The essential role of aggregation for the emulsifying ability of a fungal CYS-rich protein.

Biosurfactants are in demand by the global market as natural commodities suitable for incorporation into commercial products or utilization in environmental applications. Fungi are promising producers of these molecules and have garnered interest also for their metabolic capabilities in efficiently utilizing recalcitrant and complex substrates, like hydrocarbons, plastic, etc. Within this framework, biosurfactants produced by two Fusarium solani fungal strains, isolated from plastic waste-contaminated landfill soils, were analyzed. Mycelia of these fungi were grown in the presence of 5% olive oil to drive biosurfactant production. The characterization of the emulsifying and surfactant capacity of these extracts highlighted that two different components are involved. A protein was purified and identified as a CFEM (common in fungal extracellular membrane) containing domain, revealing a good propensity to stabilize emulsions only in its aggregate form. On the other hand, an unidentified cationic smaller molecule exhibits the ability to reduce surface tension. Based on the 3D structural model of the protein, a plausible mechanism for the formation of very stable aggregates, endowed with the emulsifying ability, is proposed. KEY POINTS: • Two Fusarium solani strains are analyzed for their surfactant production. • A cationic surfactant is produced, exhibiting the ability to remarkably reduce surface tension. • An identified protein reveals a good propensity to stabilize emulsions only in its aggregate form.

Estrogenic activity in wastewater treatment plants through in vitro effect-based assays: Insights into extraction phase.

Effluents of wastewater treatment plants can abundantly spread endocrine disrupting chemicals in the environment. To improve water quality monitoring, the use of effect-based tools that measure estrogenic activity has been suggested, however their results could be influenced by different factors. This study compared the estrogenic activity of wastewater samples extracted with two stationary phases and tested with two in vitro effect-based assays to investigate whether and how stationary phases and assays could influence biomonitoring data. During four seasonal periods, the effluents of six WWTPs located in northern Italy were sampled. After the extraction using two different stationary phases (HLB, C18), the samples (n = 72) were tested using two effect-based assays: a gene reporter luciferase assay on mammalian cells (MELN) and yeast estrogen screen assay (YES). The results showed that estrogenic activity of HLB extracts was significantly different from the activity of C18 extracts, suggesting that extraction phase can influence biomonitoring data. Moreover, the estrogenic activity was overall higher using gene reporter MELN assay than using YES assay, suggesting that, due to difference in cell membrane permeability and metabolic activation, the applied cell model can affect the biomonitoring results. Finally, from the comparison between the activity of the final effluent and the environmentally safe estrogenic levels in surface waters, MELN data suggested that the activity of this effluent may pose an environmental risk, while YES data showed that it should not be considered a threat to the receiving surface waters. This study pointed out that a standardized approach is needed to assess the estrogenic activity of waters; it reported important data to select the most suitable stationary phase for samples extraction (samples extracted with C18 sorbent showed higher estradiol equivalent concentration values) and the most appropriate bioassay (gene reporter luciferase MELN assay was more sensitive than YES assay) to assess the environmental risk, thus protecting human health.

New Phenylspirodrimanes from the Sponge-Associated Fungus Stachybotrys chartarum MUT 3308.

Two phenylspirodrimanes, never isolated before, stachybotrin J (1) and new stachybocin G (epi-stachybocin A) (2), along with the already reported stachybotrin I (3), stachybotrin H (4), stachybotrylactam (5), stachybotrylactam acetate (6), 2α-acetoxystachybotrylactam acetate (7), stachybotramide (8), chartarlactam B (9), and F1839-J (10) were isolated from the sponge-associated fungus Stachybotrys chartarum MUT 3308. Their structures were established based on extensive spectrometric (HRMS) and spectroscopic (1D and 2D NMR) analyses. Absolute configurations of the stereogenic centers of stachybotrin J (1), stachybocin G (2), and stachybotrin I (3), were determined by comparison of their experimental circular dichroism (CD) spectra with their time-dependent density functional theory (TD-DFT) circular dichroism (ECD) spectra. The putative structures of seventeen additional phenylspirodrimanes were proposed by analysis of their respective MS/MS spectra through a Feature-Based Molecular Networking approach. All the isolated compounds were evaluated for their cytotoxicity against five aggressive cancer cell lines (MP41, 786, 786R, CAL33, and CAL33RR), notably including two resistant human cancer cell lines (786R, CAL33RR), and compounds 5, 6, and 7 exhibited cytotoxicity with IC50 values in the range of 0.3-2.2 µM.

Knufia obscura sp. nov. and Knufia victoriae sp. nov., two new species from extreme environments.

Six strains of black meristematic fungi were isolated from Antarctic soils, gasoline car tanks and from the marine alga Flabellia petiolata. These fungi were characterized by morphological, physiological and phylogenetic analyses. According to the maximum-likelihood analysis reconstructed with ITS and LSU sequences, these strains belonged to the genus Knufia. Knufia obscura sp. nov. (holotype CBS 148926) and Knufia victoriae sp. nov. (holotype CBS 149015) are proposed as two novel species and descriptions of their morphological, physiological and phylogenetic features are presented. Based on the maximum-likelihood analyses, K. obscura was closely related to Knufia hypolithi (99 % bootstrap support), while K. victoriae clustered in the clade of Knufia cryptophialidica and Knufia perfecta (93 % bootstrap support). Knufia victoriae, recorded in Antarctic soil samples, had a psychrophilic behaviour, with optimal growth between 10 and 15 °C and no growth recorded at 20 °C. Knufia obscura, from a gasoline car tank and algae, displayed optimal growth between 20 and 25 °C and was more tolerant to salinity than K. victoriae.

Cerato-Platanins from Marine Fungi as Effective Protein Biosurfactants and Bioemulsifiers.

Two fungal strains, Aspergillus terreus MUT 271 and Trichoderma harzianum MUT 290, isolated from a Mediterranean marine site chronically pervaded by oil spills, can use crude oil as sole carbon source. Herein, these strains were investigated as producers of biosurfactants, apt to solubilize organic molecules as a preliminary step to metabolize them. Both fungi secreted low molecular weight proteins identified as cerato-platanins, small, conserved, hydrophobic proteins, included among the fungal surface-active proteins. Both proteins were able to stabilize emulsions, and their capacity was comparable to that of other biosurfactant proteins and to commercially available surfactants. Moreover, the cerato-platanin from T. harzianum was able to lower the surface tension value to a larger extent than the similar protein from A. terreus and other amphiphilic proteins from fungi. Both cerato-platanins were able to make hydrophilic a hydrophobic surface, such as hydrophobins, and to form a stable layer, not removable even after surface washing. To the best of our knowledge, the ability of cerato-platanins to work both as biosurfactant and bioemulsifier is herein demonstrated for the first time.

Wastewater-Agar as a selection environment: A first step towards a fungal in-situ bioaugmentation strategy.

Viable and metabolically active fungi in toxic mixed liquors, treating landfill leachates and municipal wastewaters, were identified by culture depending methods. A selective culture medium consisting of wastewater and agar (WA) restrained fungi that could be randomly present (94% of the 51 taxa retrieved on WA were sample-specific), overcoming the problem of fast growing fungi or mycoparasite fungi. Moreover, WA allowed the isolation of fungi with a possible role in the degradation of pollutants typically present in the two wastewaters. Phoma medicaginis var. medicaginis, Chaetomium globosum, and Geotrichum candidum were mainly found in municipal wastewater, whereas Pseudallescheria boydii, Scedosporium apiospermum, Aspergillus pseudodeflectus, and Scopulariopsis brevicaulis were typical of landfill leachate.

Biotransformation of industrial tannins by filamentous fungi.

Tannins are secondary metabolites that are widely distributed in the plant kingdom. They act as growth inhibitors for many microorganisms: they are released upon microbial attack, helping to fight infection in plant tissues. Extraction of tannins from plants is an active industrial sector with several applications, including oenology, animal feeding, mining, the chemical industry, and, in particular, the tanning industry. However, tannins are also considered very recalcitrant pollutants in wastewater of diverse origin. The ability to grow on plant substrates rich in tannins and on industrial tannin preparations is usually considered typical of some species of fungi. These organisms are able to tolerate the toxicity of tannins thanks to the production of enzymes that transform or degrade these substrates, mainly through hydrolysis and oxidation. Filamentous fungi capable of degrading tannins could have a strong environmental impact as bioremediation agents, in particular in the treatment of tanning wastewaters.

Fungi from industrial tannins: potential application in biotransformation and bioremediation of tannery wastewaters.

Tannins are a complex family of polyphenolic compounds, widely distributed in the plant kingdom where they act as growth inhibitors towards many microorganisms including bacteria, yeasts, and fungi. Tannins are one of the major components of tannery wastewaters and may cause serious environmental pollution. In the present study, four different tannins (the hydrolysable chestnut ellagitannin and tara gallotannin and the condensed quebracho and wattle tannins) were characterized from a mycological point of view with the aim of selecting fungal strains capable of growing in the presence of high tannin concentration and thus potentially useful in industrial biotransformations of these compounds or in the bioremediation of tannery wastewaters. A total of 125 isolates of filamentous fungi belonging to 10 species and four genera (Aspergillus, Paecilomyces, Penicillium, and Talaromyces) were isolated from the tannin industrial preparations. Miniaturized biotransformation tests were set up with 10 fungal strains and the high-performance liquid chromatography (HPLC) analysis pointed out a strong activity of all the tested fungi on both chestnut and tara tannins. Two strains (Aspergillus tubingensis MUT 990 and Paecilomyces variotii MUT 1125), tested against a real tannery wastewater, were particularly efficient in chemical oxygen demand (COD) and tannin removal (> 60%), with a detoxification above 74%. These results indicate that these fungi are potentially exploitable in the treatment of tannery wastewaters.

Stimulation of laccases from Trametes pubescens: Use in dye decolorization and cotton bleaching.

The production of laccases from Trametes pubescens was investigated along with the role of nutrients and elicitors. Copper proved to be a fundamental inducer, although productivity yields were consistently enhanced only in the presence of additional compounds (textile dyes). Using a central composite design, the optimal culture condition was examined, by taking into consideration the three distinct variables and their combinatorial effect. The 290 U ml(-1) of laccases were produced after setting nitrogen, copper, and reactive blue 19 concentration; in a bioreactor, activity recovery was lower (90 U ml(-1)) and pellet morphology was different. The activity of the laccase crude extract was maximal at 60°C and stable for 14 h at 50°C and for 2 months at pH 6 and room temperature. The biotechnological potential was assessed, confirming the capacity to decolorize single or mixed solutions of textile dyes and to enhance the whitening yield of raw cotton fibers, working in synergism with the conventional H2O2-based method.

A guide to the use of bioassays in exploration of natural resources.

Bioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are critical for optimal bioprospecting. They are used both in the early stages of compounds isolation/purification/identification, and in later stages to evaluate their safety and efficacy. In this review, we provide a comprehensive overview of the most common bioassays used in the discovery and development of new bioactive compounds with a focus on marine bioresources. We present a comprehensive list of practical considerations for selecting appropriate bioassays and discuss in detail the bioassays typically used to explore antimicrobial, antibiofilm, cytotoxic, antiviral, antioxidant, and anti-ageing potential. The concept of quality control and bioassay validation are introduced, followed by safety considerations, which are critical to advancing bioactive compounds to a higher stage of development. We conclude by providing an application-oriented view focused on the development of pharmaceuticals, food supplements, and cosmetics, the industrial pipelines where currently known marine natural products hold most potential. We highlight the importance of gaining reliable bioassay results, as these serve as a starting point for application-based development and further testing, as well as for consideration by regulatory authorities.

A Further Step toward Sustainable Development: The Case of the Edible Mushroom Supply Chain.

This study provides an accurate economic characterization of the supply of edible mushrooms throughout Italy within the European context to fill the relevant research gap and highlight barriers and opportunities that are consistent with the Sustainable Development Goals. Italian companies operating in this field were identified and economically characterized using the Chamber of Commerce's Register of Companies. A qualitative web content analysis was then conducted to extract information about the marketed products, mushroom species, and retail channels, as well as the adopted certifications. The obtained data were quantitatively analyzed through descriptive statistics and multiple correspondence analysis. The Italian market is concentrated in northern areas of the country, and the limited company size indicates fragmentation at the production level, which led to Italy not being competitive enough and, thus, heavily rely on imports. Production is limited to less than 10 species, and innovative mushroom-based products, such as burgers, have shown a limited presence on the market, although they are gaining market share online. The novelty of growing kits highlights the potential to use food production waste to create fungal substrates. Investments in training new mushroom growers and studying new formulations and new fungal species are needed; these investments could allow greater market differentiation and be a good opportunity to promote local economies and create new job opportunities, thus meeting the requirements for sustainable development.

The genomes of Scedosporium between environmental challenges and opportunism.

Emerging fungal pathogens are a global challenge for humankind. Many efforts have been made to understand the mechanisms underlying pathogenicity in bacteria, and OMICs techniques are largely responsible for those advancements. By contrast, our limited understanding of opportunism and antifungal resistance is preventing us from identifying, limiting and interpreting the emergence of fungal pathogens. The genus Scedosporium (Microascaceae) includes fungi with high tolerance to environmental pollution, whilst some species can be considered major human pathogens, such as Scedosporium apiospermum and Scedosporium boydii. However, unlike other fungal pathogens, little is known about the genome evolution of these organisms. We sequenced two novel genomes of Scedosporium aurantiacum and Scedosporium minutisporum isolated from extreme, strongly anthropized environments. We compared all the available Scedosporium and Microascaceae genomes, that we systematically annotated and characterized ex novo in most cases. The genomes in this family were integrated in a Phylum-level comparison to infer the presence of putative, shared genomic traits in filamentous ascomycetes with pathogenic potential. The analysis included the genomes of 100 environmental and clinical fungi, revealing poor evolutionary convergence of putative pathogenicity traits. By contrast, several features in Microascaceae and Scedosporium were detected that might have a dual role in responding to environmental challenges and allowing colonization of the human body, including chitin, melanin and other cell wall related genes, proteases, glutaredoxins and magnesium transporters. We found these gene families to be impacted by expansions, orthologous transposon insertions, and point mutations. With RNA-seq, we demonstrated that most of these anciently impacted genomic features responded to the stress imposed by an antifungal compound (voriconazole) in the two environmental strains S. aurantiacum MUT6114 and S. minutisporum MUT6113. Therefore, the present genomics and transcriptomics investigation stands on the edge between stress resistance and pathogenic potential, to elucidate whether fungi were pre-adapted to infect humans. We highlight the strengths and limitations of genomics applied to opportunistic human pathogens, the multifactoriality of pathogenicity and resistance to drugs, and suggest a scenario where pressures other than anthropic contributed to forge filamentous human pathogens.

Tourism affects microbial assemblages in show caves.

Anthropogenic disturbance on natural ecosystems is growing in frequency and magnitude affecting all ecosystems components. Understanding the response of different types of biocoenosis to human disturbance is urgently needed and it can be achieved by adopting a metacommunity framework. With the aid of advanced molecular techniques, we investigated sediment communities of Fungi, Bacteria and Archaea in four Italian show caves, aiming to disentangle the effects induced by tourism on their diversity and to highlight changes in the driving forces that shape their community composition. We modelled diversity measures against proxies of tourism pressure. With this approach we demonstrate that the cave tourism has a direct effect on the community of Bacteria and an indirect influence on Fungi and Archaea. By analysing the main driving forces influencing the community composition of the three microbial groups, we highlighted that stochastic factors override dispersal-related processes and environmental selection in show caves compared to undisturbed areas. Thanks to this approach, we provide new perspectives on the dynamics of microbial communities under human disturbance suggesting that a proper understanding of the underlying selective mechanisms requires a comprehensive and multi-taxonomic approach.

Influence of culture medium on fungal biomass composition and biosorption effectiveness.

Zygomycetes such as Cunninghamella elegans seem to be promising biosorbents for pollutants removal from wastewaters because of their particular cell wall characteristics. In this article the effect of ten culture media on C. elegans biomass composition was investigated by means of Fourier transform infra red spectroscopy (FTIR). Biomasses grown on starches from potatoes and cereals were characterised by high amount of chitin and polysaccharides, the glucose gave rise to a biomass rich in acidic polysaccharides and lipids. By contrast, biomasses grown on corn steep liquor were poor in acidic polysaccharides and, when N sources and micronutrients were added, rich in proteins. The lipid content of the biomass generally increased by halving nutrients. Biosorption yields of these biomasses towards four wastewater models were assessed in terms of colour, salts and toxicity reduction. The biomasses rich in proteins and acid polysaccharides were less effective in removing reactive and direct dyes, whereas those rich in cationic polysaccharides showed a higher affinity for these dyes. Both chromatography and FTIR analyses showed that biomasses cultured in halved C and N had the highest affinity for salts. The wastewaters detoxification was quite always achieved, with values often lower that the Italian legal threshold limit.

Dihydroauroglaucin Isolated from the Mediterranean Sponge Grantia compressa Endophyte Marine Fungus Eurotium chevalieri Inhibits Migration of Human Neuroblastoma Cells.

Cancer cell migration is a hallmark of the aggressiveness and progression of malignancies such as high-risk neuroblastoma. Given the lack of effective therapeutic solutions to counteract cancer progression, basic research aims to identify novel bioactive molecules with inhibitory potential on cancer cell migration. In this context, this work investigated the role of members of the salicylaldehyde secondary metabolite set from the sponge endophyte fungus Eurotium chevalieri MUT 2316 as potential inhibitors of human neuroblastoma SH-SY5Y cell migration. Since tetrahydroauroglaucin (TAG) and dihydroauroglaucin (DAG) were isolated in large amounts, both were evaluated for their anticancer properties towards SH-SY5Y cells. Both molecules were found to be non-cytotoxic by MTT assay and cytofluorimetric analysis. Moreover, DAG showed efficacy in inhibiting the highly migratory phenotype of SH-SY5Y cells by wound healing assay; whereas TAG, although structurally similar to DAG, showed no anti-migratory effect. Therefore, this work provides good reasons to conduct further in vitro and in vivo studies focusing on DAG as a potentially useful migrastatic natural marine molecule.

The Culturable Mycobiota of Sediments and Associated Microplastics: From a Harbor to a Marine Protected Area, a Comparative Study.

Fungi are an essential component of marine ecosystems, although little is known about their global distribution and underwater diversity, especially in sediments. Microplastics (MPs) are widespread contaminants worldwide and threaten the organisms present in the oceans. In this study, we investigated the fungal abundance and diversity in sediments, as well as the MPs, of three sites with different anthropogenic impacts in the Mediterranean Sea: the harbor of Livorno, the marine protected area "Secche della Meloria"; and an intermediate point, respectively. A total of 1526 isolates were cultured and identified using a polyphasic approach. For many of the fungal species this is the first record in a marine environment. A comparison with the mycobiota associated with the sediments and MPs underlined a "substrate specificity", highlighting the complexity of MP-associated fungal assemblages, potentially leading to altered microbial activities and hence changes in ecosystem functions. A further driving force that acts on the fungal communities associated with sediments and MPs is sampling sites with different anthropogenic impacts.

Cunninghamella elegans biomass optimisation for textile wastewater biosorption treatment: an analytical and ecotoxicological approach.

The effect of pre-treatments on the composition of Cunninghamella elegans biomass and on its biosorption yields in the treatment of simulated textile wastewaters was investigated. The inactivated biomass was subjected to physical treatments, such as oven drying and lyophilisation, and chemical treatments using acid or alkali. The wastewater colour, COD and toxicity variations were evaluated. The lyophilisation sped up the biosorption process, whereas the chemical pre-treatment changed the affinity of biomass for different dyes. The alkali per-treated biomass achieved the highest COD reduction in the treatment of alkali wastewaters, probably because no release of alkali-soluble biomass components occurred under the alkaline pH conditions. Accordingly, only the acid pre-treated biomass decreased the COD of the acidic effluent. The ecotoxicity test showed significant toxicity reduction after biosorption treatments, indicating that decolourisation corresponds to an actual detoxification of the treated wastewaters. Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analyses of biomasses allowed highlighting their main chemical and physical properties and the changes induced by the different pre-treatments, as well as the effect of the chemical species adsorbed from wastewaters.

Evaluation of toxicity, genotoxicity and environmental risk of simulated textile and tannery wastewaters with a battery of biotests.

Textile and tannery wastewaters are complex mixtures of toxic pollutants and only a battery of ecotoxicity tests can assess their potential environmental impact and the actual effectiveness of alternative treatments. In this work the toxicity of four simulated textile and tannery wastewaters was evaluated by means of a battery of seven bioassays, using organisms that belong to different trophic levels. Moreover, since the outputs of the bioassay battery were quite difficult to compare, a novel synthetic index for environmental risk assessment was applied to the outputs of the test battery. All four simulated wastewaters were very toxic but they showed no mutagenic effect. The alga Pseudokirchneriella subcapitata was the most sensitive organism. In addition, the use of two mathematical models pointed out the interaction effect between dyes and salts, which resulted in a synergistic effect of wastewater toxicity.

Trichoderma harzianum cerato-platanin enhances hydrolysis of lignocellulosic materials.

Considering its worldwide abundance, cellulose can be a suitable candidate to replace the fossil oil-based materials, even if its potential is still untapped, due to some scientific and technical gaps. This work offers new possibilities demonstrating for the first time the ability of a cerato-platanin, a small fungal protein, to valorize lignocellulosic Agri-food Wastes. Indeed, cerato-platanins can loosen cellulose rendering it more accessible to hydrolytic attack. The cerato-platanin ThCP from a marine strain of Trichoderma harzianum, characterized as an efficient biosurfactant protein, has proven able to efficiently pre-treat apple pomace, obtaining a sugar conversion yield of 65%. Moreover, when used in combination with a laccase enzyme, a notable increase in the sugar conversion yield was measured. Similar results were also obtained when other wastes, coffee silverskin and potato peel, were pre-treated. With respect to the widespread laccase pre-treatments, this new pre-treatment approach minimizes process time, increasing energy efficiency.

Insights on Lulworthiales Inhabiting the Mediterranean Sea and Description of Three Novel Species of the Genus Paralulworthia.

The order Lulworthiales, with its sole family Lulworthiaceae, consists of strictly marine genera found on a wide range of substrates such as seagrasses, seaweeds, and seafoam. Twenty-one unidentified Lulworthiales were isolated in previous surveys aimed at broadening our understanding of the biodiversity hosted in the Mediterranean Sea. Here, these organisms, mostly found in association with Posidonia oceanica and with submerged woods, were examined using thorough multi-locus phylogenetic analyses and morphological observations. Maximum-likelihood and Bayesian phylogeny based on nrITS, nrSSU, nrLSU, and four protein-coding genes led to the introduction of three novel species of the genus Paralulworthia: P. candida, P. elbensis, and P. mediterranea. Once again, the marine environment is a confirmed huge reservoir of novel fungal lineages with an under-investigated biotechnological potential waiting to be explored.