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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Low density polyethylene degradation by filamentous fungi.

Polyethylene (PE) is the most abundant non-degradable plastic waste, posing a constant and serious threat to the whole ecosystem. In the present study, the fungal community of plastic wastes contaminating a landfill soil has been studied. After 6 months of enrichment, 95 fungi were isolated, mostly belonging to the Ascomycota phylum. They were screened under in vitro condition: most of fungi (97%) were capable of growing in the presence of PE powder (5-10 g L-1) as sole carbon source. Fusarium strains better tolerated high concentration of PE. Up to 13 strains were chosen for further degradation trails, where the process was monitored by respirometry tests and by observing changes in PE chemical and physical structure by FTIR analysis and SEM images. Major results were observed for Fusarium oxysporum, Fusarium falciforme and Purpureocillum lilacinum, as they caused strong oxidation phenomena and changes in the PE film morphology. Results suggested that the initial oxidation mechanisms targeted first the methyl terminal groups. Changes in the infrared spectra were strongly strain-dependent, denoting the activation of different degradation pathways. Through the SEM analysis, the actual damages provoked by fungi were observed, including swellings, pits and furrows, bumps and partial exfoliations. Considering the rising concern about plastic disposal worldwide, the ability of these fungi to colonize PE and utilize it as carbon source is of great interest, as no pretreatments and pro-oxidant stimulants were needed.

Extraction of short chain chitooligosaccharides from fungal biomass and their use as promoters of arbuscular mycorrhizal symbiosis.

Short chain chitooligosaccharides (COs) are chitin derivative molecules involved in plant-fungus signaling during arbuscular mycorrhizal (AM) interactions. In host plants, COs activate a symbiotic signalling pathway that regulates AM-related gene expression. Furthermore, exogenous CO application was shown to promote AM establishment, with a major interest for agricultural applications of AM fungi as biofertilizers. Currently, the main source of commercial COs is from the shrimp processing industry, but purification costs and environmental concerns limit the convenience of this approach. In an attempt to find a low cost and low impact alternative, this work aimed to isolate, characterize and test the bioactivity of COs from selected strains of phylogenetically distant filamentous fungi: Pleurotus ostreatus, Cunninghamella bertholletiae and Trichoderma viride. Our optimized protocol successfully isolated short chain COs from lyophilized fungal biomass. Fungal COs were more acetylated and displayed a higher biological activity compared to shrimp-derived COs, a feature that-alongside low production costs-opens promising perspectives for the large scale use of COs in agriculture.

Genome Sequence of Trichoderma lixii MUT3171, A Promising Strain for Mycoremediation of PAH-Contaminated Sites.

Mono- and polycyclic aromatic hydrocarbons (PAHs) are widespread and recalcitrant pollutants that threaten both environmental and human health. By exploiting the powerful enzymatic machinery of fungi, mycoremediation in contaminated sites aims at removing a wide range of pollutants in a cost-efficient and environmentally friendly manner. Next-generation sequencing (NGS) techniques are powerful tools for understanding the molecular basis of biotransformation of PAHs by selected fungal strains, allowing genome mining to identify genetic features of biotechnological value. Trichoderma lixii MUT3171, isolated from a historically PAH-contaminated soil in Italy, can grow on phenanthrene, as a sole carbon source. Here, we report the draft genome sequence of T. lixii MUT3171 obtained with high-throughput sequencing method. The genome of T. lixii MUT3171 was compared with other 14 Trichoderma genomes, highlighting both shared and unique features that can shed a light on the biotransformation of PAHs. Moreover, the genes potentially involved in the production of important biosurfactants and bioactive molecules have been investigated. The gene repertoire of T. lixii MUT3171 indicates a high degrading potential and provides hints on putative survival strategies in a polluted environment.

Fungal Diversity in the Neptune Forest: Comparison of the Mycobiota of Posidonia oceanica, Flabellia petiolata, and Padina pavonica.

Fungi are widely distributed in the Oceans, interact with other organisms and play roles that range from pathogenic to mutualistic. The present work focuses on the characterization of the cultivable mycobiota associated with the seagrass Posidonia oceanica (L.) Delile collected off the Elba Island (Italy). We identified 102 taxa (mainly Ascomycota) by the mean of a polyphasic approach. Leaves, rhizomes, roots and matte were characterized by unique mycobiota revealing a "plant-part-specificity." The comparison with the mycobiota associated with the green alga Flabellia petiolata and the brown alga Padina pavonica underlined a "substrate specificity." Indeed, despite being part of the same phytocoenosis, these photosynthetic organisms recruit different fungal communities. The mycobiota seems to be necessary for the host's defense and protection, playing, in this way, remarkable ecological roles. Among the 61 species detected in association with P. oceanica (including two species belonging to the newly introduced genus Paralulworthia), 37 were reported for the first time from the Mediterranean Sea.

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.

The role of cosubstrate and mixing on fungal biofilm efficiency in the removal of tannins.

Tannins are polyphenolic compounds produced by plants and they are used in industrial vegetable tanning of leather. Tannins represent one of the low biodegradability substances in tannery wastewaters with high recalcitrant soluble chemical oxygen demand, furthermore high concentration of tannins can inhibit biological treatment. In the present study, four novel rotating submerged packed bed reactors were inoculated with a selected fungal strain to reach a biological degradation of tannins in non-sterile conditions. The selected fungal strain, Aspergillus tubingensis MUT 990, was immobilised in polyurethane foam cubes carriers and inserted inside a submerged rotating cage reactors. The reactors were feed with a solution composed of four tannins: Quebracho (Schinopsis spp.), Wattle (Mimosa spp.), Chestnut (Castanea spp.) and Tara (Caesalpinia spp.). Four reactors with a volume of 4 L each were used, the co-substrate was pure malt extract, the hydraulic retention time was 24 h and the pH setpoint was 5.5. The reactors configuration was chosen to allow the study of the effect of rotation and the co-substrate addition on tannins removal. The experiment lasted two months and it was achieved 80% of chemical oxygen demand and up to 90% dissolved organic carbon removal, furthermore it was detected an important tannase activity.

Ecofriendly laccases treatment to challenge micropollutants issue in municipal wastewaters.

In this study, a multidisciplinary approach investigated the enzymatic degradation of micropollutants in real, not modified, municipal wastewaters of a plant located in Italy. Stir Bar Sorptive Extraction combined to Gas Chromatography-Mass Spectrometric detection (SBSE-GC-MS) was applied to profile targeted pollutants in wastewaters collected after the primary sedimentation (W1) and the final effluent (W2). Fifteen compounds were detected at ng/L - µg/L, including pesticides, personal care products (PCPs) and drugs. The most abundant micropollutants were bis(2-ethylhexyl) phthalate, diethyl phthalate and ketoprofen. Laccases of Trametes pubescens MUT 2400 were very active against all the target micropollutants: except few cases, their concentration was reduced more than 60%. Chemical analysis and environmental risk do not always come together. To verify whether the treated wastewaters can represent a stressor for the aquatic ecosystem, toxicity was also evaluated. Raphidocelis subcapitata and Lepidium sativum tests showed a clear ecotoxicity reduction, even though they did not evenly respond. Two in vitro tests (E-screen test and MELN assay) were used to evaluate the estrogenic activity. Treatments already operating in the plant (e.g. activated sludge) partially reduced the estradiol equivalent concentration, and it was almost negligible after the laccases treatment. The results of this study suggest that laccases of T. pubescens are promising biocatalysts for the micropollutants transformation in wastewaters and surface waters.

Preservation, Characterization and Exploitation of Microbial Biodiversity: The Perspective of the Italian Network of Culture Collections.

Microorganisms represent most of the biodiversity of living organisms in every ecological habitat. They have profound effects on the functioning of any ecosystem, and therefore on the health of our planet and of human beings. Moreover, microorganisms are the main protagonists in food, medical and biotech industries, and have several environmental applications. Accordingly, the characterization and preservation of microbial biodiversity are essential not only for the maintenance of natural ecosystems but also for research purposes and biotechnological exploitation. In this context, culture collections (CCs) and microbial biological resource centres (mBRCs) are crucial for the safeguarding and circulation of biological resources, as well as for the progress of life sciences. This review deals with the expertise and services of CCs, in particular concerning preservation and characterization of microbial resources, by pointing to the advanced approaches applied to investigate a huge reservoir of microorganisms. Data sharing and web services as well as the tight interconnection between CCs and the biotechnological industry are highlighted. In addition, guidelines and regulations related to quality management systems (QMSs), biosafety and biosecurity issues are discussed according to the perspectives of CCs and mBRCs.