Use Of Filamentous Fungi Research Articles

Decolorization and Degradation of azo dyes in textile wastewater effluent by Aspergillus Niger and Aspergillus ochraceous

In this study, fungi were isolated from contaminated soil collected from textile wool factories in Thi Qar Governorate. The results showed a high potential for removing Methylene Blue dye by Aspergillus niger and Aspergillus ochraceous. The removal process was conducted under optimized conditions, including different concentrations of the azo dye, pH levels, carbon sources, and nitrogen sources. The results indicated that the fungi Aspergillus niger and Aspergillus. Ochraceus were the most common isolates. Both fungi were treated with Methylene Blue dye in solid and liquid media, and both showed the ability to degrade this dye. However, Aspergillus niger demonstrated higher activity in color removal compared to Aspergillus ochraceous, with a diameter of (9) cm in solid medium for Aspergillus niger and (8) cm for Aspergillus. ochraceus. These results were confirmed by statistical analysis, which showed significant differences between these fungi and concentrations. The decolorization percentage was confirmed by Fourier Transform Infrared (FTIR) spectroscopy of which showed complete disappearance of peaks at 500 nm and at 1500 nm, indicates the degradation of dyes due to fungal activity, The technology also confirmed that a pH of 7 was the optimal pH for dye biodegradation compared to pH 4. This was observed through the dry weight of the fungi, with Aspergillus niger reaching to (2.35) and Aspergillus ochraceous reaching to (2.15) at pH 7. Also, in pH 4, the dry weight of Aspergillus niger and Aspergillus. ochraceus reached (2.17) and (2.05) respectively. Furthermore, the study confirmed the potential use of filamentous fungi in treating dye-contaminated water, regardless of their pH values.

Anticancer Asparaginases: Perspectives in Using Filamentous Fungi as Cell Factories

The enzyme L-asparaginase (L-asparagine amidohydrolase) catalyzes the breakdown of L-asparagine into aspartate and ammonia, which leads to an anti-neoplastic activity stemming from its capacity to deplete L-asparagine concentrations in the bloodstream, and it is therefore used in cases of acute lymphoblastic leukemia (ALL) to inhibit malignant cell growth. Nowadays, this anti-cancer enzyme, largely produced by Escherichia coli, is well established on the market. However, E. coli L-asparaginase therapy has side effects such as anaphylaxis, coagulation abnormality, low plasma half-life, hepatotoxicity, pancreatitis, protease action, hyperglycemia, and cerebral dysfunction. This review provides a perspective on the use of filamentous fungi as alternative cell factories for L-asparaginase production. Filamentous fungi, such as various Aspergillus species, have superior protein secretion capacity compared to yeast and bacteria and studies show their potential for the future production of proteins with humanized N-linked glycans. This article explores the past and present applications of this important enzyme and discusses the prospects for using filamentous fungi to produce safe eukaryotic asparaginases with high production yields.

Plant Growth Promoting Filamentous Fungi and Their Application in the Fertilization of Pastures for Animal Consumption

The diversity of fungi in different terrestrial and aquatic ecosystems has made it possible to explore their use as important tools in promoting plant growth and in managing plant diseases given their high potential to replace the use of synthetic chemical products (fertilizers and pesticides). Therefore, this review compiles information on the use of filamentous fungi in promoting plant growth, highlighting the most studied fungal genera for this purpose, such as Trichoderma, Penicillum, and Aspergillus. In addition, information is compiled on the promotion of forage grass growth using filamentous fungi, which could be a sustainable and lower-cost alternative in producing pastures to help raise animals.

Assessing Edible Filamentous Fungal Carriers as Cell Supports for Growth of Yeast and Cultivated Meat.

The growth and activity of adherent cells can be enabled or enhanced through attachment to a solid surface. For food and beverage production processes, these solid supports should be food-grade, low-cost, and biocompatible with the cell of interest. Solid supports that are edible can be a part of the final product, thus simplifying downstream operations in the production of fermented beverages and lab grown meat. We provide proof of concept that edible filamentous fungal pellets can function as a solid support by assessing the attachment and growth of two model cell types: yeast, and myoblast cells. The filamentous fungus Aspergillus oryzae was cultured to produce pellets with 0.9 mm diameter. These fugal pellets were inactivated by heat or chemical methods and characterized physicochemically. Chemically inactivated pellets had the lowest dry mass and were the most hydrophobic. Scanning electron microscope images showed that both yeast and myoblast cells naturally adhered to the fungal pellets. Over 48 h of incubation, immobilized yeast increased five-fold on active pellets and six-fold on heat-inactivated pellets. Myoblast cells proliferated best on heat-treated pellets, where viable cell activity increased almost two-fold, whereas on chemically inactivated pellets myoblasts did not increase in the cell mass. These results support the use of filamentous fungi as a novel cell immobilization biomaterial for food technology applications.

Bioinspired Hydrophilic and Oleophilic Absorption Media from Biotemplated Fungi

Fungi are an incredibly diverse biological kingdom with organisms that have a wide range of morphologies, properties, and structures. Previous research has investigated the use of filamentous fungi, which have naturally porous structures created by hyphal filaments, as a means of phytoremediation. This study uses these natural fungal structures to create bioinspired materials that capture both the structure and functional absorption properties of fungi. Three types of filamentous fungi with different hyphal structures (monomitic, dimitic, and trimitic) are used as organic templates to create inorganic media using two different biotemplating methods to create silica and hybrid samples. Characterization of these samples is completed using scanning electron microscope imaging, chemical characterization, nanoindentation, and hydrophilic and oleophilic absorption tests. Biotemplated samples have similar structures as their organic templates, but contained silica, which is not present in natural, dehydrated fungal samples. Fourier transform infrared analysis shows better cross‐linking in the hybrid samples, which also have higher mechanical resistance than the silica samples. Absorption testing demonstrates that silica samples are closest to mimicking the absorption properties of natural, dehydrated samples. Of the three hyphal structures, the monomitic samples show the greatest increase in mechanical properties and maintenance of absorption properties when biotemplated.

Application of filamentous fungi in microalgae-based wastewater remediation for biomass harvesting and utilization: From mechanisms to practical application

The model of microalgae-based wastewater remediation for nutrient recovery and biomass production has been intensively studied, but high cost of biomass harvesting jeopardizes the industrial application of this model. To support the implementation of this model and the sustainable development, this work introduces the use of filamentous fungi in microalgae-based wastewater remediation for biomass harvesting and utilization. First, this work describes the research progresses of co-cultivation of fungi and microalgae and fungi-assisted algae harvesting. Second, mechanisms and key parameters of fungi-assisted microalgae harvesting are discussed. Third, an in-depth discussion of co-cultivating filamentous fungi and microalgae in wastewater for nutrient removal and biomass collection is provided. Fourth, this work introduces some high-value components, which are of importance to the economic benefits and industrial application of harvested algal-fungal biomass, in microalgae and filamentous fungi. By the end of this paper, some problems associated with the co-cultivation of algal-fungal consortia in wastewater and fungi-assisted microalgae harvesting are listed and potential solutions to these problems are proposed. It is expected that this work can help readers understand the achievements in the research of fungi-assisted microalgae and the importance of this novel model to biomass harvesting and utilization.

Kinetic modeling of fungal biomass growth and lactic acid production in Rhizopus oryzae fermentation by using grape stalk as a solid substrate.

This work proposes the use of grape stalk, a residue from the wine industry, as a solid substrate for R. oryzae NCIM 1299 fermentation to obtain lactic acid. The use of filamentous fungi on grape stalk as a fermentative solid substrate has not been studied yet making this study an innovative research work. Grape Stalk was chemically characterized, and according to its properties, it was deemed as an innovative material with great potential to be used as a fermentation substrate. R. oryzae growth kinetic determination needs the measurement of the biomass dry weight of fungus versus time, but the filamentous fungi penetration into the solid substrate makes the quantitative biomass removal from the substrate practically impossible. Therefore, fungal growth on membrane experiment was performed making gravimetric quantification of fungal biomass feasible. Two mediums were used comparatively, potato dextrose agar and a medium designed with agar and grape stalk for 5 days at 30 °C and 50%RH. Logistic, First Order and First Order Plus Dead Time models were used to model biomass growth, where these last two models have not been previously used for microbial kinetics studies. Growth kinetic parameters ( X max , μ max , T p and T 0 ) were found for all models and the First Order model showed the best fit with an R 2 equal to 91.84%. The Luedeking-Piret model was used to model lactic acid production and provided the parameters Y p/x and m p , with an R 2 equal to 90.05%. The kinetic parameters found here are also of great interest for further bioreactor designs. • Grape stalk was characterized as a fungal fermentation substrate. • Grape stalk, raw material in fungal fermentation trials for Lactic acid production. • Cell biomass and lactic acid production kinetic modeling by membrane experiments. • First Order model for fungal biomass growth showed best data fitting. • The Luedeking Piret model parameters were obtained for Lactic Acid production.

Use of filamentous fungi as biocatalysts in the oxidation of 5-(hydroxymethyl)furfural (HMF)

The objective of this study was to explore the use of filamentous fungi as oxidative biocatalysts. To that end, filamentous fungal whole-cells, comprising five different species were employed in the oxidation of 5-(hydroxymethyl)furfural (HMF). Two species (A. niger and T. reesei), which demonstrated superior HMF conversion and product accumulation, were further evaluated for growth on alternative substrates (e.g. pentoses) as well as for use in a chemo-biocatalytic reaction system. Concerning the latter, the two whole-cell biocatalysts were coupled with laccase/TEMPO in a one-pot reaction designed to enable catalysis of the three oxidative steps necessary to convert HMF into 2,5-furandicarboxylic acid (FDCA), a compound with immense potential in the production of sustainable and eco-friendly polymers. Ultimately, the optimal one-pot chemo-biocatalytic cascade system, comprising 1 g/L T. reesei whole cells coupled with 2.5 mM laccase and 20 mol% TEMPO, achieved a molar yield of 88% after 80 h.

Cleaning the Cellular Factory-Deletion of McrA in Aspergillus oryzae NSAR1 and the Generation of a Novel Kojic Acid Deficient Strain for Cleaner Heterologous Production of Secondary Metabolites.

The use of filamentous fungi as cellular factories, where natural product pathways can be refactored and expressed in a host strain, continues to aid the field of natural product discovery. Much work has been done to develop host strains which are genetically tractable, and for which there are multiple selectable markers and controllable expression systems. To fully exploit these strains, it is beneficial to understand their natural metabolic capabilities, as such knowledge can rule out host metabolites from analysis of transgenic lines and highlight any potential interplay between endogenous and exogenous pathways. Additionally, once identified, the deletion of secondary metabolite pathways from host strains can simplify the detection and purification of heterologous compounds. To this end, secondary metabolite production in Aspergillus oryzae strain NSAR1 has been investigated via the deletion of the newly discovered negative regulator of secondary metabolism, mcrA (multicluster regulator A). In all ascomycetes previously studied mcrA deletion led to an increase in secondary metabolite production. Surprisingly, the only detectable phenotypic change in NSAR1 was a doubling in the yields of kojic acid, with no novel secondary metabolites produced. This supports the previous claim that secondary metabolite production has been repressed in A. oryzae and demonstrates that such repression is not McrA-mediated. Strain NSAR1 was then modified by employing CRISPR-Cas9 technology to disrupt the production of kojic acid, generating the novel strain NSARΔK, which combines the various beneficial traits of NSAR1 with a uniquely clean secondary metabolite background.

Biological Control of Plant-Parasitic Nematodes by Filamentous Fungi Inducers of Resistance: Trichoderma, Mycorrhizal and Endophytic Fungi.

Plant-parasitic-nematodes represent a major threat to the agricultural production of different crops worldwide. Due to the high toxicity of chemical nematicides, it is necessary to develop new control strategies against nematodes. In this respect, filamentous fungi can be an interesting biocontrol alternative. The genus Trichoderma, mycorrhizal and endophytic fungi are the main groups of filamentous fungi studied and used as biological control agents (BCAs) against nematodes as resistance inducers. They are able to reduce the damage caused by plant-parasitic nematodes directly by parasitism, antibiosis, paralysis and by the production of lytic enzymes. But they also minimize harm by space and resource-competition, by providing higher nutrient and water uptake to the plant, or by modifying the root morphology, and/or rhizosphere interactions, that constitutes an advantage for the plant-growth. Besides, filamentous fungi are able to induce resistance against nematodes by activating hormone-mediated (salicylic and jasmonic acid, strigolactones among others) plant-defense mechanisms. Additionally, the alteration of the transport of chemical defense components through the plant or the synthesis of plant secondary metabolites and different enzymes can also contribute to enhancing plant defenses. Therefore, the use of filamentous fungi of the mentioned groups as BCAs is a promising durable biocontrol strategy in agriculture against plant-parasitic nematodes.

Constructive use of filamentous fungi to remove pharmaceutical substances from wastewater

The removal efficiency of diclofenac and ketoprofen for five fungal strains was examined. The synergistic effect between selected fungal strains using synthetic wastewater media was studied using both individual and mixed strains. The mixed cultures had no additional effect in reducing diclofenac and ketoprofen. In contrary, fungi removed pharmaceutical substances faster when growing individually. The results showed that T. versicolor monoculture removed diclofenac completely (>99.9 %) in non-sterile municipal wastewater within three hours of incubation and continued to show complete reduction within 14 days using fungal biofilm carriers. The mechanism of removal was proposed based on the laccase enzyme production and biosorption study which revealed that T. versicolor could used in both processes. Additionally, this study demonstrated that T. versicolor could be stable in the biofilm carriers, and thus able to compete with other microorganisms in wastewater to be a promising candidate to remove diclofenac from municipal wastewater during the treatment process.

Mutation of a Conserved Alanine Residue in Transcription Factor AraR Leads to Hyperproduction of α‐l‐Arabinofuranosidases in Penicillium oxalicum

α-l-Arabinofuranosidases are important in the degradation of plant polysaccharides and are used in several industrial processes. Although the use of filamentous fungi for the production of α-l-arabinofuranosidases is widely reported, there are few reports on strain engineering for enhanced production of these enzymes by fungi. In this study, the function of transcription factor AraR in l-arabinose release and catabolism by the fungus Penicillium oxalicum (P. oxalicum) is investigated. Also, a mutant of AraR, AraRA731V , is constructed to improve the production of α-l-arabinofuranosidases on the basis of the sequence homology between AraR and the xylanolytic gene activator XlnR. The AraRA731V -overexpressing strain can synthesize α-l-arabinofuranosidase in the absence of an inducer and shows a 54.1-fold increase in α-l-arabinofuranosidase production and a 7.4-fold increase in α-galactosidase production in the medium containing wheat bran. Determination of the transcript abundances of lignocellulolytic enzyme genes reveals significant upregulation of multiple α-l-arabinofuranosidase genes and downregulation of some cellulolytic and xylanolytic enzyme genes in the engineered strain relative to its parent. Taken together, the results suggest the conserved regulatory function of AraR in the family Trichocomaceae and provide a strategy for engineering fungal strains for enhanced α- l-arabinofuranosidase production.

Optimization of pH, temperature and carbon source for bioleaching of heavy metals by Aspergillus flavus isolated from contaminated soil

Abstract Soil contamination with heavy metal content is a growing concern throughout the world as a result of industrial, mining, agricultural and domestic activities. Fungi are the most common and efficient group of heavy metal resistant microbe family which have potential for metal bioleaching. The use of filamentous fungi in bioleaching of heavy metals from contaminated soil has been developed recently. The current study intends to isolate a strain with the ability to degrade the pH value of the liquid medium. Identification results based on morphological and molecular biological analysis gave a 98% match to Aspergillus flavus. Batch experiments were conducted to select the optimal conditions for bioleaching process which indicated that 130 mg/ L sucrose, neutral pH and temperature of 30°C were more suitable during 15-day bioleaching experiments using A. flavus. In one-step bioleaching, the bioleaching efficiencies were 18.16% for Pb, 39.77% for Cd and 58.22% for Zn+2, while two-step bioleaching showed efficiencies of 16.91% for Pb, 49.66% for Cd and 65.73% for Zn+2. Overall, this study indicates that bioleaching of heavy metals in contaminated soil using A. flavus has the potential for contaminated soil remediation.

Growth of the fungus Chaetomium aureum in the presence of lead: implications in bioremediation

Filamentous fungi were isolated from semiarid soil in which the lead (Pb) concentrations were above regulatory limits as determined by the Brazilian standards. Among these fungi, four isolates were tested for their abilities to grow in a culture medium containing lead nitrate. Chaetomium aureum was the species that presented a comparatively better performance including mycelial growth in a lead-containing medium, ability to use pectin as a carbon source, as well as basophilic and thermotolerant properties. The C. aureum inoculum with either activated or inactivated native microbiota was able to reduce the free Pb in soil (61 and 54%, respectively) after 60 days of inoculation. Although the mechanism involved in decreasing water-soluble and exchangeable lead concentrations in soil has not been studied, either the processes of biosorption by organic molecules (called oosporein, produced by this species) or fungal mineral transformation is among its possible explanations. These findings support the use of filamentous fungi as potential tools for the bioremediation of contaminated sites and highlight C. aureum as a promising tool for environmental biotechnology. In addition, the ability of this fungus (collected in a lead-contaminated area) to grow efficiently in the presence of this metal indicates that the bioprospecting strategy of the indigenous microbiota should be encouraged, as they appear to be more likely to succeed.

Light-regulated promoters for tunable, temporal, and affordable control of fungal gene expression.

Regulatable promoters are important genetic tools, particularly for assigning function to essential and redundant genes. They can also be used to control the expression of enzymes that influence metabolic flux or protein secretion, thereby optimizing product yield in bioindustry. This review will focus on regulatable systems for use in filamentous fungi, an important group of organisms whose members include key research models, devastating pathogens of plants and animals, and exploitable cell factories. Though we will begin by cataloging those promoters that are controlled by nutritional or chemical means, our primary focus will rest on those who can be controlled by a literal flip-of-the-switch: promoters of light-regulated genes. The vvd promoter of Neurospora will first serve as a paradigm for how light-driven systems can provide tight, robust, tunable, and temporal control of either autologous or heterologous fungal proteins. We will then discuss a theoretical approach to, and practical considerations for, the development of such promoters in other species. To this end, we have compiled genes from six previously published light-regulated transcriptomic studies to guide the search for suitable photoregulatable promoters in your fungus of interest.

Fungal biodiversity to biotechnology.

Fungal habitats include soil, water, and extreme environments. With around 100,000 fungus species already described, it is estimated that 5.1 million fungus species exist on our planet, making fungi one of the largest and most diverse kingdoms of eukaryotes. Fungi show remarkable metabolic features due to a sophisticated genomic network and are important for the production of biotechnological compounds that greatly impact our society in many ways. In this review, we present the current state of knowledge on fungal biodiversity, with special emphasis on filamentous fungi and the most recent discoveries in the field of identification and production of biotechnological compounds. More than 250 fungus species have been studied to produce these biotechnological compounds. This review focuses on three of the branches generally accepted in biotechnological applications, which have been identified by a color code: red, green, and white for pharmaceutical, agricultural, and industrial biotechnology, respectively. We also discuss future prospects for the use of filamentous fungi in biotechnology application.

A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.

The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, we have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. The system is simple and versatile, as RNA guided mutagenesis can be achieved by transforming a target fungus with a single plasmid. The system currently contains four CRISPR-Cas9 vectors, which are equipped with commonly used fungal markers allowing for selection in a broad range of fungi. Moreover, we have developed a script that allows identification of protospacers that target gene homologs in multiple species to facilitate introduction of common mutations in different filamentous fungi. With these tools we have performed RNA-guided mutagenesis in six species of which one has not previously been genetically engineered. Moreover, for a wild-type Aspergillus aculeatus strain, we have used our CRISPR Cas9 system to generate a strain that contains an AACU_pyrG marker and demonstrated that the resulting strain can be used for iterative gene targeting.

Potential bioremediation of mercury-contaminated substrate using filamentous fungi isolated from forest soil

The use of filamentous fungi in bioremediation of heavy metal contamination has been developed recently. This research aims to observe the capability of filamentous fungi isolated from forest soil for bioremediation of mercury contamination in a substrate. Six fungal strains were selected based on their capability to grow in 25 mg/L Hg2+-contaminated potato dextrose agar plates. Fungal strain KRP1 showed the highest ratio of growth diameter, 0.831, thus was chosen for further observation. Identification based on colony and cell morphology carried out by 18S rRNA analysis gave a 98% match to Aspergillus flavus strain KRP1. The fungal characteristics in mercury(II) contamination such as range of optimum pH, optimum temperature and tolerance level were 5.5–7 and 25–35°C and 100 mg/L respectively. The concentration of mercury in the media affected fungal growth during lag phases. The capability of the fungal strain to remove the mercury(II) contaminant was evaluated in 100 mL sterile 10 mg/L Hg2+-contaminated potato dextrose broth media in 250 mL Erlenmeyer flasks inoculated with 108 spore/mL fungal spore suspension and incubation at 30°C for 7 days. The mercury(II) utilization was observed for flasks shaken in a 130 r/min orbital shaker (shaken) and non-shaken flasks (static) treatments. Flasks containing contaminated media with no fungal spores were also provided as control. All treatments were done in triplicate. The strain was able to remove 97.50% and 98.73% mercury from shaken and static systems respectively. A. flavus strain KRP1 seems to have potential use in bioremediation of aqueous substrates containing mercury(II) through a biosorption mechanism.

Characterization of Fungal Morphology using Digital Image Analysis Techniques

The use of filamentous fungi for the production of commercially important products is old but keeps increasing during the last decades. New classes of compounds are being added in the list of products of fungal fermentations as a result of progress in methodologies and applications of biotechnology. Fungi are morphologically complex organisms that differ in structure throughout their life cycle. In submerged fermentation fungal morphology may take distinct forms ranging from dispersed filaments to densely interwoven masses of mycelium known as pellets. Each morphological form has each own characteristics that have a critical impact on the overall process outcome. Dispersed growth results in highly viscous broths with pseudoplastic behavior that have a negative impact on mass and energy transfer rates resulting in higher energy input requirements. Due to the high industrial relevance of fungal morphology there has been a substantial development of tools and techniques to characterize morphology and extract quantitative information that can be used in process control and optimization studies. Digital image analysis is the state of the art method to characterize and quantify fungal morphology in the developmental process from spores to filamentous structures to pellets. The progress made in the area since the 1990s, when the first image analysis methods were reported, is discussed in detail throughout the review.

Simple and efficient recycling of fungal selectable marker genes with the Cre-loxP recombination system via anastomosis

Reverse-genetics analysis has played a significant role in advancing fungal biology, but is limited by the number of available selectable marker genes (SMGs). The Cre-loxP recombination system has been adapted for use in filamentous fungi to overcome this limitation. Expression of the Cre recombinase results in excision of an integrated SMG that is flanked by loxP sites, allowing a subsequent round of transformation with the same SMG. However, current protocols for regulated expression or presentation of Cre require multiple time-consuming steps. During efforts to disrupt four different RNA-dependent RNA polymerase genes in a single strain of the chestnut blight fungus Cryphonectria parasitica, we tested whether Cre could successfully excise loxP-flanked SMGs when provided in trans via anastomosis. Stable Cre-producing donor strains were constructed by transformation of wild-type C. parasitica strain EP155 with the Cre-coding domain under the control of a constitutive promoter. Excision of multiple loxP-flanked SMGs was efficiently achieved by simply pairing the Cre-donor strain and the loxP-flanked SMGs-transformed recipient strain and recovering mycelia from the margin of the recipient colony near the anastomosis zone. This method was shown to be as efficient as and much less time consuming than excision by transformation-mediated expression of Cre. It also allows unlimited recycling of loxP-flanked SMGs and the generation of disruption mutant strains that are free of any foreign gene. The successful application of this method to Metarhizium robertsii suggests potential use for optimizing reverse-genetics analysis in a broad range of filamentous fungi.