Genome sequencing of Porostereum spadiceum to study the degradation of levofloxacin.

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H2O2-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.

Efficient Decolorization of the Poly-Azo Dye Sirius Grey by Coriolopsis gallica Laccase-Mediator System: Process Optimization and Toxicity Assessment.

The textile industry produces high volumes of colored effluents that require multiple treatments to remove non-adsorbed dyes, which could be recalcitrant due to their complex chemical structure. Most of the studies have dealt with the biodegradation of mono or diazo dyes but rarely with poly-azo dyes. Therefore, the aim of this paper was to study the biodegradation of a four azo-bond dye (Sirius grey) and to optimize its decolorization conditions. Laccase-containing cell-free supernatant from the culture of a newly isolated fungal strain, Coriolopsis gallica strain BS9 was used in the presence of 1-hydroxybenzotriazol (HBT) to optimize the dye decolorization conditions. A Box-Benken design with four factors, namely pH, enzyme concentration, HBT concentration, and dye concentration, was performed to determine optimal conditions for the decolorization of Sirius grey. The optimal conditions were pH 5, 1 U/mL of laccase, 1 mM of HBT, and 50 mg/L of initial dye concentration, ensuring a decolorization yield and rate of 87.56% and 2.95%/min, respectively. The decolorized dye solution showed a decrease in its phytotoxicity (Germination index GI = 80%) compared to the non-treated solution (GI = 29%). This study suggests that the laccase-mediator system could be a promising alternative for dye removal from textile wastewater.

Water implications in dialysis therapy, threats and opportunities to reduce water consumption: a call for the planet.

Water is a dwindling natural resource, and potable water is wrongly considered an unlimited resource. Dialysis, particularly hemodialysis, is a water-hungry treatment that impacts the environment. The global annual water use of hemodialysis is approximately 265 million m3/yr. In this reference estimate, two-thirds of this water is represented by reverse osmosis reject water discharged into the drain. In this review, we would like to draw attention to the complexity and importance of water saving in hemodialysis. We propose that circular water management may comply with the "3R" concept: reduce (reduce dialysis need, reduce dialysate flow, and optimize reverse osmosis performance), reuse (reuse wastewater as potable water), and recycle (dialysis effluents for agriculture and aquaponic use). Awareness and sustainability should be integrated to create positive behaviors. Effective communication is crucial for water savings because local perspectives may lead to global opportunities. Besides the positive environmental impacts, planet-friendly alternatives may have significant financial returns. Innovative policies based on the transition from linear to circular water management may lead to a paradigm shift and establish a sustainable water management model. This review seeks to support policymakers in making informed decisions about water use, avoiding wasting, and finding solutions that may be planet friendly and patient friendly in dialysis, especially in hemodialysis treatments.

Characterization of Siccibacter sp. Strain C2 a Novel Rhizobacterium that Enhances Tolerance of Barley to Salt Stress.

Plant growth promoting rhizobacteria (PGPR) arouse an increasing interest as an eco-friendly solution for improving crop tolerance to environmental stresses. In this study, we report the characterization of a novel halotolerant PGPR strain (named C2) identified in a screen of rhizospheric bacterial isolates from southeast of Tunisia. Phylogenetic analysis showed that strain C2 is most likely affiliated to the genus Siccibacter with Siccibacter turicensis as the closest species (98.19%). This strain was able to perform phosphate solubilization and production of indole acetic acid (IAA), siderophores, hydrogen cyanide (HCN), as well as different hydrolytic enzymes (proteases, amylases, cellulases, and lipases). The potential of strain C2 in enhancing salt stress tolerance of Hordeum vulgare was also investigated. Our greenhouse inoculation assays showed that strain C2 promotes barley growth in the presence of 400 mM NaCl by increasing biomass, root length, and chlorophyll contents. It has a positive effect on the photosynthetic efficiency, concomitantly with lower intercellular CO2 contents, compared to non-inoculated plants. Moreover, barley inoculation with strain C2 under salt stress, resulted in higher accumulation of proline and soluble sugars and alleviate the oxidative stress by decreasing hydrogen peroxide and malondialdehyde contents. Remarkably, this positive effect corroborates with a significant activation in the expression of a subset of barley stress responsive genes, including HVA1, HvDREB1, HvWRKY38 and HvP5CS. In summary, Siccibacter sp. strain C2 is able to enhance barley salt stress tolerance and should be leveraged in developing sustainable practices for cereal crop production.

A Comparative Study of Various Pretreatment Approaches for Bio-Ethanol Production from Willow Sawdust, Using Co-Cultures and Mono-Cultures of Different Yeast Strains.

The effect of different pretreatment approaches based on alkali (NaOH)/hydrogen peroxide (H2O2) on willow sawdust (WS) biomass, in terms of delignification efficiency, structural changes of lignocellulose and subsequent fermentation toward ethanol, was investigated. Bioethanol production was carried out using the conventional yeast Saccharomyces cerevisiae, as well as three non-conventional yeasts strains, i.e., Pichia stipitis, Pachysolen tannophilus, Wickerhamomyces anomalus X19, separately and in co-cultures. The experimental results showed that a two-stage pretreatment approach (NaOH (0.5% w/v) for 24 h and H2O2 (0.5% v/v) for 24 h) led to higher delignification (38.3 ± 0.1%) and saccharification efficiency (31.7 ± 0.3%) and higher ethanol concentration and yield. Monocultures of S. cerevisiae or W. anomalus X19 and co-cultures with P. stipitis exhibited ethanol yields in the range of 11.67 ± 0.21 to 13.81 ± 0.20 g/100 g total solids (TS). When WS was subjected to H2O2 (0.5% v/v) alone for 24 h, the lowest ethanol yields were observed for all yeast strains, due to the minor impact of this treatment on the main chemical and structural WS characteristics. In order to decide which is the best pretreatment approach, a detailed techno-economical assessment is needed, which will take into account the ethanol yields and the minimum processing cost.

Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications.

BACKGROUND: Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization. RESULTS: Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum, Stemphylium lucomagnoense and Aspergillus nidulans. Among these five isolates, one T. asperellum strain (T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator. CONCLUSIONS: The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO4 as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

Characterization of the CAZy Repertoire from the Marine-Derived Fungus Stemphylium lucomagnoense in Relation to Saline Conditions.

Even if the ocean represents a large part of Earth's surface, only a few studies describe marine-derived fungi compared to their terrestrial homologues. In this ecosystem, marine-derived fungi have had to adapt to the salinity and to the plant biomass composition. This articles studies the growth of five marine isolates and the tuning of lignocellulolytic activities under different conditions, including the salinity. A de novo transcriptome sequencing and assembly were used in combination with a proteomic approach to characterize the Carbohydrate Active Enzymes (CAZy) repertoire of one of these strains. Following these approaches, Stemphylium lucomagnoense was selected for its adapted growth on xylan in saline conditions, its high xylanase activity, and its improved laccase activities in seagrass-containing cultures with salt. De novo transcriptome sequencing and assembly indicated the presence of 51 putative lignocellulolytic enzymes. Its secretome composition was studied in detail when the fungus was grown on either a terrestrial or a marine substrate, under saline and non-saline conditions. Proteomic analysis of the four S. lucomagnoense secretomes revealed a minimal suite of extracellular enzymes for plant biomass degradation and highlighted potential enzyme targets to be further studied for their adaptation to salts and for potential biotechnological applications.

Simultaneous cleanup of Reactive Black 5 and cadmium by a desert soil bacterium.

Multi-contaminated industrial wastewaters pose serious environmental risks due to high toxicity and non-biodegradability. The work reported here evaluated the ability of Pseudomonas aeruginosa strain Gb30 isolated from desert soil to simultaneously remove cadmium (Cd) and Reactive Black 5 (RB5), both common contaminants in various industrial effluents. The strain was able to grow normally and decolorize 50 mg L-1 RB5 within 24 h of incubation in the presence of 0.629 m mol L-1 of Cd2+. In order to evaluate strain performance in RB5 detoxification, a cytotoxicity test using Human Embryonic Kidney cells (HEK293) was used. Cadmium removal from culture media was determined using atomic adsorption. Even in presence of (0.115 + 0.157 + 0.401 + 0.381) m mol L-1, respectively, of Cr6+, Cd2+, Cu2+ and Zn2+ in the growth medium, strain Gb30 successfully removed 35% of RB5 and 44%, 36%, 59% and 97%, respectively, of introduced Zn2+, Cu2+, Cr6+ and Cd2+, simultaneously. In order to understand the mechanism of Cd removal used by P. aeruginosa strain Gb30, biosorption and bioaccumulation abilities were examined. The strain was preferentially biosorbing Cd on the cell surface, as opposed to intracellular bioaccumulation. Microscopic investigations using AFM, SEM and FTIR analysis of the bacterial biomass confirmed the presence of various structural features, which enabled the strain to interact with metal ions. The study suggests that Pseudomonas aeruginosa Gb30 is a potential candidate for bioremediation of textile effluents in the presence of complex dye-metal contamination.

Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense.

Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus Stemphylium lucomagnoense. The laccase-encoding gene SlLac2 from S. lucomagnoense was cloned for heterologous expression in Aspergillus niger D15#26 for protein production in the extracellular medium of around 30 mg L-1. The extracellular recombinant enzyme SlLac2 was successfully produced and purified in three steps protocol: ultrafiltration, anion-exchange chromatography, and size exclusion chromatography, with a final recovery yield of 24%. SlLac2 was characterised by physicochemical properties, kinetic parameters, and ability to oxidise diverse phenolic substrates. We also studied its activity in the presence and absence of sea salt. The molecular mass of SlLac2 was about 75 kDa, consistent with that of most ascomycete fungal laccases. With syringaldazine as substrate, SlLac2 showed an optimal activity at pH 6 and retained nearly 100% of its activity when incubated at 50°C for 180 min. SlLac2 exhibited more than 50% of its activity with 5% wt/vol of sea salt.

Degradation of bisphenol A and acute toxicity reduction by different thermo-tolerant ascomycete strains isolated from arid soils.

Four different laccase-producing strains were isolated from arid soils and used for bisphenol A (BPA) degradation. These strains were identified as Chaetomium strumarium G5I, Thielavia arenaria CH9, Thielavia arenaria HJ22 and Thielavia arenaria SM1(III) by internal transcribed spacer 5.8 S rDNA analysis. Residual BPA was evaluated by HPLC analysis during 48 h of incubation. A complete removal of BPA was observed by the whole cell fungal cultures within different times, depending on each strain. C. strumarium G5I was the most efficient degrader, showing 100% of removal within 8 h of incubation. The degradation of BPA was accompanied by the production of laccase and dye decolorizing peroxidase (DyP) under degradation conditions. The presence of aminobenzotriazole (ABT) as an inhibitor of cytochrome P450s monooxygenases (CYP) demonstrated a slight decrease in BPA removal rate, suggesting the effective contribution of CYP in the conversion. The great involvement of laccase in BPA transformation together with cell-associated enzymes, such as CYP, was supported by the identification of hydroxylated metabolites by ultra-high performance liquid chromatography-mass spectroscopy (UHPLC-MS). The metabolic pathway of BPA transformation was proposed based on the detected metabolites. The acute toxicity of BPA and its products was investigated and showed a significant reduction, except for T. arenaria SM1(III) that did not caused reduction of toxicity (IC50 < 8%), possibly due to the presence of toxic metabolites. The results of the present study point out the potential application of the isolated ascomycetes in pollutant removal processes, especially C. strumarium G5I as an efficient degrader of BPA.

Fast activated charcoal prepurification of Fusarium solani ß-glucosidase for an efficient oleuropein bioconversion.

Fungal ß-glucosidases were extensively studied regarding their various potential biotechnology applications. Here, we report the selection of Fusarium solani strain producing high yield of ß-glucosidase activity. The effect of some factors on ß-glucosidase production was studied including: Initial pH, medium composition, concentration of carbon and nitrogen sources, and particle size of raw substrates. The optimal enzyme production was obtained with 4 units of pH. The highest ß-glucosidase activity was produced on 4% wheat bran (WB) as raw carbon sources, reaching 5 U/mL. A positive correlation between WB particle size and the ß-glucosidase production level was settled. The last one was enhanced to 13.60 U/mL in the presence of 0.5% (w/v) of ammonium sulfate. Interestingly, the activated charcoal was used as an inexpensive reagent enabling a rapid and efficient purification prior step that improved the enzyme-specific activity. Eventually, F. solani ß-glucosidase acts efficiently during the bioconversion process of oleuropein. Indeed, 82.5% of oleuropein was deglycosylated after 1 hr at 40°C. Altogether, our data showed that the ß-glucosidase of F. solani has a potential application to convert oleuropein to ameliorate food quality.

Fatty acid biosynthesis during the life cycle of Debaryomyces etchellsii.

Fatty acid biosynthesis during the life cycle of the ascomycetous yeast Debaryomyces etchellsii cultivated on a non-fermentable substrate, i.e. glycerol, in nitrogen rich media (NRM) and nitrogen limited media (NLM) has been studied. Although considerable activities of key lipogenic enzymes, such as ATP citrate lyase (ACL) and malic enzyme (ME), were detected in vegetative cells during asexual proliferation (which occurred in the first growth stages in both NRM and NLM), lipid accumulation was restricted due to the high activities of NAD+-isocitrate dehydrogenase (NAD+-ICDH). A similar enzymatic profile has been found in ascii and free ascospores produced in NRM; thus lipid accumulation was low. On the contrary, very high activities of both ACL and ME and low activities of NAD+-ICDH were detected in ascii and free ascospores produced in NLM resulting in lipid accumulation. Neutral lipids (NL) were the predominant fraction of cellular lipids produced in vegetative cells and ascospores in both NRM and NLM. On the other hand, phospholipids (P) were the major polar lipids while glycolipids (G) were synthesized in low proportions. During transition from asexual to sexual phase, the percentage of NL increased with a significant decrease of P and, to a lesser extent, of G. High quantities of linoleic acid were found esterified in polar lipids, especially in P, during the vegetative stage of growth, while, with a few exceptions, during transition from asexual to sexual stage, linoleic acid concentration decreased markedly, mainly in P, while oleic acid concentration increased.

Optimized decolorization of two poly azo dyes Sirius Red and Sirius Blue using laccase-mediator system.

Factors, namely pH, laccase-like activity, dyes concentration as well as 1-Hydroxybenzotriazole (HBT) concentration was examined. The results indicated that the maximum decolorization yield and rate reached 98.30 ± 0.10% and 5.84 ± 0.01%/min, respectively for Sirius Blue, and 99.34 ± 0.47% and 5.85 ± 0.12%/min, respectively for Sirius Red after 4 h. The presence of the redox mediator 1-hydroxybenzotriazole (HBT) greatly improved the decolorization levels. The optimum concentrations of HBT, dyes, and laccase were 0.62 mM, 50 mg/L, and 0.89 U/mL respectively at pH 4.58 for both dyes. Phytotoxicity tests using treated and untreated dyes proved that the applied treatment slightly decreased the toxicity of the by-products. However, the germination index (GI) increased from 14.6 to 36.08% and from 31.6 to 36.96% for Sirius Red and Sirius Blue, respectively. The present study focused on the treatment of two recalcitrant azo dyes, namely: Sirius Blue (Direct Blue 71) and Sirius Red (Direct Red 80). The decolorization was performed using cell-free supernatant from Coriolopsis gallica culture with high laccase activity. Response surface methodology (RSM) and Box-Behnken design were applied to optimize the decolorization of the two tested dyes. The effect of four.

Fungal Bioremediation of the ß-Lactam Antibiotic Ampicillin under Laccase-Induced Conditions.

Due to widespread overuse, pharmaceutical compounds, such as antibiotics, are becoming increasingly prevalent in greater concentrations in aquatic ecosystems. In this study, we investigated the capacity of the white-rot fungus, Coriolopsis gallica (a high-laccase-producing fungus), to biodegrade ampicillin under different cultivation conditions. The biodegradation of the antibiotic was confirmed using high-performance liquid chromatography, and its antibacterial activity was evaluated using the bacterial growth inhibition agar well diffusion method, with Escherichia coli as an ampicillin-sensitive test strain. C. gallica successfully eliminated ampicillin (50 mg L-1) after 6 days of incubation in a liquid medium. The best results were achieved with a 9-day-old fungal culture, which treated a high concentration (500 mg L-1) of ampicillin within 3 days. This higher antibiotic removal rate was concomitant with the maximum laccase production in the culture supernatant. Meanwhile, four consecutive doses of 500 mg L-1 of ampicillin were removed by the same fungal culture within 24 days. After that, the fungus failed to remove the antibiotic. The measurement of the ligninolytic enzyme activity showed that C. gallica laccase might participate in the bioremediation of ampicillin.

Valorizing fungal diversity for the degradation of fluoroquinolones.

Continued widespread use of antibiotics, especially fluoroquinolones, raises environmental concerns, as its driving bacterial resistance and disrupts microbial ecosystems. Here we investigate the biodegradation of ten fluoroquinolone antibiotics (six for medical use and four for veterinary use) by ligninolytic fungi, including Trametes versicolor, Bjerkandera adusta, Porosterum spadiceum, Irpex lacteus, Pleuroteus ostreatus, Phanerochaete chrysosporium, Pycnoporus cinnabarinus, Ganoderma lucidum, and Gloeophyllum trabeum. The results show significant variations between strains in the efficiency of antibiotic transformation. B. adusta and P. spadiceum were the fungi that most efficiently reduced antibiotic concentrations and were able to totally degrade eight and six antibiotics, respectively, within a 15-day period. T. versicolor and P. ostreatus also showed the ability to effectively degrade antibiotics. Specifically, T. versicolor degraded six out of the ten fluoroquinolone antibiotics by more than 70 %, while P. ostreatus degraded the tested antibiotics between 43 % and 100 %. The remaining antibiotic activity did not always correlate with a reduction in antibiotic concentrations, which points to the presence of post-transformation antimicrobial metabolites. This study also explores the potential mechanisms used by these fungi to remove selected models of fluroquinolones via enzymatic routes, such as oxidation by laccases, heme-peroxidases, and cytochrome P450, or via adsorption on fungal biomass.

Kinetic properties of a novel Fusarium solani (phospho)lipase: a monolayer study.

Using the monomolecular film technique, we studied interfacial properties of Fusarium solani lipase (FSL). This lipolytic enzyme was found to be unique among the fungal lipases possessing not only a lipase activity but also a high phospholipase one.The FSL was able to hydrolyze dicaprin films at various surface pressures. The surface pressure dependency, the stereospecificity, and the regioselectivity of FSL were performed using optically pure stereoisomers of diglyceride (1,2-sn- dicaprin and 2,3-sn-dicaprin) and a prochiral isomer (1,3-sn-dicaprin) spread as monomolecular films at the air-water interface. The FSL prefers adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin) at low and high surface pressures. Furthermore, FSL was found to be markedly stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin at both low and high surface pressures.Moreover, FSL shows high activities on phospholipids monolayers. However, this enzyme displays high preference to zwitterionic phospholipids compared to the negatively charged ones.

Decolorization of the metal textile dye Lanaset Grey G by immobilized white-rot fungi.

In this paper we studied the ability of four Tunisian-isolated fungi (i.e. Coriolopsis gallica, Bjerkandera adusta, Trametes versicolor and Trametes trogii) immobilized into Ca-alginate beads to decolorize the metal textile dye Lanaset Grey G (LG). The effect of different operational conditions, such as initial dye concentration, temperature, pH, beads/medium ratio and agitation, on dye decolorization by the immobilized fungi was investigated. Maximal decolorization percentages of 88.7%, 89.3%, 82.1% and 81.3% for C. gallica, B. adusta, T. versicolor and T. trogii were attained, respectively, when operating at an initial LG concentration of 150 mg/L, pH values of 5.0-6.0, temperatures of 40-45 °C and a beads/medium ratio of 20% (w/v) in static conditions after 72 h of incubation. Subsequently, the re-usability of the immobilized fungi was evaluated. After three decolorization cycles, the decolorization percentage of free cell cultures dropped to values below 36%, while decolorization percentages of about 75%, 70%, 60% and 68% were obtained by the immobilized cultures of C. gallica, B. adusta, T. versicolor and T. trogii, respectively.

Purification and biochemical characterization of a new alkali-stable laccase from Trametes sp. isolated in Tunisia: role of the enzyme in olive mill waste water treatment.

A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes sp, was selected in a broad plate screening because of its ability to decolorize and dephenolize olive oil mill wastewater (OMW) efficiently. The major laccase was purified and characterized as a monomeric protein with apparent molecular mass of 61 kDa (SDS-PAGE). It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 4.0 and a temperature of 60 °C. The purified laccase is stable at alkaline pH values. The enzyme retained 50 % of its activity after 90 min of incubation at 55 °C. Using ABTS, this laccase presented K m and V max values of 0.05 mM and 212.73 µmoL min(-1) mg(-1), respectively. It has shown a degrading activity towards a variety of phenolic compounds. The purified laccase was partially inhibited by Fe(2+), Zn(2+), Cd(2+) and Mn(2+), while Cu(2+) acted as inducer. EDTA (10 mM) and NaN3 (10 mM) were found to completely inhibit its activity. 73 % OMW was dephenolized after 315 min incubation at 30 °C with 2 U mL(-1) of laccase and 2 mM HBT.