Cost-effective optimized scleroglucan production by Sclerotium rolfsii ATCC 201126 at bioreactor scale. A quantity-quality assessment.

Exopolysaccharide (EPS) secretion by Sclerotium rolfsii ATCC 201126 in submerged cultures, already identified as high-osmolarity responsive, was assessed by reducing C-source without compromising EPS yields. A designed medium with 80 g sucrose L-1 (MOPT80) was tested at 3 L-bioreactor scale at different temperature, agitation, aeration and pH (uncontrolled vs. controlled) values. Optimal operative conditions (200 rpm, 28 °C, 0.5 vvm and initial pH -pHi- 4.5) were validated, as well as the possibility to work at pHi 5.5 to reduce biomass production. Purified EPSs produced in MOPT80 at optimal and other valid operative conditions exhibited refined grade (<1 % proteins and ash, 3-4 % reducing sugars, 87-99 % total sugars). EPS purity, MW and rheological parameters led to discourage pH controlled at 4.5. Relatively constant MW (6-8 × 106 Da) and outstanding viscosifying ability were found. Polyphasic EPS analysis (titre, purity, macromolecular features and rheological fitness) would support to properly select production conditions.

Microbial production of scleroglucan and downstream processing.

Synthetic petroleum-based polymers and natural plant polymers have the disadvantage of restricted sources, in addition to the non-biodegradability of the former ones. In contrast, eco-sustainable microbial polysaccharides, of low-cost and standardized production, represent an alternative to address this situation. With a strong global market, they attracted worldwide attention because of their novel and unique physico-chemical properties as well as varied industrial applications, and many of them are promptly becoming economically competitive. Scleroglucan, a ß-1,3-ß-1,6-glucan secreted by Sclerotium fungi, exhibits high potential for commercialization and may show different branching frequency, side-chain length, and/or molecular weight depending on the producing strain or culture conditions. Water-solubility, viscosifying ability and wide stability over temperature, pH and salinity make scleroglucan useful for different biotechnological (enhanced oil recovery, food additives, drug delivery, cosmetic and pharmaceutical products, biocompatible materials, etc.), and biomedical (immunoceutical, antitumor, etc.) applications. It can be copiously produced at bioreactor scale under standardized conditions, where a high exopolysaccharide concentration normally governs the process optimization. Operative and nutritional conditions, as well as the incidence of scleroglucan downstream processing will be discussed in this chapter. The relevance of using standardized inocula from selected strains and experiences concerning the intricate scleroglucan scaling-up will be also herein outlined.

Effects of thermal, alkaline and ultrasonic treatments on scleroglucan stability and flow behavior.

Aqueous solutions (0.2%, w/v) of scleroglucans from Sclerotium rolfsii ATCC 201126 from different cultivation time or purification protocol (EPS I, EPS II, EPSi) as well as a commercial scleroglucan (LSCL) exhibited different sensitivity against thermal (65, 95 and 150°C), ultrasonic (1, 5 and 10 min; 20% amplitude) or alkaline (0.01-0.2 N NaOH) treatments. Scleroglucan triple helix usually showed signs of denaturation at 150°C or with 0.2 NaOH with a pronounced decrease in apparent viscosity and loss of pseudoplastic behavior. Differences in sensitivity could be noted depending on the scleroglucan sample, which may be likely related to polysaccharide conformational features, and these latter to production and/or downstream processing conditions. Transmission electron microscopy showed scleroglucan topologies in accordance with thermal and alkaline denaturation. Size exclusion chromatography of control scleroglucans revealed elution profiles compatible with macromolecular aggregates which tended to diminish or disappear as thermal, alkali or sonication treatments progressed. Scleroglucan granule dissolution process took ∼8-14 s, according to DIC-light microscopy, and showed to be facilitated by addition of NaOH.

Scleroglucan compatibility with thickeners, alcohols and polyalcohols and downstream processing implications.

Thickening capacity and compatibility of scleroglucan with commercial thickeners (corn starch, gum arabic, carboxymethylcellulose, gelatin, xanthan and pectin), glycols (ethylene glycol and polyethylene glycol), alcohols (methanol, ethanol, 1-propanol and isopropanol) and polyalcohols (sorbitol, xylitol and mannitol) was explored. Exopolysaccharides (EPSs) from Sclerotium rolfsii ATCC 201126 and a commercial scleroglucan were compared. Compatibility and synergism were evaluated taking into account rheology, pH and sensory properties of different thickener/scleroglucan mixtures in comparison with pure solutions. S. rolfsii ATCC 201126 EPSs induced or increased pseudoplastic behaviour with a better performance than commercial scleroglucan, showing compatibility and synergy particularly with corn starch, xanthan, pectin and carboxymethylcellulose. Compatibility and a slight synergistic behaviour were also observed with 30% (w/v) ethylene glycol whereas mixtures with polyethylene glycol (PEG) precipitated. Scleroglucan was compatible with polyalcohols, whilst lower alcohols led to scleroglucan precipitation at 20% (v/v) and above. PEG-based scleroglucan downstream processing was compared to the usual alcohol precipitation. Downstream processed EPSi (with isopropanol) and EPS-p (with PEG) were evaluated on their yield, purity, rheological properties and visual aspect pointing to alcohol downstream processing as the best methodology, whilst PEG recovery would be unsuitable. The highest purified EPSi attained a recovery yield of ~23%, similar to ethanol purification, with a high degree of purity (88%, w/w vs. EPS-p, 8%, w/w) and exhibited optimal rheological properties, water solubility and appearance. With a narrower molecular weight distribution (M(w), 2.66×10(6) g/mol) and a radius of gyration (R(w), 245 nm) slightly lower than ethanol-purified EPSs, isopropanol downstream processing showed to be a proper methodology for obtaining a refined-grade scleroglucan.

Actividad antifúngica sinérgica de combinaciones de estatinas y azólicos revelada mediante bioanálisis con Saccharomyces cerevisiae y Candida utilis y cuantificación de ergosterol / Synergistic antifungal activity of statin–azole associations as witnessed by Saccharomyces cerevisiae- and Candida utilis-bioassays and ergosterol quantification

Fundamento. La frecuencia de micosis oportunistas y la resistencia a los antimicóticos convencionales han fomentado la búsqueda de nuevas alternativas terapéuticas, como las combinaciones de antimicóticos. Objetivos. El presente estudio trató de detectar el sinergismo antifúngico entre las estatinas y los azólicos mediante un bioanálisis de difusión en pocillos de agar, utilizando Saccharomyces cerevisiae (S. cerevisiae) ATCC 32051 y Candida utilis (C. utilis) PR1-2 como cepas de control. Métodos. Los efectos antifúngicos sinérgicos se examinaron mediante la adición simultánea de una concentración sub-inhibitoria (CSI) de estatina (atorvastatina, lovastatina, pravastatina, rosuvastatina o simvastatina) más una concentración mínima inhibitoria (CMI) de un azólico (clotrimazol, fluconazol, itraconazol, ketoconazol o miconazol) a placas de agar YNB con las levaduras sembradas por inclusión. Un resultado positivo correspondió a un diámetro del halo de inhibición del crecimiento de la levadura mayor que el producido por la CMI del azólico exclusivo. Para confirmar el sinergismo estatina-azólico, se cuantificó el ergosterol de la membrana celular de las levaduras con cromatografía líquida de alto rendimiento (HPLC-RP). Para valorar la inhibición de la síntesis de ergosterol inducida por estatinas, se emplearon bioanálisis de rescate de ergosterol. Resultados. La inhibición del crecimiento aumentó significativamente cuando se combinaron clotrimazol, fluconazol, itraconazol, ketoconazol y miconazol con atorvastatina, lovastatina, rosuvastatina y simvastatina. Los mayores incrementos de la inhibición del crecimiento se observaron en S. cerevisiae (77,5%) y C. utilis (43,2%) con una CSI de simvastatina y una CMI de miconazol de 4+2,4mg/ml o 20+4,8mg/ml, respectivamente. Para pravastatina apenas se identificaron efectos significativos (incremento de la inhibición del 0-7,6%). Los mayores cocientes de interacción correspondieron a la combinación de simvastatina y miconazol y fueron indicativos de sinergismo. Este también se confirmó por la mayor disminución de los niveles celulares de ergosterol (S. cerevisiae, 40% y C. utilis, 22%). La inhibición de la síntesis de ergosterol inducida por estatinas se corroboró mediante bioanálisis de rescate de ergosterol, donde la inhibición por pravastatina se abolió con facilidad, mientras que la de rosuvastatina fue la más refractaria. Conclusiones. Las combinaciones seleccionadas de estatinas y azólicos podrían ser alternativas viables para el manejo terapéutico de las micosis, en dosis más bajas o con una mayor eficiencia(AU)

Background. Frequent opportunist fungal infections and the resistance to available antifungal drugs promoted the development of new alternatives for treatment, like antifungal drug combinations. Aims. This work aimed to detect the antifungal synergism between statins and azoles by means of an agar-well diffusion bioassay with Saccharomyces cerevisiae ATCC 32051 and Candida utilis Pr1–2 as test strains. Methods. Synergistic antifungal effects were tested by simultaneously adding a sub inhibitory concentration (SIC) of statin (atorvastatin, lovastatin, pravastatin, rosuvastatin or simvastatin) plus a minimal inhibitory concentration (MIC) of azole (clotrimazole, fluconazole, itraconazole, ketoconazole or miconazole) to yeast-embedded YNB agar plates, and a positive result corresponded to a yeast growth inhibition halo higher than that produced by the MIC of the azole alone. Yeast cell ergosterol quantification by RP-HPLC was used to confirm statin–azole synergism, and ergosterol rescue bioassays were performed for evaluating statin-induced ergosterol synthesis blockage. Results. Growth inhibition was significantly increased when clotrimazole, fluconazole, itraconazole, ketoconazole and miconazole were combined with atorvastatin, lovastatin, rosuvastatin and simvastatin. Highest growth inhibition increments were observed on S. cerevisiae (77.5%) and C. utilis (43.2%) with a SIC of simvastatin plus a MIC of miconazole, i.e. 4+2.4mg/ml or 20+4.8mg/ml, respectively. Pravastatin showed almost no significant effects (0–7.6% inhibition increase). Highest interaction ratios between antifungal agents corresponded to simvastatin–miconazole combinations and were indicative of synergism. Synergism was also confirmed by the increased reduction in cellular ergosterol levels (S. cerevisiae, 40% and C. utilis, 22%). Statin-induced ergosterol synthesis blockage was corroborated by means of ergosterol rescue bioassays, pravastatin being the most easily abolished inhibition whilst rosuvastatin being the most ergosterol-refractory. Conclusions. Selected statin–azole combinations might be viable alternatives for the therapeutic management of mycosis at lower administration doses or with a higher efficiency(AU)

Synergistic antifungal activity of statin-azole associations as witnessed by Saccharomyces cerevisiae- and Candida utilis-bioassays and ergosterol quantification.

BACKGROUND: Frequent opportunist fungal infections and the resistance to available antifungal drugs promoted the development of new alternatives for treatment, like antifungal drug combinations. AIMS: This work aimed to detect the antifungal synergism between statins and azoles by means of an agar-well diffusion bioassay with Saccharomyces cerevisiae ATCC 32051 and Candida utilis Pr(1-2) as test strains. METHODS: Synergistic antifungal effects were tested by simultaneously adding a sub inhibitory concentration (SIC) of statin (atorvastatin, lovastatin, pravastatin, rosuvastatin or simvastatin) plus a minimal inhibitory concentration (MIC) of azole (clotrimazole, fluconazole, itraconazole, ketoconazole or miconazole) to yeast-embedded YNB agar plates, and a positive result corresponded to a yeast growth inhibition halo higher than that produced by the MIC of the azole alone. Yeast cell ergosterol quantification by RP-HPLC was used to confirm statin-azole synergism, and ergosterol rescue bioassays were performed for evaluating statin-induced ergosterol synthesis blockage. RESULTS: Growth inhibition was significantly increased when clotrimazole, fluconazole, itraconazole, ketoconazole and miconazole were combined with atorvastatin, lovastatin, rosuvastatin and simvastatin. Highest growth inhibition increments were observed on S. cerevisiae (77.5%) and C. utilis (43.2%) with a SIC of simvastatin plus a MIC of miconazole, i.e. 4 + 2.4 µg/ml or 20 + 4.8 µg/ml, respectively. Pravastatin showed almost no significant effects (0-7.6% inhibition increase). Highest interaction ratios between antifungal agents corresponded to simvastatin-miconazole combinations and were indicative of synergism. Synergism was also confirmed by the increased reduction in cellular ergosterol levels (S. cerevisiae, 40% and C. utilis, 22%). Statin-induced ergosterol synthesis blockage was corroborated by means of ergosterol rescue bioassays, pravastatin being the most easily abolished inhibition whilst rosuvastatin being the most ergosterol-refractory. CONCLUSIONS: Selected statin-azole combinations might be viable alternatives for the therapeutic management of mycosis at lower administration doses or with a higher efficiency.

Removal efficiency of Cr6+ by indigenous Pichia sp. isolated from textile factory effluent.

Resistance of the indigenous strains P. jadinii M9 and P. anomala M10, to high Cr(6+) concentrations and their ability to reduce chromium in culture medium was studied. The isolates were able to tolerate chromium concentrations up to 104 µg mL(-1). Growth and reduction of Cr(6+) were dependent on incubation temperature, agitation, Cr(6+) concentration, and pH. Thus, in both studied strains the chromium removal was increased at 30 °C with agitation. The optimum pH was different, with values of pH 3.0 and pH 7.0 in the case of P. anomala M10 and pH 7.0 using P. jadinii M9. Chromate reduction occurred both in intact cells (grown in culture medium) as well as in cell-free extracts. Chromate reductase activity could be related to cytosolic or membrane-associated proteins. The presence of a chromate reductase activity points out a possible role of an enzyme in Cr(6+) reduction.

Unraveling the decolourizing ability of yeast isolates from dye-polluted and virgin environments: an ecological and taxonomical overview.

Microcosm assays with dye-amended culture media under a shot-feeding strategy allowed us to obtain 100 yeast isolates from the wastewater outfall channel of a dyeing textile factory in Tucumán (Argentina). Meanwhile, 63 yeast isolates were obtained from Phoebe porphyria (Laurel del monte) samples collected from Las Yungas rainforest (Tucumán), via a classical isolation scheme. Isolated yeasts, both from dye-polluted and virgin environments, were compared for their textile dye decolourization ability when cultured on solid and liquid media. Nine isolates from wastewater and 17 from Las Yungas showed the highest decolourization potential on agar plates containing six different reactive dyes, either alone or as a mixture. Five yeasts from each environment were further selected on the basis of their high dye removal rate in Vilmafix(®) Red 7B-HE- or Vilmafix(®) Blue RR-BB-amended liquid cultures. Yeasts from wastewater showed slightly higher decolourization percentages after 36 h of culture than yeasts from Las Yungas (98-100% vs. 91-95%, respectively). However, isolates from Las Yungas exhibited higher specific decolourization rates than isolates from effluents (1.8-3.0 vs. 0.9-1.3 mg g(-1)h(-1), respectively). All selected isolates were first grouped according to microsatellite-PCR analysis and representative isolates from each group were subsequently identified based on the 26S rRNA gene sequence analysis. Yeasts from wastewater were identified as the ascomycetous Pichia kudriavzevii (100%) and closely related to Candida sorbophila (99.8%), whilst yeasts from Las Yungas were identified as the basidiomycetous Trichosporon akiyoshidainum and Trichosporon multisporum. It is suggested that findings concerning yeast selection during screening programs for dye-decolourizing yeasts may be explained in the light of the copiotroph-oligotroph microorganisms rationale.

Phenotypical and genetic characterization of Trichosporon sp. HP-2023. A yeast isolate from Las Yungas rainforest (Tucumán, Argentina) with dye-decolorizing ability.

A basidiomycetous yeast isolated from Las Yungas rainforest (Tucumán, Argentina) and arbitrarily named HP-2023 was selected based on its outstanding textile dye decolorizing ability. Complete decolorization of Vilmafix Red 7B-HE and Vilmafix Blue RR-BB (200 mg/l) was achieved after 16 h of cultivation. Yeast characterization was accomplished by means of both traditional and molecular methods. Results concerning molecular fingerprinting and phenotypic characterization led to identify it as Trichosporon sp., closely related to the T. multisporum-T. laibachii complex. The present work represents the first description of a Trichosporon yeast involved in reactive textile dye decolorization processes.

Sclerotium rolfsii scleroglucan: the promising behavior of a natural polysaccharide as a drug delivery vehicle, suspension stabilizer and emulsifier.

Gel matrices of scleroglucans from Sclerotium rolfsii ATCC 201126 (EPS I and EPS II, from 48-h and 72-h fermentations, respectively) were evaluated on their release kinetics of theophylline (Th). Equivalent polymer (2%, w/w) and Th (0.2%, w/w) concentrations showed almost coincident drug release patterns, independently of polymer molecular weight or the microstructural properties of gel matrices. Dynamic rheological studies of scleroglucan hydrogel structures (storage, G', and loss, G'', moduli) indicated a solid-like behavior. Differences on pore size dimensions (EPS I=20 microm and EPS II=7 microm) were in accordance to the differences in G' (EPS I=113 Pa and EPS II=161 Pa), a fact likely related to variations in the cross-linking density of polymer networks. Compared to already known biopolymers, EPS I and EPS II at 0.5 g/L showed a good dispersing ability against particulate suspensions of activated charcoal, bentonite, CaCO(3), celite and quartz powder. Emulsifying ability of both EPSs at 2g/L was high (E=56-60%) when tested with kerosene, moderate ( approximately 30%) with hexadecane, and negligible in the presence of olive oil-in-water emulsions.