White-rot fungi with selective lignin-degrading ability can improve the conversion efficiency of lignocellulose to biofuels. Understanding the fungal deconstruction process is critical for developing efficient and mild fungal pretreatment technologies. This study reveals the role of selective delignification with Echinodontium taxodii in overcoming saccharification recalcitrance of bamboo culm through cellulase adsorption experiments, surface-property and porosity measurements, and chemical structural analysis. Selective removal of hydrophobic lignin coating cellulose increased substrate hydrophilicity and enlarged the volume of accessible pores of 5–10 nm diameter, and both interunit linkages of lignin and cross-linkages between lignin and xylan were extensively cleaved by E. taxodii. This allowed more cellulase to infiltrate the lignocellulosic matrix to access the cellulose, thus markedly improving the saccharification of the bamboo culms. Fungal delignification increased nonproductive adsorption of cellulase onto residual lignin, but not sufficiently to inhibit saccharification. Compared to harsh thermochemical processes, this natural system for delignification provides a deconstruction strategy that can significantly reduce the rigid lignin barrier of recalcitrant biomass without increasing cellulase inhibition by residual lignin.Keywords: Bamboo culms; Cellulose accessibility; Nonproductive adsorption; Recalcitrance; Saccharification; Selective delignification; White rot fungus;

Manganese peroxidase is one of the Class II fungal peroxidases that are able to oxidize the low redox potential phenolic lignin compounds. For high redox potential non-phenolic lignin degradation, mediators such as GSH and unsaturated fatty acids are required in the reaction. However, it is not known whether carboxylic acids are a mediator for non-phenolic lignin degradation.The white rot fungus Irpex lacteus is one of the most potent fungi in degradation of lignocellulose and xenobiotics. Two manganese peroxidases (IlMnP1 and IlMnP2) from I. lacteus CD2 were over-expressed in Escherichia coli and successfully refolded from inclusion bodies. Both IlMnP1 and IlMnP2 oxidized the phenolic compounds efficiently. Surprisingly, they could degrade veratryl alcohol, a non-phenolic lignin compound, in a Mn2+-dependent fashion. Malonate or oxalate was found to be also essential in this degradation. The oxidation of non-phenolic lignin was further confirmed by analysis of the reaction products using LC–MS/MS. We proved that Mn2+ and a certain carboxylate are indispensable in oxidation and that the radicals generated under this condition, specifically superoxide radical, are at least partially involved in lignin oxidative degradation. IlMnP1 and IlMnP2 can also efficiently decolorize dyes with different structures.We provide evidence that a carboxylic acid may mediate oxidation of non-phenolic lignin through the action of radicals. MnPs, but not LiP, VP, or DyP, are predominant peroxidases secreted by some white rot fungi such as I. lacteus and the selective lignocellulose degrader Ceriporiopsis subvermispora. Our finding will help understand how these fungi can utilize MnPs and an excreted organic acid, which is usually a normal metabolite, to efficiently degrade the non-phenolic lignin. The unique properties of IlMnP1 and IlMnP2 make them good candidates for exploring molecular mechanisms underlying non-phenolic lignin compounds oxidation by MnPs and for applications in lignocellulose degradation and environmental remediation.

Lignin utilization during biomass conversion has been a major challenge for lignocellulosic biofuel. In particular, the conversion of lignin along with carbohydrate for fungible fuels and chemicals will both improve the overall carbon efficiency and reduce the need for chemical pretreatments. However, few biomass-converting microorganisms have the capacity to degrade all cell wall components including lignin, cellulose, and hemicellulose. We hereby evaluated a unique oleaginous fungus strain, Cunninghamella echinulata FR3, for its capacity to degrade lignin during biomass conversion to lipid, and the potential to carry out consolidated fermentation without chemical pretreatment, especially when combined with sorghum (Sorghum bicolor) bmr mutants with reduced lignin content. The study clearly showed that lignin was consumed together with carbohydrate during biomass conversion for all sorghum samples, which indicates that this organism has the potential for biomass conversion without chemical pretreatment. Even though dilute acid pretreatment of biomass resulted in more weight loss during fungal fermentation than untreated biomass, the lipid yields were comparable for untreated bmr6/bmr12 double mutant and dilute acid-pretreated wild-type biomass samples. The mechanisms for lignin degradation in oleaginous fungi were further elucidated through transcriptomics and chemical analysis. The studies showed that in C. echinulata FR3, the Fenton reaction may play an important role in lignin degradation. This discovery is among the first to show that a mechanism for lignin degradation similar to those found in white and brown rot basidiomycetous fungi exists in an oleaginous fungus. This study suggests that oleaginous fungi such as C. echinulata FR3 can be employed for complete biomass utilization in a consolidated platform without chemical pretreatment or can be used to convert lignin waste into lipids.

The feasibility of biological pretreatment for subsequent saccharification largely depends upon an effective pretreatment system. A significant enhancement of saccharification was discovered with corn stover pretreated by white rot fungus Irpex lacteus CD2. The highest saccharification ratio reached 66.4%, which was significantly higher than what was reported. Hemicellulose was first destroyed in the process and then lignin. Lignin and hemicellulose were selectively degraded over cellulose, respectively, resulting in increased crystallinity. Enhanced saccharification and the fluctuation in crystallinity together indicated the destruction of the cellulose crystalline structure. Additionally, further studies revealed the disruption of the cell wall and the vital increase of large pores in the pretreated samples, which might be caused by the selective degradation of amorphous components and fungal penetration. Results suggest that I. lacteus has a more efficient degradation system than other reported white rot fungi and can be further explored as an alternative to the existing thermochemical processes.

The solubility of genistein in water, methanol, ethanol, propan-2-ol, 1-butanol, and ethyl acetate was measured at temperatures ranging from (280 to 333) K under atmospheric pressure using high-performance liquid chromatography (HPLC). Ethanol could be the most suitable solvent for purification and crystallization of geinstein. The experimental data were well-correlated with the Apelblat equation, which could be used as a useful model in the purification process of genistein.

Kummerowia striata (Thunb.) Schindl has long been used as a fork herb in inflammation-related therapy. This study was undertaken to determine the anti-inflammatory effect of the plant. High performance liquid chromatography (HPLC) was used for evaluating the extract. While dexamethasone (DM) was used as a positive control, the effects of ethanol extract on the production of IL-1β, IL-6, NO, COX-2 and TNF-α, the expression of iNOS mRNA, TNF-α mRNA, COX-2 mRNA, protein production of COX-2 and HO-1, NF-κB and I-κB of LPS-stimulated RAW 264.7 cells were studied by sandwich ELISA, real-time PCR, Western blot analysis and immunocytochemistry assay respectively. The results showed that K. striata (Thunb.) Schindl had a good anti-inflammatory effect on LPS-stimulated RAW264.7 cell. On one hand, it could significantly inhibit the production of IL-1β, IL-6, NO, TNF-α, COX-2 in LPS-stimulated cell than that of single LPS stimulated cell (p < 0.01 or p < 0.05). On the other hand, it could increase the production of IL-10 and HO-1 than that of single LPS intervention cell (p < 0.01 or p < 0.05). Furthermore, the extract also could inhibit the production of NF-κB and I-κB compared to single LPS stimulated cell. In a word, it suggested that the anti-inflammatory actions of K. striata (Thunb.) Schindl ethanol extract might be due to the down-regulation of IL-1β, IL-6, NO, TNF-α and COX-2 via the suppression of NF-κB activation and conversation of I-κB production, and another pathway was up regulating the production of IL-10 and HO-1.

•A novel MnP (manganese peroxidase) from white rot fungus E. taxodii 2538 was reported for the first time.•The MnP was stable at its optimum pH and had strong tolerance in acidic environment.•The MnP could oxidize both phenolic and nonphenolic lignin units.•The MnP combined with laccase from E. taxodii 2538 degraded lignin more efficiently.A novel manganese peroxidase (MnP) was isolated and characterized in this study. The MnP was produced by a new selective linguini-degrading white-rot fungus Echinodontium taxodii 2538 (E. taxodii 2538) on natural lignocellulose medium of moso bamboo. The purified MnP had an estimated molecular mass of 53.4 kDa, showing a single band on sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE), and composed of an amino acid sequence of GTTPSNGVVVP at N-terminal. The enzyme showed maximum activity when incubated at pH 3.5 or 55 °C and could maintain a high enzymatic activity after 24 h incubation under a broad range of pH (2.0–6.0) and temperature (below 45 °C). The kinetic parameters revealed that the MnP had the highest affinity toward MnSO4 (Km values was 0.35 μM) among all the substrates. Degradation of different types of lignin model compounds by MnP was investigated. It revealed that the MnP could oxidize both phenolic and nonphenolic lignin units. In addition, the MnP combined with laccase (Lac) from E. taxodii 2538 could degrade lignin more efficiently. In summary, this study provided a potential enzyme and a promising path for more efficient lignin modification.MnP from E. taxodii 2538 was able to degrade both nonphenolic and phenolic lignin model compounds, while Lac from the same fungus could degrade phenolic lignin model compounds only. Moreover in vitro, these two ligninolytic enzymes from E. taxodii 2538 degraded complicated lignin more efficiently when they acted in combination.Download high-res image (191KB)Download full-size image

Laccase belongs to a family of multi-copper oxidases which is especially useful for biotechnological and industrial applications. A laccase-producing white-rot fungi strain designated as Trametes sp. 5930 was nearly isolated from Shennongjia Nature Reserve in China. Trametes sp. 5930 had the high yield of laccase and was capable of decolorizing different dyes efficiently. Laccase played a very important role in the decolorization of different dyes by this fungus. The laccase gene lac5930-1 and its corresponding full-length cDNA were then cloned and characterized from Trametes sp. 5930. The 1563 bp full-length cDNA of lac5930-1 encoded a mature laccase protein consisting of 499 amino acids preceded by a signal peptide of 21 amino acids. lac5930-1 gene was successfully expressed in Pichia pastoris, which verified the function of lac5930-1 encoding active laccase by means of gene expression. The recombinant laccase produced by the yeast transformant in which lac5930-1 was efficiently expressed, conferred the ability to decolorize different dyes. The capability of decolorizing different dyes was positively related to the laccase activity, which provided strong evidence for the important function of laccase used in decolorizing industrial dyes.Highlights► Trametes sp. 5930 was isolated from the virgin forest of Shennongjia Nature Reserve in China. ► The laccase gene lac5930-1 and its corresponding full-length cDNA were cloned and characterized. ► lac5930-1 gene was successfully expressed in the heterologous Pichia pastoris system. ► The capability of decolorizing different dyes was positively related to the laccase activity.