Native Fungi Research Articles

Fungal biomass and ectomycorrhizal community assessment of phosphorus responsive Pinus taeda plantations.

Ectomycorrhizal fungi and non-ectomycorrhizal fungi are responsive to changes in environmental and nutrient availabilities. Although many species of ectomycorrhizas are known to enhance the uptake of phosphorus and other nutrients for Pinus taeda, it is not understood how to optimize these communities to have tangible effects on plantation silviculture and P use efficiency. The first step of this process is the identification of native fungi present in the system that are associated with P. taeda and influence P uptake efficiency. We used sand-filled mesh bags baited with finely ground apatite to sample ectomycorrhizal and non-ectomycorrhizal fungi associated with the rhizosphere of P-responsive P. taeda under several field conditions. Mesh bags were assessed for biomass accumulation over three years using a single three-month burial period pre-harvest and three six-month burial periods post-planting. Amplicon sequencing assessed ectomycorrhizal and non-ectomycorrhizal communities between phosphorus treatments, sites, mesh bags, and the rhizosphere of actively growing P. taeda in the field. We found biomass accumulation within the mesh bags was inversely related to increasing phosphorus fertilization (carryover) rates from pre-harvest to post-planting. Up to 25% increases in total biomass within the bags were observed for bags baited with P. Taxonomic richness was highest in Alfisol soils treated with phosphorus from the previous rotation and lowest in the Spodosol regardless of phosphorus treatment.

Coriolopsis strumosa as an Orchid Endophytic Fungus and Its Spatial Distribution in Epidendrum sp. (Orchidaceae).

Coriolopsis spp. are wood-decaying fungi that inhabit forests. They are mainly distributed in tropical and subtropical areas. Strain Epi910 was isolated from the asymbiotically germinated protocorm of Epidendrum sp. and identified as Coriolopsis strumosa. Symbiotic germination and high-throughput sequencing of the endophytic fungal communities of different parts were performed to characterize the function and spatial distribution of the Epi910 isolate. Under symbiotic germination, Epi910 promoted seed germination and seedling formation as an endophytic native fungus of Epidendrum sp. Endophytic fungal communities from seven different parts of Epidendrum sp. were characterized. In total, 645 OTUs were identified; 30 OTUs were shared among all seven parts. The internal transcribed spacer sequence of Epi910 was identical to that of a dominant shared OTU (OTU6). The relative abundance of OTU6 in the seven parts was identified as follows: capsule pericarp > seed > root > asymbiotically germinated protocorm > epiphytic root > ovary > rachis. Our results suggest that the isolate belonging to Coriolopsis strumosa could promote the germination of Epidendrum sp. There may, therefore, be endophytic fungi other than common orchid mycorrhizal fungi with the ability to enhance germination in orchids.

Synthesis and characterization of electrospun nanofibers for the immobilization of a cocktail of native laccases from Pycnoporus sanguineus CS:43 and their evaluation in the biotransformation of 2,4,6-trinitrotoluene

Enzymatic biotransformation stands as an eco-friendly strategy for eliminating toxic contaminants like 2,4,6-trinitrotoluene (TNT). Moreover, the application of immobilized biocatalysis is relevant for industrial applications due to the improved stability and reusability of the immobilized enzymes. In the present study, an enzymatic cocktail containing laccases from a native fungus from the northeastern of Mexico (Pycnoporus Ssanguineus CS43) was immobilized in nanofibers. The study explores three biopolymer mixtures—polyalcohol vinyl (PVA), PVA/alginate (PVA/SA), and PVA/chitosan (PVA/CS)—for nanofiber preparation and employs three distinct immobilization strategies: entrapment, adsorption, and covalent bonding. Among them entrapped laccases in PVA/SA nanofibers showed the highest immobilization rate (96.92%) and retained 77.85% activity after five cycles using 2,2-azino- bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate. Preliminary kinetic parameters and optimal pH/temperature were determined, comparing the cocktail of immobilized laccases to free ones. The application of this system resulted in a substantial 93.43% TNT removal at pH 7 and 25 °C within four days, preserving 24.13% activity after five cycles. Significantly, this study marks the pioneering use of immobilized laccase for TNT biotransformation, offering a promising eco-friendly strategy for tackling persistent pollutants such as TNT.

Bioengineered xylanase from Misiones Argentina rainforest: A bakery enhancement approach.

In this study, we pursued the heterologous expression of the xylanase gene from Trichoderma atroviride, a native fungus in the province of Misiones, and used it to enhance the textural properties of baked goods through varying enzymatic concentrations. This marks the inaugural exploration into its functionality in the context of bread production. The recombinant xylanase exhibited improved activity, reaching 36,292UL-1, achieved by supplementing the culture medium with dextrose. Following the optimization of recombinant xylanase concentration, promising results emerged, notably reducing hardness and chewiness parameters of bread significantly. Our findings underscore the potential of this native fungal enzyme for industrial processes, offering a sustainable and efficient means to enhance the quality of baked goods with broad implications for the food industry. No prior research has been documented on the heterologous expression of the xylanase gene derived from T. atroviride, from the Misiones rainforest, expressed in Kluyveromyces lactis. PRACTICAL APPLICATION: This research, focusing on the isolation and cloning of xylanase enzyme from Trichoderma atroviride, a native fungus in the province of Misiones, offers a valuable tool for improving the texture of bakery products. By optimizing enzyme concentrations, our findings present a practical approach for the food industry, offering a viable solution to improve the overall quality and consumer satisfaction of bakery products.

The aquastatin biosynthetic gene cluster encodes a versatile polyketide synthase capable of synthesising heteromeric depsides with diverse alkyl side chains.

Depsides have garnered substantial interest due to the diverse biological activities exhibited by members of this class. Among these are the antibacterial aquastatins, glycosylated heteromeric depsides formed through the condensation of orsellinic acid with corticiolic acid. In this work, we isolated aquastatins and the recently described geministatins, along with several novel aquastatin-related depsides with different alkyl side chains from the fungus Austroacremonium gemini MST-FP2131. The structures were determined through comprehensive spectroscopic analysis and chemical degradation. Genome mining and heterologous expression in Aspergillus nidulans and Saccharomyces cerevisiae revealed that aquastatin biosynthesis requires only two genes: a non-reducing polyketide synthase (SAT-KS-AT-PT-ACP-TE) and a glycosyltransferase. We demonstrated that the single polyketide synthase can synthesise an acetyl-primed orsellinic acid and alkylresorcylate with various chain lengths (C14, C16, or C18) by incorporating different long-chain acyl-CoAs as starter units, and then join these as heteromeric depsides. Using chemical degradation, we generated a series of analogues and showed that several aglycone depsides exhibit antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), as well as antifungal and cytotoxic activities. Interestingly, heterologous expression of the aquastatin gene cluster in A. nidulans produced higher levels of geministatins with Δ15,16 and Δ18,19 double bonds, which have superior bioactivities compared to the aquastatins but are only present as minor compounds in the native fungus A. gemini.

Application of trichoderma and aspergillus as biofertilizers in eco-friendly ratoon rice cultivation

The study’s goal is to find the best native fungus from rice husk waste so that a solid biofertilizer can be made with high-husk flour as a carrier material and an inert agent. This study was conducted on agricultural land in Seloliman Village, Trawas District, and Mojokerto Regency. Biofertilization and biological agent formulation activities carried out at Muhammadiyah University of Sidoarjo’s Microbiology Laboratory aided the research. The experiment was conducted using a factorial randomized block design. The first factor consisted of three treatments: no fungi, Trichoderma sp., and Aspergillus sp. The second factor consists of soil treatment and apical treatment. The six treatment combinations were repeated four times (24 samples). The variables measured comprised plant height, number of panicles, weight of grain per plant, weight of 100 grams of grain, and the efficacy of biological agents in improving plant growth and productivity. All data underwent analysis of variety and then an HSD test at the 5% significance level to identify disparities among treatments. The study reveals that isolates Tc-013 and As-022 were identified as Trichoderma esperellum and Aspergillus flavus or A. oryzae, respectively. The application of Trichoderma and Aspergillus caused a decrease in the intensity of disease symptoms, reaching 64.7% and 37.3%, an increase in plant height and number of panicles, and an increase in the weight of 100 grains of 59.89 and 49.35%, respectively, as compared to the control treatment where the fungus was not applied.

The Eucalyptus snout beetle in Colombia: Selection and evaluation of entomopathogenic fungi as bioinsecticides against Gonipterus platensis

The Eucalyptus snout beetles, which belong to the Gonipterus scutellatus species complex, cause heavy damage to Eucalyptus spp. worldwide. The species was reported in Antioquia, Colombia for the first time in 2016, threatening more than 115,000 ha of planted Eucalyptus spp. Elsewhere, its damage has been controlled using Anaphes spp. as classical biological control, but Anaphes spp. are not available in Colombia, where there are no documented control methods for this species to date. We identified the Gonipterus species using molecular methods and isolated entomopathogenic fungi from naturally infected beetles collected from the current distribution area in Colombia. We characterised the fungal isolates, selecting the best to develop a biological control agent. We considered insecticidal activity, UV-B radiation tolerance, yield, germination, and enzyme activity of conidia in making our selection. We confirmed the presence of G. platensis in Colombia, with adults naturally infected by Beauveria spp. and Metarhizium spp. From the isolated native fungi, M. brunneum (CA-3), M. robertsii (RI-1), B. bassiana (CA-1) and (CA-2), B. pseudobassiana (SP-1) were identified. We found remarkable differences in response to different variables between the selected isolates and SP-1 and CA-3 were the most virulent. The SP-1 isolate stood out from the rest for being the most tolerant to UV-B radiation, alkaline media, and low temperatures, germinating faster and producing elevated levels of lipases, chitinases, and proteases. The identified promising native fungus isolate, naturally occurring on field individuals, could be used to develop a biopesticide for controlling G. platensis in Eucalyptus spp. plantations in Colombia.

Antagonistic interactions between native fungi of Minnesota and the root rot pathogen Heterobasidion irregulare

AbstractThe forest pathogen, Heterobasidion irregulare, is a serious threat to conifers in North America including Minnesota. Fungi native to Minnesota were isolated and tested in laboratory and field assays to evaluate their antagonism towards H. irregulare. One management strategy for plant pathogens, and especially H. irregulare, is to use fungi as biological control agents. A successful biological control agent used to manage root rot disease caused by H. irregulare is the fungus Phlebiopsis gigantea. The goal of this research was to screen different native fungi, including P. gigantea, against H. irregulare and examine and quantify their interactions in vitro and ex vitro. A set of four different antagonism assays were conducted. These assays served as a screening process involving both the laboratory and the field. Interactions were first examined with dual inoculation studies on media and wood discs of red pine (Pinus resinosa). These assays demonstrated strong inhibition and limited growth of H. irregulare by select fungi, including Phanerochaete livescens and P. gigantea. Another assay involved using soil microcosms and wood wedges of red pine. This allowed for a different examination, as wood wedges were inoculated with a candidate antagonistic fungus and placed in soil microcosms with H. irregulare. The opposite interaction was also examined with wedges inoculated with H. irregulare and then placed in soil microcosms containing different candidate fungi. In the field, large wood discs were placed around stumps and inoculated with candidate fungi in a red pine plantation infected with H. irregulare. Certain fungi performed well in different assays, but across all assays, P. gigantea performed the best. The antagonism of P. gigantea was most noticeable on wood discs and wood wedges used in vitro, as H. irregulare was not able to be reisolated from these substrates. Overall, these results provide more information on the fungi that appear to be acting as antagonists in forests to prevent H. irregulare from colonizing and provide new information on potential candidate fungi that could be used as a new biological control agent.

Bioremediation of Triphenylmethane and Sulfonated Azo Dyes Using an Indigenous Strain of Trichoderma Asperellum: Mechanisms and Efficacy Assessment

This study aimed to isolate a native fungal strain that was potent enough to curb the toxicity of azo dyes in textile effluent drain sludge. The native fungus, which has the potential to degrade several azo dyes under ‘non-sterile’ conditions, was isolated and identified as Trichoderma asperellum (TA). It was observed that the isolated strain was capable of decolorizing up to 71.42 %, 60.19 %, and 89.65 % of azo dyes (methyl orange, malachite green, and crystal violet, respectively) within a span of 14 days with an extensive range of temperatures (15°C-40°C) and pH (5-8) before optimization. The targeted dyes at concentrations of 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, and 500 mg/L were decolorized after optimization. The maximum decolorization of methyl orange (MO), malachite green (MG), and crystal violet (CV) was 88.89 %, 71.09 %, and 92.75 %, respectively, at a dye concentration of 100 mg/L, pH of 6.5, and temperature of 25°C. The fungal isolate Trichoderma asperellum frequently contains chitinases, cellulose, and laccases. The findings of this study validated the potential of TA as an effective biocatalyst for the remediation of methyl orange, malachite green, and crystal violet in textile wastewater. The degradation products showed decreased toxicity to sprout seeds, such as Cicer arietinum (chickpea) and Vigna radiata (mung seeds), when compared to untreated wastewater.

Studying the effects of Aspergillus niger (MF431834) dead biomass on water defluoridation in batch and bed column: Adsorption kinetics, characterization, genotoxicity studies

Fluoride pollution in water caused by biological and anthropogenic activities has been recorded as a significant global issue, posing severe risks to the ecosystem. The present study focuses on the isolated fungus Aspergillus niger (MF431834)-dead biomass served as novel adsorbents for defluorination. The dead fungal mycelium of A. niger was used to study the biosorption of fluoride from an aqueous medium while varying the pH, fluoride concentrations, bio-sorbent concentrations, temperatures, contact periods, and coexisting ions. After the batch experimentation, the maximum fluoride adsorption capacity of 93.85 % was achieved by 0.2 g dosage, a 50-min equilibrium time was predicted as a contact time, considerable fluoride adsorption was observed at 15 mg/L of initial fluoride concentration, the maximum fluoride adsorption of 92 % was achieved by pH 2.0, and NaOH was the best desorption agent. The biosorption of fluoride ion well pseudo-second-order (R20.99) compared to pseudo-first-order and the biosorption equilibrium data were more suited by the Langmuir isotherm than the Freund isotherm model. Thermodynamic parameters such as ΔG°, ΔS°, and ΔH° the biosorption of fluoride ions by endothermic nature. Energy Dispersive Spectroscopy (SEM-EDX) and Fourier transform infrared (FTIR) spectroscopy examine the biosorption of fluoride by fungal bio-sorbent. Fluoride removal was also significantly attained in the column study with bio-sorbent. The findings demonstrated that A. cepa subjected to untreated polluted fluoride water exhibited chromosome abnormalities, including disorganized, disturbed metaphase, chromosomal displacement in anaphase, aberrant telophase, spindle disturbances, and binucleate cells. The highest chromosomal aberrations and mitotic inhibition were found in roots exposed to contaminated fluoride water. The novelty of this research can be used as a biomarker to identify the genotoxic effects of fluoride water contamination on biota. Hence, the native fungus A. niger exhibits as an effective adsorbent for fluoride elimination, being valuable in the theory and practical management of environmental pollution.

Recent advances in the production of malic acid by native fungi and engineered microbes.

Malic acid is mainly produced by chemical methods which lead to various environmental sustainability concerns associated with CO2 emissions and resulting global warming. Since malic acid is naturally synthesized, microorganisms offer an eco-friendly and cost-effective alternative for its production. An additional advantage of microbial production is the synthesis of pure L-form of malic acid. Due to its numerous applications, biotechnologically- produced L-malic acid is a much sought-after platform chemical. Malic acid can be produced by microbial fermentation via oxidative/reductive TCA and glyoxylate pathways. This article elaborates the potential and limitations of high malic acid producing native fungi belonging to Aspergillus, Penicillium, Ustilago and Aureobasidium spp. The utilization of industrial side streams and low value renewable substrates such as crude glycerol and lignocellulosic biomass is also discussed with a view to develop a competitive bio-based production process. The major impediments present in the form of toxic compounds from lignocellulosic residues or synthesized during fermentation along with their remedial measures are also described. The article also focuses on production of polymalic acid from renewable substrates which opens up a cost-cutting dimension in production of this biodegradable polymer. Finally, the recent strategies being employed for its production in recombinant organisms have also been covered.

Viability of Entomopathogenic Fungi in Oil Suspensions and Their Effectiveness against the Agave Pest Scyphophorus acupunctatus under Laboratory Conditions

Oaxaca, Mexico, is home to over 30 species of the genus Agave, and its cultivation is of great economic and social importance for the mezcal industry, which depends on its production. The incidence of the pest Scyphophorus acupunctatus causes severe losses and damage. Agrochemicals are used for its control, but a viable alternative is microbial control. The objectives of this study were to determine the natural occurrence of the entomopathogenic fungi (EPF) Beauveria bassiana and Metarhizium anisopliae, isolated from S. acupunctatus in agave crops, and to evaluate the effect of vegetable oil in water emulsions containing conidia from the native fungi against adults of S. acupunctatus under laboratory conditions. Viability of the fungal isolates was determined at a concentration of ×108 conidia/mL in avocado (Persea americana), mamey (Mammea americana), chia (Salvia hispanica), or olive (Olea europaea) oil in water emulsions at two concentrations, 20% and 40%. The most effective oil emulsion on S. acupunctatus adults was determined under laboratory conditions. Naturally occurring fungi in 900 field collected insects was 2.21%. Ninety-six hours after preparing a 20% emulsion in P. americana oil, B. bassiana and M. anisopliae had a viability of 75% and 66.5%, respectively, while the control conidia suspended in distilled water remained viable for only 48 h. Twenty-four hours after applying M. anisopliae conidia in a 40% P. americana oil emulsion, effectiveness was 100% on S. acupunctatus, followed by M. anisopliae in 20% P. americana oil emulsion with 75% effectiveness. At 72 h post-application, all fungus in oil emulsions achieved an accumulated insect mortality of 100%, while the control showed no effect on adult S. acupunctatus. The most promising combination was 40% P. americana oil emulsion, which achieved 50% viable B. bassiana or M. anisopliae conidia up to 96 h after preparation, and its accumulated effectiveness on S. acupunctatus adults was 87.5% after 24 h.

Transcriptomic Profile of Penicillium digitatum Reveals Novel Aspects of the Mode of Action of the Antifungal Protein AfpB.

Antifungal proteins (AFPs) from filamentous fungi are promising biomolecules to control fungal pathogens. Understanding their biological role and mode of action is essential for their future application. AfpB from the citrus fruit pathogen Penicillium digitatum is highly active against fungal phytopathogens, including its native fungus. Our previous data showed that AfpB acts through a multitargeted three-stage process: interaction with the outer mannosylated cell wall, energy-dependent cell internalization, and intracellular actions that result in cell death. Here, we extend these findings by characterizing the functional role of AfpB and its interaction with P. digitatum through transcriptomic studies. For this, we compared the transcriptomic response of AfpB-treated P. digitatum wild type, a ΔafpB mutant, and an AfpB-overproducing strain. Transcriptomic data suggest a multifaceted role for AfpB. Data from the ΔafpB mutant suggested that the afpB gene contributes to the overall homeostasis of the cell. Additionally, these data showed that AfpB represses toxin-encoding genes, and they suggest a link to apoptotic processes. Gene expression and knockout mutants confirmed that genes coding for acetolactate synthase (ALS) and acetolactate decarboxylase (ALD), which belong to the acetoin biosynthetic pathway, contribute to the inhibitory activity of AfpB. Moreover, a gene encoding a previously uncharacterized extracellular tandem repeat peptide (TRP) protein showed high induction in the presence of AfpB, whereas its TRP monomer enhanced AfpB activity. Overall, our study offers a rich source of information to further advance in the characterization of the multifaceted mode of action of AFPs. IMPORTANCE Fungal infections threaten human health worldwide and have a negative impact on food security, damaging crop production and causing animal diseases. At present, only a few classes of fungicides are available due to the complexity of targeting fungi without affecting plant, animal, or human hosts. Moreover, the intensive use of fungicides in agriculture has led to the development of resistance. Therefore, there is an urgent need to develop antifungal biomolecules with new modes of action to fight human-, animal-, and plant-pathogenic fungi. Fungal antifungal proteins (AFPs) offer great potential as new biofungicides to control deleterious fungi. However, current knowledge about their killing mechanism is still limited, which hampers their potential applicability. AfpB from P. digitatum is a promising molecule with potent and specific fungicidal activity. This study further characterizes its mode of action, opening avenues for the development of new antifungals.

Host population effects on ectomycorrhizal fungi vary between low and high phosphorus soils of temperate rainforests.

Geographic distinctions in the affinity of tree populations for select ectomycorrhizal fungi (EMF) may occur where strong edaphic pressures act on fungal communities and their hosts. We examine this premise for Pseudotsuga menziesii var. menziesii of southwest British Columbia, using ten native seedlots collected from a range of mean annual precipitation (MAP), as a proxy for podzolization extent and phosphorus (P) deficiencies, and evaluated in contrasting low P and high P soils. After two growing seasons, seedling biomass in the high P soil dwarfed that of the low P soil, and better growth rates under high P were detected for populations from very dry and very wet origins. EMF communities on the high P soil displayed more symmetry among host populations than the low P soil (average community dissimilarity of 0.20% vs. 0.39%, respectively). Seedling foliar P% differed slightly but significantly in relation to MAP of origin. EMF species richness varied significantly among host populations but independently of climatic parameters. There were significant shifts in EMF species abundance related to seedlot MAP, particularly on the low P soil where nonlinear relationships were found for Wilcoxina mikolae, Hyaloscypha finlandica, and Rhizopogon villosulus. Despite efforts to enhance colonization by native fungi, the predominance of ruderal EMF species hindered a realistic evaluation of local adaptation among host-fungi populations. Nevertheless, the shifting affinity in taxa abundance and wider community disparity on low P soil reflected the potential for a consequential host genetic effect related to geographical patterns in P availability across temperate rainforests.

New insight into the mechanisms of autochthonous fungal bioaugmentation of phenanthrene in petroleum contaminated soil by stable isotope probing

Autochthonous fungal bioaugmentation (AFB) is considered a reliable bioremediation approach for polycyclic aromatic hydrocarbon (PAH) contamination, but little is known about its mechanisms in contaminated soils. Here, a microcosm experiment was performed to explore the AFB mechanisms associated with two highly efficient phenanthrene degrading agents of fungi (with laccase-producing Scedosporium aurantiacum GIG-3 and non-laccase-producing Aspergillus fumigatus LJD-29), using stable-isotope-probing (SIP) and high-throughput sequencing. The results showed that each fungus markedly improved phenanthrene removal, and microcosms with both fungi exhibited the best phenanthrene removal performance among all microcosms. Additionally, AFB markedly shifted the composition of the microbial community, particularly the phenanthrene-degrading bacterial taxa. Interestingly, based on SIP results, strains GIG-3 and LJD-29 did not assimilate phenanthrene directly during AFB, but instead played key roles in the preliminary decomposition of phenanthrene though secretion of different extracellular enzymes to oxidize the benzene ring (GIG-3 bioaugmentation with laccase, and LJD-29 bioaugmentation with manganese and lignin peroxidases). In addition, all functional degraders directly involved in phenanthrene assimilation were indigenous bacteria, while native fungi rarely participated in the direct phenanthrene mineralization. Our findings provide a new mechanism of AFB with multiple fungi, and support AFB as a promising strategy for the in situ bioremediation of PAH-contaminated soil.

Entomopathogenic fungi for the control of larvae and adults of Aedes aegypti (Diptera: Culicidae) vector of dengue, chikungunya and Zika viruses in Mexico

Objective. To assess larvicide and adulticide activity of different native strains of fungi on Aedes aegypti. Materials and methods. Third instar larvae were exposed for 72 h at a concentration of 1x108 conidia/ml of 15 fungi; only fungi that significantly affected the larvae were evaluated against the adult phase at a concentration of 2x1010 conidia/ml. Mortality readings were performed at 24, 48, and 72 h for larvae, and every day to 30 days for adults. Results. Trichoderma longibrachiatum, Aspergillus aculeatus, and Metarhizium anisopliae had the best larvicidal activity at 24 h of exposure (p<0.05), causing mortalities of 100, 72, and 62%, respectively. Adult mosquitoes were more affected by Gliocladium virens (45% mortality), M. anisopliae (30% mortality), and T. longibrachiatum (23.33% mortality). Conclusion. The larval stage of Ae. aegypti was more susceptible than the adult phase to the pathogenic action of native fungi, with T. longibrachiatum being with the highest virulence.

Purification and characterization of a fungal laccase expressed in Kluyveromyces lactis suitable for baking.

Laccase enzyme can replace chemical additives to improve texture properties and the volume of bread. Laccase encoding gene from Phlebia brevispora, a native fungus from Misiones, Argentina, was expressed in the generally recognized as safe yeast Kluyveromyces lactis. To improve laccase activity, medium conditions were optimized. The use of iron sulfate at a concentration of 1mM led to optimum laccase activity (1289 U·L-1 ) on the fourth day of incubation. SDS-PAGE analysis revealed that the molecular mass of purified laccase was about 180kDa. Optimum pH for the enzyme was 4 and optimum temperature was 40°C. Laccase exhibited high stability at low pH and high temperature. The application of recombinant laccase to bread decreased hardness, gumminess, and chewiness and increased bread volume. Based on these results, recombinant laccase from P. brevispora with improved yield is a good option for application as an improver of the physicochemical quality of bread at the industrial level. Besides, it will allow us to advance toward our goal of developing healthy alternatives for the bakery industry. No previous work has been reported concerning the heterologous expression of the laccase gene native to the province of Misiones, Argentina, with an aim for application in baking. PRACTICAL APPLICATION: Healthy bakeries became a trend in recent years. The use of the laccase enzyme increases the specific volume and decreases the hardness of bread, being thus an alternative for the replacement of chemical additives in the bakery industry.

Integration of agricultural residues as biomass source to saccharification bioprocess and for the production of cellulases from filamentous fungi.

The production of second-generation bioethanol has several challenges, among them finding cheap and efficient enzymes for a sustainable process. In this work, we analyzed two native fungi, Cladosporium cladosporioides and Penicillium funiculosum, as a source of cellulolytic enzyme production, and corn stover, wheat bran, chickpeas, and bean straw as a carbon source in two fermentation systems: submerged and solid fermentation. Corn stover was selected for cellulase production in both fermentation systems, because we found the highest enzymatic activities when carboxymethyl cellulase activity (CMCase) was assessed using CMC as substrate. C. cladosporioides showed the highest CMCase activity (1.6U/mL), while P. funiculosum had the highest filter paper activity (Fpase) (0.39U/mL). The ß-glucosidase activities produced by both fungi were similar in submerged fermentation using corn stover as substrate. Through in-gel zymography, three polypeptides with cellulolytic activities were identified in each fungus: with molecular weights of ~ 38, 45 and 70kDa in C. cladosporioides and ~ 21, 63 and 100kDa in P. funiculosum. The best results for saccharification (10.11g/L of reducing sugars) of diluted acid pretreated corn stover were obtained after 36h of the hydrolytic process at pH 5 and 50°C using the enzyme extract of P. funiculosum. This is the first report of cellulase identification in C. cladosporioides and the saccharification of corn stover using enzymes of this fungus. Enzymatic extracts of C. cladosporioides and P. funiculosum obtained from low-cost lignocellulosic biomass have great potential for use in the production of second-generation bioethanol.

Copper nanoparticles embedded fungal chitosan as a rational and sustainable bionanozyme with robust laccase activity for catalytic oxidation of phenolic pollutants.

Despite their potential for oxidation of persistent environmental pollutants, the development of rational and sustainable laccase nanozymes with efficient catalytic performance remains a challenge. Herein, fungal-produced chitosan-copper (CsCu) is proposed as a rational and sustainable bionanozyme with intrinsic laccase activity. The CsCu nanozyme was prepared by in situ reduction of copper on chitosan extracted from Irpex sp. isolate AWK2 a native fungus, from traditional fermented foods, yielding a low molecular weight chitosan with a 70% degree of deacetylation. Characterizations of the nanozyme using SEM-EDX, XRD, and XPS confirmed the presence of a multi-oxidation state copper on the chitosan matrix which is consistent with the composition of natural laccase. The laccase memetic activity was investigated using 2,4-DP as a substrate which oxidized to form a reddish-pink color with 4-AP (λmax = 510 nm). The CsCu nanozyme showed 38% higher laccase activity than the pristine Cu NPs at pH 9, indicating enhanced activity in the presence of chitosan structure. Further, CsCu showed significant stability in harsh conditions and exhibited a lower Km (0.26 mM) which is competitive with that reported for natural laccase. Notably, the nanozyme converted 92% of different phenolic substrates in 5 h, signifying a robust performance for environmental remediation purposes.

Application of mineral carriers for immobilization of Trichoderma viride

There is no doubt in the fact that preparations containing Trichoderma as the main component are currently a real alternative to synthetic agrochemicals as antagonists of soil-borne plant diseases and growth stimulators. The use of this kind of drugs does not always give sufficiently stable results. In addition, technological problems can often arise, which are associated with the short shelf life of liquid preparations based on Trichoderma. The use of the solid form of the drug often causes the problem of self-inhibition of conidia germination. And therefore a more careful calculation of the optimal dose for each specific strain is required. The development of multifunctional bio-preparations that are more stable in their effectiveness, especially preparations that include Trichoderma, is one of the most important tasks in the system of biotechnology development, which determined the relevance of the research. The aim of the work is to establish the optimal carrier for the immobilization of Trichoderma viride cells and the effect of immobilization on the stability of drugs. It has been experimentally established that the growth and development of the immobilized fungus Trichoderma viride is influenced by the nature and method of modification of the carrier. It was established that when the fungus immobilized on native and modified carriers (particle size 0.5 μm – 2.5 mm) was seeded on a sterile nutrient medium, a decrease in the growth rate of Trichoderma viride was found, compared to the control one where the native fungus was used. It has been proven that the optimal amount of grinding of both native and modified media is – 150 μm – 1.5 mm. It has been found that saponite modified with starch is the optimal carrier for Trichoderma viride immobilization. It has been proven that upon immobilization of the fungus Trichoderma viride, the latter becomes more resistant to the negative factors of the growth environment (antagonistic action of the natural conglomerate of microorganisms residing on spoiled alfalfa hay). Key words: fungi, cell immobilization, carriers, modified starch, humic acids, saponite, zeolite, zeolite-containing basalt tuff.