Application of ESI FT-ICR MS to Study Kraft Lignin Modification by the Exoenzymes of the White Rot Basidiomycete Fungus TrametesHirsutaLE-BIN 072

Trameteshirsuta is a wood rotting fungus that possesses a vast array of lignin degrading enzymes, including7 laccases, 7 ligninolyticmanganese peroxidases, 9 lignin peroxidases and 2 versatile peroxidases. In this study,electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS)was used to examine kraft lignin modification by the enzymatic system of this fungus.The observed pattern of lignin modification suggested that before the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungus started to degrade less oxidized, more recalcitrant, compounds, converting them into the more oxidized forms. The altered pattern of lignin modification enabled changes in the fungal enzymatic system. These changes were further attributed to the appearance of the particular ligninolyticmanganese peroxides enzyme(MnP7), which was added by the fungus to the mixture of enzymes that had already been secreted (VP2 and MnP5). Keywords: wood rotting fungi, kraft lignin, mass spectrometry, peroxidases

Biodegradation Potential of SteccherinumOchraceum: Growth on Different Wood Types and Preliminary Evaluation of Enzymatic Activities

White-rot fungi isa source of a great variety of oxidative and hydrolytic enzymes suitable for biotechnological applications, e.g. in pulp and paper, textile and food industries, bioethanol production, degradation of recalcitrant environmental pollutants,and others. Steccherinumochraceum is a xylotrophicwhite-rot basidiomycetethat can be found in variousclimatic zones on different woody substrates (mostly well decayed). For this research, seventeenstrains of S. ochraceumwere collected in different regions of Russia from various wood substrates (aspen, alder, oak, hazel, birch and willow). Phylogeneticanalyseswere performedbasedon the nucleotide sequences of ITS1, ITS2, 5.8S rRNA, 28S rRNA, β-tubulin and tef1.Oxidaseandcellulaseactivitieswereassessedbyplate-tests with ABTS and CMC. Forevaluation of biodegradation potential,solid state fermentation on alder and pine sawdust wasperformed. Weightanddensitylossaswellas the C:Nratioweremeasuredafter 90 days of cultivation.All S. ochraceum strains exhibited high oxidative activity towards ABTS, indicating secretion of oxidative enzymes (i.e. laccases and class II peroxidases). Cellulase activity was medium or low for most strains and in some strains – absent. Allstrainswereabletodegradealderandpinesawdust. There was no correlation between the enzymatic activity, biodegradation potential and geographic origin of S. ochraceum strains. However, S. ochraceum strains isolated from the same wood substrates exhibited similar characteristics in most cases. Strain LE-BIN 3398 was the most effective for degrading both alder and pine sawdust and could be regarded as a promising source of oxidative enzymes for biotechnology. Keywords: basidiomycetes, biodegradation, solid state fermentation, oxidase activity, Steccherinumochraceum

Transcriptomic Analysis of the White-rot Basidiomycete Lentinus Squarrosulus to Provide Insights Into Its Lignocellulose Biodegradation Ability

BackgroundBasidiomycetes are of special interest in biotechnological research for their versatile potential in the degradation of lignocellulosic biomass, chiefly attributed to ligninolytic enzymes along with exo, endo β-glucanases, xylanases, esterases, pectinases, mannanases, cellobiohydrolases, polysaccharide monooxygenases. Relatively little is known about the metabolic process and the subsequent polysaccharide degradation. Transcriptomic analysis of lignicolous fungi grown on different substrates, although attempted by researchers, has focused on a fairly small group of species reporting the expression of fungal genes in response to lignocellulosic biomass as a substrate. This study accordingly reports analysis of transcriptome of a white-rot Basidiomycete L.squarrosulus grown in simple potato dextrose broth supplemented with aromatic compound, reactive black dye to gain an insight into the degradation ability of the fungus. RNA was sequenced using Illumina NextSeq 500 to obtain 6,679,162 high-quality paired-end reads that were assembled de novo using CLC assembly cells to generate 25,244 contigs. Putative functions were assigned for the 10,494 transcripts based on sequence similarities through BLAST2GO 5.2 and Function annotator.ResultsFunctional assignments revealed enhanced oxidoreductase activity through the expression of diverse biomass-degrading enzymes and their corresponding coregulators. CAZyme analysis through dbCAN and CUPP revealed the presence of 6 families of polysaccharide lyases, 51 families of glycoside hydrolases, 23 families of glycoside transferases, 7 families of carbohydrate esterases and 10 families of auxiliary activities. Genes encoding ligninolytic enzymes and auxiliary activities among the transcript sequences were identified through gene prediction by AUGUSTUS and FGENESH. Biochemical analysis of several biomass-degrading enzymes substantiated the functional predictions.ConclusionIn essence, L. squarrosulus grown in a simple medium devoid of lignocellulosic substrate demonstrated the presence of a repertoire of lignocellulose-degrading enzymes, simplying that a source of lignocellulose is not required for the expression of these biomass-degrading enzymes. This study on the transcriptome analysis of L. squarrosulus revealed significant facts on this front and will definitely enhance the knowledge about the biodegradative ability of this fungus, potentially paving the way for efficient biotechnological applications utilizing its potency in biomass degradation and its future functional exploitation in biomass conversion applications.

Immune-Enhancing Effect of Submerged Culture of Ceriporia lacerata Mycelia on Cyclophosphamide-Induced Immunosuppressed Mice and the Underlying Mechanisms in Macrophages

The white-rot fungi Ceriporia lacerata is used in bioremediation, such as lignocellulose degradation, in nature. Submerged cultures and extracts of C. lacerata mycelia (CLM) have been reported to contain various active ingredients, including β-glucan and extracellular polysaccharides, and to exert anti-diabetogenic properties in mice and cell lines. However, the immunostimulatory effects have not yet been reported. This study aimed to identify the immunomodulatory effects, and underlying mechanisms thereof, of submerged cultures of CLM using RAW264.7 macrophages and cyclophosphamide (CTX)-induced immunosuppression in mice. Compared to CTX-induced immunosuppressed mice, the spleen and thymus indexes in mice orally administered CLM were significantly increased; body weight loss was alleviated; and natural killer (NK) cytotoxicity, lymphocyte proliferation, and cytokine (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, and interleukin [IL]-2) production were elevated in the serum. In RAW264.7 macrophages, treatment with CLM induced phagocytic activity, increased the production of nitric oxide (NO), and promoted mRNA expression of the immunomodulatory cytokines TNF-α, IFN-γ, IL-1β, IL-6, IL-10, and IL-12. In addition, CLM increased the inducible NO synthase (iNOS) concentration in macrophages, similar to lipopolysaccharide (LPS) stimulation. Mechanistic studies showed that CLM induced the activation of the NF-κB, PI3k/Akt, ERK1/2, and JNK1/2 pathways. Moreover, the phosphorylation of NF-κB and IκB induced by CLM in RAW264.7 cells was suppressed by specific MAPKs and PI3K inhibitors. Further experiments with a TLR4 inhibitor demonstrated that the production of TNF-α, IL-1β, and IL-6 induced by CLM was decreased after TLR4 was blocked. Overall, CLM protected against CTX-induced adverse reactions by enhancing humoral and cellular immune functions, and has potential as an immunomodulatory agent.

Enzymatic Activities, Characteristics of Wood-Decay and Wood Substrate Specificity within Genera of Some Wood-Rotting Basidiomycetes from Cameroon and Tropical Africa

A total of 72 species of wood-rotting Basidiomycetes belonging to 40 genera, 13 families and 5 orders, were investigated in this study of which 46 for the first time as far as type of wood-rot is concerned. 61 of the 72 cause white rot (W) representing 84.72 % of the total and 11 cause brown rot (B) or just 15.27 %, confirming the predominance already known of white rot (W) on brown rot (B) as shown in other studies. Results recorded show that even though most species belonging to same genera display the same type of rot (W or B), species of some few other genera were found to rather display different types of rot (W and B) in species within the same genera. Other results show that when determining the type of wood-rot caused by some species of fungi, in case of negative (-) test using tincture of guaiac which is known as the key test to determine the type of rot, syringaldazine must also be used as a differential test before concluding whether the species is a white (W) or brown rot (B) fungus. The level of activity of tyrosinase and peroxidase shows important variations between species of some genera, whereby some species of a given genus show varying potential level (+, ++, +++, ++++) of activities of these two enzymes, whereas other species of the same genus show no sign (-) of activity of the same enzymes. Therefore, our results led to the conclusion that the presence (+, ++, +++, ++++) or absence (-) of activity of peroxidase and tyrosinase can from now onwards also be used as an enzyme linked taxonomic criteria to distinguish between species of some genera. About detection of laccases activity, if a negative (-) result occurs during detection test in a wood-rotting fungus using syringaldazine, α-naphtol must also be used as a differential test before concluding on whether the species produces laccases or not. Based on the overall results recorded in the detection of enzymes activities, it appears necessary to use, where indicated, several substrates with different chemical sensitivities to detect the existence of an enzyme and its potential activity level in a fungal species. Additionally, preliminary lists of wood-decay fungi with potentially strong (+++, ++++) capacity to produce different types of polyphenol oxidases potentially usable in paper making industries, wastewater treatment and soil remediation, are provided. With regard to the study of substrate specificity which aimed at finding out the existence or not of a specificity between polyporales and tree wood species on which they grow, the first results recorded on a limited number of trees investigated led to the preliminary conclusion that, although some few tree species may serve as hosts for only a single species of polyporales, there is rather a greater tendency of finding several species growing on different species of wood as well as the same species of wood hosting several species of polyporales. These field observations led to the remark that a much larger inventory including a greater number of tree species in various tropical ecosystems is necessary in order to come out with a final conclusion.

Biodegradation of low-density polyethylene plastics by fungi isolated from waste disposal site at district Peshawar, Pakistan

Plastics are resistant to microbial attack, which has become a major cause of environmental pollution. The current study aimed to identify the fungi, capable of the biodegradation of low-density polyethylene plastics from different waste disposal sites at Peshawar, Pakistan. A total of 15 soil samples were collected from different waste disposable sites of Peshawar. From these samples, five fungal isolates Aspergillus Niger, Aspergillus flavus, White rot, and Brown rot fungi were identified based on their colony morphology and microscopic examination. The biodegradation ability of these isolates against low-density polyethylene plastics was studied through the weight loss percentage method on 30 days of incubation. The weight loss percentage showed that Aspegillus Niger, Aspergillus flavus, Brown rot, and white rot fungi showed 22.9%, 16.1%, 18.4%, and 22.7% biodegradation, respectively. This study concluded that Aspergillus Niger, Aspergillus flavus, White rot, and Brown rot fungi are capable to degrade polyethylene plastics.Low density polyethylene, Plastics, Fungi, Biodegradation

Mushroom Quality Related with Various Substrates’ Bioaccumulation and Translocation of Heavy Metals

Mushrooms are popular due to the nutrition contents in the fruit bodies and are relatively easy to cultivate. Mushrooms from the white-rot fungi group can be cultivated on agricultural biomass such as sawdust, paddy straw, wheat straw, oil palm frond, oil palm empty fruit bunches, oil palm bark, corn silage, corn cobs, banana leaves, coconut husk, pineapple peel, pineapple leaves, cotton stalk, sugarcane bagasse and various other agricultural biomass. Mushrooms are exceptional decomposers that play important roles in the food web to balance the ecosystems. They can uptake various minerals, including essential and non-essential minerals provided by the substrates. However, the agricultural biomass used for mushroom cultivation is sometimes polluted by heavy metals because of the increased anthropogenic activities occurring in line with urbanisation. Due to their role in mycoremediation, the mushrooms also absorb pollutants from the substrates into their fruit bodies. This article reviews the sources of agricultural biomass for mushroom cultivation that could track how the environmental heavy metals are accumulated and translocated into mushroom fruit bodies. This review also discusses the possible health risks from prolonged uptakes of heavy metal-contaminated mushrooms to highlight the importance of early contaminants’ detection for food security.

Biodegradation Of Iron Industry And Gold Industrial Wastewater By White Rot Fungi – Calocybe Indica & Agaricus Bisporus Comparative Study

Increasing discharge and improper management of liquid and solid industrial wastes have created a great concern among industrialists and the scientific community over their economic treatment and safe disposal. Hence, there is a growing need for the development of novel, efficient, eco-friendly, and cost-effective approach for the remediation for these industries released into the environment and to safeguard the ecosystem. In this regard, recent advances in wastewater of heavy metal have propelled bioremediation as a prospective alternative to conventional techniques. Heavy metals are toxic and dangerous to the ecosystem. White rot fungi (WRF) are versatile and robust organisms having enormous potential for oxidative bioremediation of a variety of toxic chemical pollutants due to high tolerance to toxic substances in the environment. The decolorization and detoxification potential of WRF can be harnessed thanks to emerging knowledge of the physiology of these organisms as well as of the bio catalysis and stability characteristics of their enzymes. This knowledge will need to be transformed into reliable and robust waste treatment processes.

Dimensional stability and decay resistance of clay treated, furfurylated, and clay-reinforced furfurylated poplar wood

Plantation-grown poplar (Populus cathayana) is regarded as a source of low-quality wood, with poor dimensional stability and low decay resistance. In this study, poplar wood was impregnated with sodium montmorillonite (Na-MMT) or organo-montmorillonite (O-MMT), furfuryl alcohol (FA, at concentrations of 15%, 30% and 50%), separately or in their combinations to prepare clay treated, furfurylated, and clay-reinforced furfurylated wood, respectively. The two-step method by introducing Na-MMT first and then FA and organic modifier was feasible to achieve a reasonable penetration. These components could entirely enter the wood cell lumen or partly enter the wood cell wall, and thus initiate a series of reactions. Compared with Na-MMT reinforced furfurylated wood (M-F), the O-MMT reinforced furfurylated wood (O-F) exhibited better dimensional stability (ASE up to 71%) and decay resistance (3.2% mass loss). Moreover, O-MMT played a predominant role in decay resistance of O-MMT reinforced furfurylated wood. Even at low O-MMT loadings, the modified wood had a significant inhibitory effect on the white-rot decay fungus Trametes versicolor. Based on an overall evaluation, O-MMT reinforced furfurylated wood seemed to provide an optimal choice for both moist or wet conditions.