Lignocellulolytic extremozymes and their biotechnological applications.

Lignocellulolytic enzymes are used in different industrial and environmental processes. The rigorous operating circumstances of these industries, however, might prevent these enzymes from performing as intended. On the other side, extremozymes are enzymes produced by extremophiles that can function in extremely acidic or basic; hot or cold; under high or low salinity conditions. These severe conditions might denature the normal enzymes that are produced by mesophilic microorganisms. The increased stability of these enzymes has been contributed to a number of conformational modifications in their structures. These modifications may result from a few amino acid substitutions, an improved hydrophobic core, the existence of extra ion pairs and salt bridges, an increase in compactness, or an increase in positively charged amino acids. These enzymes are the best option for industrial and bioremediation activities that must be carried out under difficult conditions due to their improved stability. The review, therefore, discusses lignocellulolytic extremozymes, their structure and mechanisms along with industrial and biotechnological applications.

A thermostable bacterial catalase-peroxidase oxidizes phenolic compounds derived from lignins.

Lignocellulosic biomass is rich in lignins, which represent a bottomless natural source of aromatic compounds. Due to the high chemical complexity of these aromatic polymers, their biological fractionation remains challenging for biorefinery. The production of aromatics from the biological valorization of lignins requires the action of ligninolytic peroxidases and laccases produced by fungi and bacteria. Therefore, identification of efficient ligninolytic enzymes with high stability represents a promising route for lignins biorefining. Our strategy consists in exploiting the enzymatic potential of the thermophilic bacterium Thermobacillus xylanilyticus to produce robust and thermostable ligninolytic enzymes. In this context, a gene encoding a putative catalase-peroxidase was identified from the bacterial genome. The present work describes the production of the recombinant protein, its biochemical characterization, and ligninolytic potential. Our results show that the catalase-peroxidase from T. xylanilyticus is thermostable and exhibits catalase-peroxidase and manganese peroxidase activities. The electrochemical characterization using intermittent pulse amperometry showed the ability of the enzyme to oxidize small aromatic compounds derived from lignins. This promising methodology allows the fast screening of the catalase-peroxidase activity towards small phenolic molecules, suggesting its potential role in lignin transformation. KEY POINTS: • Production and characterization of a new thermostable bacterial catalase-peroxidase • The enzyme is able to oxidize many phenolic monomers derived from lignins • Intermittent pulse amperometry is promising to screen ligninolytic enzyme.

Exoproteomic Study and Transcriptional Responses of Laccase and Ligninolytic Peroxidase Genes of White-Rot Fungus Trametes hirsuta LE-BIN 072 Grown in the Presence of Monolignol-Related Phenolic Compounds.

Being an abundant renewable source of aromatic compounds, lignin is an important component of future bio-based economy. Currently, biotechnological processing of lignin through low molecular weight compounds is one of the conceptually promising ways for its valorization. To obtain lignin fragments suitable for further inclusion into microbial metabolism, it is proposed to use a ligninolytic system of white-rot fungi, which mainly comprises laccases and peroxidases. However, laccase and peroxidase genes are almost always represented by many non-allelic copies that form multigene families within the genome of white-rot fungi, and the contributions of exact family members to the overall process of lignin degradation has not yet been determined. In this article, the response of the Trametes hirsuta LE-BIN 072 ligninolytic system to the presence of various monolignol-related phenolic compounds (veratryl alcohol, p-coumaric acid, vanillic acid, and syringic acid) in culture media was monitored at the level of gene transcription and protein secretion. By showing which isozymes contribute to the overall functioning of the ligninolytic system of the T. hirsuta LE-BIN 072, the data obtained in this study will greatly contribute to the possible application of this fungus and its ligninolytic enzymes in lignin depolymerization processes.

Production of high-value oxidative enzymes by Cyathus bulleri on agricultural and agri-food wastes for application in the textile sector.

Ligninolytic and other oxidative enzymes have emerged as promising biocatalysts in several industries. Since their production at a low cost is necessary for any large-scale application, we demonstrate the use of rice bran (RB), an agricultural waste and agri-food wastes such as potato peelings (PP), banana peelings (BP), and green pea peelings (GPP) for their production. High activity of laccase (12 U/ml), manganese peroxidase (16.11 ± 1.43 U/ml), and aryl alcohol oxidase (1.25 U/ml) was obtained on the PP on the 12th day of growth and ~ 6 U/ml of lytic polysaccharide monooxygenase was obtained on the 14th day of growth demonstrating PP to be a good substrate for their production. RB served as the next best substrate for the production of these enzymes. While the GPP was effective for the production of laccase (9.2 U/ml), this and the BP were not good substrates for the production of other enzymes. Efficient (48-82%) decolorization of several azo-, triarylmethane- dyes, and real textile effluent, without the addition of any mediator, demonstrated the high oxidative ability of the crude culture filtrate produced on the PP (CF-PP), which was a significant improvement compared to the treatment given by the previously reported culture filtrate obtained on wheat bran (CF-WB). An extensive breakdown of Reactive Orange (RO) 16 was demonstrated using CF-PP resulting in the formation of a new product at m/z of 294.05 (6-acetamido-3,4-dioxo-3,4-dihydronapthalene-2-sulfonate), previously reported to be produced on ozonation/advanced oxidation of RO16. The predominant laccase and manganese peroxidase isoforms produced on the PP were also identified.

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity.

As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates-namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose-methanol-choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases-we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways and mapped the appearance of the different enzyme types in a time-calibrated species tree.

Degradation and decolourization potential of ligninolytic enzyme producing Bacillus paramycoides BL2 and Micrococcus luteus BL3 for pulp paper industrial effluent and its toxicity evaluation.

Aim of this study was to optimize the production of Ligninolytic enzyme for the degradation of complex pollutants present in pulp paper industrial effluent (PPIE). Two ligninolytic enzyme-producing bacterial strains were isolated from PPIE and identified as Bacillus paramycoides strain BL2 (MZ676667) and Micrococcus luteus strains BL3 (MZ676668). The identified bacterial strain Bacillus paramycoides strain BL2 showed optimum production of LiP (4.30 U/ml), MnP (3.38 U/ml) at 72 h of incubation, while laccase (4.43 U/ml) at 96 h of incubation. While, Micrococcus luteus strains BL3 produced maximum LiP (3.98) and MnP (3.85 U/ml) at 96 h of incubation and maximum laccase (3.85 U/ml) at 72 h of incubation, pH 7-8, and temperatures of 30-35 °C. Furthermore, in the presence of glucose (1.0%) and peptone (0.5%) as nutrient sources, the enzyme activity of consortium leads to reduction of lignin (70%), colour (63%) along with COD (71%) and BOD (58%). The pollutants detected in control i.e. 3.6-Dioxa-2,7-disilaoctane, 2-Heptnoic acid,trimethylsilyl ester, 7-Methyldinaphtho [2,1-b,1',2'-d] silole, Hexadeconoic acid, trimethylysilyl ester, Methyl1(Z)-3,3-dipheny.1-4-hexenoale, 2,6,10,14,18,22-Tetracosahexane,2,2-dimethylpropyl(2Z,6E)-10,11epoxy5,6 Dihyrostigmasterol, acetate were completely diminished. The toxicity of PPIE was reduced up to 75%. Hence, knowledge of this study will be very useful for industrial sector for treatment of complex wastewater.

Plant growth promoting strain Bacillus cereus (RCS-4 MZ520573.1) enhances phytoremediation potential of Cynodon dactylon L. in distillery sludge.

Elevated levels of physico-chemical pollution including organic pollutants, metals and metalloids were detected in distillery sludges despite of the anaerobic digestion treatment prior to disposal. The concentrations of the metals were (in mg kg-1): Fe (400.98 ± 3.11), Zn (17.21 ± 0.54), Mn (8.32 ± 0.42), Ni (8.00 ± 0.98), Pb (5.09 ± 0.43), Cr (4.00 ± 0.98), and Cu (3.00 ± 0.10). An invasive grass species, Cynodon dactylon L., demonstrated its ability to remediate the distillery waste sludge (DWS) in the field study. All the physico-chemical parameters of the sludge significantly improved (up to 70-75%) in the presence of Cynodon dactylon L. (p < 0.001) than the control with no plant growth. The highest phytoremediation capacity was associated with the uptake of Fe in the root and shoot. Sludge samples collected near the rhizosphere also showed lower amount of organic compounds compared to control sludge samples. Metal resistant Bacillus cereus (RCS-4 MZ520573.1) was isolated from the rhizosphere of Cynodon dactylon L. and showed potential to enhance the process of phytoremediation via plant growth promoting activities such as production of high level of ligninolytic enzymes: manganese peroxidase (35.98 U), lignin peroxidase (23.98 U) and laccase (12.78 U), indole acetic acid (45.87(mgL-1), phosphatase activity (25.76 mg L-1) and siderophore production (23.09 mg L-1). This study presents information on the performance of Cynodon dactylon L., an abundant invasive perennial grass species and its associated plant growth promoting rhizobacteria demonstrated good capacity to remediate and restore contaminated soil contained complex organic and inorganic pollutants, they could be integrated into the disposal system of distillery sludge to improve the treatment efficiency.

Application of Deconica castanella ligninolytic enzymatic system in the degradation of hexachlorobenzene in soil.

Hexachlorobenzene (HCB) is a pollutant still found in the environment despite being widely banned. Considering that basidiomycetes are useful to degrade a variety of organochlorinated pollutants, we therefore report the influence of HCB on the ligninolytic enzymatic system of Deconica castanella. The inoculum was prepared with sugarcane bagasse and soybean flour and was added in soil with and without HCB (2000 mg kg soil-1 ), 5% emulsion containing soybean oil and Tween 20 at proportion 9:1, v:v; with 70% moisture at 25°C. Fungal biomass was quantified by widely acknowledged growth biomarker ergosterol. The extraction of the enzymatic complex was performed and laccase, Mn-dependent peroxidase (MnP), and lignin peroxidase (LiP) activities were determined. Furthermore, HCB and its metabolites were quantified by gas chromatography and chlorides by potentiometric titration. Results evidenced that HCB did not interfere in fungal growth, though the only detected enzymatic activity was laccase. MnP and Lip were not detected during D. castanella growth in soil. The peak of laccase enzymatic activity occurred in the presence of HCB. In addition, the laccase exhibited thermostability. Therefore, we hereby shed light on the role of laccase in the degradation of HCB by an efficient low-cost and environmentally safe detoxification mechanism.

Transcriptome Analysis of Podoscypha petalodes Strain GGF6 Reveals the Diversity of Proteins Involved in Lignocellulose Degradation and Ligninolytic Function.

The present study reports transcriptomic profiling of a Basidiomycota fungus, Podoscypha petalodes strain GGF6 belonging to the family Podoscyphaceae, isolated from the North-Western Himalayan ranges in Himachal Pradesh, India. Podoscypha petalodes strain GGF6 possesses significant biotechnological potential as it has been reported for endocellulase, laccase, and other lignocellulolytic enzymes under submerged fermentation conditions. The present study attempts to enhance our knowledge of its lignocellulolytic potential as no previous omics-based analysis is available for this white-rot fungus. The transcriptomic analysis of P. petalodes GGF6 reveals the presence of 280 CAZy proteins. Furthermore, bioprospecting transcriptome signatures in the fungi revealed a diverse array of proteins associated with cellulose, hemicellulose, pectin, and lignin degradation. Interestingly, two copper-dependent lytic polysaccharide monooxygenases (AA14) and one pyrroloquinolinequinone-dependent oxidoreductase (AA12) were also identified, which are known to help in the lignocellulosic plant biomass degradation. Overall, this transcriptome profiling-based study provides deeper molecular-level insights into this Basidiomycota fungi, P. petalodes, for its potential application in diverse biotechnological applications, not only in the biofuel industry but also in the environmental biodegradation of recalcitrant molecules. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01037-6.

Genome-based engineering of ligninolytic enzymes in fungi.

BACKGROUND: Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes. SHORT CONCLUSION: Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.

Biodegradation of lignin monomers and bioconversion of ferulic acid to vanillic acid by Paraburkholderia aromaticivorans AR20-38 isolated from Alpine forest soil.

Lignin bio-valorization is an emerging field of applied biotechnology and has not yet been studied at low temperatures. Paraburkholderia aromaticivorans AR20-38 was examined for its potential to degrade six selected lignin monomers (syringic acid, p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid, vanillic acid, benzoic acid) from different upper funneling aromatic pathways. The strain degraded four of these compounds at 10°C, 20°C, and 30°C; syringic acid and vanillic acid were not utilized as sole carbon source. The degradation of 5 mM and 10 mM ferulic acid was accompanied by the stable accumulation of high amounts of the value-added product vanillic acid (85-89% molar yield; 760 and 1540 mg l-1, respectively) over the whole temperature range tested. The presence of essential genes required for reactions in the upper funneling pathways was confirmed in the genome. This is the first report on biodegradation of lignin monomers and the stable vanillic acid production at low and moderate temperatures by P. aromaticivorans. KEY POINTS: • Paraburkholderia aromaticivorans AR20-38 successfully degrades four lignin monomers. • Successful degradation study at low (10°C) and moderate temperatures (20-30°C). • Biotechnological value: high yield of vanillic acid produced from ferulic acid.

Lignin valorization using biological approach.

Due to the structural complexity and recalcitrance nature of lignin, its depolymerization into monomeric units becomes one of the biggest challenges in the bioconversion of lignin into value-added products. Depolymerization of lignin produces a blend of many compounds that are problematic for isolating components in a cost-effective way. Lignin valorization using a biological approach facilitates sustainable and commercially viable biorefineries. The use of microbes for the conversion of depolymerized lignin compounds into target products can be a solution to the heterogeneity issue. Several studies have been carried out to develop robust strains that can utilize all relevant lignin-derived compounds, but constructing these strains is difficult. As an alternative, designing multiple microbes to convert a mixture of various compounds into the desired product seems realistic. This review provides an overview of lignin bioconversion using various approaches such as metabolic engineering and synthetic biology. Ligninolytic strains have a broad enzymatic machine for depolymerization of lignin and its conversion into intermediates such as catechol or protocatechuate. These intermediates can be further converted to metabolite products such as polyhydroxyalkanoates and triacylglycerol. Synthetic biology offers encouraging methodologies to construct pathways for lignin conversion and to engineer ligninolytic microbes as prospective strains for lignin bioconversion.

Dual effects of entomopathogenic fungi on control of the pest Lobesia botrana and the pathogenic fungus Eutypella microtheca on grapevine.

BACKGROUND: Entomopathogenic fungi (EPF) are the natural enemies of insect pests. Nevertheless, research on the use of EPF for simultaneous prevention of pest and disease agents on the same crop is limited. In this study, we explored the potential dual effects of three strains of the EPF Metarhizium anisopliae on the control of detrimental agents of Vitis vinifera L., including different developmental stages (larvae, pupae, and adult) of the insect pest Lobesia botrana and the phytopathogenic fungus Eutypella microtheca. METHODS: Laboratory pathogenicity trials were performed to examine the effects of the three M. anisopliae strains on the mortality rate of L. botrana. In addition, field trials were conducted to assess the biocontrol potential of one selected M. anisopliae strain on the larval stage of L. botrana. Moreover, inhibitory effects of the three EPF strains on E. microtheca growth were examined in vitro. RESULTS: All the M. anisopliae strains were highly effective, killing all stages of L. botrana as well as inhibiting the growth of E. microtheca. The in vitro mortality of larvae treated with the strains was over 75%, whereas that of treated pupae and adults was over 85%. The three EPF strains showed similar efficacy against larvae and adult stages; nevertheless, pupal mortality was observed to be strain dependent. Mortality of L. botrana larvae ranged from 64 to 91% at field conditions. Inhibition of E. microtheca growth reached 50% in comparison to the control. CONCLUSIONS: Our study showed that M. anisopliae strains were highly effective in ensuring control of two different detrimental agents of V. vinifera L., providing new evidence to support the dual effects of entomopathogenic fungi.

Assessment of polyethylene degradation by biosurfactant producing ligninolytic bacterium.

Accumulation of plastic waste has become an environmental threat and a global problem. In this research, polyethylene degrading ligninolytic bacteria were isolated from plastic waste polluted soil. Two bacterial isolates, namely PE2 and PE3 have been obtained from the soil samples. Polyethylene degrading ability of the isolates has been assessed individually in a synthetic media containing polyethylene as a carbon source. The results indicated that maximum weight reduction of polyethylene (6.68%) was found in PE3 inoculated media after thirty days of incubation. Fourier Transform Infrared Spectroscopic results showed the appearance of carbonyl peaks. 16S rRNA gene sequencing studies revealed that the potential isolate PE3 belongs to the genus Bacillus and it was named Bacillus sp. strain PE3. From the scanning electron microscopic results, it is inferred that Bacillus sp. strain PE3 could colonize on the polyethylene surface and form a biofilm. Besides, the viable Bacillus sp. strain PE3 on polyethylene surface was confirmed by fluorescence microscopic analysis. Alkanes and fatty acids were identified in the degraded products by gas chromatography-mass spectrometer analysis. From the results of native polyacrylamide gel electrophoresis, the activities of laccase and lignin peroxidase were noticed. Furthermore, extracellular production of biosurfactant has been observed in the Bacillus sp. strain PE3 inoculated mineral salt media and synthetic media with glucose and polyethylene as the carbon source respectively. The characterization studies of crude biosurfactant have confirmed that lipopeptide nature biosurfactant. The present study demonstrates that the ligninolytic enzymes laccase, lignin peroxidase, and lipopeptide type biosurfactant are produced by Bacillus sp. strain PE3 in the media with polyethylene as a carbon source.

Role of microalgal ligninolytic enzymes in industrial dye decolorization.

In this study, the decolorization efficiency of seven microalgae isolates; Nostoc muscorum, Nostoc humifusum, Spirulina platensis, Anabaena oryzae, Wollea saccata, Oscillatoria sp. and Chlorella vulgaris was investigated for dye decolorization. The highest decolorization percentages of Brazilwood, Orange G, and Naphthol Green B dyes (99.5%, 99.5%, and 98.5%, respectively) were achieved by Chlorella vulgaris. However, the maximum efficiency for dye decolorization percentages of CV and malachite green dyes were exhibited by A. oryzae (97.4%) and W. saccata (93.3%). Ligninolytic enzymes activity assay was carried out for laccase and lignin peroxidase enzymes, which revealed a high efficiency of the C. vulgaris, A. oryzae and W. saccata to lignin containing compound degradation. The highest laccase production recorded by C. vulgaris with Brazilwood, Orange G, and Naphthol Green B dyes (665.0, 678.6, and 659.5 U/ml, respectively). Similarly, C. vulgaris gave a high lignin peroxidase enzyme production with the above three dyes respectively (306.00, 298.34, and 311.45 U/ml). In addition, A. oryzae and W. saccata showed the highest production of the laccase enzyme (634.6 and 577.45 U/ml, respectively) with CV and malachite green dyes. The degradation products have been characterized after decolorization and verified using FTIR analysis. The high decolorization percentages achieved by C. vulgaris, A. oryzae and W. saccata make them potential candidates for bioremediation and pre-processing to remove dyes from textile effluents.

Evaluation of a Trametes pubescens laccase concentrated extract on allura red AC decolorization without the addition of synthetic mediators.

Synthetic dye bioremediation is a topic of great importance since these pollutants possess toxic effects, and huge quantities of them are being discharged into water bodies. Ligninolytic enzyme treatment stands out for being a cost-effective methodology, capable of obtaining high decolorization levels. In this work, a laccase enzyme treatment was evaluated to effectively perform a cycle of dye bioremediation. Furthermore, a dye decolorization improvement was also assessed through laccase immobilization. Particularly, a Trametes pubescens enzyme extract was concentrated, immobilized onto calcium alginate beads, and characterized to assess its dye decolorization potential. Ammonium sulfate precipitation and vacuum evaporation were evaluated to concentrate the crude extract and to decolorize allura red AC. Both treatments reached a high enzyme yield recovery (>90%), but only the vacuum-evaporated extract achieved a high allura red AC decolorization level after 16 h of contact time. This suggested that essential compounds for allura red AC decolorization were present in the crude extract, implying that neither a complete laccase purification process nor an addition of synthetic mediators are necessary. Under optimized immobilization conditions, 94.6% immobilization efficiency and 49.8% activity recovery were obtained with 0:1 alginate:enzyme (v/v), 100 mM CaCl2, and 5.0% w/v sodium alginate. Furthermore, by immobilizing the laccase concentrated extract, both the pH and temperature stabilities were improved. The decolorization of allura red AC by free and immobilized laccase was 68.4% and 4.6%, respectively, showing that although the enzyme stability was improved, dye decolorization was negatively affected. Thus, an efficient allura red AC decolorization was obtained with concentrated-free laccase by a feasible and low-cost methodology.

A proposed stepwise screening framework for the selection of polycyclic aromatic hydrocarbon (PAH)-degrading white rot fungi.

This study suggests a simple three-step screening protocol for the selection of white rot fungi (WRF) capable of degrading polycyclic aromatic hydrocarbons (PAHs), which combines easily applicable bioassay techniques, and verifies that protocol by evaluating the PAH degradation activity, ligninolytic enzyme secretion, and relevant gene expressions of the selected PAH-degraders. Using 120 fungal strains, a sequence of bioassay techniques was applied: Bavendamm's reaction (Step 1), remazol brilliant blue R (RBBR) decolorization (Step 2); assays for tolerance to four mixed PAHs-phenanthrene, anthracene, fluoranthene, and pyrene (Step 3). This stepwise protocol selected 14 PAH-degrading WRF, including Microporus vernicipes, Peniophora incarnata, Perenniporia subacida, Phanerochaete sordida, Phlebia acerina, and Phlebia radiata. Of these, P. incarnata exhibited the highest PAH degradative activity, ranging from 40 to > 90%, which was related to the time-variable secretions of three extracellular ligninolytic enzymes: laccase, manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP). Laccase and MnP production by P. incarnata tended to be greater in the early stages of PAH degradation, whereas its LiP production became intensified with decreasing laccase and MnP production. Pilc1 and pimp1 genes encoding laccase and MnP were expressed, indicating the occurrence of extracellular enzyme-driven biodegradation of PAH by the fungal strains.

Effect of Ferulic Acid, a Phenolic Inducer of Fungal Laccase, on 26S Proteasome Activities In Vitro.

The 26S proteasome is an ATP-dependent protease complex (2.5 MDa) that degrades most cellular proteins in Eukaryotes, typically those modified by a polyubiquitin chain. The proteasome-mediated proteolysis regulates a variety of critical cellular processes such as transcriptional control, cell cycle, oncogenesis, apoptosis, protein quality control, and stress response. Previous studies conducted in our laboratory have shown that 26S proteasomes are involved in the regulation of ligninolytic enzymes (such as laccase) in white-rot fungi in response to nutrient starvation, cadmium exposure, and ER stress. Laccases are useful biocatalysts for a wide range of biotechnological applications. The goal of the current study was to determine the effect of ferulic acid (4-hydroxy-3-methoxycinnamic acid), a phenolic compound known to induce some ligninolytic enzymes, on proteasomes isolated from mycelia of the wood-decomposing basidiomycete Trametes versicolor. The peptidase activities of 26S proteasomes were assayed by measuring the hydrolysis of fluorogenic peptide substrates specific for each active site: Suc-LLVY-AMC, Z-GGR-AMC and Z-LLE-AMC for chymotrypsin-like, trypsin-like, and caspase-like site, respectively. Ferulic acid affected all peptidase activities of the 26S fungal proteasomes in a concentration-dependent manner. A possible inhibitory effect of ferulic acid on peptidase activities of the 26S human proteasomes was tested as well. Moreover, the ability of ferulic acid to inhibit (at concentrations known to induce laccase activity in white-rot fungi) the rate of 26S proteasome-catalyzed degradation of a model full-length protein substrate (ß-casein) was demonstrated by a fluorescamine assay and by a gel-electrophoretic analysis. Our findings provide new insights into the role of ferulic acid in lignin-degrading fungi. However, the detailed molecular mechanisms involved remain to be elucidated by future studies.

Modification of post-industrial lignin by fungal strains of the genus Trichoderma isolated from different composting stages.

One of the links in the environmental management chain is the environmentally friendly utilization of the emerging post-industrial waste and improvement of the methods of processing thereof. The aim and novelty of this research was to evaluate the potential of fungi to purify wastewater containing post-industrial lignin, i.e. waste originating from the pulp and paper industry. Trichoderma were dominant in the composts with different qualities and quantities of lignocellulosic compounds. The Trichoderma strains used in the research were isolated from two lignocellulosic composts at three different time points (from 10-, 20- and 30-week-old composting mass). Eighteen strains of the genus Trichoderma were tested for their ability to biodegrade 0.2% post-industrial lignin. It was evaluated by determination of decolorization, activities of ligninolytic enzymes, and concentration of phenolic compounds in the post-culture liquid. The Trichoderma strains isolated from 10-week-old compost I and 30-week-old compost II showed the highest decolorization activity and biotransformation of dark post-industrial lignin. All strains secreted horseradish-like peroxidase (HRP-like), superoxide dismutase-like (SOD-like), xylanase, and phenolic compounds. Strains isolated from 30-week-old compost I and from 10-week-old compost II released the greatest amounts of phenolic compounds into the culture liquid containing post-industrial lignin. The strains isolated from 10- and 20-week-old compost were characterized by high SOD-like and HRP-like activity, respectively. The concentration of phenolic compounds measured with HPLC in Trichoderma fungus culture VII from compost I corresponded with the decolorization degree and high HRP-like activity. The study results indicate that the genus Trichoderma with decolorization activity isolated from the first composting stages can be used in the biotransformation of post-industrial lignin waste.

Evaluation of several white-rot fungi for the decolorization of a binary mixture of anionic dyes and characterization of the residual biomass as potential organic soil amendment.

Industrial pollution is a great concern for modern society and developing cyclic processes is one of the major challenges. As far as we know, this work is the first to report the use of multiple white-rot fungi species for degrading a binary mixture of anionic dyes under solid state fermentation (SSF) conditions and a further physicochemical characterization of the residual biomass. First, eight white-rot fungi decolorized the dye mixture of brilliant blue FCF and allura red AC adsorbed onto corncob, reaching decolorizations between 11.47% and 87.64%. Then, I. lacteus, B. adusta and T. versicolor, based on the decolorization yield, were selected to evaluate the effect of carbon:nitrogen ratio, moisture content and inoculum quantity on the decolorization percentage. The factorial designs showed that C:N ratio had a negative effect while moisture and inoculum quantity a positive effect. In terms of the kinetics, the three white-rot fungi achieved their maximum decolorization level, around 80.11-86.04%, after 10-12 days. I. lacteus exhibited the highest decolorization percentage, even though only the enzyme manganese peroxidase was detected, with a maximum activity of 6.62 U gds-1 at day 14. Besides, T. versicolor was the only species with laccase activity, with a maximum of 15.94 U gds-1 at day 6 of fermentation. The physicochemical characterization of the biomass allowed to conclude that these aggregates represent a potential organic amendment, as for their significant oxidizable organic carbon (more than 9.5% on wet basis) and essential nutrients content, as well as for their low ash content (less than 1% on wet basis). Finally, for outlining an efficient bioremediation cycle, a cheap and effective methodology for drying the biomass at the end of the SSF process is required.