Application of fungal-based microbial fuel cells for biodegradation of pharmaceuticals: Comparative study of individual vs. mixed contaminant solutions.

The present study focuses on the application of fungal-based microbial fuel cells (FMFC) for the degradation of organic pollutants including Acetaminophen (APAP), Para-aminophenol (PAP), Sulfanilamide (SFA), and finally Methylene Blue (MB). The objective is to investigate the patterns of degradation (both individually and as a mixture solution) of the four compounds in response to fungal metabolic processes, with an emphasis on evaluating the possibility of generating energy. Linear Sweep Voltammetry (LSV) has been used for electrochemical analysis of the targeted compounds on a Glassy Carbon Electrode (GCE). A dual chamber MFC has been applied wherein the cathodic compartment, the reduction reaction of oxygen was catalyzed by an elaborated biofilm of Trametes trogii, and the anodic chamber consists of a mixed solution of 200 mg L-1 APAP, PAP, MB, and SFA in 0.1 M PBS and an elaborated biofilm of Trichoderma harzianum. The obtained results showed that all the tested molecules were degraded over time by the Trichoderma harzianum. The biodegradation kinetics of all the tested molecules were found to be in the pseudo-first-order. The results of half-lives and the degradation rate reveal that APAP in its individual form degrades relatively slower (0.0213 h-1) and has a half-life of 33 h compared to its degradation in a mixed solution with a half-life of 20 h. SFA showed the longest half-life in the mixed condition (98 h) which is the opposite of its degradation as individual molecules (20 h) as the fastest molecule compared to other pollutants. The maximum power density of the developed MFC dropped from 0.65 mW m-2 to 0.32 mW m-2 after 45.5 h, showing that the decrease of the residual concentration of molecules in the anodic compartment leads to the decrease of the MFC performance.

Enhanced biodegradation of benzo[a]pyrene with Trametes versicolor stimulated by citric acid.

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants with carcinogenic, mutagenic and teratogenic effects. The white-rot fungi in the fungal group have significant degradation ability for high molecular weight organic pollutants. However, exogenous fungi are easily antagonized by indigenous microorganisms. Low molecular weight organic acids, a small molecular organic matter secreted by plants, can provide carbon sources for soil microorganisms. Combining organic acids with white rot fungi may improve the nutritional environment of fungi. In this study, immobilized Trametes versicolor was used to degrade benzo[a]pyrene in soil, and its effect on removing benzo[a]pyrene in soil mediated by different low molecular weight organic acids was investigated. The results showed that when the degradation was 35 days, the removal effect of the experimental group with citric acid was the best, reaching 43.7%. The degradation effect of Trametes versicolor on benzo[a]pyrene was further investigated in the liquid medium when citric acid was added, and the effects of citric acid on the biomass, extracellular protein concentration and laccase activity of Trametes versicolor were investigated by controlling different concentrations of citric acid. In general, citric acid can act as a carbon source for Trametes versicolor and promote its extracellular protein secretion and laccase activity, thereby accelerating the mineralization of benzo[a]pyrene by Trametes versicolor. Therefore, citric acid can be used as a biostimulant in the remediation of PAHs contaminated soil with Trametes versicolor.

Improved productivity and dye removal performance of Trametes versicolor pellets using rice husk as a co-substrate.

Pellet production represents a critical step for several processes requiring fungal biomass, nevertheless, its optimization is seldom reported. The use of finely ground rice husk as a microcarrier and co-substrate permitted a marked increase (≈ 2.7×) in the productivity of fungal pellet production using Trametes versicolor compared to traditional production methods. The pellets show similar structure and smaller size compared to typical sole-mycelium pellets, as well as comparable laccase activity. The efficiency of the pellets for biodegradation was confirmed by the removal of the crystal violet dye, achieving significantly faster decolorization rates compared to the traditionally produced pellets. The use of these pellets during the continuous treatment of the dye in a stirred tank bioreactor resulted in 97% decolorization operating at a hydraulic residence time of 4.5 d.

Morphological, sterical, and localized thermodynamics in the adsorption of CO2 by activated biocarbon from the white rot fungi Trametes gibbosa.

The capture of CO2 by biochar has recently become one of the cornerstones of circular economy models for a sustainable society. In this work, we synthesized an activated biocarbon using Trametes gibbosa (BioACTG) in a one-step synthesis. We investigated CO2 adsorption mechanisms under five different temperatures using a statistical physics approach. The data was better represented by the multilayer model with two distinguished energies, providing more accurate values for the estimated parameters. According to the number of carbon dioxide molecules per site (n) and the densities of the receptor sites (Dzif), the tendency to form a second layer increased as the temperature increased. The adsorption of CO2 on BioACTG was exothermic (the values of Qasat = 15.5 mmol/g at 273 K decrease to 10.5 mmol/g at 353 K), and the temperature influenced CO2 as well as the morphological features of the process. A computational approach was used to investigate the electronic properties of the adsorbate, showing that its lowest unoccupied orbital (LUMO) heavily contributed to the high efficiency of the process which was ruled by pore diffusion mechanisms driven by energetic fluctuations. Other molecules present in CO2-rich mixtures were also investigated, showing that their concentration limited their competitiveness with CO2.

Assessment of textile effluent treatment by immobilized Trametes pubescens MB 89 for plant growth promotion.

Industrialization and the ever-increasing world population have diminished high-quality water resources for sustainable agriculture. It is imperative to effectively treat industrial effluent to render the treated water available for crop cultivation. This study aimed to assess the effectiveness of textile effluent treated with Trametes pubescens MB 89 in supporting maize cultivation. The fungal treatment reduced the amounts of Co, Pb and As in the textile effluent. The biological oxygen demand, total dissolved solids and total suspended solids were within the permissible limits in the treated effluent. The data indicated that the irrigation of maize with fungal-treated textile effluent improved the growth parameters of the plant including root, shoot length, leaf area and chlorophyll content. Moreover, better antioxidant activity, total phenol content and protein content in roots, stems and leaves of maize plants were obtained. Photosynthetic parameters (potential quantum yield, electron transport rate and fluorescence yield of non-photochemical losses other than heat) were also improved in the plants irrigated with treated effluent as compared to the control groups. In conclusion, the treatment of textile effluent with the immobilized T. pubescens presents a sustainable solution to minimize chemical pollution and effectively utilize water resources.

Exploring the formation of heterodimers of barley hydroxycinnamoylagmatines by oxidative enzymes.

Dimers of hydroxycinnamoylagmatines are phenolic compounds found in barley and beer. Although they are bioactive and sensory-active compounds, systematic reports on their structure-property relationships are missing. This is partly due to lack of protocols to obtain a diverse set of hydroxycinnamoylagmatine homo- and heterodimers. To better understand dimer formation in complex systems, combinations of the monomers coumaroylagmatine (CouAgm), feruloylagmatine (FerAgm), and sinapoylagmatine (SinAgm) were incubated with horseradish peroxidase. For all combinations, the main oxidative coupling products were homodimers. Additionally, minor amounts of heterodimers were formed, except for the combination of FerAgm and CouAgm. Oxidative coupling was also performed with laccases from Agaricus bisporus and Trametes versicolor, resulting in formation of the same coupling products and no formation of CouAgm-FerAgm heterodimers. Our protocol for oxidative coupling combinations of hydroxycinnamoylagmatines yielded a structurally diverse set of coupling products, facilitating production of dimers for future research on their structure-property relationships.

Identification of redox activators for continuous reactivation of glyoxal oxidase from Trametes versicolor in a two-enzyme reaction cascade.

Glyoxal oxidases, belonging to the group of copper radical oxidases (CROs), oxidize aldehydes to carboxylic acids, while reducing O2 to H2O2. Their activity on furan derivatives like 5-hydroxymethylfurfural (HMF) makes these enzymes promising biocatalysts for the environmentally friendly synthesis of the bioplastics precursor 2,5-furandicarboxylic acid (FDCA). However, glyoxal oxidases suffer from inactivation, which requires the identification of suitable redox activators for efficient substrate conversion. Furthermore, only a few glyoxal oxidases have been expressed and characterized so far. Here, we report on a new glyoxal oxidase from Trametes versicolor (TvGLOX) that was expressed at high levels in Pichia pastoris (reclassified as Komagataella phaffii). TvGLOX was found to catalyze the oxidation of aldehyde groups in glyoxylic acid, methyl glyoxal, HMF, 2,5-diformylfuran (DFF) and 5-formyl-2-furancarboxylic acid (FFCA), but barely accepted alcohol groups as in 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), preventing formation of FDCA from HMF. Various redox activators were tested for TvGLOX reactivation during catalyzed reactions. Among them, a combination of horseradish peroxidase and its substrate 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) most efficiently reactivated TvGLOX. Through continuous reactivation of TvGLOX in a two-enzyme system employing a recombinant Moesziomyces antarcticus aryl-alcohol oxidase (MaAAO) almost complete conversion of 8 mM HMF to FDCA was achieved within 24 h.

Under explored roles of microbial ligninolytic enzymes in aerobic polychlorinated biphenyl transformation.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants in the environment that are responsible for many adverse health effects. Bioremediation appears to be a healthy and cost-effective alternative for remediating PCB-contaminated environments. While some microbial species have been observed to be capable of transforming PCBs, only two different microbial pathways (rdh and bph pathways) have been described to be involved in PCB transformations. Ligninolytic enzymes have been observed or are under suspicion in some microbial PCB transformations. However, the role of these promising PCB-transforming enzymes, which are produced by fungi and some aerobic bacteria, is still unclear. The present review describes their role by identifying microbial PCB-transforming species and their reported ligninolytic enzymes whether proven or suspected to be involved in PCB transformations. There are several lines of evidence that ligninolytic enzymes are responsible for PCB transformations such as (1) the ability of purified laccases from Myceliophthora thermophila, Pycnoporus cinnabarinus, Trametes versicolor, Cladosporium sp, and Coprinus cumatus to transform hydroxy-PCBs; (2) the increased production of laccases and peroxidases by many fungi in the presence of PCBs; and (3) the enhanced PCB transformation by Pseudomonas stutzeri and Sinorhizobium meliloti NM after the addition of ligninolytic enzyme enhancers. However, if the involvement of ligninolytic enzymes in PCB transformation is clearly demonstrated in some fungal species, it does not seem to be implicated in all microbial species suggesting other still unknown metabolic pathways involved in PCB transformation and different from the bph and rdh pathways. Therefore, PCB transformation may involve several metabolic pathways, some involving ligninolytic enzymes, bph or rdh genes, and some still unknown, depending on the microbial species. In addition, current knowledge does not fully clarify the role of ligninolytic enzymes in PCB oxidation and dechlorination. Therefore, further studies focusing on purified ligninolytic enzymes are needed to clearly elucidate their role in PCB transformation.

Immobilization of recombinant Trametes versicolor aflatoxin B1-degrading enzyme (TV-AFB1D) with montmorillonite for absorption and in situ degradation of aflatoxin B1.

Aflatoxin B1 is a highly carcinogenic and teratogenic substance mainly produced by toxin-producing strains such as Aspergillus flavus and Aspergillus parasitic. The efficient decomposition of aflatoxin is an important means to reduce its harm to humans and livestock. In this study, Trametes versicolor aflatoxin B1-degrading enzyme (TV-AFB1D) was recombinantly expressed in Bacillus subtilis (B. subtilis) 168. MMT-CTAB-AFB1D complex was prepared by the immobilization of TV-AFB1D and montmorillonite (MMT) by cross-linking glutaraldehyde. The results indicated that TV-AFB1D could recombinantly express in engineered B. subtilis 168 with a size of approximately 77 kDa. The immobilization efficiency of MMT-CTAB-AFB1D reached 98.63% when the concentration of glutaraldehyde was 5% (v/v). The relative activity of TV-AFB1D decreased to 72.36% after reusing for 10 times. The content of AFB1 in MMT-CTAB-AFB1D-AFB1 decreased to 1.1 µg/g from the initial 5.6 µg/g after incubation at 50 °C for 6 h. The amount of 80.4% AFB1 in the MMT-CTAB-AFB1D-AFB1 complex was degraded by in situ catalytic degradation. Thus, the strategy of combining adsorption and in situ degradation could effectively reduce the content of AFB1 residue in the MMT-CTAB-AFB1D complex.

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Optimizing the degradation of aflatoxin B1 in corn by Trametes versicolor and improving the nutritional composition of corn.

BACKGROUND: Corn, being an important grain, is prone to contamination by aflatoxin B1 (AFB1 ), and AFB1 -contaminated corn severely endangers the health of humans and livestock. Trametes versicolor, a fungus that can grow in corn, possesses the ability to directly degrade AFB1 through its laccase. This study aimed to optimize the fermentation conditions for T. versicolor to degrade AFB1 in corn and investigate the effect of T. versicolor fermentation on the nutritional composition of corn. AFB1 -contaminated corn was used as the culture substrate for T. versicolor. A combination of single-factor experiments and response surface methodology was employed to identify the optimal conditions of AFB1 degradation. RESULTS: The optimal conditions of AFB1 degradation were as follows: 9 days of fermentation, a fermentation temperature of 26.7 °C, a moisture content of 70.5% and an inoculation amount of 4.9 mL (containing 51.99 mg of T. versicolor mycelia). With the optimal conditions, the degradation rate of AFB1 in corn could reach 93.01%, and the dry basis content of protein and dietary fiber in the fermented corn was significantly increased. More importantly, the lysine content in the fermented corn was also significantly increased. CONCLUSION: This is the first report that direct fermentation of AFB1 -contaminated corn by T. versicolor not only efficiently degrades AFB1 but also improves the nutritional composition of corn. These findings suggest that the fermentation of corn by T. versicolor is a promising, environmentally friendly and efficient approach to degrade AFB1 and improve the nutritional value of corn. © 2023 Society of Chemical Industry.

Bioremediation of Diesel-Contaminated Soil by Fungal Solid-State Fermentation.

To address the poor removal of diesel in soil by indigenous microorganisms, we proposed a fungal solid-state fermentation (SSF) method for bioremediation. We screened Pycnoporus sanguineus 5.815, Trametes versicolor 5.996, and Trametes gibbosa 5.952 for their diesel-degrading abilities, with Trametes versicolor 5.996 showing the most promise. The fungal inoculum was obtained through SSF using wood chips and bran. Trametes versicolor 5.996 was applied to two treatments: natural attenuation (NA, diesel-contaminated soil) and bioremediation (BR, 10% SSF added to diesel-contaminated soil). Over 20 days, NA removed 12.9% of the diesel, while BR achieved a significantly higher 38.3% degradation rate. BR also increased CO2 and CH4 emissions but reduced N2O emissions. High-throughput sequencing indicated SSF significantly enriched known diesel-degrading microorganisms like Ascomycota (83.82%), Proteobacteria (46.10%), Actinobacteria (27.88%), Firmicutes (10.35%), and Bacteroidota (4.66%). This study provides theoretical support for the application of fungal remediation technology for diesel and improves understanding of microbiologically mediated diesel degradation and soil greenhouse gas emissions.