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.

Pretreatment of Grape Stalks by Fungi: Effect on Bioactive Compounds, Fiber Composition, Saccharification Kinetics and Monosaccharides Ratio.

Grape stalks, an inedible lignocellulosic residue from winemaking and agro-industrial grape juice production, can be valorized as a source of bioactive compounds and as feedstock for the saccharification and bioconversion of soluble sugars. Solid-state fermentation (SSF) by six white-rot fungi was applied as pretreatment. Fiber composition, free radical scavenging activity, four ligninolytic, and three hydrolytic enzyme activities were determined. Saccharification kinetics, yield, and productivity were evaluated and complemented with scanning electron microscopy (SEM), high performance liquid chromatography (HPLC) quantification of monosaccharides, and principal component analysis (PCA). After SSF, the biomass exhibited a drastic free radical scavenging activity decrease and the main enzymes produced were manganese-dependent peroxidase and xylanase. Scanning electron microscopy revealed the erosion of cell walls, and PCA exhibited a negative correlation between saccharification, and neutral detergent fiber and acid detergent lignin. Phlebia rufa pretreated biomass gave the highest sugars yield and productivity, representing a nearly three-fold increase compared to untreated samples. Also, monosaccharides quantification revealed that the 1:1 ratio of glucose to the sum of xylose plus galactose changes to the value of 2:1 after pretreatment. In this work, and for the first time, P. rufa proved to be an effective pretreatment of grape stalks for the saccharification and further bioconversion into value-added chemicals. In addition, lignocellulolytic enzymes were also produced through SSF.

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.