White Rot Fungus Irpex Lacteus Research Articles

Valorization of the Caragana waste via two-stage bioaugmentation: Optimizing nutrition composition, palatability, and microbial contaminant control

Caragana korshinskii kom. (CKK) waste, a common forestry byproduct in northwest of China, presents challenges in its transformation into alternative ruminant feed due to its initial nutritional limitations and unappealing palatability. Conventional strategies, such as ensiling and fungal-based solid-state fermentation (SSF) cannot effectively address this issue in practice. Herein, a two-stage bioaugmentation (TBA) process was devised, leveraging the benefits of ensiling and SSF. During the anaerobic ensiling phase, CKK waste was inoculated with Lactiplantibacillus plantarum LP1, effectively suppressing potential animal pathogens such as Aspergillus and Nocardiopsis while enriching the material with potential probiotics like Pediococcus and Lactiplantibacillus, reaching an abundance of 95.7%. In the subsequent aerobic SSF stage, the ensiled CKK underwent inoculation with the white-rot fungus Irpex lacteus F17, which became enriched to 87.9%. Comprehensive multi-omics analysis identified Irpex as the key taxon, possessing an extensive redox enzyme system that led to the improvement in nutrient composition, reduction of astringent phenolic substances, and mitigation of mycotoxins. As a result, the crude protein content of the CKK increased by 39.2%, while lignin, total phenolic substances, and tannic acid content decreased by 24.4%, 52.2%, and 51.4%, respectively. The mycotoxin levels, including aflatoxin B1, zearalenone, and vomitoxin, were rendered negligible, confirming the safety. Overall, this study demonstrates the TBA strategy can successfully transform challenging and unpalatable CKK waste into a nutrient-enriched and safe mycelium-based bioproduct, thereby enabling the valorization of a previously underutilized forestry resource as a promising alternative feed.

The potential of white and soft-rot fungi for biodegradation of multi-walled carbon nanotubes (MWCNTs): characterization and enzyme analysis

ABSTRACT Multi-walled carbon nanotubes (MWCNTs) have significant environmental concerns for soil, water and ecosystems. Since their harmful effects on human health, including respiratory disease, they are considered dangerous substances. Thus, it is required to discover a constructive solution to decrease of MWCNT toxicity. Due to their high degradability, white rot and soft rot fungi have been used in the biodegradation of various pollutants. In this study, we evaluated the ability of native Iranian white and soft rot fungi to degrade MWCNTs mainly by means of dynamic light scattering (DLS) and surface charge measurement. In this regard, growth kinetics, CO2 and protein production, size, surface charge and pH change were measured. Continuous Raman spectroscopy, scanning electron microscope (SEM) and transmission electron microscope (TEM) imaging were carried out to detect the process of degradation. The results showed that the morphology, arrangement, size and diameter of MWCNTs were modified due to fungal degradation. The cytotoxicity of treated MWCNTs was determined by the MTT test. The results indicated that native Iranian white and soft rot fungi have a significant ability for degradation of MWCNTs. Furthermore, it could be concluded that fungal treatment could reduce the toxicity of MWCNTs and the best result was achieved in 500 ppm MWCNTs for Trichoderma sp. The activities of oxidative enzymes such as laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) were measured to determine the mechanism of degradation. Enzyme assays have suggested that these oxidising enzymes especially laccase might play a key role in the degradation of MWCNTs in white rot and soft rot fungi. The overall results confirmed the reproductive ability of Trichoderma sp. WF29 as a soft rot and two white rot fungi Irpex lacteus WF36 and Trametes versicolor to degrade MWCNT.

Towards industrial application of fungal pretreatment in 2G biorefinery: scale-up of solid-state fermentation of wheat straw

Fungal pretreatment of lignocellulosic biomass for bioethanol production is an environmental-friendly alternative to steam explosion. However, this biological pretreatment has been tested on a small scale, where most of the typical problems of solid-state fermentations (SSF), such as limited aeration or temperature control, are not observed. The main objective of this study was to assess the feasibility of the fungal pretreatment of lignocellulosic biomass (wheat straw) at a demonstration scale using the white-rot fungus Irpex lacteus to improve straw digestibility. Different configurations were evaluated for the design of a 22 L SSF reactor, but a versatile vertical design that can operate as a packed-bed and as a tray reactor was selected. The wheat straw digestibility obtained in the SSF bioreactor after 21 days of pretreatment (60.6%) was similar to that achieved on a small scale (57.9%). In addition, the most common online monitoring variables (temperature and CO2 production) correlate with the fungal action on wheat straw. As well as the weight loss, obtaining comparable results at flask and reactor scale (30 and 34.5%, respectively).Graphical abstract

Biodegradability of Dental Care Antimicrobial Agents Chlorhexidine and Octenidine by Ligninolytic Fungi.

Chlorhexidine (CHX) and octenidine (OCT), antimicrobial compounds used in oral care products (toothpastes and mouthwashes), were recently revealed to interfere with human sex hormone receptor pathways. Experiments employing model organisms—white-rot fungi Irpex lacteus and Pleurotus ostreatus—were carried out in order to investigate the biodegradability of these endocrine-disrupting compounds and the capability of the fungi and their extracellular enzyme apparatuses to biodegrade CHX and OCT. Up to 70% ± 6% of CHX was eliminated in comparison with a heat-killed control after 21 days of in vivo incubation. An additional in vitro experiment confirmed manganese-dependent peroxidase and laccase are partially responsible for the removal of CHX. Up to 48% ± 7% of OCT was removed in the same in vivo experiment, but the strong sorption of OCT on fungal biomass prevented a clear evaluation of the involvement of the fungi or extracellular enzymes. On the other hand, metabolites indicating the enzymatic transformation of both CHX and OCT were detected and their chemical structures were proposed by means of liquid chromatography–mass spectrometry. Complete biodegradation by the ligninolytic fungi was not achieved for any of the studied analytes, which emphasizes their recalcitrant character with low possibility to be removed from the environment.

Comparison of the Ability of Several White-rot Fungi to Biobleach Acacia Oxygen-delignified Kraft Pulp

Previous screening analyses demonstrated that the in vivo biobleaching activities of the white-rot fungi Irpex lacteus KB-1.1 and Lentinus tigrinus LP-7 are higher than those of Phanerochaete chrysosporium and Trametes versicolor. The purpose of the current study was to examine the production of extracellular enzymes of these four white-rot fungi grown on three types of low-cost media containing agricultural and forestry waste, and to evaluate the ability of the produced extracellular enzymes to biobleach Acacia oxygen-delignified kraft pulp (A-OKP). The biobleaching activity of extracellular fractions of I. lacteus, L. tigrinus, T. versicolor, and P. chrysosporium cultures was the most pronounced after 3 days of incubation with Acacia mangium wood powder supplemented with rice bran and 1% glucose (WRBG) with resultant Kappa number reduction of 4.4%, 6.7%, 3.3%, and 3.3%, respectively. Therefore, biobleaching ability of I. lacteus and L. tigrinus have been shown to be higher than of T. versicolor and P. chrysosporium, both in vivo and in vitro.

Dye-decolorizing peroxidases in Irpex lacteus combining the catalytic properties of heme peroxidases and laccase play important roles in ligninolytic system

BackgroundThe white rot fungus Irpex lacteus exhibits a great potential in biopretreatment of lignocellulose as well as in biodegradation of xenobiotic compounds by extracellular ligninolytic enzymes. Among these enzymes, the possible involvement of dye-decolorizing peroxidase (DyP) in lignin degradation is not clear yet.ResultsBased on the extracellular enzyme activities and secretome analysis, I. lacteus CD2 produced DyPs as the main ligninolytic enzymes when grown in Kirk’s medium supplemented with lignin. Further transcriptome analysis revealed that induced transcription of genes encoding DyPs was accompanied by the increased expression of transcripts for H2O2-generating enzymes such as alcohol oxidase, pyranose 2-oxidase, and glyoxal oxidases. Meanwhile, accumulation of transcripts for glycoside hydrolase and protease was observed, in agreement with abundant proteins. Moreover, the biochemical analysis of IlDyP2 and IlDyP1 confirmed that DyPs were able to catalyze the oxidation of typical peroxidases substrates ABTS, phenolic lignin compounds DMP, and guaiacol as well as non-phenolic lignin compound, veratryl alcohol. More importantly, IlDyP1 enhanced catalytic activity for veratryl alcohol oxidation in the presence of mediator 1-hydroxybenzotriazole, which was similar to the laccase/1-hydroxybenzotriazole system.ConclusionsThe results proved for the first time that DyPs depolymerized lignin individually, combining catalytic features of different peroxidases on the functional level. Therefore, DyPs may be considered an important part of ligninolytic system in wood-decaying fungi.

Deciphering lignocellulose deconstruction by the white rot fungus Irpex lacteus based on genomic and transcriptomic analyses

BackgroundIrpex lacteus is one of the most potent white rot fungi for biological pretreatment of lignocellulose for second biofuel production. To elucidate the underlying molecular mechanism involved in lignocellulose deconstruction, genomic and transcriptomic analyses were carried out for I. lacteus CD2 grown in submerged fermentation using ball-milled corn stover as the carbon source.ResultsIrpex lacteus CD2 efficiently decomposed 74.9% lignin, 86.3% cellulose, and 83.5% hemicellulose in corn stover within 9 days. Manganese peroxidases were rapidly induced, followed by accumulation of cellulase and hemicellulase. Genomic analysis revealed that I. lacteus CD2 possessed a complete set of lignocellulose-degrading enzyme system composed mainly of class II peroxidases, dye-decolorizing peroxidases, auxiliary enzymes, and 182 glycoside hydrolases. Comparative transcriptomic analysis substantiated the notion of a selection mode of degradation. These analyses also suggested that free radicals, derived either from MnP-organic acid interplay or from Fenton reaction involving Fe2+ and H2O2, could play an important role in lignocellulose degradation.ConclusionsThe selective strategy employed by I. lacteus CD2, in combination with low extracellular glycosidases cleaving plant cell wall polysaccharides into fermentable sugars, may account for high pretreatment efficiency of I. lacteus. Our study also hints the importance of free radicals for future designing of novel, robust lignocellulose-degrading enzyme cocktails.

Enhancement of Environmental Hazard Degradation in the Presence of Lignin: a Proteomics Study

Proteomics studies of fungal systems have progressed dramatically based on the availability of more fungal genome sequences in recent years. Different proteomics strategies have been applied toward characterization of fungal proteome and revealed important gene functions and proteome dynamics. Presented here is the application of shot-gun proteomic technology to study the bio-remediation of environmental hazards by white-rot fungus. Lignin, a naturally abundant component of the plant biomass, is discovered to promote the degradation of Azo dye by white-rot fungus Irpex lacteus CD2 in the lignin/dye/fungus system. Shotgun proteomics technique was used to understand degradation mechanism at the protein level for the lignin/dye/fungus system. Our proteomics study can identify about two thousand proteins (one third of the predicted white-rot fungal proteome) in a single experiment, as one of the most powerful proteomics platforms to study the fungal system to date. The study shows a significant enrichment of oxidoreduction functional category under the dye/lignin combined treatment. An in vitro validation is performed and supports our hypothesis that the synergy of Fenton reaction and manganese peroxidase might play an important role in DR5B dye degradation. The results could guide the development of effective bioremediation strategies and efficient lignocellulosic biomass conversion.

Oxidation of a non-phenolic lignin model compound by two Irpex lacteus manganese peroxidases: evidence for implication of carboxylate and radicals

BackgroundManganese 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.ResultsThe 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.ConclusionsWe 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.

Enhanced simultaneous saccharification and fermentation of pretreated beech wood by in situ treatment with the white rot fungus Irpex lacteus in a membrane aerated biofilm reactor

The aim of the present study was to investigate the combination of steam pretreatment and biological treatment with lignin degrading fungal strains in order to enable efficient bioprocessing of beech wood to ethanol. In a sequential process of steam and fungal pretreatment followed by enzymatic hydrolysis, Irpex lacteus almost doubled the glucose yield for mildly pretreated beech wood, but could not improve yields for more severely pretreated substrates. However, when simultaneous saccharification and fermentation is combined with in situ I. lacteus treatment, which is enabled by the application of a membrane aerated biofilm reactor, ethanol yields of optimally steam pretreated beech could be improved from 65 to 80%. Generally, in situ fungal treatment during bioprocessing of lignocellulose is an interesting method to harness the versatile abilities of white rot fungi.

Decolorization of Reactive Orange 16 in Rotating Drum Biological Contactor

A laboratory scale Rotating Drum Biological Contactor (RDBC; drum length×diameter: 255×130mm) filled by randomly arranged particulate carriers with mycelium of the white rot fungus Irpex lacteus was tested for Reactive Orange 16 (RO16) decolorization. Five mycelium carriers were pre-screened for fungus immobilization and the dye degradation in static cultures. The best mycelium growth was observed with the straw, wooden shavings and polyether foam cylinders. Considerable dye adsorption (up to 30%) was observed with natural carriers; very low adsorption was observed with carrier particles made of plastics. Natural mycelium carriers with attached I. lacteus yielded the highest RO16 decolorization efficiency (50–90%). The straw, wooden shavings and polyether foam particles were tested in the RDBC in batch dye degradation experiments and the straw was also tested in continuous experiments. The straw yielded the fastest and stable dye decolorization in the subsequent batches. With the straw, wooden shavings and polyether foam particles RO16 decolorization higher than 90% was achieved within 48h in batch experiments and better than 80% decolorization was attained at liquid mean residence time of 33.3h in continuous experiments. Dye degradation rate of 3.5–4.0gm−3h−1 was achieved in the batch RDBC and the rate of 1.1–1.5gm−3h−1 in the continuously operated reactor.

Fungal pretreatment of agricultural residues for bioethanol production

The biological pretreatment of lignocellulosic biomass for the production of ethanol is an environmentally friendly process that needs to be evaluated with different feedstocks in order to avoid dependence on a single feedstock. In this study, four agricultural residues (corn stover, barley straw, corncob and wheat straw), selected in terms of their composition and geographic availability, were pretreated using the white-rot fungus Irpex lacteus. After the fungal pretreatment, the biggest reduction in lignin content (45.8±3.5%), lowest sugars consumption (11.5±1.4%) and highest lignin selectivity removal (2.1±0.15) were achieved with corn stover. Moreover, total holocellulose digestibility was significantly increased after the biological pretreatment with all the substrates (37–103%), excepting corncob. In this study, fungal pretreatment was successfully applied to three of the most common agricultural residues available in Europe for producing ethanol, demonstrating that it is capable of handling feedstocks of variable origin.

Degradation and detoxification of the triphenylmethane dye malachite green catalyzed by crude manganese peroxidase from Irpex lacteus F17.

Malachite green (MG), a recalcitrant, carcinogenic, and mutagenic triphenylmethane dye, was decolorized and detoxified using crude manganese peroxidase (MnP) prepared from the white rot fungus Irpex lacteus F17. In this study, the key factors (pH, temperature, MG, Mn(2+), H2O2, MnP) in these processes were investigated. Under optimal conditions, 96% of 200mgL(-1) of MG was decolorized when 66.32UL(-1) of MnP was added for 1h. The K m, V max, and k cat values were 109.9μmolL(-1), 152.8μmolL(-1)min(-1), and 44.5s(-1), respectively. The decolorization of MG by MnP followed first-order reaction kinetics with a kinetic rate constant of 0.0129h(-1). UV-vis and UPLC analysis revealed degradation of MG. Furthermore, seven different intermediates formed during the MnP treatment of 0.5h were identified by LC-TOF-MS. These degradation products were generated via two different routes by either N-demethylation of MG or the oxidative cleavage of the C-C double bond in MG. Based on ecotoxicity analyses performed on bacteria and algae, it was confirmed that MG metabolites produced by the MnP-catalyzed system were appreciably less toxic than the parent compound. These studies indicate the potential use of this enzyme system in the clean-up of aquatic and terrestrial environments.

Alkali treatment of fungal pretreated wheat straw for bioethanol production

AbstractBioconversion of lignocellulosic materials into ethanol requires an intermediate pretreatment step for conditioning biomass. Sugar yields from wheat straw were previously improved by the addition of a mild alkali pretreatment step before bioconversion by the white-rot fungus Irpex lacteus. In this work, an alternative alkaline treatment, which significantly reduces water consumption, was implemented and optimized. Sugar recovery increased 117% with respect to the previously developed alkaline wash process at optimal process conditions (30°C, 30 minutes and 35.7% (w/w) of NaOH). In order to further reduce operational costs, a system for alkali recycling was implemented. This resulted in the treatment of 150% more wheat straw using the same amount of NaOH. Finally, enzymatic hydrolysis was optimized and resulted in a reduction of enzyme dose of 33%.

Degradation of Bunker C Fuel Oil by White-Rot Fungi in Sawdust Cultures Suggests Potential Applications in Bioremediation.

Fungal lignocellulolytic enzymes are promising agents for oxidizing pollutants. This study investigated degradation of Number 6 “Bunker C” fuel oil compounds by the white-rot fungi Irpex lacteus, Trichaptum biforme, Phlebia radiata, Trametes versicolor, and Pleurotus ostreatus (Basidiomycota, Agaricomycetes). Averaging across all studied species, 98.1%, 48.6%, and 76.4% of the initial Bunker C C10 alkane, C14 alkane, and phenanthrene, respectively were degraded after 180 days of fungal growth on pine media. This study also investigated whether Bunker C oil induces changes in gene expression in the white-rot fungus Punctularia strigosozonata, for which a complete reference genome is available. After 20 days of growth, a monokaryon P. strigosozonata strain degraded 99% of the initial C10 alkane in both pine and aspen media but did not affect the amounts of the C14 alkane or phenanthrene. Differential gene expression analysis identified 119 genes with ≥ log2(2-fold) greater expression in one or more treatment comparisons. Six genes were significantly upregulated in media containing oil; these genes included three enzymes with potential roles in xenobiotic biotransformation. Carbohydrate metabolism genes showing differential expression significantly accumulated transcripts on aspen vs. pine substrates, perhaps reflecting white-rot adaptations to growth on hardwood substrates. The mechanisms by which P. strigosozonata may degrade complex oil compounds remain obscure, but degradation results of the 180-day cultures suggest that diverse white-rot fungi have promise for bioremediation of petroleum fuels.

Statistical Optimization of Medium Components of Xylanase Production by Irpex lacteus BAFC 1168 strain F under liquid fermentation

The enhanced xylanase production of white rot fungus Irpex lacteus BAFC 1168 strain F using a liquid fermentation culture was investigated. The media nutrients were screened by a Plackett-Burman design and from the analysis of variance, carboxy-methylcellulose, urea and peptone were found to be the most positive and significant nutrient components. Hence, these three variables were selected for further optimization using a five-level central composite design in response surface methodology. The validation of the model experiment was conducted reaching 8.29 U/mL of xylanase activity when the composition medium was 1.6 g/L of CMC, 0.84 g/L of urea and 2 g/L of peptone. Optimum temperature and pH conditions on xylanase activity were also determined using a central composite design. It was reached a maximum xylanase activity of 8.36 U/mL when temperature and pH were 42.8°C and 5.6, respectively.

Induction, Purification and Characterization of a Novel Manganese Peroxidase from Irpex lacteus CD2 and Its Application in the Decolorization of Different Types of Dye

Manganese peroxidase (MnP) is the one of the important ligninolytic enzymes produced by lignin-degrading fungi which has the great application value in the field of environmental biotechnology. Searching for new MnP with stronger tolerance to metal ions and organic solvents is important for the maximization of potential of MnP in the biodegradation of recalcitrant xenobiotics. In this study, it was found that oxalic acid, veratryl alcohol and 2,6-Dimehoxyphenol could stimulate the synthesis of MnP in the white-rot fungus Irpex lacteus CD2. A novel manganese peroxidase named as CD2-MnP was purified and characterized from this fungus. CD2-MnP had a strong capability for tolerating different metal ions such as Ca2+, Cd2+, Co2+, Mg2+, Ni2+ and Zn2+ as well as organic solvents such as methanol, ethanol, DMSO, ethylene glycol, isopropyl alcohol, butanediol and glycerin. The different types of dyes including the azo dye (Remazol Brilliant Violet 5R, Direct Red 5B), anthraquinone dye (Remazol Brilliant Blue R), indigo dye (Indigo Carmine) and triphenylmethane dye (Methyl Green) as well as simulated textile wastewater could be efficiently decolorized by CD2-MnP. CD2-MnP also had a strong ability of decolorizing different dyes with the coexistence of metal ions and organic solvents. In summary, CD2-MnP from Irpex lacteus CD2 could effectively degrade a broad range of synthetic dyes and exhibit a great potential for environmental biotechnology.

Purification of pyranose oxidase from the white rot fungus Irpex lacteus and its cooperation with laccase in lignin degradation

Laccase and peroxidases mainly cause polymerization of lignin in vitro due to the random coupling of the phenoxy radicals or quinoid intermediates. White rot fungi may avoid polymerization in vivo by reduction of these intermediates. Pyranose oxidase is suggested to play such a role based on its quinone-reducing activity, but direct evidence has been lacking. In this study, a pyranose oxidase was purified from the white rot fungus Irpex lacteus and partially characterized. The enzyme is composed of four subunits of 71kDa as determined by SDS-PAGE. It exhibits maximum activity at pH 6.5 and 55°C and is rather stable. d-glucose is the preferred substrate, but d-galactose, l-sorbose and d-xylose are also readily oxidized. In addition to O2, the enzyme can also transfer electrons to various quinones and the ABTS [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] cation radical. Laccase-generated quinoids are also reduced by the enzyme. Four different technical lignins were treated with laccase with and without pyranose oxidase. Subsequent gel permeation chromatography analysis demonstrated that the pyranose oxidase efficiently inhibited the polymerization of lignin caused by laccase and even brought about degradation.

Rotating Drum Biological Contactor for Immobilization of the White-rot Fungus Irpex Lacteus and Degradation of Textile Dyes

Rotating Drum Biological Contactor for Immobilization of the White-rot Fungus Irpex Lacteus and Degradation of Textile Dyes

Biobleaching of Acacia kraft pulp with extracellular enzymes secreted by Irpex lacteus KB-1.1 and Lentinus tigrinus LP-7 using low-cost media

The white-rot fungi Irpex lacteus KB-1.1 and Lentinus tigrinus LP-7 have been shown in previous studies to have high biobleaching activity in vivo. The aim of this study was to investigate the activities and stabilities of extracellular enzymes, prepared from I. lacteus and L. tigrinus culture grown in three types of economical media of agricultural and forestry wastes, for biobleaching of Acacia oxygen-delignified kraft pulp using kappa number reduction as an indicator of delignification. After 3 days of incubation, the extracellular enzymes preparations from I. lacteus and L. tigrinus cultures in media of Acacia mangium wood powder supplemented with rice bran and addition 1 % glucose (WRBG), resulted in significant decrease of 4.4 and 6.7 %, respectively. A slightly higher kappa number reduction (7.4 %) was achieved with the combine extracellular enzymes from I. lacteus and L. tigrinus. One of the strategies for reducing the cost of enzyme production for treatment processes in the pulp and paper industry is the utilization of agricultural and forestry waste. Thus, WRBG has potential as a culture medium for producing stable lignolytic enzymes simply and economically.