of the global lignin-degrading microbiome remains elusive. The greatest barrier has been absence of a comprehensive and accurate functional gene database. Here, we first developed a curated functional gene database (LCdb) for metagenomic profiling of lignin degrading microbial consortia. Via the LCdb, we draw a clear picture describing the global biogeography of communities with lignin-degrading potential. They exhibit clear niche differentiation at the levels of taxonomy and functional traits. The terrestrial microbiomes showed the highest diversity, yet the lowest correlations. In particular, there were few correlations between genes involved in aerobic and anaerobic degradation pathways, showing a clear functional redundancy property. In contrast, enhanced correlations, especially closer inter-connections between anaerobic and aerobic groups, were observed in aquatic consortia in response to the lower diversity. Specifically, dypB and dypA, are widespread on Earth, indicating their essential roles in lignin depolymerization. Estuarine and marine consortia featured the laccase and mnsod genes, respectively. Notably, the roles of archaea in lignin degradation were revealed in marine ecosystems. Environmental factors strongly influenced functional traits, but weakly shaped taxonomic groups. Null mode analysis further verified that composition of functional traits was deterministic, while taxonomic composition was highly stochastic, demonstrating that the environment selects functional genes rather than taxonomic groups. Our study not only develops a useful tool to study lignin degrading microbial communities via metagenome sequencing but also advances our understanding of ecological traits of these global microbiomes., (© 2024 John Wiley & Sons Ltd.)

Fiber film have received widespread attention due to its green friendliness. We can use microorganisms to degrade lignin in straw to obtain cellulose and make fiber films. Herein, a group of high-temperature (50 °C) lignin degrading bacterial consortium (LDH) was enriched and culture conditions for lignin degradation were optimized. Combined with high-throughput sequencing technology, the synergistic effect of LDH-composited bacteria was analyzed. Then LDH was used to treat rice straw for the bio-pulping experiment. The results showed that the lignin of rice straw was degraded 32.4 % by LDH at 50 °C for 10 d, and after the optimization of culture conditions, lignin degradation rate increased by 9.05 % (P < 0.001). The bacteria that compose in LDH can synergistically degrade lignin. Paenibacillus can encode all lignin-degrading enzymes present in the LDH. Preliminary tests of LDH in the pulping industry have been completed. This study is the first to use high temperature lignin degrading bacteria to fabricate fiber film., Competing Interests: Declaration of competing interest Authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Lignin is an intricate phenolic polymer found in plant cell walls that has tremendous potential for being converted into value-added products with the possibility of significantly increasing the economics of bio-refineries. Although lignin in nature is bio-degradable, its biocatalytic conversion is challenging due to its stable complex structure and recalcitrance. In this context, an understanding of strain's genomics, enzymes, and degradation pathways can provide a solution for breaking down lignin to unlock the full potential of lignin as a dominant valuable bioresource. A gammaproteobacterial strain AORB19 has been isolated previously from decomposed wood based on its high laccase production. This work then focused on the detailed genomic and functional characterization of this strain based on whole genome sequencing, the identification of lignin degradation products, and the strain's laccase production capabilities on various agro-industrial residues., Results: Lignin degrading bacterial strain AORB19 was identified as Serratia quinivorans based on whole genome sequencing and core genome phylogeny. The strain comprised a total of 123 annotated CAZyme genes, including ten cellulases, four hemicellulases, five predicted carbohydrate esterase genes, and eight lignin-degrading enzyme genes. Strain AORB19 was also found to possess genes associated with metabolic pathways such as the β-ketoadipate, gentisate, anthranilate, homogentisic, and phenylacetate CoA pathways. LC-UV analysis demonstrated the presence of p-hydroxybenzaldehyde and vanillin in the culture media which constitutes potent biosignatures indicating the strain's capability to degrade lignin. Finally, the study evaluated the laccase production of Serratia AORB19 grown with various industrial raw materials, with the highest activity detected on flax seed meal (257.71 U/L), followed by pea hull (230.11 U/L), canola meal (209.56 U/L), okara (187.67 U/L), and barley malt sprouts (169.27 U/L)., Conclusions: The whole genome analysis of Serratia quinivorans AORB19, elucidated a repertoire of genes, pathways and enzymes vital for lignin degradation that widens the understanding of ligninolytic metabolism among bacterial lignin degraders. The LC-UV analysis of the lignin degradation products coupled with the ability of S. quinivorans AORB19 to produce laccase on diverse agro-industrial residues underscores its versatility and its potential to contribute to the economic viability of bio-refineries., (© 2024. Crown.)

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a β-5 phenylcoumaran lignin dimer. The reaction products did not contain glutathione, indicating a catalytic role for reduced glutathione in this enzyme. Three reaction products were identified: the major product was a cis-stilbene arising from C-C fragmentation involving loss of formaldehyde; two minor products were an alkene arising from elimination of glutathione, and an oxidised ketone, proposed to arise from reaction of an intermediate with molecular oxygen. Testing of the recombinant enzyme against a soda lignin revealed the formation of new signals by two-dimensional NMR analysis, whose chemical shifts are consistent with the formation of a stilbene unit in polymeric lignin., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

To improve the titre of lignin-derived pyridine-dicarboxylic acid (PDCA) products in engineered Rhodococcus jostii RHA1 strains, plasmid-based overexpression of seven endogenous and exogenous lignin-degrading genes was tested. Overexpression of endogenous multi-copper oxidases mcoA, mcoB, and mcoC was found to enhance 2,4-PDCA production by 2.5-, 1.4-, and 3.5-fold, respectively, while overexpression of dye-decolorizing peroxidase dypB was found to enhance titre by 1.4-fold, and overexpression of Streptomyces viridosporus laccase enhanced titre by 1.3-fold. The genomic context of the R. jostii mcoA gene suggests involvement in 4-hydroxybenzoate utilization, which was consistent with enhanced whole cell biotransformation of 4-hydroxybenzoate by R. jostii pTipQC2-mcoA. These data support the role of multi-copper oxidases in bacterial lignin degradation, and provide an opportunity to enhance titres of lignin-derived bioproducts., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Microbial pretreatment of lignocellulosic biomass holds significant promise for environmentally friendly biofuel production, offering an alternative to fossil fuels. This study focused on the isolation and characterization of two novel delignifying bacteria, GIET1 and GIET2, to enhance cellulose accessibility by lignin degradation. Molecular characterization confirmed their genetic identities, providing valuable microbial resources for biofuel production. Our results revealed distinct preferences for temperature, pH, and incubation period for the two bacteria. Bacillus haynesii exhibited optimal performance under moderate conditions and shorter incubation period, making it suitable for rice straw and sugarcane bagasse pretreatment. In contrast, Paenibacillus alvei thrived at higher temperatures and slightly alkaline pH, requiring a longer incubation period ideal for corn stalk pretreatment. These strain-specific requirements highlight the importance of tailoring pretreatment conditions to specific feedstocks. Structural, chemical, and morphological analyses demonstrated that microbial pretreatment reduced the amorphous lignin, increasing cellulose crystallinity and accessibility. These findings underscore the potential of microbial pretreatment to enhance biofuel production by modifying the lignocellulosic biomass. Such environmentally friendly bioconversion processes offer sustainable and cleaner energy solutions. Further research to optimize these methods for scalability and broader application is necessary in the pursuit for more efficient and greener biofuel production., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Gut microbiota plays a vital role in obtaining nutrition from bamboo for giant pandas. However, low cellulase activity has been observed in the panda's gut. Besides, no specific pathway has been implicated in lignin digestion by gut microbiota of pandas. Therefore, the mechanism by which they obtain nutrients is still controversial. It is necessary to elucidate the precise pathways employed by gut microbiota of pandas to degrade lignin. Here, the metabolic pathways for lignin degradation in pandas were explored by comparing 209 metagenomic sequencing data from wild species with different feeding habits. Lignin degradation central pathways, including beta-ketoadipate and homogentisate pathway, were enriched in the gut of wild bamboo-eating pandas. The gut microbiome of wild bamboo-eating specialists was enriched with genes from pathways implicated in degrading ferulate and p -coumarate into acetyl-CoA and succinyl-CoA, which can potentially provide the raw materials for metabolism in pandas. Specifically, Pseudomonas , as the most dominant gut bacteria genus, was found to be the main bacteria to provide genes involved in lignin or lignin derivative degradation. Herein, three Pseudomonas -associated strains isolated from the feces of wild pandas showed the laccase, lignin peroxidase, and manganese peroxidase activity and extracellular lignin degradation ability in vitro . A potential mechanism for pandas to obtain nutrition from bamboo was proposed based on the results. This study provides novel insights into the adaptive evolution of pandas from the perspective of lignin metabolism., Importance: Although giant pandas only feed on bamboo, the mechanism of lignin digestion in pandas is unclear. Here, the metabolic pathways for lignin degradation in wild pandas were explored by comparing gut metagenomic from species with different feeding habits. Results showed that lignin degradation central pathways, including beta-ketoadipate and homogentisate pathway, were enriched in the gut of wild bamboo-eating pandas. Genes from pathways involved in degrading ferulate and p -coumarate via beta-ketoadipate pathway were also enriched in bamboo-eating pandas. The final products of the above process, such as acetyl-CoA, can potentially provide the raw materials for metabolism in pandas. Specifically, Pseudomonas , as the most dominant gut bacteria genus, mainly provides genes involved in lignin degradation. Herein, Pseudomonas -associated strains isolated from the feces of pandas could degrade extracellular lignin. These findings suggest that gut microbiome of pandas is crucial in obtaining nutrition from lignin via Pseudomonas , as the main lignin-degrading bacteria., Competing Interests: The authors declare no conflict of interest.

Rhodococcus opacus PD630 is a high oil-producing strain with the ability to convert lignin-derived aromatics to high values, but limited research has been done to elucidate its conversion pathway, especially the upper pathways. In this study, we focused on the upper pathways and demethylation mechanism of lignin-derived aromatics metabolism by R. opacus PD630. The results of the aromatic carbon resource utilization screening showed that R. opacus PD630 had a strong degradation capacity to the lignin-derived methoxy-containing aromatics, such as guaiacol, 3,4-veratric acid, anisic acid, isovanillic acid, and vanillic acid. The gene of gcoA R , which encodes cytochrome P450, showed significant up-regulation when R. opacus PD630 grew on diverse aromatics. Deletion mutants of gcoA R and its partner protein gcoB R resulted in the strain losing the ability to grow on guaiacol, but no significant difference to the other aromatics. Only co-complementation alone of gcoA R and gcoB R restored the strain's ability to utilize guaiacol, demonstrating that both genes were equally important in the utilization of guaiacol. In vitro assays further revealed that GcoA R could convert guaiacol and anisole to catechol and phenol, respectively, with the production of formaldehyde as a by-product. The study provided robust evidence to reveal the molecular mechanism of R. opacus PD630 on guaiacol metabolism and offered a promising study model for dissecting the demethylation process of lignin-derived aromatics in microbes.IMPORTANCEAryl- O -demethylation is believed to be the key rate-limiting step in the catabolism of heterogeneous lignin-derived aromatics in both native and engineered microbes. However, the mechanisms of O -demethylation in lignin-derived aromatic catabolism remain unclear. Notably, guaiacol, the primary component unit of lignin, lacks in situ demonstration and illustration of the molecular mechanism of guaiacol O -demethylation in lignin-degrading bacteria. This is the first study to illustrate the mechanism of guaiacol metabolism by R. opacus PD630 in situ as well as characterize the purified key O -demethylase in vitro . This study provided further insight into the lignin metabolic pathway of R. opacus PD630 and could guide the design of an efficient biocatalytic system for lignin valorization., Competing Interests: The authors declare no conflict of interest.

Lignocellulose, as the most abundant natural organic carbon on earth, plays a key role in regulating the global carbon cycle, but there have been only few studies in marine ecosystems. Little information is available about the extant lignin-degrading bacteria in coastal wetlands, limiting our understanding of their ecological roles and traits in lignocellulose degradation. We utilized in situ lignocellulose enrichment experiments coupled with 16S rRNA amplicon and shotgun metagenomics sequencing to identify and characterize bacterial consortia attributed to different lignin/lignocellulosic substrates in the southern-east intertidal zone of East China Sea. We found the consortia enriched on woody lignocellulose showed higher diversity than those on herbaceous substrate. This also revealed substrate-dependent taxonomic groups. A time-dissimilarity pattern with increased alpha diversity over time was observed. Additionally, this study identified a comprehensive set of genes associated with lignin degradation potential, containing 23 gene families involved in lignin depolymerization, and 371 gene families involved in aerobic/anaerobic lignin-derived aromatic compound pathways, challenging the traditional view of lignin recalcitrance within marine ecosystems. In contrast to similar cellulase genes among the lignocellulose substrates, significantly different ligninolytic gene groups were observed between consortia under woody and herbaceous substrates. Importantly, we not only observed synergistic degradation of lignin and hemi-/cellulose, but also pinpointed the potential biological actors at the levels of taxa and functional genes, which indicated that the alternation of aerobic and anaerobic catabolism could facilitate lignocellulose degradation. Our study advances the understanding of coastal bacterial community assembly and metabolic potential for lignocellulose substrates. IMPORTANCE It is essential for the global carbon cycle that microorganisms drive lignocellulose transformation, due to its high abundance. Previous studies were primarily constrained to terrestrial ecosystems, with limited information about the role of microbes in marine ecosystems. Through in situ lignocellulose enrichment experiment coupled with high-throughput sequencing, this study demonstrated different impacts that substrates and exposure times had on long-term bacterial community assembly and pinpointed comprehensive, yet versatile, potential decomposers at the levels of taxa and functional genes in response to different lignocellulose substrates. Moreover, the links between ligninolytic functional traits and taxonomic groups of substrate-specific populations were revealed. It showed that the synergistic effect of lignin and hemi-/cellulose degradation could enhance lignocellulose degradation under alternation of aerobic and anaerobic conditions. This study provides valuable taxonomic and genomic insights into coastal bacterial consortia for lignocellulose degradation., Competing Interests: The authors declare no conflict of interest.

Paddy straw (PS) and pine needles (PN) are one of the challenging biomasses in terms of disposal and compost making due to their high silica and tannin contents. Particulate air pollution, loss of biodiversity and respiratory impairments are some of disastrous outcomes caused by burning. However, high percentage of cellulose and hemicellulose makes them potential substrate for paper and pulp industries. The main aim of work was to study and utilize a combinatorial approach of weak chemical treatment and lignin degrading fungal species as agents of effective production of lignin modifying enzymes (LME's) for lignin depolymerisation from the biomasses. Phanerochaete chrysosporium was found to be the best degrader of lignin (47.11 % in PS + PN in 28 days) with maximum LME's production between 10th-17th days. Efficient lignin degradation in the PS and PN biomass will aid further application in pulp production supporting the transition to a circular economy in a greener way., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Thermoalkaliphilic laccase (CtLac) from the Caldalkalibacillus thermarum strain TA2.A1 has advantageous properties with potential industrial applications, such as high enzyme activity and stability at 70 °C and pH 8.0. In the present study, a directed evolution approach using a combination of random and site-directed mutagenesis was adopted to enhance the laccase activity of CtLac. Spectrophotometric assay and real-time oxygen measurement techniques were employed to compare and evaluate the enzyme activity among mutants. V243 was targeted for site-directed mutagenesis based on library screening. V243D showed a 25-35% higher laccase activity than wild-type CtLac in the spectrophotometric assay and oxygen consumption measurement results. V243D also showed higher catalytic efficiency than wild-type CtLac with decreased K m and increased k cat values. In addition, V243D enhanced oxidative degradation of the lignin model compound, guaiacylglycerol-β-guaiacyl ether (GGGE), by 10% and produced a 5-30% increase in high-value aldehydes than wild-type CtLac under optimal enzymatic conditions (i.e., 70 °C and pH 8.0). Considering the lack of protein structural information, we used the directed evolution approach to predict Val at the 243 position of CtLac as one of the critical amino acids contributing to the catalytic efficiency of the enzyme. Moreover, it found that the real-time oxygen measurement technique could overcome the limitations of the spectrophotometric assay, and apply to evaluate oxidase activity in mutagenesis research. KEY POINTS: • CtLac was engineered for enhanced laccase activity through directed evolution approach • V243D showed higher catalytic efficiency (k cat /K m ) than wild-type CtLac • V243D produced higher amounts of high-value aldehydes from rice straw than wild-type CtLac., (© 2022. The Author(s).)

Lignin, an abundant aromatic heteropolymer in secondary plant cell walls, is the single largest source of renewable aromatics in the biosphere. Leveraging this resource for renewable bioproducts through targeted microbial action depends on lignin fragment uptake by microbial hosts and subsequent enzymatic action to obtain the desired product. Recent computational work has emphasized that bacterial inner membranes are permeable to many aromatic compounds expected from lignin depolymerization processes. In this study, we expand on these findings through simulations for 42 lignin-related compounds across a gram-negative bacterial outer membrane model. Unbiased simulation trajectories indicate that spontaneous crossing for the full outer membrane is relatively rare at molecular simulation timescales, primarily due to preferential membrane partitioning and slow diffusion within the lipopolysaccharide layer within the outer membrane. Membrane partitioning and permeability coefficients were determined through replica exchange umbrella sampling simulations to overcome sampling limitations. We find that the glycosylated lipopolysaccharides found in the outer membrane increase the permeation barrier to many lignin-related compounds, particularly the most hydrophobic compounds. However, the effect is relatively modest; at industrially relevant concentrations, uncharged lignin-related compounds will readily diffuse across the outer membrane without the need for specific porins. Together, our results provide insight into the permeability of the bacterial outer membrane for assessing lignin fragment uptake and the future production of renewable bioproducts., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In present research, a potent fungal strain was isolated from paper mill effluent (black liquor) in order to investigate its potential for the biodegradation of lignin. Two step strategy was used to screen most efficient fungal strain having ability to growin MSM-black liquor medium and to degrade alkali lignin.The results of initial screening indicated that the strain M-2 produced comparatively higher ligninolytic zone on MSN agar plates supplemented with black liquor (BL) and alkali ligninase compared to the other isolates.The results of 18S rRNA gene sequencing revealed that strain M-2 showed ≥ 99% sequence homology with Dipodasceus australiansis.The process for the biodegradation of lignin was optimized using Taguchi Orthogonal Array design. Under optimized conditions of pH 9, 40 °C and 4% inoculum, a maximum of 89% lignin was degraded with 41% color reduction after 8 days of incubation period by Dipodasceus australiansis M-2. The pH and temperature were found to be significant terms with the p-values of 0.002 and 0.001 respectively. The laccase activity of the Dipodascus australiensis was found to be maximum of 1.511 U/mL. The HPLC analysis of lignin biodegradation indicated sharp transformation of peaks as compared to the control. Our results suggested that the strain Dipodascus australiensis M-2 possess excellent lignin degradation and color reduction capability and can be applied in waste treatment systems for pulp and paper mill effluent. In present work we are reporting first hand information regarding biodegradation of lignin by a potent strain of Dipodascus australiensis and statistical optimization of the bioprocess., Competing Interests: Conflict of interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Lignocellulosic biorefinery based on sugar-platform has been considered as an efficient strategy to replace fossil fuel-based refinery. In the bioconversion process, pretreatment is an essential step to firstly open up lignocellulose cell wall structure and enhance the accessibility of carbohydrates to hydrolytic enzymes. However, various lignin and/or carbohydrates degradation products (e.g. phenolics, 5-hydroxymethylfurfural, furfural) are also generated during pretreatment, which severely inhibit the following enzymatic hydrolysis and the downstream fermentation process. Among them, the lignin derived phenolics have been considered as the most inhibitory compounds and their inhibitory effects are highly dependent on the source of biomass and the type of pretreatment strategy. Although liquid-solid separation and subsequent washing can remove the lignin derived phenolics and other inhibitors, this is undesirable in the realistic industrial application where the whole slurry of pretreated biomass needs to be directly used in the hydrolysis process. This review summarizes the phenolics formation mechanism for various commonly applied pretreatment methods and discusses the key factors that affect the inhibitory effect of phenolics on cellulose hydrolysis. In addition, the recent achievements on the rational design of inhibition mitigation strategies to boost cellulose hydrolysis for sugar-platform biorefinery are also introduced. This review also provides guidance for rationally designing detoxification strategies to facilitate whole slurry hydrolysis which helps to realize the industrialization of lignocellulose biorefinery., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Five yeast fungi strains (i.e., two Cryptococcus albidus, one Candida guillermondii, and two Candida tropicalis) were isolated from sugarcane and tested for their use of lignin as sole carbon source and their potential to grow in the presence of phenol and phenol derivatives (i.e., pentachlorophenol and p-nitrophenol). The full set of isolated yeasts showed ligninolytic activity, achieving at least 36% lignin degradation after 25 days. The C. albidus JS-B1 strain had the highest ligninolytic activity, achieving 27% lignin degradation within 4 days. This increased activity was associated with the production of ligninolytic laccase enzymes. All the tested yeast fungi strains showed growth in the presence of high concentrations of phenolic compounds (i.e., 900 mg/L phenol, 200 mg/L p-nitrophenol, 50 mg/L pentachlorophenol) and showed significant potential for lignin and lignin by-product degradation. Each of these five strains has the potential to be used in biological treatment processes for contaminated effluents from paper pulping and bleaching or phenol and phenol-derivative biodegradation processes for other industrial wastewater effluents., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Background: To date, fungus-assisted pretreatment of agricultural residue has not become the preferred method to produce protein-enriched and ruminally digestible animal feed because of low time efficiency of fungal delignification and protein production, i.e. the long solid-state fermentation period, and because of laccase as a potential inhibitor of cellulose activity. In this study, response surface methodology was employed to optimize the parameters in the process of producing nutritious animal feed from wheat straw with Inonotus obliquus pretreatment., Results: The mineral salt solution containing (w/v) (NH 4 ) 2 SO 4 1%, MgSO 4 ·7H 2 O 0.03%, KH 2 PO 4 0.011%, Tween-80 0.4%, and corn starch 10% with pH of 7.4 was optimized. Inonotus obliquus rapidly and completely colonized on wheat straw with an ergosterol content of 280 μg g -1 dry matter, consuming 45% of lignin after 15 days of fermentation, producing maximums of lignin peroxidase (1729 IU g -1 ), manganese peroxidase (610 IU g -1 ) and laccase (98 IU g -1 ) on days 5, 15, and 25, respectively. The crude protein (102.4 g kg -1 ) of 15-day fermented wheat straw increased by ~132%. After hydrolysis, the essential protein-bound amino acids (15.3 g kg -1 ) increased by ~47%, within which Met and Lys measured ~1070% and ~60% higher. The treatment with I. obliquus also improved the in vitro gas production after 72 h (IVGP 72 ) of wheat straw to 178.8 mL g -1 organic matter (~43% increase)., Conclusion: For the first time, we found that I. obliquus is an effective white rot fungus turning wheat straw into ruminally digestible animal feed without laccase inhibitor., (© 2021 Society of Chemical Industry.)

To improve the lignin degradation efficiency, we established a co-culture consortium (LDFC) consisting of Trametes hirsuta BYL-3, Trametes versicolor BYL-7 and Trametes hirsuta BYL-8. The testing results showed that the constructed consortium showed improved the lignin degradation rate by fungi. The optimal cultivation conditions were mixture at 1:1:1 vol ratio of each fungus, 7% (w/v) of inoculum amount, culture temperature at 26 °C, pH was 6.9 and 10 days of culturing time. Under these conditions, the degradation rate of lignin was 39.7%, which was 9.3% higher than those before optimization (30.4%). Using rice straw for treatment by LDFC to papermaking, the paper tensile strength was 8 N, and the ring pressure index was 2.46 N·m/g, which meets the standards for the production of corrugated paper for packaging. These results indicate that LDFC has potential application value to convert rice straw resources for bio-pulping to make papers., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lignin is a major byproduct of pulp and paper industries, which is resistant to depolymerization due to its heterogeneous structure. The enzymes peroxidases can be utilized as potent bio-catalysts to degrade lignin. In the current study, an Efeb gene of 1251bp encoding DyP-type peroxidase from Bacillus sp. strain BL5 (DyPBL5) was amplified, cloned into a pET-28a (+) vector and expressed in Escherichia coli BL21 (DE3) cells. A 46 kDa protein of DyPBL5 was purified through ion-exchange chromatography. Purified DyPBL5 was active at wide temperature (25-50 °C) and pH (3.0-8.0) range with optimum activity at 35 °C and pH 5.0. Effects of different chemicals on DyPBL5 were determined. The enzyme activity was strongly inhibited by SDS, DDT and β-mercaptoethanol, whereas stimulated in the presence of organic solvents such as methanol and ethanol. The kinetic parameters were determined and K m , V max and K ca t , respectively. Docking of DyPBL5 with ABTS revealed that, Asn 244, Arg 339, Asp 383 and Thr 389 are putative amino acids, taking part in the oxidation of ABTS. The recombinant DyPBL5 resulted in the reduction of lignin contents up to 26.04 %. The SEM and FT-IR analysis of test samples gave some indications about degradation of lignin by DyPBL5. Various low molecular weight lignin degradation products were detected by analyzing the samples through gas chromatography mass spectrometry. High catalytic efficiency and lignin degradation rate make DyPBL5 an ideal bio-catalyst for remediation of lignin-contaminated sites.1 , respectively. Docking of DyPBL5 with ABTS revealed that, Asn 244, Arg 339, Asp 383 and Thr 389 are putative amino acids, taking part in the oxidation of ABTS. The recombinant DyPBL5 resulted in the reduction of lignin contents up to 26.04 %. The SEM and FT-IR analysis of test samples gave some indications about degradation of lignin by DyPBL5. Various low molecular weight lignin degradation products were detected by analyzing the samples through gas chromatography mass spectrometry. High catalytic efficiency and lignin degradation rate make DyPBL5 an ideal bio-catalyst for remediation of lignin-contaminated sites., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The degradation of lignin is the main rate-limiting step in the bio-pulping of rice straw. A lignin-degrading bacterial consortium LDC, which can efficiently degrade lignin of reed, was screened in the early stage of our laboratory work. In present study, 7-day incubation of LDC can degrade rice straw lignin by 31.18% in mineral salt medium. The communities' structure of different incubation phases varied greatly, in which high abundance (44.78%) of Anaerocolumna was first found. The expression levels of lignin degradation enzyme class II peroxidase (AA2), vanillyl alcohol oxidase (AA4) and 1,4-benzoquinone reductase (AA6) during peak phase (48 h) were significantly up-regulated than initial phase (24 h), increasing by 112%, 165% and 67%, respectively, and 42.86% AA2 was from Thaurea; 100% AA4 was from Clostridium; 62.5% AA6 was from Pseudomonas. These provide microbial resources and data support for the industrialization of rice straw bio-pulping., (Copyright © 2021. Published by Elsevier Ltd.)

Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that Termitomyces employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that Termitomyces induces hydroquinone-mediated Fenton chemistry (Fe 2+ + H 2 O 2 + H + → Fe 3+ + • OH + H 2 O) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MH 2 Q, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect's agricultural symbiosis is accomplished. IMPORTANCE Fungus-growing termites have optimized the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a codiversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here, we provide genomic, transcriptomic, and enzymatic evidence that Termitomyces also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton chemistry-based hydroquinone-catalyzed lignin degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms reveal new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.

In this review, we chart the major milestones in the research progress on the DyP-type peroxidase family over the past decade. Though mainly distributed among bacteria and fungi, this family actually exhibits more widespread diversity. Advanced tertiary structural analyses have revealed common and different features among members of this family. Notably, the catalytic cycle for the peroxidase activity of DyP-type peroxidases appears to be different from that of other ubiquitous heme peroxidases. DyP-type peroxidases have also been reported to possess activities in addition to peroxidase function, including hydrolase or oxidase activity. They also show various cellular distributions, functioning not only inside cells but also outside of cells. Some are also cargo proteins of encapsulin. Unique, noteworthy functions include a key role in life-cycle switching in Streptomyces and the operation of an iron transport system in Staphylococcus aureus, Bacillus subtilis and Escherichia coli . We also present several probable physiological roles of DyP-type peroxidases that reflect the widespread distribution and function of these enzymes. Lignin degradation is the most common function attributed to DyP-type peroxidases, but their activity is not high compared with that of standard lignin-degrading enzymes. From an environmental standpoint, degradation of natural antifungal anthraquinone compounds is a specific focus of DyP-type peroxidase research. Considered in its totality, the DyP-type peroxidase family offers a rich source of diverse and attractive materials for research scientists.

Lignin, which is a component of wood, is difficult to degrade in nature. However, serious decay caused by microbial consortia can happen to wooden antiques during the preservation process. This study successfully screened four microbial consortia with lignin degradation capabilities (J-1, J-6, J-8 and J-15) from decayed wooden antiques. Their compositions were identified by genomic sequencing, while the degradation products were analyzed by GC-MS. The lignin degradation efficiency of J-6 reached 54% after 48 h with an initial lignin concentration of 0.5 g/L at pH 4 and rotation speed of 200 rpm. The fungal consortium of J-6 contained Saccharomycetales (98.92%) and Ascomycota (0.56%), which accounted for 31% of the total biomass. The main bacteria in J-6 were Shinella sp. (47.38%), Cupriavidus sp. (29.84%), and Bosea sp. (7.96%). The strongest degradation performance of J-6 corresponded to its composition, where Saccharomycetales likely adapted to the system and improved lignin degradation enzymes activities, and the abundant bacterial consortium accelerated lignin decomposition. Our work demonstrated the potential utilization of microbial consortia via the synergy of microbial consortia, which may overcome the shortcomings of traditional lignin biodegradation when using a single strain, and the potential use of J-6 for lignin degradation/removal applications.

Lignin modifying or extracellular enzymes secreted by the white rot fungi have the ability of degrading wide range of lignocellulosic substrates and organic pollutants. Lignocellulosic biomass, despite being a renewable source of energy, is difficult to hydrolyse (hydrolysis being rate-limiting stage in anaerobic digestion process). Various pre-treatment techniques like physical, chemical, thermo-chemical and biological to enhance the accessibility of microbes to carbohydrates have been studied. Recently, usage of white- rot fungi in a biological pre-treatment technique have received renewed interest due to its low cost and eco-friendly nature. This review deals with: a) lignocellulosic biomass recalcitrance, b) various pre-treatment techniques and its economic feasibility, c) delignification and hydrolysis mechanism using white-rot fungi, d) factors controlling white-rot fungi pre- treatment process, and e) improvement in methane production through solid-state anaerobic digestion of white-rot fungi pre-treated lignocellulosic biomass. Finally a future perspective is also included., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Genetic modification of Rhodococcus jostii RHA1 was carried out in order to optimise the production of pyridine-2,4-dicarboxylic acid and pyridine-2,5-dicarboxylic acid bioproducts from lignin or lignocellulose breakdown, via insertion of either the Sphingobium SYK-6 ligAB genes or Paenibacillus praA gene respectively. Insertion of inducible plasmid pTipQC2 expression vector containing either ligAB or praA genes into a ΔpcaHG R. jostii RHA1 gene deletion strain gave 2-threefold higher titres of PDCA production from lignocellulose (200-287 mg/L), compared to plasmid expression in wild-type R. jostii RHA1. The ligAB genes were inserted in place of the chromosomal pcaHG genes encoding protocatechuate 3,4-dioxygenase, under the control of inducible P icl or P nitA promoters, or a constitutive P tpc5 promoter, producing 2,4-PDCA products using either wheat straw lignocellulose or commercial soda lignin as carbon source. Insertion of Amycolatopsis sp. 75iv2 dyp2 gene on a pTipQC2 expression plasmid led to enhanced titres of 2,4-PDCA products, due to enhanced rate of lignin degradation. Growth in minimal media containing wheat straw lignocellulose led to the production of 2,4-PDCA in 330 mg/L titre in 40 h, with > tenfold enhanced productivity, compared with plasmid-based expression of ligAB genes in wild-type R. jostii RHA1. Production of 2,4-PDCA was also observed using several different polymeric lignins as carbon sources, and a titre of 240 mg/L was observed using a commercially available soda lignin as feedstock.

Present study focuses on the organic solvent and ionic liquid compatibility of lignolytic enzymes produced from H. aswanensis strain ABC&#95;IITR. Lignin peroxidase (LiP), Manganese Peroxidase (MnP) and Laccase (Lac) obtained by Solid State Fermentation (SSF) of wheat bran. In this work, lignolytic enzymes subjected to 20-40% (v/v) organic solvents, metals ions, and cholinium laurate based ionic liquid (CLIL). Use of 40% (v/v) of pyridine along with 1.5 M NaCl and 0.15 mM CLIL in reaction system increased the bio-catalytic activity of lignolytic enzymes whereas metal ions like Fe increased LiP and MnP activities, and Cu enhanced laccase activity compared to control. The inherent stability of lignolytic enzymes in CLIL was not affected significantly whereas it decreased in pyridine reaction system. Further, in Kalson lignin degradation study, higher degradation achieved in CLIL (generate less saline waste) as compared to 40% (v/v) pyridine reaction system. In MnP catalyzed system, use of Glutathione (GSH) as mediator had resulted in maximum reduction of lignin weight of 40.84 and 31.83% in 40% (v/v) pyridine (1.5 NaCl) and 0.15 mM CLIL, respectively. This is the first report on lignolytic enzymes of haloarchaea with capability to get activated in organic solvent and cholinium laurate based ionic liquid., (Copyright © 2020. Published by Elsevier B.V.)

Lignin serves as the most abundant source of aromatic high-value products, but it has remained underexploited due to its inert and recalcitrant nature. White-rot basidiomycetes degrade lignin by secreting a set of lignin-modifying enzymes, including lignin peroxidase (LiP), manganese peroxidase, versatile peroxidase, laccase and various auxiliary enzymes. Among these, LiP presents significant potential for application in various industrial sectors such as second-generation biofuels, cosmetics, food, bio-pulping and biobleaching. However, the lack of commercial LiP preparations has hindered its industrial application. In addition, they are unstable at high temperatures, deactivated by solvents, susceptible to inactivation by hydrogen peroxide and challenging to produce in ample quantities. Several expression systems have been investigated to produce LiP, with fungal hosts that have shown the most promise to date. We discuss the progress and challenges of producing native and recombinant LiPs, and offer some future prospects on strategies to enhance the recombinant production of LiPs for industrial and biotechnological applications., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Deletion of the pcaHG genes, encoding protocatechuate 3,4-dioxygenase in Rhodococcus jostii RHA1, gives a gene deletion strain still able to grow on protocatechuic acid as the sole carbon source, indicating a second degradation pathway for protocatechuic acid. Metabolite analysis of wild-type R. jostii RHA1 grown on medium containing vanillin or protocatechuic acid indicated the formation of hydroxyquinol (benzene-1,2,4-triol) as a downstream product. Gene cluster ro01857-ro01860 in Rhodococcus jostii RHA1 contains genes encoding hydroxyquinol 1,2-dioxygenase and maleylacetate reductase for degradation of hydroxyquinol but also putative mono-oxygenase (ro01860) and putative decarboxylase (ro01859) genes, and a similar gene cluster is found in the genome of lignin-degrading Agrobacterium species. Recombinant R. jostii mono-oxygenase and decarboxylase enzymes in combination were found to convert protocatechuic acid to hydroxyquinol. Hence, an alternative pathway for degradation of protocatechuic acid via oxidative decarboxylation to hydroxyquinol is proposed. IMPORTANCE There is a well-established paradigm for degradation of protocatechuic acid via the β-ketoadipate pathway in a range of soil bacteria. In this study, we have found the existence of a second pathway for degradation of protocatechuic acid in Rhodococcus jostii RHA1, via hydroxyquinol (benzene-1,2,4-triol), which establishes a metabolic link between protocatechuic acid and hydroxyquinol. The presence of this pathway in a lignin-degrading Agrobacterium sp. strain suggests the involvement of the hydroxyquinol pathway in the metabolism of degraded lignin fragments., (Copyright © 2020 American Society for Microbiology.)

An alkali lignin-degrading, Gram-stain-negative, rod-shaped, non-motile and facultatively anaerobic bacterium, designated BM&#95;7 T , was isolated from mangrove sediment of the supralittoral zone in the Jiulong river estuary, PR China. The cells of strain BM&#95;7 T were 0.4-0.6 µm wide and 1.0-8.5 µm long. Oxidase and catalase activities were positive. Strain BM&#95;7 T could grow at 10-37 °C (optimum, 25-28 °C), at pH 6.0-8.0 (optimum, pH 7.0) and in the presence of 0.5-6 % (w/v) NaCl (optimum, 2%). Phylogenetic analysis of 16S rRNA gene sequences indicated that strain BM&#95;7 T belonged to the genus Mangrovibacterium of the family Prolixibacteraceae . It showed the highest similarity to Mangrovibacterium diazotrophicum JCM 19152 T (96.8 %), followed by Mangrovibacterium marinum KCTC 42253 T (96.1%). The values of average nucleotide identity and DNA-DNA hybridization were calculated as 76.9, 24.3 and 76.1, 17.4 % between strain BM&#95;7 T with M. diazotrophicum JCM 19152 T and M. marinum KCTC 42253 T , respectively. The major respiratory quinone of strain BM&#95;7 T was MK-7. The polar lipids were detected as phosphatidylethanolamine, three unidentified phospholipids and four unidentified aminolipids. The dominant fatty acids consisted of iso-C 15 : 0 , anteiso-C 15 : 0 , C 15 : 1   ω6 c , iso-C 17 : 0 3-OH, C 17 : 1  ω 6 c, C 17 : 0 3-OH and C 17 : 0 . The genome size of strain BM&#95;7 T is 5.6 Mb, with G+C content of 43.4 mol%. Based on the phylogenetic and phenotypic characteristics, strain BM&#95;7 T was considered to represent a novel species of the genus Mangrovibacterium , and the name Mangrovibacterium lignilyticum sp. nov. is proposed. The type strain is BM&#95;7 T (=MCCC 1A15882 T =KCTC 72696 T ).

The laccase has the ability to oxidize substituted phenols and the water is the sole byproduct, thus it has been employed to remove and/or modify the lignin in lignocellulosic material. A putative laccase gene, LacSM, from Sordaria macrospora k-hell was screened by a genome mining approach. Then, it was cloned and highly expressed in Escherichia coli. The molecular weight of recombinant LacSM was ~ 67 kDa. The optimal pH values for the LacSM oxidation of guaiacol, syringaldazine, 2,6-dimethoxyphenol, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were 6, 7, 5, and 5, respectively. The optimal activity of laccase was observed at 60, 55, 55, and 50 °C for four respective substrates. LacSM remained stable at pH 5-8 and thermostable at 60 °C with guaiacol as the substrate. 1 mM K + , Na + , or Mn 2+ ions slightly stimulated laccase activity. In addition, LacSM was moderately tolerant to the Cl - ion and showed an ability to remove and/or modify lignin. Thus, LacSM was a potential candidate for industrial applications, such as lignin degradation of lignocellulosic biomass.

This study examine ways to make biorefinery lignin accessible for anaerobic digestion. The raw material was the residue after removing carbohydrates by Wet Explosion pretreatment at 190 °C and 7.5% O 2 followed by enzymatic hydrolysis. The residual solid was mainly composed of lignin and was the raw material for a second WEx pretreatment operated at 220 °C with 4% oxygen and variable concentrations of NaOH (0-2%). Lignin B was the residue after pretreated without NaOH, Lignin C was pretreated at 1% NaOH, and Lignin E at 2% NaOH. Anaerobic digestion was carried out on all lignin fractions (Lignin A, B, C and E) at thermophilic conditions (52 °C) by mixing 70% of each lignin fractions with 30% clarified manure. The results showed that the lignin samples were demethoxylated as part of the biodegradation and that the highest severity pretreatment (with oxygen and 2% NaOH) resulted in the highest methane yield., (Published by Elsevier Ltd.)

Oil palm empty fruit bunches (OPEFB) are the most abundant, inexpensive, and environmentally friendly lignocellulosic biomass in Malaysia. Investigations on the microbial diversity of decaying OPEFB may reveal microbes with complex enzymes that have the potential to enhance the conversion of lignocellulose into second-generation biofuels as well as the production of other value-added products. In the present study, fungal and bacterial diversities in decaying OPEFB were identified using Illumina MiSeq sequencing of the V3 region of the 16S rRNA gene and V4 region of the 18S rRNA gene. Fungal diversity in decaying OPEFB was dominated by the phylum Ascomycota (14.43%), while most of the bacterial sequences retrieved belonged to Proteobacteria (76.71%). Three bacterial strains isolated from decaying OPEFB, designated as S18, S20, and S36, appeared to grow with extracted OPEFB-lignin and Kraft lignin (KL) as the sole carbon source. 16S rRNA gene sequencing identified the 3 isolates as Paenibacillus sp.. The molecular weight distribution of KL before and after degradation showed significant depolymerization when treated with bacterial strains S18, S20, and S36. The presence of low-molecular-weight lignin-related compounds, such as vanillin and 2-methoxyphenol derivatives, which were detected by a GC-MS analysis, confirmed the KL-degrading activities of isolated Paenibacillus strains.

Lytic polysaccharide monooxygenases (LPMOs), a class of copper-dependent enzymes, play a crucial role in boosting the enzymatic decomposition of polysaccharides. Here, we reveal that LPMOs might be associated with a lignin degradation pathway. An LPMO from white-rot fungus Pleurotus ostreatus , LPMO9A ( Po LPMO9A), was shown to be able to efficiently drive the activity of class II lignin-degrading peroxidases in vitro through H 2 O 2 production regardless of the presence or absence of a cellulose substrate. An LPMO-driven peroxidase reaction can degrade β-O-4 and 5-5' types of lignin dimer with 46.5% and 37.7% degradation, respectively, as well as alter the structure of natural lignin and kraft lignin. H 2 O 2 generated by Po LPMO9A was preferentially utilized for the peroxidase from Physisporinus sp. strain P18 ( Ps VP) reaction rather than cellulose oxidation, indicating that white-rot fungi may have a strategy for preferential degradation of resistant lignin. This discovery shows that LPMOs may be involved in lignin oxidation as auxiliary enzymes of lignin-degrading peroxidases during the white-rot fungal decay process. IMPORTANCE The enzymatic biodegradation of structural polysaccharides is affected by the degree of delignification of lignocellulose during the white-rot fungal decay process. The lignin matrix decreases accessibility to the substrates for LPMOs. H 2 O 2 has been studied as a cosubstrate for LPMOs, but the formation and utilization of H 2 O 2 in the reactions still represent an intriguing focus of current research. Lignin-degrading peroxidases and LPMOs usually coexist during fungal decay, and therefore, the relationship between H 2 O 2 -dependent lignin-degrading peroxidases and LPMOs should be considered during the wood decay process. The current study revealed that white-rot fungal LPMOs may be involved in the degradation of lignin through driving a versatile form of peroxidase activity in vitro and that H 2 O 2 generated by Po LPMO9A was preferentially used for lignin oxidation by lignin-degrading peroxidase ( Ps VP). These findings reveal a potential relationship between LPMOs and lignin degradation, which will be of great significance for further understanding the contribution of LPMOs to the white-rot fungal decay process., (Copyright © 2019 American Society for Microbiology.)

In this work, potassium iodide (KI) and graphene oxide (GO) were utilized to promote the selectivity of photocatalytic process for alkali lignin oxidation over ZnO. Different concertation of GO was added during the microwave synthesis procedure of ZnO, and the characterization results revealed that graphene can shift the conduction band to more reducing potential, resulting to higher production of superoxide anion radicals (O 2 ) compared to OH. Response Surface Methodology revealed the most suitable interaction among loading of GO, KI and irradiation time on lignin and total phenolic compound (TPC) degradation. Specifically, the optimal conditions (i.e. maximum lignin (52%) and minimum TPC (55%) degradation) were at [KI] = 0.64 mM; GO content into ZnO 1.2 mg/mL; 240 min of irradiation time. The results showed that higher addition of graphene into structure of ZnO could preserve more phenolics from degradation due to less production of OH. Furthermore, the addition of KI at optimized conditions could enhance the selectivity of degradation of lignin and phenolics via producing I radicals and quenching the excess amount of generated OH, respectively. The lower generation of OH at optimized conditions was quantitatively confirmed by a photoluminescence simplified technique. In addition, the effect of the photocatalytic process on substrate's anaerobic degradability was examined in order to evaluate the suitability of the pretreated solution for energy recovery. Indeed, besides the higher TPC concentration, the biogas production of treated straw at optimized conditions was increased by 35% compared to the untreated sample.- ) compared to OH. Response Surface Methodology revealed the most suitable interaction among loading of GO, KI and irradiation time on lignin and total phenolic compound (TPC) degradation. Specifically, the optimal conditions (i.e. maximum lignin (52%) and minimum TPC (55%) degradation) were at [KI] = 0.64 mM; GO content into ZnO 1.2 mg/mL; 240 min of irradiation time. The results showed that higher addition of graphene into structure of ZnO could preserve more phenolics from degradation due to less production of OH. Furthermore, the addition of KI at optimized conditions could enhance the selectivity of degradation of lignin and phenolics via producing I radicals and quenching the excess amount of generated OH, respectively. The lower generation of OH at optimized conditions was quantitatively confirmed by a photoluminescence simplified technique. In addition, the effect of the photocatalytic process on substrate's anaerobic degradability was examined in order to evaluate the suitability of the pretreated solution for energy recovery. Indeed, besides the higher TPC concentration, the biogas production of treated straw at optimized conditions was increased by 35% compared to the untreated sample., (Copyright © 2018. Published by Elsevier Ltd.)

Lignin is a heterogeneous polymer of aromatic subunits that is a major component of lignocellulosic plant biomass. Understanding how microorganisms deconstruct lignin is important for understanding the global carbon cycle and could aid in developing systems for processing plant biomass into valuable commodities. Sphingomonad bacteria use stereospecific glutathione S -transferases (GSTs) called β-etherases to cleave the β-aryl ether (β-O-4) bond, the most common bond between aromatic subunits in lignin. Previously characterized bacterial β-etherases are homodimers that fall into two distinct GST subclasses: LigE homologues, which cleave the β( R ) stereoisomer of the bond, and LigF homologues, which cleave the β( S ) stereoisomer. Here, we report on a heterodimeric β-etherase (BaeAB) from the sphingomonad Novosphingobium aromaticivorans that stereospecifically cleaves the β( R )-aryl ether bond of the di-aromatic compound β-(2-methoxyphenoxy)-γ-hydroxypropiovanillone (MPHPV). BaeAB's subunits are phylogenetically distinct from each other and from other β-etherases, although they are evolutionarily related to LigF, despite the fact that BaeAB and LigF cleave different β-aryl ether bond stereoisomers. We identify amino acid residues in BaeAB's BaeA subunit important for substrate binding and catalysis, including an asparagine that is proposed to activate the GSH cofactor. We also show that BaeAB homologues from other sphingomonads can cleave β( R )-MPHPV and that they may be as common in bacteria as LigE homologues. Our results suggest that the ability to cleave the β-aryl ether bond arose independently at least twice in GSTs and that BaeAB homologues may be important for cleaving the β( R )-aryl ether bonds of lignin-derived oligomers in nature., (© 2019 Kontur et al.)

This chapter provides an overview of the biosynthesis and structure of lignin. Moreover, examples of the commercial use of lignin and its promising future implementation are briefly described. Many applications are still hampered by the properties of technical lignins. Thus, the major challenge is the conversion of lignins into suitable building blocks or aromatics in order to open up new avenues for the usage of this renewable raw material. This chapter focuses on details about natural lignin degradation by fungi and bacteria, which harbor potential tools for lignin degradation and modification, which might help to develop eco-efficient processes for lignin utilization.

The degradation of lignin has attracted much attention since it represents approximately 30% of all non-fossil carbon sources and constitutes a sustainable bio-resource for fuels and aromatic derivatives. Here we investigated the degradation of lignin by laccase-catalysed reactions using 2,2'-Azino-bis(3-ethybenzothiazoline-6-sulfonic acid) (ABTS) as a mediator coupled with the carbon material graphene. Results indicated that there was a significant, two-fold, increase in the catalytic activity of lignin degradation in laccase-ABTS systems in the presence of graphene. Analysis suggested that the enhancement of lignin degradation could be attributed to graphene acting as an electron transfer conductor, thereby accelerating electron transfer, which facilitated the formation of intermediate oxidation states of ABTS and rendered the reactions between lignin and ABTS intermediates more efficient. This study could promote the development of novel enzymatic lignin degradation systems coupled with the carbon-based material graphene., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Members of the archaeal phylum Bathyarchaeota are among the most abundant microorganisms on Earth. Although versatile metabolic capabilities such as acetogenesis, methanogenesis, and fermentation have been suggested for bathyarchaeotal members, no direct confirmation of these metabolic functions has been achieved through growth of Bathyarchaeota in the laboratory. Here we demonstrate, on the basis of gene-copy numbers and probing of archaeal lipids, the growth of Bathyarchaeota subgroup Bathy-8 in enrichments of estuarine sediments with the biopolymer lignin. Other organic substrates (casein, oleic acid, cellulose, and phenol) did not significantly stimulate growth of Bathyarchaeota Meanwhile, putative bathyarchaeotal tetraether lipids incorporated 13 C from 13 C-bicarbonate only when added in concert with lignin. Our results are consistent with organoautotrophic growth of a bathyarchaeotal group with lignin as an energy source and bicarbonate as a carbon source and shed light into the cycling of one of Earth's most abundant biopolymers in anoxic marine sediment., Competing Interests: The authors declare no conflict of interest.

As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-guaiacyl ether (GGE) and found that Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate β-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S -transferase NaGST Nu is the only enzyme needed to remove glutathione from both ( R )- and ( S )-GS-HPV in N. aromaticivorans We solved the crystal structure of NaGST Nu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr 51 , Tyr 166 , and Tyr 224 ) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6 (which can also break the β-aryl ether bond) and Escherichia coli (which cannot break the β-aryl ether bond) can also cleave ( R )- and ( S )-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S -transferases., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Extensive research efforts have been dedicated to describing degradation of wood, which is a complex process; hence, microorganisms have evolved different enzymatic and non-enzymatic strategies to utilize this plentiful plant material. This review describes a number of fungal and bacterial organisms which have developed both competitive and mutualistic strategies for the decomposition of wood and to thrive in different ecological niches. Through the analysis of the enzymatic machinery engaged in wood degradation, it was possible to elucidate different strategies of wood decomposition which often depend on ecological niches inhabited by given organism. Moreover, a detailed description of low molecular weight compounds is presented, which gives these organisms not only an advantage in wood degradation processes, but seems rather to be a new evolutionatory alternative to enzymatic combustion. Through analysis of genomics and secretomic data, it was possible to underline the probable importance of certain wood-degrading enzymes produced by different fungal organisms, potentially giving them advantage in their ecological niches. The paper highlights different fungal strategies of wood degradation, which possibly correlates to the number of genes coding for secretory enzymes. Furthermore, investigation of the evolution of wood-degrading organisms has been described., (© FEMS 2017.)

This study examined the white rot fungus I. obliquus on the degradation of three types of straw biomass and the production of extracellular lignocellulolytic enzymes under submerged fermentation. The fungus process resulted in a highest lignin loss of 72%, 39%, and 47% in wheat straw, rice straw, and corn stover within 12days, respectively. In merely two days, the fungus selectively degraded wheat straw lignin by 37%, with only limited cellulose degradation (13%). Fourier transform infrared spectroscopy revealed that the fungus most effectively degraded the wheat straw lignin and rice straw crystalline cellulose. Scanning electronic microscopy showed the most pronounced structural changes in wheat straw. High activities of manganese peroxidase (159.0U/mL) and lignin peroxidase (123.4U/mL) were observed in wheat straw culture on Day 2 and 4, respectively. Rice straw was the best substrate to induce the production of cellulase and xylanase., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Sweet sorghum (Sorghum sp.) has high biomass yield. Hydrolysis of lignocellulosic sweet sorghum bagasse (SSB) to fermentable sugar could be useful for manufacture of biofuel or other fermentation products. Pretreatment of lignocellulosic biomass to degrade lignin before enzymatic hydrolysis is a key step. Fungal pretreatment of SSB with combined CuSO 4 -gallic acid supplements in solid-state fermentation (SSF) to achieve higher lignin degradation, selectivity value (SV), and enzymatic hydrolysis to sugar was studied. Coriolus versicolor was selected due to high activities of ligninolytic enzymes laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), polyphenol oxidase (PPO), and arylalcohol oxidase (AAO) and low activities of cellulolytic enzymes CMCase, FPase, and β-glucosidase with high lignin degradation and SV in 20 days. CuSO 4 /gallic acid increased the activities of ligninolytic enzymes resulting in enhanced lignin degradations and SVs. Cumulative/synergistic effect of combined supplements further increased the activities of laccase, LiP, MnP, PPO, and AAO by 7.6, 14.6, 2.67, 2.06, and 2.15-folds, respectively (than control), resulting in highest lignin degradation 31.1 ± 1.4% w/w (1.56-fold) and SV 2.33 (3.58-fold). Enzymatic hydrolysis of pretreated SSB yielded higher (~2.2 times) fermentable sugar. The study showed combined supplements can improve fungal pretreatment of lignocellulosic biomass. XRD, SEM, FTIR, and TGA/DTG of SSB confirmed the results.

The objective of this work was to study the increase in multiple lignolytic enzyme productions through the use of supplements in combination in pretreatment of sweet sorghum bagasse (SSB) by Coriolus versicolor such that enzymes act synergistically to maximize the lignin degradation and selectivity. Enzyme activities were enhanced by metallic salts and phenolic compound supplements in SSF. Supplement of syringic acid increased the activities of LiP, AAO and laccase; gallic acid increased MnP; CuSO 4 increased laccase and PPO to improve the lignin degradations and selectivity individually, higher than control. Combination of supplements optimized by RSM increased the production of laccase, LiP, MnP, PPO and AAO by 17.2, 45.5, 3.5, 2.4 and 3.6 folds respectively for synergistic action leading to highest lignin degradation (2.3 folds) and selectivity (7.1 folds). Enzymatic hydrolysis of pretreated SSB yielded ∼2.43 times fermentable sugar. This technique could be widely applied for pretreatment and enzyme productions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To increase the efficiency of winery-derived biomass biodegradation, grape pomace was ultrasonicated for 20min in the presence of 0.25M, 0.5Mand1.0MKOH and 1.0MNaOH. This was followed by treatment with a 1:1 (v/v) mix of crude enzyme preparation derived from Phanerochaete chrysosporium and Trametes versicolor for 18h and a further 18h treatment with a 60:14:4:2 percent ratio combination of enzymes derived from Aspergillus niger: Penicillium chrysogenum: Trichoderma harzianum: P. citrinum, repsectively. Process efficiency was evaluated by its comparison to biological only mixed fungal degradation over 16days. Ultrasonication treatment with 0.5MKOH followed by mixed enzyme treatment yielded the highest lignin degradation of about 13%. Cellulase, β-glucosidase, xylanase, laccase and lignin peroxidase activities of 77.9, 476, 5,390.5, 66.7 and 29,230.7U/mL, respectively, were observed during biomass degradation. Gas chromatography-mass spectrometry (GC-MS) analysis of the degraded material identified commercially important compounds such as gallic acid, lithocholic acid, glycolic acid and lactic acid which were generated in considerable quantities. Thus, the combination of sonication pre-treatment and enzymatic degradation has the potential to considerably improve the breakdown of agricultural biomass and produce commercially useful compounds in markedly less time (<40h) with respect to biological only degradation (16days)., (Copyright © 2016 Elsevier Inc. All rights reserved.)