Wastewater-based epidemiology (WBE) is a useful tool that has the potential to act as a complementary approach to monitor the presence of SARS-CoV-2 in the community and as an early alarm system for COVID-19 outbreak. Many studies reported low concentrations of SARS-CoV-2 in sewage and also revealed the need for methodological validation for enveloped viruses concentration in wastewater. The aim of this study was to evaluate different methodologies for the concentration of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. A total of 11 concentration techniques based on different principles were evaluated: adsorption-elution protocols with negatively charged membranes followed by polyethylene glycol (PEG) precipitation (Methods 1-2), PEG precipitation (Methods 3-7), aluminum polychloride (PAC) flocculation (Method 8), ultrafiltration (Method 9), skim milk flocculation (Method 10) and adsorption-elution with negatively charged membrane followed by ultrafiltration (Method 11). To evaluate the performance of these concentration techniques, feline calicivirus (FCV) was used as a process control in order to avoid the risk associated with handling SARS-CoV-2. Two protocols, one based on PEG precipitation and the other on PAC flocculation, showed high efficiency for FCV recovery from wastewater (62.2 % and 45.0 %, respectively). These two methods were then tested for the specific recovery of SARS-CoV-2. Both techniques could recover SARS-CoV-2 from wastewater, PAC flocculation showed a lower limit of detection (4.3 × 10 2 GC/mL) than PEG precipitation (4.3 × 10 3 GC/mL). This work provides a critical overview of current methods used for virus concentration in wastewaters and the analysis of sensitivity for the specific recovery of SARS-CoV-2 in sewage. The data obtained here highlights the viability of WBE for the surveillance of COVID-19 infections in the community.

The effect of yeast concentration on ultraviolet (UV) inactivation of five strains of Escherichia coli O157: H7 from different sources, inoculated both individually and simultaneously in orange juice, was analyzed and mathematically modeled. The presence of yeast cells in orange juice decreases the performance of UV radiation on E. coli inactivation. UV absorption coefficients in the juice increased with increasing yeast concentration, and higher UV doses were necessary to inactivate bacterial strains. UV intensities of I=3.00±0.3 mW/cm 2 and exposure times (t) between 0 and 10 min were applied; radiation doses (energy, E=I×t) ranging between 0 and 2 J/cm 2 were measured using a UV digital radiometer. All the tested individual strains showed higher resistance to the treatment when UV radiation was applied at 4°C in comparison to 20°C. UV inactivation of E. coli O157:H7 individual strain was satisfactory fitted with a first order kinetic model. A linear relationship was found between UV absorptivities and D values (radiation doses required to decrease microbial population by 90%) for each strain. The dose required to reach 5-log reduction for the most unfavorable conditions that is the most UV resistant strain, and maximum background yeast concentration was 2.19 J/cm 2 at 4°C (corresponding to 11 min of UV treatment) and 2.09 J/cm 2 at 20°C (corresponding to 10.55 min of UV treatment). When a cocktail of strains was inoculated in orange juice, the logistic equation was the best model that fits the experimental results due to the deviation from the log-linear kinetics. The UV resistance between strain cocktail and single strain were mathematically compared. Slopes of the decline curves for strain cocktail at high UV doses were lower than the slopes of the log-linear equation calculated for the individual strains, even for the most resistant one. Therefore, microbial inactivation tests using a cocktail of strains are particularly important to determine the performance of the UV inactivation treatment.

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The efficacy of UV light for inactivating E. coli (ATCC 25922) and E. coli O157:H7 (EDL 933) was examined in fruit juices (orange, apple, and multifruit) with different absorptivities under several operating conditions (liquid film thickness and agitation rate). The juices were inoculated with two bacterial concentrations (10(5) and 10(7) CFU/ml) and were treated using a UV desinfection unit at 254 nm; UV doses ranged from 0 to 6 J/cm2. The effect of the culture medium, tryptone soy agar (TSA) and sorbitol MacConkey agar (SMAC), on the recovery of E. coli strains exposed to UV radiation was also analyzed. The most suitable culture medium for recovery of E. coli strains in juices exposed to UV radiation was TSA. Values of D (radiation dose [joules per square centimeter] necessary to decrease the microbial population by 90%) obtained in all juices assessed were higher in TSA than in SMAC. In the juices analyzed, stirring of the medium exposed to UV radiation and reducing liquid film thickness (to 0.7 mm) produced the highest bactericidal effect. A linear relationship was found between the D-values obtained and the absorptivity coefficients for all the juices. The higher the absorbance of the medium, the greater the values of D required to inactivate E. coli strains by UV radiation. An equation was developed to describe the relationship of the fraction of energy absorbed by the system (absorbed energy factor [AEF]), the thickness of the film exposed to UV radiation, and the absorptivity coefficient of the juices. A linear relationship was found between D and AEF in the different juices tested.

Interspecific hybrids among species in the Saccharomyces genus are frequently detected in anthropic habitats and can also be obtained easily in the laboratory. This occurs because the most important genetic barriers among Saccharomyces species are postzygotic. Depending on several factors, including the involved strains, the hybridization mechanism and stabilization conditions, hybrids that bear differential genomic constitutions, and hence phenotypic variability, can be obtained. In the present study, Saccharomyces cerevisiae × Saccharomyces uvarum hybrids were constructed using genetically and physiologically different S. uvarum parents at distinct temperatures (13 and 20°C). The effect of those variables on the main oenological features of the wines obtained with these hybrids was evaluated. Hybrids were successfully obtained in all cases. However, genetic stabilization based on successive fermentations in white wine at 13°C was significantly longer than that at 20°C. Our results demonstrated that, irrespective of the S. uvarum parent and temperature used for hybrid generation and stabilization, similar physicochemical and aromatic features were found in wines. The hybrids generated herein were characterized by low ethanol production, high glycerol synthesis and the capacity to grow at low temperature and to produce malic acid with particular aroma profiles. These features make these hybrids useful for the new winemaking industry within the climate change era frame.

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We aimed at isolating lactic acid bacteria (LAB) from different plant materials to study their crossed-fermentation capacity in silos and to find strains able to confer enhanced aerobic stability to silage. A total of 129 LAB isolates were obtained from lucerne (alfalfa), maize, sorghum, ryegrass, rice, barley, canola, Gatton panic, Melilotus albus, soy, white clover, wheat, sunflower, oat, and moha. Four Lactiplantibacillus plantarum subsp. plantarum strains (isolated from oat, lucerne, sorghum, or maize) were selected for their growth capacity. Identity (16S sequencing) and diversity (RAPD-PCR) were confirmed. Fermentative capacity (inoculated at 104, 105, 106, 107 CFU/g) was studied in maize silage and their cross-fermentation capacity was assessed in oat, lucerne, sorghum, and maize. Heterofermentative strains with the highest acetic acid production capacity conferred higher aerobic stability to maize silages. Regardless the source of isolation, L. plantarum strains, inoculated at a rate of 106 CFU/g, were effective to produce silage from different plant materials. From more than 100 isolates obtained, the application of a succession of experiments allowed us to narrow down the number of potential candidates of silage inoculants to two strains. Based on the studies made, L. plantarum LpM15 and Limosilactobacillus fermentum LfM1 showed potential to be used as inoculants, however further studies are needed to determine their performance when inoculated together. The former because it positively influenced different quality parameters in oat, lucerne, sorghum, and maize silage, and the latter because of its capacity to confer enhanced aerobic stability to maize silage. The rest of the strains constitute a valuable collection of autochthonous strains that will be further studied in the future for new applications in animal or human foods.