Bioprospecting of Microalgae Isolated from the Adriatic Sea: Characterization of Biomass, Pigment, Lipid and Fatty Acid Composition, and Antioxidant and Antimicrobial Activity.

Marine microalgae and cyanobacteria are sources of diverse bioactive compounds with potential biotechnological applications in food, feed, nutraceutical, pharmaceutical, cosmetic and biofuel industries. In this study, five microalgae, Nitzschia sp. S5, Nanofrustulum shiloi D1, Picochlorum sp. D3, Tetraselmis sp. Z3 and Tetraselmis sp. C6, and the cyanobacterium Euhalothece sp. C1 were isolated from the Adriatic Sea and characterized regarding their growth kinetics, biomass composition and specific products content (fatty acids, pigments, antioxidants, neutral and polar lipids). The strain Picochlorum sp. D3, showing the highest specific growth rate (0.009 h-1), had biomass productivity of 33.98 ± 0.02 mg L-1 day-1. Proteins were the most abundant macromolecule in the biomass (32.83-57.94%, g g-1). Nanofrustulum shiloi D1 contained significant amounts of neutral lipids (68.36%), while the biomass of Picochlorum sp. D3, Tetraselmis sp. Z3, Tetraselmis sp. C6 and Euhalothece sp. C1 was rich in glycolipids and phospholipids (75%). The lipids of all studied microalgae predominantly contained unsaturated fatty acids. Carotenoids were the most abundant pigments with the highest content of lutein and neoxanthin in representatives of Chlorophyta and fucoxanthin in strains belonging to the Bacillariophyta. All microalgal extracts showed antioxidant activity and antimicrobial activity against Gram-negative E. coli and S. typhimurium and Gram-positive S. aureus.

The Cultivation Method Affects the Transcriptomic Response of Aspergillus niger to Growth on Sugar Beet Pulp.

In nature, filamentous fungi are exposed to diverse nutritional sources and changes in substrate availability. Conversely, in submerged cultures, mycelia are continuously exposed to the existing substrates, which are depleted over time. Submerged cultures are the preferred choice for experimental setups in laboratory and industry and are often used for understanding the physiology of fungi. However, to what extent the cultivation method affects fungal physiology, with respect to utilization of natural substrates, has not been addressed in detail. Here, we compared the transcriptomic responses of Aspergillus niger grown in submerged culture and solid culture, both containing sugar beet pulp (SBP) as a carbon source. The results showed that expression of CAZy (Carbohydrate Active enZyme)-encoding and sugar catabolic genes in liquid SBP was time dependent. Moreover, additional components of SBP delayed the A. niger response to the degradation of pectin present in SBP. In addition, we demonstrated that liquid cultures induced wider transcriptome variability than solid cultures. Although there was a correlation regarding sugar metabolic gene expression patterns between liquid and solid cultures, it decreased in the case of CAZyme-encoding genes. In conclusion, the transcriptomic response of A. niger to SBP is influenced by the culturing method, limiting the value of liquid cultures for understanding the behavior of fungi in natural habitats. IMPORTANCE Understanding the interaction between filamentous fungi and their natural and biotechnological environments has been of great interest for the scientific community. Submerged cultures are preferred over solid cultures at a laboratory scale to study the natural response of fungi to different stimuli found in nature (e.g., carbon/nitrogen sources, pH). However, whether and to what extent submerged cultures introduce variation in the physiology of fungi during growth on plant biomass have not been studied in detail. In this study, we compared the transcriptomic responses of Aspergillus niger to growth on liquid and solid cultures containing sugar beet pulp (a by-product of the sugar industry) as a carbon source. We demonstrate that the transcriptomic response of A. niger was highly affected by the culture condition, since the transcriptomic response obtained in a liquid environment could not fully explain the behavior of the fungus in a solid environment. This could partially explain the differences often observed between the phenotypes on plates compared to liquid cultures.

Solid-State Cultivation of Aspergillus niger-Trichoderma reesei from Sugarcane Bagasse with Vinasse in Bench Packed-Bed Column Bioreactor.

Solid-state cultivation (SSC) is microbial growth on solid supports under limited water conditions. Citric acid is a microbial aerobic metabolic product with several industrial applications, with production potential that can be obtained by SSF. Several wastes from agro-industries are used in SSF, such as sugarcane bagasse and vinasse. Cultures of mixed fungi or co-cultures are used in this SSF in order to complement the inoculum's xylanolytic enzymes for action on the lignocellulosic material (bagasse). Thus, this study aims to evaluate the effect of inoculum (Aspergillus niger and Trichoderma reesei consortium) in the production of citric acid from sugarcane bagasse impregnated with vinasse using bench packed-bed reactors (PBR). The results show the importance of T. reesei and A. niger in inoculum at a ratio of 50:50 and 25:75, suggesting the use of solid support due to the complementation of the hydrolytic enzymes. The highest concentration of citric acid, approximately 1000 mg L-1, was obtained for 100 mm of bed height in 48 and 72 h, with maximum glucose yield in citric acid (2.2 mg citric acid mg glucose-1). kLa indicates that maintaining solid moisture and liquid film thickness is important to keep the oxygen transfer in SSC.

Repeated Exposure of Aspergillus niger Spores to the Antifungal Bacterium Collimonas fungivorans Ter331 Selects for Delayed Spore Germination.

The bacterial strain Collimonas fungivorans Ter331 (CfTer331) inhibits mycelial growth and spore germination in Aspergillus niger N402 (AnN402). The mechanisms underlying this antagonistic bacterial-fungal interaction have been extensively studied, but knowledge on the long-term outcome of this interaction is currently lacking. Here, we used experimental evolution to explore the dynamics of fungal adaptation to recurrent exposure to CfTer331. Specifically, five single-spore isolates (SSIs) of AnN402 were evolved under three selection scenarios in liquid culture, i.e., (i) in the presence of CfTer331 for 80 growth cycles, (ii) in the absence of the bacterium for 80 cycles, and (iii) in the presence of CfTer331 for 40 cycles and then in its absence for 40 cycles. The evolved SSI lineages were then evaluated for phenotypic changes from the founder fungal strain, such as germinability with or without CfTer331. The analysis showed that recurrent exposure to CfTer331 selected for fungal lineages with reduced germinability and slower germination, even in the absence of CfTer331. In contrast, when AnN402 evolved in the absence of the bacteria, lineages with increased germinability and faster germination were favored. SSIs that were first evolved in the presence of CfTer331 and then in its absence showed intermediate phenotypes but overall were more similar to SSIs that evolved in the absence of CfTer331 for 80 cycles. This suggests that traits acquired from exposure to CfTer331 were reversible upon removal of the selection pressure. Overall, our study provides insights into the effects on fungi from the long-term coculture with bacteria. IMPORTANCE The use of antagonistic bacteria for managing fungal diseases is becoming increasingly popular, and thus there is a need to understand the implications of their long-term use against fungi. Most efforts have so far focused on characterizing the antifungal properties and mode of action of the bacterial antagonists, but the possible outcomes of the persisting interaction between antagonistic bacteria and fungi are not well understood. In this study, we used experimental evolution in order to explore the evolutionary aspects of an antagonistic bacterial-fungal interaction, using the antifungal bacterium Collimonas fungivorans and the fungus Aspergillus niger as a model system. We show that evolution in the presence or absence of the bacteria selects for fungal lineages with opposing and conditionally beneficial traits, such as slow and fast spore germination, respectively. Overall, our studies reveal that fungal responses to biotic factors related to antagonism could be to some extent predictable and reversible.

Loss of function of the carbon catabolite repressor CreA leads to low but inducer-independent expression from the feruloyl esterase B promoter in Aspergillus niger.

OBJECTIVE: With the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB, a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter. RESULT: PfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively. The genetic screen on the non-inducing carbon source D-fructose yielded in total 111 trans-acting mutants. The genome of one of the mutants was sequenced and revealed several SNPs, including a point mutation in the creA gene encoding a transcription factor known to be involved in carbon catabolite repression. Subsequently, all mutants were analyzed for defects in carbon catabolite repression by determining sensitivity towards allyl alcohol. All except four of the 111 mutants were sensitive to allyl alcohol, indicating that the vast majority of the mutants are defective in carbon catabolite repression. The creA gene of 32 allyl alcohol sensitive mutants was sequenced and 27 of them indeed contained a mutation in the creA gene. Targeted deletion of creA in the reporter strain confirmed that the loss of CreA results in constitutive expression from the faeB promoter. CONCLUSION: Loss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.

Dynamic response of Aspergillus niger to periodical glucose pulse stimuli in chemostat cultures.

In industrial large-scale bioreactors, microorganisms encounter heterogeneous substrate concentration conditions, which can impact growth or product formation. Here we carried out an extended (12 h) experiment of repeated glucose pulsing with a 10-min period to simulate fluctuating glucose concentrations with Aspergillus niger producing glucoamylase, and investigated its dynamic response by rapid sampling and quantitative metabolomics. The 10-min period represents worst-case conditions, as in industrial bioreactors the average cycling duration is usually in the order of 1 min. We found that cell growth and the glucoamylase productivity were not significantly affected, despite striking metabolomic dynamics. Periodical dynamic responses were found across all central carbon metabolism pathways, with different time scales, and the frequently reported ATP paradox was confirmed for this A. niger strain under the dynamic conditions. A thermodynamics analysis revealed that several reactions of the central carbon metabolism remained in equilibrium even under periodical dynamic conditions. The dynamic response profiles of the intracellular metabolites did not change during the pulse exposure, showing no significant adaptation of the strain to the more than 60 perturbation cycles applied. The apparent high tolerance of the glucoamylase producing A. niger strain for extreme variations in the glucose availability presents valuable information for the design of robust industrial microbial hosts.

Metabolomic and secretomic approach to the resistance features of the fungus Aspergillus niger IOC 4687 to copper stress.

Metabolomic and secretomic analyses of Aspergillus niger IOC 4687 indicated the features of resistance of this strain to copper stress. To investigate the metabolites produced under oxidative stress conditions, gas chromatography-mass spectrometry analysis was performed. The secretome principal component analysis results showed that mannitol could be the main metabolite responsible for conferring resistance to the fungus, and gluconic acid is the possible cause of copper desorption because of its chelating ability. The meta-analysis of the metabolome of A. niger IOC 4687 indicated that a low concentration of sorbitol and ribonolactone during growth may be an indicator of oxidative stress.

Light Signaling Regulates Aspergillus niger Biofilm Formation by Affecting Melanin and Extracellular Polysaccharide Biosynthesis.

Light is an important signal source in nature, which regulates the physiological cycle, morphogenetic pathways, and secondary metabolites of fungi. As an external pressure on Aspergillus niger, light signaling transmits stress signals into the cell via the mitogen-activated protein kinase (MAPK) signaling pathway. Studying the effect of light on the biofilm of A. niger will provide a theoretical basis for light in the cultivation of filamentous fungi and industrial applications. Here, the characterization of A. niger biofilm under different light intensities confirmed the effects of light signaling. Our results indicated that A. niger intensely accumulated protective mycelial melanin under light illumination. We also discovered that the RlmA transcription factor in the MAPK signaling pathway is activated by light signaling to promote the synthesis of melanin, chitin, and other exopolysaccharides. However, the importance of melanin to A. niger biofilm is rarely reported; therefore, we knocked out key genes of the melanin biosynthetic pathway-Abr1 and Ayg1 Changes in hydrophobicity and electrostatic forces resulted in the decrease of biofilm caused by the decrease of melanin in mutants.IMPORTANCE As an important industrial filamentous fungus, Aspergillus niger can perceive light. The link between light signaling and A. niger biofilm is worthy of further study since reports are lacking in this area. This study found that light signaling promotes biofilm production in A. niger, wherein melanin plays an important role. It was further discovered that the RlmA transcription factor in the mitogen-activated protein kinase (MAPK) signaling pathway was mediated by light signaling to promote the synthesis of melanin and extracellular polysaccharides. These findings set the stage for light signal regulation of biofilm in filamentous fungi and provide a theoretical basis for the development of a new light-controlled biofilm method to improve biofilm-based industrial fermentation.

Continuous production of fructooligosaccharides by recycling of the thermal-stable ß-fructofuranosidase produced by Aspergillus niger.

OBJECTIVE: To achieve continuous production of fructooligosaccharides (FOS) by recycling of the mycelial cells containing the thermal-stable ß-fructofuranosidase in Aspergillus niger without immobilization. RESULTS: The thermal-stable ß-fructofuranosidase FopA-V1 was successfully expressed in A. niger ATCC 20611 under the control of the constitutive promoter PgpdA. The engineered A. niger strain FV1-11 produced the ß-fructofuranosidase with improved thermostability, which remained 91.2% of initial activity at 50 °C for 30 h. Then its mycelial ß-fructofuranosidase was recycled for the synthesis of FOS. It was found that the enzyme still had 79.3% of initial activity after being reused for six consecutive cycles, whereas only 62.3% ß-fructofuranosidase activity was detected in the parental strain ATCC 20611. Meanwhile, the FOS yield of FV1-11 after six consecutive cycles reached 57.1% (w/w), but only 51.0% FOS yield was detected in ATCC 20611. CONCLUSIONS: The thermal-stable ß-fructofuranosidase produced by A. niger can be recycled to achieve continuous synthesis of FOS with high efficiency, providing a powerful and economical strategy for the industrial production of FOS.

Production and characterization of a novel, thermotolerant fungal phytase from agro-industrial byproducts for cattle feed.

OBJECTIVE: The application of phytases helps in releasing bound phosphorus and other nutrients in cattle feed eventually reducing the need for supplementations. However, high production cost owing to the unavailability of cheaper sources of phytases has limited their usage in developing countries. Herein, firstly isolation, identification of a phytase from fungal isolate, Aspergillus niger NT7 was carried out followed by optimizing of all production parameters, through solid-state fermentation (SSF). Secondly, crude phytase was characterized and potential applicability of crude phytase was evaluated for dephytinization of wheat bran. RESULTS: The highest phytase production (208.30 ± 0.22 U/gds) was achieved using wheat bran as cheap agro-industrial substrate for SSF. The various physiological parameters were optimized including inoculum age and level (3-day old inoculum and 15 × 107 spores/ml), temperature (35 °C), a moistening agent (distilled water), medium pH (5), and supplementation of various biochemicals like sugar (Mannitol), nitrogen (ammonium sulphate) and detergent (Tween 80). Process optimization through one variable at a time (OVAT) approach increased the difference in productivity to more than 200%. The crude phytase of A. niger NT7 was thermostable, with optimal activity at 60 °C and also displayed optimal activity over a broad range of acidic pH. Further, enhancement in phytase activity was found specifically in the presence of Ca2+, Zn2+, and Co2+ ions, while other metal ions including Fe2+, Fe3+, Mn2+, Mg2+and Cu2+ inhibited its activity. Finally, the phytase showed efficient and sustained release of inorganic phosphate, proteins, and reducing sugars (> 60 h) from livestock feed. CONCLUSION: Overall, our report highlights the production of an efficient and thermotolerant phytase with potential as a low-cost animal feed supplement.

Caprylic acid enhances hydroxyhexylitaconic acid production in Aspergillus niger S17-5.

AIM: Aspergillus niger S17-5 produces two alkylitaconic acids, 9-hydroxyhexylitaconic acid (9-HHIA) and 10-hydroxyhexylitaconic acid (10-HHIA), which have cytotoxic and polymer building block properties. In this study, we characterized the production of 9-HHIA and 10-HHIA by addition of their expected precursor, caprylic acid, to a culture of A. niger S17-5, and demonstrated batch fermentation of 9-HHIA and 10-HHIA in a jar fermenter with DO-stat. METHODS AND RESULTS: Production titres of 9-HHIA and 10-HHIA from 3% glucose in a flask after 25 days cultivation were 0·35 and 1·01 g l-1 respectively. Addition of 0·22 g l-1 of caprylic acid to a suspension of resting cells of A. niger S17-5 led to 32% enhancement of total 9-HHIA and 10-HHIA production compared to no addition. No enhancement of the production of 9-HHIA or 10-HHIA by the addition of oxaloacetic acid was observed. Addition of caprylic acid to the culture at mid-growth phase was more suitable for 9-HHIA and 10-HHIA production due to less cell growth inhibition by caprylic acid. DO-stat batch fermentation with 3% glucose and 14·4 g l-1 of caprylic acid in a 1·5 l jar fermenter resulted in the production titres of 9-HHIA and 10-HHIA being 0·48 and 1·54 g l-1 respectively after 10 days of cultivation. CONCLUSIONS: Addition of caprylic acid to the culture of A. niger S17-5 enhances 9-HHIA and 10-HHIA production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that 9-HHIA and 10-HHIA are synthesized with octanoyl-CoA derived from caprylic acid, and that the supply of octanoyl-CoA is a rate-limiting step in 9-HHIA and 10-HHIA production. To the best of our knowledge, this is the first report regarding the fermentation of naturally occurring itaconic acid derivatives in a jar fermenter.

Determination of mold contamination using ergosterol imprinted particles.

Ergosterol is a key biochemical marker for fungal mycelial growth. In this study, molecularly ergosterol imprinted particles (Erg-MIPs) were newly synthesized for the selective detection of ergosterol in mold samples. Erg-MIPs were characterized via scanning electron microscopy, swelling studies, and surface area measurements. Maximum selective ergosterol adsorption achieved as 28.50 mg/g Erg-MIP. Selectivity studies showed that Erg-MIPs adsorbed Erg 2.01 and 3.27 times higher than that of cholesterol and stigmasterol, respectively. Erg adsorption from Aspergillus niger was found as 23.87 mg/g. Reusability of Erg-MIPs was studied and decrease in Erg adsorption capacity of the particles was negligible (3%). Erg-MIPs are good affinity materials for the selective Erg detection from food samples, prior to use in food industry.

Synthesis, characterization, antimicrobial, antioxidant and computational evaluation of N-acyl-morpholine-4-carbothioamides.

The present research paper reports the convenient synthesis, successful characterization, in vitro antibacterial, antifungal, antioxidant potency and biocompatibility of N-acyl-morpholine-4-carbothioamides (5a-5j). The biocompatible derivatives were found to be highly active against the tested bacterial and fungal strains. Moreover, some of the screened N-acyl-morpholine-4-carbothioamides exhibited excellent antioxidant potential. Docking simulation provided additional information about possibilities of their inhibitory potential against RNA. It has been predicted by in silico investigation of the binding pattern that compounds 5a and 5j can serve as the potential surrogate for design of novel and potent antibacterial agents. The results for the in vitro bioassays were promising with the identification of compounds 5a and 5j as the lead and selective candidate for RNA inhibition. Results of the docking computations further ascertained the inhibitory potential of compound 5a. Based on the in silico studies, it can be suggested that compounds 5a and 5j can serve as a structural model for the design of antibacterial agents with better inhibitory potential. Binding mode of compound 5j inside the active site of RNA in 3D space. 5j displayed highest antibacterial potential than the reference drug ampicillin with ZOI 10.50 mm against Staphylococcus aureus. 5j also displayed highest antifungal potential than the reference drug amphotericin B with ZOI 18.20 mm against Fusarium solani.

Tolerance of three fungal species to lithium and cobalt: Implications for bioleaching of spent rechargeable Li-ion batteries.

AIMS: This paper aims to quantify the growth and organic acid production of Aspergillus niger, Penicillium chrysogenum and Penicillium simplicissimum when these fungi are exposed to varying levels of lithium (Li) and cobalt (Co). The study also tests whether pre-exposing the fungi to these metals enables the fungi to develop tolerance to Li or Co. METHODS AND RESULTS: When cultures of A. niger, P. chrysogenum or P. simplicissimum were exposed to 250 mg l-1 of Li or Co, biomass production and excretion of organic acids were significantly inhibited after 5 days of growth compared to cultures grown in the absence of these metals. Pre-exposing cultures of A. niger to 250 mg l-1 of Li or Co for 20 days significantly increased biomass production when the fungus was subsequently sub-cultured into 250 or 500 mg l-1 of Li or Co. However, pre-exposure of P. chrysogenum or P. simplicissimum to 250 mg l-1 of Li or Co for 20 days did not increase biomass production. CONCLUSIONS: Aspergillus niger, but not the Penicillium species, developed tolerance to Li and to Co during the 20-day pre-exposure period. Therefore, processes that utilize fungal bioleaching with A. niger to mobilize and recover valuable metals such as Li or Co should consider a pre-exposure step for fungi to improve their tolerance to metal toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: Fungi may have the ability to extract valuable metals such as Li and Co from spent rechargeable batteries. However, the toxicity of the extracted metals can inhibit fungal growth and organic acid production. Pre-exposure to metals may alleviate toxicity for some fungal species. This knowledge can be used to improve the design of bioleaching protocols, increasing the potential for fungal bioleaching to become an economical and environmentally friendly method of recovering Li and Co from spent batteries.

Biomass of unconventional plants from Brazilian semiarid as substrate for hydrolytic enzymes production by Aspergillus niger under solid and submerged fermentation

Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C

Validation of a vapor-phase advanced oxidation process for inactivating Listeria monocytogenes, its surrogate Lactobacillus fructivorans, and spoilage molds associated with green or red table grapes.

A method based on vapor-phase advanced oxidation process (AOP) for decontaminating red or green grapes was validated for inactivating Listeria monocytogenes and spoilage molds. A Central Composite Design (CCD) and Response Surface Methodology (RSM) were applied to determine the contribution of UV-C (254 nm) dose, hydrogen peroxide, and ozone concentration on the lethality toward Aspergillus niger spores (biodensiometer) and changes to the grape quality (firmness and color over 14-day post-treatment storage at 4 °C). A high UV-C dose (>129 mJ/cm2 ) or >4.0 % v/v hydrogen peroxide induced-blistering and darkening of grapes at the end of the storage period. Yet, an optimized AOP treatment (with regards to preserving grape quality) was derived to be 1.3% v/v hydrogen peroxide (5 mL/10 berries) with 9-mg ozone gas and a UV-C dose of 123 mJ/cm2 (10 s at UV-C intensity of 12 mW/cm2 ). A predictive model was constructed and verified based on the log reduction of A. niger spores and changes in quality characteristics of red grapes. The optimal AOP treatment supported a 1.6-log CFU/g reduction of Aspergillus spores and decreased L. monocytogenes counts by 3.92 ± 0.17 and 4.77 ± 0.30 log CFU/g on green and red grapes, respectively, that were not significantly different to the surrogate, Lactobacillus fructivorans. There was no significant difference in the reduction of L. monocytogenes with grapes arranged in a single or double layer. Botrytis cinerea counts were reduced by 1.08 to 1.35 log CFU/g using the optimized AOP treatment with no change in grape color or firmness during storage. A sensory panel could not differentiate AOP-treated grapes from nontreated controls although 3 of 15 panelists did note subtle flavor notes. PRACTICAL APPLICATION: Postharvest washing of fresh produce has limited efficacy in removing foodborne pathogens and spoilage microbes. This is especially relevant to berries, such as grapes, that are susceptible to spoilage following washing. The vapor-phase AOP treatment provides a supplemental or alternative approach for produce decontamination. However, the operating parameters need to be optimized to ensure that decontamination of grapes is not at the expense of quality. In the current study, this was achieved by ensuring a balance between hydrogen peroxide, ozone, and UV-C dose that form the elements of an AOP treatment.

Enhanced bio-recovery of aluminum from low-grade bauxite using adapted fungal strains.

Filamentous fungi have been proved to have a pronounced capability to recover metals from mineral ores. However, the metal recovery yield is reduced due to toxic effects triggered by various heavy metals present in the ore. The current study highlights the fungal adaptations to the toxic effects of metals at higher pulp densities for the enhanced bio-recovery of aluminum from low-grade bauxite. In the previous studies, a drastic decrease in the aluminum dissolution was observed when the bauxite pulp density was increased from 1 to 10% (w/v) due to the high metal toxicity and low tolerance of Aspergillus niger and Penicillium simplicissium to heavy metals. These fungi were adapted in order to increase heavy metal tolerance of these fungal strains and also to get maximum Al dissolution. A novel approach was employed for the adaptation of fungal strains using a liquid growth medium containing 5% bauxite pulp density supplemented with molasses as an energy source. The mycelia of adapted strains were harvested and subsequently cultured in a low-cost oat-agar medium. Batch experiments were performed to compare the aluminum leaching efficiencies in the direct one-step and the direct two-step bioleaching processes. FE-SEM analysis revealed the direct destructive and corrosive action by the bauxite-tolerant strains due to the extension and penetration of the vegetative mycelium filaments into the bauxite matrix. XRD analysis of the bioleached bauxite samples showed a considerable decline in oxide minerals such as corundum and gibbsite. Results showed a high amount of total Al (≥ 98%) was successfully bioleached and solubilized from low-grade bauxite by the adapted fungal strains grown in the presence of 5% pulp density and molasses as a low-cost substrate. Graphical abstract.

Secretomic analysis of cheap enzymatic cocktails of Aspergillus niger LBM 134 grown on cassava bagasse and sugarcane bagasse.

Currently, agroindustrial wastes are little used for generating value-added products; hence, their use of these waste to produce enzymatic cocktails for the conversion of lignocellulosic biomass to fermentable sugars is a very interesting alternative in the second-generation bioethanol process. The Ascomycota fungus Aspergillus niger LBM 134 produces hydrolytic enzymes in large proportions. In this work, A. niger LBM 134 was grown on sugarcane and cassava bagasses under optimized conditions. To identify the extracellular enzymes involved in the degradation of these agroindustrial wastes, the secretomes of the culture supernatants of the fungus were analyzed and validated by biochemical assays of the enzymatic activities. A. niger LBM 134 secreted higher quantities of xylanases and accessory hemicellulases when it grew on sugarcane bagasse, whereas more cellulases, amylases, and pectinases were secreted when it grew on cassava bagasse. These findings suggest two promising enzyme cocktails for the hydrolysis of lignocellulose carbohydrate polymers to fermentable sugars. These bioinformatic analysis were functional validates through enzymatic biochemical assays that confirm the biotechnological potential of A. niger LBM 134 for the bioconversion of hemicellulosic substrates such as sugarcane and cassava bagasses.

Antifungal activity of endophytic fungi from Cupressaceae against human pathogenic Aspergillus fumigatus and Aspergillus niger.

Aspergillus is a fungal genus that strongly affects health of humans, animals, and plants worldwide. Endophytes are now widely considered as a rich source of bioproducts with potential uses in medicine, agriculture, and bioindustry. Cupressaceae plant family hosts a variety of bioactive ascomycetous endophytes. In this study, antifungal activity of a number of such endophytes were investigated against human pathogenic fungi Aspergillus fumigatus and Aspergillus niger. To this end, 16 superior bioactive endophytic fungi from Cupressaceae were used, including Alternaria alternata, Alternaria pellucida, Ascorhizoctonia sp., Aspergillus fumigatus, Aspergillus niger, Aurobasidium sp., Cladosporium porophorum, Fusarium oxysporum, Penicillium viridicatum, Phoma herbarum, Phoma sp., Pyrenochaeta sp., Trichoderma atroviride, Trichoderma atroviride and Trichoderma koningii. In vitro bioassays indicated anti-Asperilli activity of the endophytic fungi in dual cultures. Most notably, Trichoderma koningii CSE32 and Trichoderma atroviride JCE33 showed complete growth inhibition of both A. niger and A. fumigatus, within 3 to 7 days. Also, volatile compouds (VOCs) of T. koningii CSE32 and T. atroviride JCE33 exhibited 33-100% growth inhibition of A. niger, whithin 3 days. Moreover, on the day 7, growth of A. niger was less affected than that of A. fumigatus. In general, it appears that there is a direct relationship between the exposure time and the inhibitory activity of endophytes VOCs on the growth of target Aspergillus species. Furthremore, the extracellular secondary metabolites (SMs) of four selected fungal endophytes exhibited anti-Aspergillus activity at all treatment levels as shown by Agar-diffusion assay. SMs from T. koningii CSE32 and Pyrenochaeta CSE134 showed strongest activities against A. niger, and SMs from T. koningii CSE32 and F. oxysporum CAE14 showed strongest activities against A. fumigatus. In conclusion, given the globally recognized issue of antibiotic resistance and the urge to discover new antimicrobial substances, our findings provide new insights into the potential use of Cupressaceae's endophytic fungi in antifungal-based drug discovery programs.

Kolliphor® HS 15-cyclodextrin Complex for the Delivery of Voriconazole: Preparation, Characterization, and Antifungal Activity.

BACKGROUND: This study aimed to reduce the amount of sulfobutylether-ß-cyclodextrin (SBECD) used in the marketed voriconazole injections to meet the clinical needs of patients with moderate-to-severe renal impairment (creatinine clearance rate <50 mL/min). OBJECTIVE: This study found that the surfactant Kolliphor® HS 15 (HS 15) and SBECD had significant synergistic effects on solubilizing voriconazole, and a novel voriconazole complex delivery system (VRC-CD/HS 15) was established. METHODS: The complex system was characterized, and its antifungal activity was studied by dynamic light scattering, dialysis bag method, disk diffusion, and broth microdilution. RESULTS: Compared with the control, its encapsulation efficiency (90.07±0.48%), drug loading (7.37±0.25%) and zeta potential (-4.36±1.37 mV) were increased by 1.54%, 41.19%, and 296.36%, respectively; its average particle size (13.92±0.00 nm) was reduced by 15.69%, so the complex system had better stability. Simultaneously, its drug release behavior was similar to that of the control, and it was a first-order kinetic model. Antifungal studies indicated that the complex system had noticeable antifungal effects. With the increase of drug concentration, the inhibition zone increased. The minimum inhibitory concentrations of the complex system against Cryptococcus neoformans, Aspergillus niger and Candida albicans were 0.0313 µg/mL, 1 µg/mL and 128 µg/mL, respectively. CONCLUSION: It showed a significant inhibitory effect on C. neoformans and had a visible therapeutic effect on Kunming mice infected with C. neoformans. Consequently, VRC-CD/HS 15 had better physicochemical properties and still had an apparent antifungal effect, and was promising as a potential alternative drug for clinical application.