Mechanistic insight in the selective delignification of wheat straw by three white-rot fungal species through quantitative 13C-IS py-GC-MS and whole cell wall HSQC NMR.
ABSTRACT: Background:The white-rot fungi Ceriporiopsis subvermispora (Cs), Pleurotus eryngii (Pe), and Lentinula edodes (Le) have been shown to be high-potential species for selective delignification of plant biomass. This delignification improves polysaccharide degradability, which currently limits the efficient lignocellulose conversion into biochemicals, biofuels, and animal feed. Since selectivity and time efficiency of fungal delignification still need optimization, detailed understanding of the underlying mechanisms at molecular level is required. The recently developed methodologies for lignin quantification and characterization now allow for the in-depth mapping of fungal modification and degradation of lignin and, thereby, enable resolving underlying mechanisms. Results:Wheat straw treated by two strains of Cs (Cs1 and Cs12), Pe (Pe3 and Pe6) and Le (Le8 and Le10) was characterized using semi-quantitative py-GC-MS during fungal growth (1, 3, and 7 weeks). The remaining lignin after 7 weeks was quantified and characterized using 13C lignin internal standard based py-GC-MS and whole cell wall HSQC NMR. Strains of the same species showed similar patterns of lignin removal and degradation. Cs and Le outperformed Pe in terms of extent and selectivity of delignification (Cs???Le?>>?Pe). The highest lignin removal [66% (w/w); Cs1] was obtained after 7 weeks, without extensive carbohydrate degradation (factor 3 increased carbohydrate-to-lignin ratio). Furthermore, though after treatment with Cs and Le comparable amounts of lignin remained, the structure of the residual lignin vastly differed. For example, C?-oxidized substructures accumulated in Cs treated lignin up to 24% of the total aromatic lignin, a factor two higher than in Le-treated lignin. Contrarily, ferulic acid substructures were preferentially targeted by Le (and Pe). Interestingly, Pe-spent lignin was specifically depleted of tricin (40% reduction). The overall subunit composition (H:G:S) was not affected by fungal treatment. Conclusions:Cs and Le are both able to effectively and selectively delignify wheat straw, though the underlying mechanisms are fundamentally different. We are the first to identify that Cs degrades the major ?-O-4 ether linkage in grass lignin mainly via C?-O-aryl cleavage, while C?-C? cleavage of inter-unit linkages predominated for Le. Our research provides a new insight on how fungi degrade lignin, which contributes to further optimizing the biological upgrading of lignocellulose.
Project description:Wheat straw is the major crop residue in European countries which makes it the most promising material for bioconversion into biofuels. However, cellulose and hemicellulose are protected with lignin, so delignification is an inevitable phase in lignocellulose processing. The organisms predominantly responsible for its degradation are white-rot fungi and among them Trametes species represent promising degraders due to a well-developed ligninolytic enzyme system. Although numerous studies have confirmed that low molecular weight compounds can induce the production and activity of ligninolytic enzymes it is not clear how this reflects on the extent of delignification. The aim of the study was to assess the capacity of p-anisidine and veratryl alcohol to induce the production and activity of Mn-oxidizing peroxidases and laccases, and wheat straw delignification by six Trametes species. Significant inter- and intraspecific variations in activity and features of these enzymes were found, as well as differences in the potential of lignocellulose degradation in the presence or absence of inducers. Differences in the catalytic properties of synthesized enzyme isoforms strongly affected lignin degradation. Apart from enhanced lignin degradation, the addition of p-anisidine could significantly improve the selectivity of wheat straw ligninolysis, which was especially evident for T. hirsuta strains.
Project description:Background:The ascomycete fungus Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome. Results:Quantitative 13C lignin internal standard py-GC-MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC-MS and HSQC NMR analyses demonstrated that C?-oxidized substructures significantly increased through fungal action, while intact ?-O-4' aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H2O2-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis. Conclusions:For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms.
Project description:Background:Plant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of P. brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide. Results:We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners. Conclusion:As a peculiar feature of P. brumalis, we observed gene family extension, up-regulation and secretion of an abundant set of versatile peroxidases and manganese peroxidases, compared with other Polyporales species. The orchestrated secretion of an abundant set of these delignifying enzymes and redox cycling enzymatic partners could contribute to the delignification capabilities of the fungus. Our findings highlight the diversity of wood decay mechanisms present in Polyporales and the potentiality of further exploring this taxonomic order for enzymatic functions of biotechnological interest.
Project description:In this study, two bacterial strains capable of degrading lignin, cellulose, and hemicellulose were isolated from wood feeding termite. The isolates were identified by 16S rRNA gene sequencing. A bacterium Ochrobactrum oryzae BMP03 capable of degrading lignin was isolated on alkali lignin medium and Bacillus sp. BMP01 strain capable of degrading cellulose and hemicellulose were isolated on carboxymethyl cellulose and xylan media. The efficiency of bacterial degradation was studied by evaluating the composition of rice straw both before and after degradation. The appearance of new cellulose bands at 1382, 1276, 1200, and 871 cm-1, and the absence of former lignin bands at 1726, 1307, and 1246 cm-1 was observed after biodelignification. This was further confirmed by the formation of channeling and layering of the microstructure of biodelignified rice straw observed under electron microscope. Maximum lignin removal was achieved in separate biodelignification and hydrolysis process after the 14th day of treatment by Ochrobactrum oryzae BMP03 (53.74% lignin removal). Hydrolysis of the biodelignified rice straw released 69.96% of total reducing sugars after the 14th day hydrolysis by Bacillus sp. BMP01. In simultaneous delignification and hydrolysis process, about 58.67% of total reducing sugars were obtained after the 13th day of biotreatment. Separate delignification and hydrolysis process were found to be effective in lignin removal and sugar released than the simultaneous process. The bacteria, Bacillus sp. BMP01, has the ability to degrade hemicellulose and cellulose simultaneously. Overall, these results demonstrate that the possibility of rice straw bioconversion into reducing sugars by bacteria from termite gut.
Project description:BACKGROUND:The selective lignin-degrading white-rot fungi are regarded to be the best lignin degraders and have been widely used for reducing the saccharification recalcitrance of lignocellulose. However, the biological delignification and conversion of lignocellulose in biorefinery is still limited. It is necessary to develop novel and more efficient bio-delignification systems. RESULTS:Physisporinus vitreus relies on a new versatile peroxidase (VP)-based delignification strategy to remove enzymatic recalcitrance of corn stover efficiently, so that saccharification of corn stover was significantly enhanced to 349.1 mg/g biomass (yield of glucose) and 91.5% (hydrolysis yield of cellulose) at 28 days, as high as levels reached by thermochemical treatment. Analysis of the lignin structure using pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) showed that the total abundance of lignin-derived compounds decreased by 54.0% and revealed a notable demethylation during lignin degradation by P. vitreus. Monomeric and dimeric lignin model compounds were used to confirm the ligninolytic capabilities of extracellular ligninases secreted by P. vitreus. The laccase (Lac) from P. vitreus could not oxidize nonphenolic lignin compounds and polymerized ?-O-4 and 5-5' dimers to precipitate which had a negative effect on the enzymatic hydrolysis of corn stover in vitro. However, the VP from P. vitreus could oxidize both phenolic and nonphenolic lignin model compounds as well as break the ?-O-4 and 5-5' dimers into monomeric compounds, which were measured by high-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Moreover, we showed that addition of purified VP in vitro improved the enzymatic hydrolysis of corn stover by 14.1%. CONCLUSIONS:From the highly efficient system of enzymatic recalcitrance removal by new white-rot fungus, we identified a new delignification strategy based on VP which could oxidize both phenolic and nonphenolic lignin units and break different linkages in lignin. In addition, this is the first evidence that VP could break 5-5' linkage efficiently in vitro. Moreover, VP improved the enzymatic hydrolysis of corn stover in vitro. The remarkable lignin-degradative potential makes VP attractive for biotechnological applications.
Project description:A sustainable strategy for synergistic surface engineering of lignocellulose and cellulose fibers derived from wood by synergistic combination of metal-free catalysis and renewable polyelectrolyte (PE) complexes is disclosed. The strategy allows for improvement and introduction of important properties such as strength, water resistance, and fluorescence to the renewable fibers and cellulosic materials. For example, the "green" surface engineering significantly increases the strength properties (up to 100% in <i>Z</i>-strength) of chemi-thermomechanical pulp (CTMP) and bleached sulphite pulp (BSP)-derived sheets. Next, performing an organocatalytic silylation with a nontoxic organic acid makes the corresponding lignocellulose and cellulose sheets hydrophobic. A selective color modification of polysaccharides is developed by combining metal-free catalysis and thiol-ene click chemistry. Next, fluorescent PE complexes based on cationic starch (CS) and carboxymethylcellulose (CMC) are prepared and used for modification of CTMP or BSP in the presence of a metal-free catalyst. Laser-scanning confocal microscopy reveals that the PE-strength additive is evenly distributed on the CTMP and heterogeneously on the BSP. The fluorescent CS distribution on the CTMP follows the lignin distribution of the lignocellulosic fibers.
Project description: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 (PoLPMO9A), was shown to be able to efficiently drive the activity of class II lignin-degrading peroxidases in vitro through H2O2 production regardless of the presence or absence of a cellulose substrate. An LPMO-driven peroxidase reaction can degrade ?-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. H2O2 generated by PoLPMO9A was preferentially utilized for the peroxidase from Physisporinus sp. strain P18 (PsVP) 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. H2O2 has been studied as a cosubstrate for LPMOs, but the formation and utilization of H2O2 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 H2O2-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 H2O2 generated by PoLPMO9A was preferentially used for lignin oxidation by lignin-degrading peroxidase (PsVP). 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.
Project description:Background:The efficient utilization of lignocellulosic biomass for biofuel production has received increasing attention. Previous studies have investigated the pretreatment process of biomass, but the detailed enzymatic hydrolysis process of pretreated biomass remains largely unclear. Thus, this study investigated the pretreatment efficiency of dilute alkali, acid, hydrogen peroxide and its ultimate effects on enzymatic hydrolysis. Furthermore, to better understand the enzymatic digestion process of alkali-pretreated sweet sorghum straw (SSS), multimodal microscopy techniques were used to visualize the enzymatic hydrolysis process. Result:After pretreatment with alkali, an enzymatic hydrolysis efficiency of 86.44% was obtained, which increased by 99.54% compared to the untreated straw (43.23%). The FTIR, XRD and SEM characterization revealed a sequence of microstructural changes occurring in plant cell walls after pretreatment, including the destruction of lignin-polysaccharide interactions, the increase of porosity and crystallinity, and reduction of recalcitrance. During the course of hydrolysis, the cellulase dissolved the cell walls in the same manner and the digestion firstly occurred from the middle of cell walls and then toward the cell wall corners. The CLSM coupled with fluorescent labeling demonstrated that the sclerenchyma cells and vascular bundles in natural SSS were highly lignified, which caused the nonproductive bindings of cellulase on lignin. However, the efficient delignification significantly increased the accessibility and digestibility of cellulase to biomass, thereby improving the saccharification efficiency. Conclusion:This work will be helpful in investigating the biomass pretreatment and its structural characterization. In addition, the visualization results of the enzymatic hydrolysis process of pretreated lignocellulose could be used for guidance to explore the lignocellulosic biomass processing and large-scale biofuel production.