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. 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 β-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 β-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%. 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.

Background: The efficiency of biological systems as an option for pretreating lignocellulosic biomass has to be improved to make the process practical. Fungal treatment with manganese (Mn) addition for improving lignocellulosic biomass fractionation and enzyme accessibility were investigated in this study. The broad-spectrum effect was tested on two different types of feedstocks with three fungal species. Since the physicochemical and structural properties of biomass were the main changes caused by fungal degradation, detailed characterization of biomass structural features was conducted to understand the mechanism of Mn-enhanced biomass saccharification. Results: The glucose yields of fungal-treated poplar and wheat straw increased by 2.97- and 5.71-fold, respectively, after Mn addition. Particularly, over 90% of glucose yield was achieved in Mn-assisted Pleurotus ostreatus-treated wheat straw. A comparison study using pyrolysis gas chromatography mass spectrometry (Py-GC/MS) and two-dimensional ¹H–¹³C heteronuclear single quantum coherence (2D HSQC) nuclear magnetic resonance (NMR) spectroscopy was conducted to elucidate the role of Mn addition on fungal disruption of the cross-linked structure of whole plant cell wall. The increased C_α-oxidized products was consistent with the enhanced cleavage of the major β-O-4 ether linkages in poplar and wheat straw lignin or in the wheat straw lignin–carbohydrate complexes (LCCs), which led to the reduced condensation degree in lignin and decreased lignin content in Mn-assisted fungal-treated biomass. The correlation analysis and principal component analysis (PCA) further demonstrated that Mn addition to fungal treatment enhanced bond cleavage in lignin, especially the β-O-4 ether linkage cleavage played the dominant role in removing the biomass recalcitrance and contributing to the glucose yield enhancement. Meanwhile, enhanced deconstruction of LCCs was important in reducing wheat straw recalcitrance. The findings provided not only mechanistic insights into the Mn-enhanced biomass digestibility by fungus, but also a strategy for improving biological pretreatment efficiency of lignocellulose. Conclusion: The mechanism of enhanced saccharification of biomass by Mn-assisted fungal treatment mainly through C_α-oxidative cleavage of β-O-4 ether linkages further led to the decreased condensation degree in lignin, as a result, biomass recalcitrance was significantly reduced by Mn addition. Graphic

Producing polyhydroxyalkanoate (PHA) from lignin through biological conversion has great further potential, but is held by its high heterogeneous characteristic toxicity of the depolymerized products, and low bioconversion of the depolymerized products. In this study, a Fenton-like reaction, which is inspired by fungal decay strategy, was reported to cleave Kraft lignin linkages and produce low toxic mono-aromatic and low molecular organic compounds for microbial conversion. The treatment improved the bioconversion of lignin to PHA by 141.7% compared to Kraft lignin. The bond cleavage of Kraft lignin was characterized by Py-GC/MS and 2D NMR. Seven major depolymerized products were chosen to test their toxicity effect on bacterial fermentation. Furthermore, 920.4 mg of PHA was obtained from 1-L black liquor after Fenton-like reaction treatment. This is a novel attempt mimicking fungal decay strategy coupled with the microbial conversion of lignin into high-value PHA with a sustainable future.