Enological and genetic traits of Saccharomyces cerevisiae isolated from former and modern wineries

Saccharomyces cerevisiae strains isolated from two different wineries in central Italy were subjected to enological and molecular characterization to investigate the influence of the winery environment. One of the selected wineries is a modern, working winery, whereas the second one was abandoned since 1914 and was located in an artificial cavern. The results obtained by our analysis of the fermentation traits underline the selectivity of the winery environment (winery effect), since strains isolated from the industrial winery showed higher values for characters typically subjected to selective pressure, such as maximum capability to produce ethanol, fermentation rate and SO(2) resistance. Pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD)-PCR and SAU-PCR were carried out to assesss genetic differences between the two populations studied. Only RAPD-PCR could distinguish between the two populations based on their provenience, whereas PFGE and SAU-PCR gave profiles shared between strains isolated from the industrial and former winery. Moreover, analysis of the karyotypes suggested the presence of chromosomal-length polymorphism; differences in the size and number of chromosomes between the two groups of isolates, as well as within each group, were observed.     

Biotechnology of natural and winery-associated strains of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

A new body of evidence challenges the original consolidated theory of Pasteur on the natural (vineyard) origin of wine strains of Saccharomyces cerevisiae and instead indicates a local, winery-restricted life cycle. The findings open novel biotechnological perspectives for obtaining autochthonous selected starters for the wine industry. A local, individual, and specific fermenting yeast flora, mass selected year after year through many generations of S. cerevisiae in grape must, is present on the surfaces of every winery. These yeast strains are endowed with exceptional enological properties and capable of producing an assortment of volatile compounds apparently contributing to the specific bouquet of locally produced wines.     

Detection and quantification of Erysiphe necator DNA in wine grapes and resultant must and juice

Powdery mildew of grapevines is difficult to assess visually at the weighbridge, particularly in large consignments of machine-harvested fruit. To facilitate accurate methods for the detection and quantification of the disease in grape samples obtained from both the vineyard and winery, we developed a DNA probe for the pathogen Erysiphe necator. The E. necator-specific 450 bp DNA fragment pEnA1, targets highly repetitive sequences and was isolated from a partial genomic library. In screening for species specificity, clone pEnA1 was used in slot-blot hybridization and detected E. necator DNA from grapes and resultant must and juice, but not from clarified juice and wine. The detection threshold was approximately 50 pg ofE. necator DNA per 100 ng total DNA of grape sample and was equivalent to 1–5 % of a grape bunch visually affected by powdery mildew. Disease severity, expressed as the percentage of surface area of a bunch with powdery mildew, and E. necator DNA content were highly correlated, r² = 0.955, P < 0.001. The DNA-based hybridization assay has the potential to predict the severity of powdery mildew in grape samples from the vineyard and in must and juice samples at the winery. The DNA sequence of clone pEnA1 was used to design species-specific primers, the results maintaining the same specificity patterns observed in the initial hybridization assays. The PCR-based assay was sensitive enough to detect approximately 1 pg DNA, being equivalent to 1 conidium per sample. This is the first report to date of the detection of all known phenetic groups of E. necator DNA and of the quantification of DNA from grape samples at the winery. Accurate information on the extent of powdery mildew contamination of grape lots would enable wineries to make more informed decisions about the use of fruit and must.     

Alleviation of the effects of nitrogen limitation in high gravity worts through increased inoculation rates

Summary There are few inexpensive, practical methods to increase the usable nitrogen level in a substrate to be fermented to a potable alcohol product, but the provision of adequate assimilable nitrogen to a fermentation medium is critical for rapid and full wort attenuation. One practical solution to circumvent the problem may be to increase the inoculation rate to much higher than recommended levels. In this work, an increase in the pitching rate from 1.6×10⁷ cfu/ml to 8×10⁷ cfu/ml was shown to alleviate fermentation problems caused by nitrogen limitation. Attenuation and ethanol production rates became independent of the initial wort-free amino nitrogen (FAN) concentration, as did yeast viability and maximal yeast cell number. However, the final total cell mass was lower if the wort was nitrogen-deficient, regardless of the pitching rate. These cells were smaller and/or lighter and contained less protein at the end of fermentation. Such yeast could cause problems in subsequent fermentations if reuse of yeast (common in brewing) was considered.     

Potential for methane production from anaerobic co-digestion of swine manure with winery wastewater

This work examines the methane production potential for the anaerobic co-digestion of swine manure (SM) with winery wastewater (WW). Batch and semi-continuous experiments were carried out under mesophilic conditions. Batch experiments revealed that the highest specific methane yield was 348 mL CH₄ g⁻¹ COD added, obtained at 85.4% of WW and 0.7 g COD g⁻¹ VS. Specific methane yield from SM alone was 27 mL CH₄ g⁻¹ COD added d⁻¹. Furthermore, specific methane yields were 49, 87 and 107 mL CH₄ g⁻¹ COD added d⁻¹ for the reactors co-digesting mixtures with 10% WW, 25% WW and 40% WW, respectively. Co-digestion with 40% WW improved the removal efficiencies up to 52% (TCOD), 132% (SCOD) and 61% (VSS) compared to SM alone. These results suggest that methane can be produced very efficiently by the co-digestion of swine manure with winery wastewater.     

Strategies for recycling and valorization of grape marc

Grapes are one of the most cultivated fruit crops worldwide. Either for wine or juice production, grape processing generates a large amount of residues that must be treated, disposed of or reused properly to reduce their pollution load before being applied to the soil. In this review, a special focus is given to the treatment and valorization of the winemaking by-product like grape marc via anaerobic digestion, composting and vermicomposting at laboratory, pilot, and industrial scales. The impact of the final products (digestates, composts, and vermicomposts) on soil properties is briefly addressed. Moreover, the role of grape marc and seeds as a valuable source of natural phytochemicals that include polyphenols and other bioactive compounds of interest for pharmaceutical, cosmetic, and food industries is also discussed. This is of paramount importance given the fact that sustainability requires the use of management and valorization strategies that allow the recovery of valuable compounds (e.g. antioxidants) with minimum disposal of waste streams.     

Microbial characterisation of activated sludge in jet-loop bioreactors treating winery wastewaters

Jet-loop reactors (JLR) used as biological waste treatment processes introduce an additional selective pressure on the natural microbial flora of the incoming effluent. Several high-performing microbial inocula were tested for winery wastewater treatment and the microbial composition was analysed. A microbial consortium was enriched and selected for use with a new type of aerobic JLR. The reactor was operated continuously for more than 1 year using winery wastewaters collected in different seasons. Chemical oxygen demand (COD) removal efficiency was on average greater than 80%, with retention times of 0.8–1 day. Microbial populations were sampled for characterisation after 6 months and at the end of the study. Isolates were identified at genus and/or species level. Almost all isolates belonged to the genera Pseudomonas and Bacillus. Saccharomyces cerevisiae was also found but no filamentous fungi. These results show that a highly adapted population develops in JLRs treating winery effluents as compared to other bioreactors. Aerobic JLRs impose a stringent selective criterion on the composition of the microbial biomass.     

Anaerobic treatment of winery wastewater using laboratory-scale multi- and single-fed filters at ambient temperatures

A lab-scale investigation was conducted to examine the effectiveness of a multi-fed upflow anaerobic filter process for the methane production from a rice winery effluent at ambient temperatures. The experiment was carried in two identical 3.0-l upflow filters, a single-fed reactor and a multi-fed reactor. The results showed that the multi-fed reactor, operated at the ambient temperatures of 19–27 °C and influent chemical oxygen demand (COD) of 8.34–25.76 g/l, could remove over 82% of COD even at an organic loading rate (OLR) of 37.68 g-COD/l d and a short hydraulic retention time (HRT) of 8 h. This reactor produced biogas with a methane yield of 0.30–0.35 l-CH₄/g-COD_removed. The multi-fed upflow anaerobic filter was proved to be more efficient than the single-fed reactor in terms of COD removal efficiency and stability against hydraulic loading shocks. A linear-regression model with influent COD concentration and HRT terms adequately described the multi-fed upflow anaerobic filter system for the treatment of rice winery wastewater at ambient temperatures.