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

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, Cα-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 β-O-4 ether linkage in grass lignin mainly via Cβ–O–aryl cleavage, while Cα–Cβ 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. Electronic supplementary material The online version of this article (10.1186/s13068-018-1259-9) contains supplementary material, which is available to authorized users

Mass spectrometric fragmentation patterns discriminate C1- and C4-oxidised cello-oligosaccharides from their non-oxidised and reduced forms

Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that degrade recalcitrant polysaccharides, such as cellulose. However, the identification of LPMO-generated C1- and/or C4-oxidised oligosaccharides is far from straightforward. In particular, their fragmentation patterns have not been well established when using mass spectrometry. Hence, we studied the fragmentation behaviours of non-, C1- and C4-oxidised cello-oligosaccharides, including their sodium borodeuteride-reduced forms, by using hydrophilic interaction chromatography and negative ion mode collision induced dissociation - mass spectrometry. Non-oxidised cello-oligosaccharides showed predominantly C- and A-type cleavages. In comparison, C4-oxidised ones underwent B-/Y- and X-cleavage close to the oxidised non-reducing end, while closer to the reducing end C-/Z- and A-fragmentation predominated. C1-oxidised cello-oligosaccharides showed extensively A-cleavage. Reduced oligosaccharides showed predominant glycosidic bond cleavage, both B-/Y- and C-/Z-, close to the non-reducing end. Our findings provide signature mass spectrometric fragmentation patterns to unambiguously elucidate the catalytic behaviour and classification of LPMOs.</p

Quantitative mapping of lignin : Comprehensive insight into fungal delignification of plant biomass

Plant biomass delignification is crucial for terrestrial carbon cycling and is essential for incentives aiming at the valorization of lignocellulose. For understanding this central process in nature and biorefinery, we need to elucidate and comprehend the mechanisms that govern recalcitrance and conversion at the molecular level. This insight can only be obtained by accurate analysis of the molecules involved, both contentwise and structurewise. This research, therefore, aimed to advance the quantitative mapping of lignin and employ the developed analytical toolkit to unravel the underlying mechanisms of a process that has largely remained elusive to date: the fungal delignification of plant biomass. We demonstrate through careful method validation that py-GC-MS analysis can be used for the concurrent quantification and structural characterization of grass, hardwood and softwood lignin, when employing uniformly 13C labeled lignin internal standards and relative response factors for the individual pyrolysis products. The developed method was used to assess three white-rot fungal species in terms of delignification effectivity and selectivity. In both important traits, Ceriporiopsis subvermispora outperformed Lentinula edodes and Pleurotus eryngii. Comprehensive structural analyses of the residual lignin after growth of C. subvermispora allowed us to reconstruct various degradation routes of lignin&rsquo;s most abundant &beta;-O-4&rsquo; ethers and determine the relative susceptibility of various &beta;-O-4&rsquo; substructures. Our results imply that one-electron oxidation initiates in situ ligninolysis, which then cascades into the cleavage of Ca-Cb, Cb-O and O-4&rsquo;-aryl bonds. Ligninolysis was shown to depend on the electron density of the 4&rsquo;-O&shy;-subunit, diastereochemistry and &gamma;-acylation. In addition to white-rot basidiomycete fungi, we studied the ligninolytic capacity of the ascomycete fungus P. anserina. Substrate characterization unambiguously confirmed lignin degradation and secretome analysis suggested that laccases and H2O2 producing enzymes were likely involved in the observed ligninolysis

The solubility of primary plant cell wall polysaccharides in LiCl-DMSO

In order to understand the architecture of the primary plant cell wall, knowledge on its polysaccharides and their interactions is of importance. In this study, further architectural insight was obtained by sequential LiCl-DMSO and buffer extractions after planetary ball milling. After milling, up to 50% of all polysaccharides in the Chelating agent Unextractable Solids (ChUS) from carrot, tomato and strawberry solubilised in LiCl-DMSO without loss of structural information. Approximately 30% of all pectin was LiCl-DMSO insoluble but solubilised in the subsequent buffer extraction, and these populations had higher HG:RG-I ratios than LiCl-DMSO soluble populations. The degree of methyl-esterification (DM) of pure pectins highly determined its solubility in LiCl-DMSO. However, solubility of cell wall pectin was governed by more factors since both soluble and insoluble pectin were substantially methyl-esterified and acetylated. Digestion of LiCl-DMSO soluble and insoluble fractions by pectinases confirmed the presence of acetylated HG-regions for carrot and strawberry pectin.</p

Breeding Targets to Improve Biomass Quality in Miscanthus

Lignocellulosic crops are attractive bioresources for energy and chemicals production within a sustainable, carbon circular society. Miscanthus is one of the perennial grasses that exhibits great potential as a dedicated feedstock for conversion to biobased products in integrated biorefineries. The current biorefinery strategies are primarily focused on polysaccharide valorization and require severe pretreatments to overcome the lignin barrier. The need for such pretreatments represents an economic burden and impacts the overall sustainability of the biorefinery. Hence, increasing its efficiency has been a topic of great interest. Inversely, though pretreatment will remain an essential step, there is room to reduce its severity by optimizing the biomass composition rendering it more exploitable. Extensive studies have examined the miscanthus cell wall structures in great detail, and pinpointed those components that affect biomass digestibility under various pretreatments. Although lignin content has been identified as the most important factor limiting cell wall deconstruction, the effect of polysaccharides and interaction between the different constituents play an important role as well. The natural variation that is available within different miscanthus species and increased understanding of biosynthetic cell wall pathways have specified the potential to create novel accessions with improved digestibility through breeding or genetic modification. This review discusses the contribution of the main cell wall components on biomass degradation in relation to hydrothermal, dilute acid and alkaline pretreatments. Furthermore, traits worth advancing through breeding will be discussed in light of past, present and future breeding efforts.</p

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

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, Cα-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 β-O-4 ether linkage in grass lignin mainly via Cβ–O–aryl cleavage, while Cα–Cβ 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

Uniformly ¹³C Labeled Lignin Internal Standards for Quantitative Pyrolysis-GC-MS Analysis of Grass and Wood

With the ever-advancing lignocellulose valorization strategies, lignin analyses need to advance as well. However, lignin quantification still heavily relies on unspecific, time- and sample-consuming gravimetric, and spectrophotometric analyses. Here, we demonstrate that lignin isolates from uniformly 13C-labeled wheat straw, willow, and douglas fir serve as "ideal" internal standards for pyrolysis gas chromatography high-resolution mass spectrometry (py-GC-HR-MS) analyses of plant biomass, allowing the accurate and precise quantification and structural characterization of lignin in grasses, hardwoods, and softwoods. The 13C lignin internal standards were comprehensively structurally characterized by HSQC NMR and py-GC-HR-MS analyses, and their application for lignin quantification was validated in biomass model systems and in actual plant biomass. For all botanical origins and species, the lignin content was determined within 5% relative deviation of the Klason benchmark. These results establish the capability of the developed analytical platform to selectively quantify and structurally characterize lignin simultaneously and demonstrate a valuable addition to the lignin analysis toolbox.</p

RP-UHPLC-UV-ESI-MS/MS analysis of LPMO generated C4-oxidized gluco-oligosaccharides after non-reductive labeling with 2-aminobenzamide

Lytic polysaccharide monooxygenases (LPMOs) are able to cleave recalcitrant polysaccharides, such as cellulose, by oxidizing the C1 and/or C4 atoms. The analysis of the resulting products requires a variety of analytical techniques. Up to now, these techniques mainly focused on the identification of non-oxidized and C1-oxidized oligosaccharides. The analysis of C4-oxidized gluco-oligosaccharides is mostly performed by using high pressure anion exchange chromatography (HPAEC). However, the alkaline conditions used during HPAEC analysis lead to tautomerization of C4-oxidized gluco-oligosaccharides, which limits the use of this technique. Here, we describe the use of reverse phase-ultra high performance liquid chromatography (RP-UHPLC) in combination with non-reductive 2-aminobenzamide (2-AB) labeling. Non-reductive 2-AB labeling enabled separation of C4-oxidized gluco-oligosaccharides from their non-oxidized counterparts. Moreover, RP-UHPLC does not require buffered mobile phases, which reduce mass spectrometry (MS) sensitivity. The latter is seen as an advantage over other techniques such as hydrophilic interaction liquid chromatography and porous graphitized carbon coupled to MS. RP-UHPLC coupled to UV detection and mass spectrometry allowed the identification of both labeled non-oxidized and C4-oxidized oligosaccharides. Non-reductive labeling kept the ketone at the C4-position of LPMO oxidized oligosaccharides intact, while selective reducing agents such as sodium triacetoxyborohydride (STAB) reduced this ketone group. Our results show that RP-UHPLC-UV-ESI-MS in combination with non-reductively 2-AB labeling is a suitable technique for the separation and identification of LPMO-generated C4-oxidized gluco-oligosaccharides

Understanding laccase/HBT-catalyzed grass delignification at the molecular level

Laccase-mediator systems (LMS) are potential green tools to improve the valorization of lignocellulosic biomass by selective degradation of lignin. Despite extensive attention devoted to lignin degradation by LMS in literature, knowledge on the underlying mechanisms is largely limited to model compound studies. Here, we report a mechanistic study on the delignification of wheat straw (WS) and corn stover (CS) by a laccase/HBT system. Quantitative 13C-IS py-GC-MS analysis revealed that WS and CS were delignified in the range of 28-51% (w/w). Based on a combination of py-GC-MS, 2D NMR, SEC and RP-UHPLC-MS, extensive structural characterization of both residual and solubilized lignin structures was performed, from which we reconstructed the degradation pathway of native lignin by laccase/HBT. For the first time, we show that degradation of native lignin in the plant cell wall matrix by LMS occurs via both Cα-Cβ cleavage and ether cleavage of β-O-4′ aryl ethers, and that the latter primarily occurs via cleavage of the β-O bond. Cγ-Coumaroylated substructures were found to be more recalcitrant towards degradation than non-acylated substructures. In addition to lignin degradation, our results provide evidence for grafting of HBT onto lignin.</p

Uniformly ¹³C Labeled Lignin Internal Standards for Quantitative Pyrolysis-GC-MS Analysis of Grass and Wood

With the ever-advancing lignocellulose valorization strategies, lignin analyses need to advance as well. However, lignin quantification still heavily relies on unspecific, time- and sample-consuming gravimetric, and spectrophotometric analyses. Here, we demonstrate that lignin isolates from uniformly 13C-labeled wheat straw, willow, and douglas fir serve as "ideal" internal standards for pyrolysis gas chromatography high-resolution mass spectrometry (py-GC-HR-MS) analyses of plant biomass, allowing the accurate and precise quantification and structural characterization of lignin in grasses, hardwoods, and softwoods. The 13C lignin internal standards were comprehensively structurally characterized by HSQC NMR and py-GC-HR-MS analyses, and their application for lignin quantification was validated in biomass model systems and in actual plant biomass. For all botanical origins and species, the lignin content was determined within 5% relative deviation of the Klason benchmark. These results establish the capability of the developed analytical platform to selectively quantify and structurally characterize lignin simultaneously and demonstrate a valuable addition to the lignin analysis toolbox.</p