Removal pattern of vinasse phenolics by Phlebia rufa, characterization of an induced laccase and inhibition kinetics modeling.

Vinasse from the distillation of winemaking residues is a wastewater characterized by high levels of aromatic compounds. Batch cultures of Phlebia rufa showed a significant (p < 0.05) correlation between laccase activity and initial vinasse concentration. The pattern of biodegradation of hydroxybenzoic acids, hydroxycinnamic acids and flavonoids, assessed by HPLC-DAD, revealed that p-hydroxybenzoic acid is the most recalcitrant compound. Vinasse-induced laccase showed electrophoretic homogeneity and molecular weight of 62 kDa after being purified 21-fold. Optimum pH for oxidation of 2,6-dimethoxyphenol (2,6-DMP) was 3.5 and optimum temperature was 50 °C, with an activation energy of 42.8 kJ mol-1. Catalytic efficiency of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) oxidation is about two orders of magnitude higher than 2,6-DMP oxidation, being their Km values 36.2 ± 2.6 µM and 303.0 ± 44.7 µM, respectively and kcat values 486.1 s-1 and 179.6 s-1, respectively. Akaike information criterion and Akaike weights were used to discriminate inhibition models that best fitted 2,6-DMP oxidation in the presence of inhibitors. Inhibition constants of mixed-type inhibitors azide and fluoride, and competitive-type inhibitor chloride, showed the following inhibitors potency: azide > fluoride > chloride. Taken together, this study is consistent with the assumption that P. rufa could be a useful tool for aerobic degradation of phenolic-rich wastewaters.

Fungal biodegradation and multi-level toxicity assessment of vinasse from distillation of winemaking by-products.

The wastewaters from distilleries of winemaking by-products, a scarcely studied type of vinasse, were treated by white-rot fungal strains from species Irpex lacteus, Ganoderma resinaceum, Trametes versicolor, Phlebia rufa and Bjerkandera adusta. The main objectives of this study were to evaluate fungal performance during vinasse biodegradation, their enzyme patterns and ecotoxicity evolution throughout treatment. Despite all strains were able to promote strong (>80%) dephenolization and reduction of total organic carbon (TOC), P. rufa was less affected by vinasse toxicity and exhibit better decolorization. In batch cultures at 28 °C and pH 4.0, the first phase of P. rufa biodegradation kinetics was characterized by strong metabolic activity with simultaneous depletion of TOC, phenolics and sugars. The main events of second phase are the increase of peroxidases production after the peak of laccase activity, and strong color removal. At the end of treatment, it was observed highly significant (p < 0.001) abatement of pollution parameters (83-100% removal). Since water reclamation and reuse for e.g. crop irrigation is a priority issue, vinasse ecotoxicity was assessed with bioindicators representing three different phylogenetic and trophic levels: a marine bacterium (Aliivibrio fischeri), a freshwater microcrustacean (Daphnia magna) and a dicotyledonous macrophyte (Lepidium sativum). It was observed significant (p < 0.05) reduction of initial vinasse toxicity, as evaluated by these bioindicators, deserving special mention an almost complete phytotoxicity elimination.

Hazardous impact of vinasse from distilled winemaking by-products in terrestrial plants and aquatic organisms.

Vinasses obtained from the distillation of winemaking by-products (WDV) are complex effluents with variable physicochemical properties. Frequently, WDVs are used to irrigate agricultural soil, and/or discharged into aquatic bodies, which may result in serious environmental pollution, due to the presence of organic acids and recalcitrant compounds (polyphenols, tannins and metals). The present study aimed to evaluate the toxicity impact of an untreated WDV on terrestrial and aquatic organisms, at different levels of biological organization. The effluent was collected at the distillation column exit and characterized according to several physicochemical properties. The WDV potential phytotoxicity was assessed by germination inhibition assays on six agricultural crops, and its acute toxicity was assessed on Aliivibrio fisheri (microtox assay), Daphnia magna neonates (freshwater crustacean), and zebrafish Danio rerio (fish embryo toxicity test, FET). The WDV presented a low pH (3.88), high levels of electrical conductivity, ECond (6.36 dS m-1) and salinity (3.3 ppt), besides high level of potassium (2.1 g L-1) and organic compounds (TOC = 17.7 g L-1), namely polyphenols (1.7 g L-1). The diluted WDV displayed variable inhibitory effects on the plant endpoints (percentage of inhibition of germination and radicle elongation and germination index). Overall, plants' susceptibility to increasing concentrations of WDV were differential (onion ≈ garden cress ≥ tomato > lettuce > maize > green beans) and the germination index EC50 varied from 10.9 to 64.4% v/v. Also, the acute negative effects toward aquatic organisms were determined, decreasing from the more complex organism to the simpler one: zebrafish embryos (96 h-LC50 = 0.34% v/v)>D. magna (48 h-LC50 = 4.8% v/v)>A. fisheri (30min-EC50 = 7.0% v/v). In conclusion, the findings suggest that WDVs might have a high toxicological impact on both terrestrial plants and aquatic organisms, even at high dilution levels, reinforcing the need for appropriate treatments before considering its discharge or reuse.

Endopolysaccharides from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor affect differently the proliferation rate of HepG2 cells.

Fungi have been used for medicinal purposes for long time by Asian countries, being a putative source of powerful new phytopharmaceuticals such as polysaccharides. The aim of this study was to extract endopolysaccharides (IPS) from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor, grown under submerged culture, to compare crude IPS production, total carbohydrate, and protein yield, and to study the effect of these IPS on HepG2 cells proliferation rate. Total biomass produced by G. resinaceum, P. rufa, and T. versicolor was (in gram per liter) 3.32 ± 0.80, 5.42 ± 0.58, and 4.2 ± 1.29 and the IPS yield (as the biomass percent) was 9.9 ± 0.05, 29.0 ± 6.3, and 9.1 ± 3.1 %, respectively. Characterization of IPS has shown different proportion between total sugar and protein being, on average 6.04, 10.74, and 22.62, for G. resinaceum, T. versicolor, and P. rufa, respectively. The IPS effect, at 50, 100, and 200 µg mL(-1) on HepG2 cell growth and viability was negligible for G. resinaceum and P. rufa but, in the case of T. versicolor, 200 µg mL(-1) of IPS evoked 40 % reduction on cell growth. The results suggest that the intracellular polysaccharides from T. versicolor are a potential source for bioactive molecules with anti-proliferative properties.