Air quality in a small city: criteria pollutants, volatile organic compounds, metals, and microbes.

This work presents a year-long integral study of air quality parameters in Ciudad Real, a small city in the center of Spain, and its influence on the nearby national park, Las Tablas de Daimiel. The study covers meteorological parameters and criteria pollutants such as O3, NO, NO2, SO2, and PM10. Additionally, for each month, a 1-week campaign was performed sampling air in sorbent tubes with 8-h time resolution to analyze anthropogenic volatile organic compounds and the effects of seasons, daytime, and working-weekend days. During these campaigns, 24-h PM2.5 samples were also collected to measure the load of bacteria and fungi, as well as the trace concentrations of elements.The city and the national park NOx profiles showed that emissions from the town had a non-perceivable effect on the protected area. PM10 levels in Ciudad Real were influenced by Saharan intrusions, as was the national park; however, Ciudad Real had a higher contribution from anthropogenic sources. Ozone levels were lower in the city during the cold season due to the higher concentration of NOx and have not changed significantly in the last decade.The VOCs with higher average concentrations were toluene, m,p-xylene, benzene, methylene chloride, and o-xylene, with traffic being the main source of these pollutants in the city. For benzene and carbon tetrachloride levels, weak carcinogenic risks were estimated. In PM2.5, the most abundant metals were Na, Zn, Mg, Ca, Al, Fe, and K. The carcinogenic and non-carcinogenic risks estimated from the levels of the studied metals were negligible. Bacterial and fungal counts positively correlated with the concentration of PM2.5. Microbial community composition showed seasonal variability, with the dominance of human pathogenic bacteria which correlated with certain pollutants such as SO2. Bacillus and Cutibacterium were the most abundant genera.

Effects of massive desiccation of olive waste residues on air quality.

New industries are proliferating in the recovery of agri-food wastes, such as those involved in the revaluation of alperujo, generated in the production of olive oil. Despite the potential environmental benefits, their activity is not exempt from new forms of emissions, aggravated by the massification of waste treatments. This work reports a six-month field campaign carried out in an alperujo desiccation plant which can serve as a proxy for these emerging industries in the Mediterranean countries. The study focused on air quality parameters, covering criteria pollutants, metals and microbiological load of particulate matter and the characterization of volatile organic compounds (VOCs). The results show a slight contribution of the factory to the NOx levels in the surroundings (3.0-12.5 µg/m3). Statistically significant effects were not observed for ozone, CO, SO2, or PM10. Concerning the levels of metals, concentrations were low and calculated health risk indexes indicated safe conditions in the area. The most abundant elements were Na (6.5 × 102 ng/m3), K (4.0 × 102 ng/m3), Al (2.7 × 102 ng/m3), Zn (2.1 × 102 ng/m3), Ca (2.16 × 102 ng/m3), Fe (3.6 × 101 ng/m3) and Mg (3.2 × 101 ng/m3). Bacterial counts, with a mean value of 15.9 CFU/m3, showed a seasonal shift, mainly explained by weather (air moisture and temperature) and PM2.5 concentration. The genomic analysis showed Cutibacterium as the dominant genus during the cold months while Bacillus predominated in the warm season. The VOCs with higher average concentrations were acetic acid (130 µg/m3), nonanoic acid (124 µg/m3), benzoic acid (29.7 µg/m3), octanoic acid (19.9 µg/m3) and nonanal (4.70 µg/m3), with the rest of compounds in concentrations below 4 µg/m3. Odorant pollutants with the greatest contribution to olfactory nuisance were aldehydes (from pentanaldehyde to decanaldehyde), acetic acid and phenol. Although the observable effects of the waste treatments were low, several parameters showed an influence on the environment which should be assessed to foresee and prevent long-term consequences.

Ozone modified hypothalamic signaling enhancing thermogenesis in the TDP-43A315T transgenic model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS), a devastating progressive neurodegenerative disease, has no effective treatment. Recent evidence supports a strong metabolic component in ALS pathogenesis. Indeed, metabolic abnormalities in ALS correlate to disease susceptibility and progression, raising additional therapeutic targets against ALS. Ozone (O3), a natural bioactive molecule, has been shown to elicit beneficial effects to reduce metabolic disturbances and improved motor behavior in TDP-43A315T mice. However, it is fundamental to determine the mechanism through which O3 acts in ALS. To characterize the association between O3 exposure and disease-associated weight loss in ALS, we assessed the mRNA and protein expression profile of molecular pathways with a main role in the regulation of the metabolic homeostasis on the hypothalamus and the brown adipose tissue (BAT) at the disease end-stage, in TDP-43A315T mice compared to age-matched WT littermates. In addition, the impact of O3 exposure on the faecal bacterial community diversity, by Illumina sequencing, and on the neuromuscular junctions (NMJs), by confocal imaging, were analysed. Our findings suggest the effectiveness of O3 exposure to induce metabolic effects in the hypothalamus and BAT of TDP-43A315T mice and could be a new complementary non-pharmacological approach for ALS therapy.

Are the Portable Air Cleaners (PAC) really effective to terminate airborne SARS-CoV-2?

The transmission of SARS-CoV-2 virus through aerosols has become an outstanding issue, where plenty of spread aspects are being analyzed. Portable Air Cleaners (PAC) with high-efficiency particulate air (HEPA) filters have been discussed as an adjunctive means for indoor environments coronavirus decontamination. This study evaluates, first, the air and surfaces SARS-COV-2 RNA contamination due to positive patients in households, and second, the efficiency of a PAC with HEPA filter to eliminate virus. A total of 29 air and surface samples were collected inside 9 households, by using an air portable collector with gelatin filters and swabs. SARS-CoV-2 RNA detection was performed using real-time reverse transcription polymerase chain reaction (RT-PCR). Overall, all the air samples collected before using PAC and 75% of swab samples were positive for SARS-CoV-2. After the PAC usage, all samples except one were negative, displaying a 80% device effectiveness. Portable HEPA cleaners usage allowed the removal of SARS CoV-2 and, therefore, they could be recommended for places with inadequate ventilation, considering the limitations and functionality of the device.

Temporal variability measurements of PM2.5 and its associated metals and microorganisms on a suburban atmosphere in the central Iberian Peninsula.

A novel and multidisciplinary observational analysis of atmospheric components in the Central Iberian Peninsula is presented here. PM2.5 concentrations and both populations of cultivable and non-cultivable microorganisms and concentrations of a wide range of trace elements associated have been simultaneously studied during multiple events along one year. The aim has been to characterize their potential relations and dependencies, and their seasonal, daily and hourly evolution. Tools that could explain the atmospheric mechanisms and sources from all these elements have been also evaluated. As it would be expected from a suburban environment, absolute levels obtained were not close to legislation limits. Anthropogenic and natural sources, such as heating home, soil resuspension, or Sahara dust intrusion; and atmospheric factors are responsible for higher PM2.5 and metals concentrations in months with both low and high temperatures. Daily and hourly evolution depends on University Campus activity, especially on traffic flow and resuspended dust due to human transit. No statistical significant differences on daily or seasonal scales between cultivable counts of fungi and bacteria were displayed. However, using the q-PCR technique, the bacterial population was lower in winter. Positive correlations between PM2.5 and relative humidity; and PM2.5 and cultivable microorganism have been established. It was also the case among 7 of the 11 trace elements, indicating then common natural or anthropogenic sources. In summary, this work illustrates the interest of a combined inspection of elements, interactions and dependencies when studying the unique and continuous atmospheric environment, which are typically analysed separately.

Factors Affecting Levels of Airborne Bacteria in Dairy Farms: A Review.

This review attempts to reflect the importance of different factors that affect the environmental quality of dairy farms and must, therefore, be taken into account when considering the importance of environmental microbiology as a tool in the improvement of the quality of milk and dairy products. The effect of a factor such as temperature is vital for the dairy farm environment, especially when the temperatures are extreme, because a proper choice of temperature range improves the quality of the air and, thus, animal welfare. Similarly, the appropriate level of relative humidity in the environment should be taken into consideration to avoid the proliferation of microorganisms on the farm. Air quality, well-designed livestock housing, proper hygienic practices on the farm, stocking density, and the materials used in the livestock houses are all important factors in the concentration of microorganisms in the environment, promoting better welfare for the animals. In addition, a ventilation system is required to prevent the pollution of the farm environment. It is demonstrated that proper ventilation reduces the microbial load of the environment of dairy farms, enhancing the quality of the air and, therefore, the wellbeing of the animals. All this information is very useful to establish certain standards on dairy farms to improve the quality of the environment and, thereby, achieve better quality milk and dairy products.

Is staphylococci population from milk of healthy goats safe?

The aim of this study was to assess the species and the genetic diversity of the staphylococci population in raw milk from healthy goats. Isolates representative of all genotypes were screened for their potential pathogenicity by the occurrence of some relevant safety-related properties, such as antibiotic resistance, presence of virulence factor genes, biofilm formation ability and biogenic amine production. A total of 314 staphylococci were isolated, and randomly amplified polymorphic DNA-PCR analysis displayed 48 genotypes. Isolates were identified as belonging to S. epidermidis (87.5%), S. caprae (6.2%), S. aureus (4.2%) and S. simulans (2.1%) species. The antibiotic resistance varied strongly with strains, with S. epidermidis and S. aureus strains showing resistance to more number of antibiotics. A high occurrence of strains harbouring hemolysin genes was also found in both species. On the contrary, none of the strains assayed harboured enterotoxin or amino acid decarboxylase genes, and, although a moderate or high biofilm formation was observed in 29% of the strains, they did not harbour icaA or icaD genes. This study gives a first and extensive picture of safety-related properties within Staphylococcus species isolated from milk of healthy goats, displaying that these species can act as a reservoir for spreading genes related to safety.

Genomic diversity of Oenococcus oeni populations from Castilla La Mancha and La Rioja Tempranillo red wines.

The Oenococcus oeni populations of Tempranillo wines from Castilla La Mancha and La Rioja winemaking regions were analysed from one to three years and up to ten wineries. The objective was to evaluate the genetic variability and the O. oeni population structure. For this purpose a MLST scheme based on four loci (gyrB, purK, pgm and recP genes) and PFGE with SfiI restriction enzyme were developed for later combination. The results showed an O. oeni population completely adapted to winemaking regions. A purifying selection influenced the genes evolution, especially recP that along with purK were the most interesting loci to analyse the genetic variability of the isolates. In this way linkage disequilibrium and intergenic and intragenic recombination were determined between isolates. PFGE typing with UPGMA data were not coincident with the phylograms assessed for MLST by Maximum likelihood and combination of both techniques differentiated all the isolates as strains. Those results led the research to conclude that O. oeni population from CM and LR was a panmictic population with a slight clonal evolution, so subpopulations could not be described. A broader study including more winemaking regions with different grape varieties and distinct ways of elaborating would be interesting to complete the knowledge about O. oeni populations.

Sequential inoculation versus co-inoculation in Cabernet Franc wine fermentation.

A study has been carried out in order to determine the effect of the lactic acid bacteria inoculation time on the kinetic of vinification and on chemical and sensory characteristics of Cabernet Franc wines. Traditional vinifications, with lactic acid bacteria inoculated after completion of alcoholic fermentation were compared with vinifications where yeast and bacteria were co-inoculated at the beginning of vinification. One commercial yeast strain and an autochthonous Oenococcus oeni strain (C22L9), previously identified and selected at our laboratory, were used. Monitoring of alcoholic and malolactic fermentations was carried out by yeast and lactic acid bacteria counts and by measuring l-malic acid concentration. Wines were chemically characterized and analysed for volatile compounds content. A sensory analysis, consisting of a descriptive and a triangular test, was also carried out. Results from this study showed that the concurrent yeast/bacteria inoculation of musts at the beginning of vinification produced a reduction in duration of the process without an excessive increase in volatile acidity. Differences in volatile compounds content and the corresponding impact on the sensorial profile of wines were also displayed. These results suggest that co-inoculation is a worthwhile alternative for winemaking of Cabernet Franc wines, if compared with traditional post-alcoholic fermentation lactic acid bacteria inoculation.

Microbial communities in air and wine of a winery at two consecutive vintages.

The aim of this study was to assess, both quantitatively and qualitatively, the populations of lactic acid bacteria (LAB) and yeasts in air and wine of a winery, in order to evaluate the possible exchange of microorganisms between them. Samples were taken in a winery located in Castilla-La Mancha (Spain) during the winemaking period of two consecutive vintages (2011 and 2012). The microbial composition was determined by using both a culture-dependent method and a culture-independent method, PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In addition, genetic characterization of isolates from plates was carried out. A high diversity of species was detected in air and wine samples from both vintages. Leuconostoc mesenteroides was the predominant lactic acid bacteria in air from both vintages while Oenococcus oeni was the predominant in wine. Saccharomyces cerevisiae was the most frequently isolated yeast in both air and wine. Typing of O. oeni and S. cerevisiae isolates from air and wine samples showed the presence of coincident genotypes in both samples, that would confirm the exchange of microorganisms between the two environments, air and wine, and furthermore some of these genotypes were also found at samples taken at different vintages, indicating that they would remain in the winery. The results display the influence of the activity taking place in the winery and the moment of fermentation of the wines in tanks, on the microorganisms present in the air and the role of the air for the dispersal of microorganisms within the winery.

Are Enterococcus populations present during malolactic fermentation of red wine safe?

The aim of this study was the genetic characterisation and safety evaluation of 129 Enterococcus isolates obtained from wine undergoing malolactic fermentation. Genetic characterisation by randomly amplified polymorphic DNA-PCR displayed 23 genotypes. 25 isolates representative of all genotypes were identified as Enterococcus faecium by species-specific PCR and assayed for antibiotic resistance, presence of virulence genes and aminobiogenic capacity, both in decarboxylase medium and wine. The aminobiogenic capacity in wine was analysed in presence (assay 1) and absence (assay 2) of Oenococcus oeni CECT 7621. Resistance to tetracycline, cotrimoxazol, vancomycin and teicoplanin was exhibited by 96% of the strains, but none of them harboured the assayed virulence genes. All of the strains harboured the tyrosine decarboxylase (tdc) gene, while 44% were positive for tyramine in decarboxylase medium. Only five out of 25 strains survived in wine after seven days of incubation, and when concentrations of biogenic amines in wines were determined by HPLC, only those wines in which the five surviving strains occurred contained biogenic amines. Histamine, putrescine and cadaverine were detected in wines from both assays, although concentrations were higher in assay 2. Tyramine and phenylethylamine were detected only in absence of O. oeni. This research contributes for the knowledge of safety aspects of enterococci related to winemaking.

Esterase activity of lactic acid bacteria isolated from malolactic fermentation of red wines.

The goal of this study was to examine the esterase activity of 243 lactic acid bacteria (LAB) strains from wines of different red grape varieties, belonging to the genera Oenococcus, Lactobacillus, Pediococcus and Enterococcus. p-Nitrophenyl octanoate was used as substrate. All strains presented esterase activity in the first screening, but only those showing higher activity were used in subsequent studies to determine the cellular location of this activity, the influence of pH, temperature and the presence of ethanol and the substrate specificity. For the thirteen selected strains, the highest activity was observed in the intracellular fraction. Responses to pH, temperature and ethanol were strain-dependent, but for all the strains, a marked decrease in activity in presence of ethanol was observed. When the influence of pH and ethanol acting together was studied at 25 °C and 37 °C, temperature-dependent differences were not observed for any of the strains except for Oen6. In the substrate specificity assay, the majority of strains of all genera displayed a trend to more readily hydrolyse ester substrates from C8 and longer.

Malolactic fermentation and secondary metabolite production by Oenoccocus oeni strains in low pH wines.

UNLABELLED: Different wine varieties, including some with low pH, were studied to determine the ability to grow and produce secondary metabolites of a previously selected autochthonous Oenococcus oeni strain (C22L9), compared with a commercial strain. Monitoring of malolactic fermentation (MLF) was carried out by microbiological and chemical analysis of wines. The concentration of some major volatile compounds and biogenic amines in wines before and after malolactic fermentation was also determined. The results showed major differences in MLF duration both between wines and strains, although the differences between strains were slight for most of the analyzed compounds. Statistically significant differences in citric acid degradation were found in all wine varieties and it was confirmed that O. oeni C22L9 is a poor degrader of citric acid; this means that MLF can be prolonged without the risk of producing high concentrations of diacetyl and acetoin. Sensory analysis of wines after MLF showed similar characteristics in wines from both strains. This study thus shows that O. oeni C22L9 possesses even better sensory and fermentation properties than the commercial strain and can be used in wines with different characteristics, which makes it highly valuable for industrial use. PRACTICAL APPLICATION: The increasingly use of grape varieties of low pH in winemaking and the higher alcohol content of wines, as a consequence of the climatic change, make interesting the study of the behavior during MLF of O. oeni strains in order to determine their ability to grow, when growth conditions are not optimal, and to produce secondary metabolites. A comparative study was conducted using an autochthonous O. oeni strain (C22L9) and a commercial O. oeni strain and 4 wine varieties.

Screening for glycosidase activities of lactic acid bacteria as a biotechnological tool in oenology.

The aim of this study was to evaluate the ability from a number of lactic acid bacteria isolated from different sources to produce glycosidase enzymes. Representative isolates (225) from clusters obtained after genotyping, using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis, of 1,464 isolates, were screened for ß-D-glucosidase activity. Thirty-five of them were selected for subsequent analysis. These strains were able to hydrolyze α-D-glucopyranoside, ß-D-xylopyranoside and α-L-arabinofuranoside although ß-D-glucosidase activity was the predominant activity for 22 of the selected strains. Only some of them did so with α-L-rhamnopyranoside. All of these were from wine samples and were identified as belonging to the Oenococcus oeni species using Amplification and Restriction Analysis of 16S-rRNA gene (16S-ARDRA). When the influence of pH, temperature and ethanol or sugars content on ß-D-glucosidase activity was assayed, a strain-dependent response was observed. The ß-D-glucosidase activity occurred in both whole and sonicated cells but not in the supernatants from cultures or obtained after cell sonication. Strains 10, 17, 21, and 23 retained the most ß-D-glucosidase activity when they were assayed at the conditions of temperature, pH, ethanol and sugar content used in winemaking. These results suggest that these strains could be used as a source of glycosidase enzymes for use in winemaking.

Enterococcus populations in artisanal Manchego cheese: biodiversity, technological and safety aspects.

Enterococci represent a considerable proportion of the microbiota in Manchego cheeses. In this study, a total of 132 enterococci isolated from good quality Manchego cheeses from two dairies at different ripening times were genotypically characterized and identified using molecular techniques. Representative isolates from the clusters obtained after genotyping were assayed for some enzymatic activities considered to have a potential role in cheese ripening, and for 2,3-butanedione and acetoin production, evaluation of odor intensity and appearance in milk and safety evaluation. Enterococcus faecalis was the predominant specie, accounting for 81.8% of the total isolates, while Enterococcus faecium, Enterococcus hirae and Enterococcus avium were present in low proportions. The number of genotypes involved at each ripening time varied both between dairies and with the ripening times; genotype E. faecalis Q1 being present in almost all the samples from both dairies. Eight isolates showed a higher proteolytic activity and 3 isolates produced high quantities of acetoin-diacetyl, for which reason they are interesting from a technological standpoint. A low antibiotic resistance was found and almost all the strains were susceptible to clinically important antibiotics. On the contrary, only four isolates (E. faecalis C4W1 and N0W5, and E. faecium N32W1 and C16W2) did not harbor some of the virulence genes assayed.

Bacterial biodiversity and dynamics during malolactic fermentation of Tempranillo wines as determined by a culture-independent method (PCR-DGGE).

The bacterial population during malolactic fermentation of Tempranillo wine was studied using the polymerase chain reaction-denaturing gradient gel electrophoresis, a culture-independent method successfully used for identification and monitoring of bacterial population in different habitats included food fermentations. The results showed that Oenococcus oeni was the predominant species in the malolactic fermentation of Tempranillo wines, although the presence of Gluconobacter oxydans, Asaia siamensis, Serratia sp., and Enterobacter sp. was also observed. These results were partly coincidental with those obtained from a culture-dependent method, using a selective medium. Therefore, it may be concluded that for a more complete knowledge of the bacterial community present during malolactic fermentation of Tempranillo wine, an approach that combines a culture-independent method and a culture-dependent method would be advisable.

Selection of autochthonous Oenococcus oeni strains according to their oenological properties and vinification results.

The goal of this study is to carry out a characterization of 84 Oenococcus oeni strains isolated from Tempranillo wine samples taken at the cellars in Castilla-La Mancha, in order to select those showing the highest potential as oenological starter cultures. Various oenological properties were analyzed and the ability of some of these strains to grow and undergo MLF in simulated laboratory microvinifications was tested. Twenty-two strains were selected on the basis of fermentation assays and the eight that produced the best results in the chemical analysis of the wines were chosen for further assays. None of the eight strains was either able to produce biogenic amines or displayed tannase or anthocyanase activities. On the other hand all presented activity against p-NP-beta Glucopyranoside, p-NP-alpha Glucopyranoside and p-NP-beta xylopyranoside. Randomly Amplified Polymorphic DNA (RAPD)-PCR was used to determine the colonizing ability of the inoculated strains. C22L9 and D13L13 strains showed the highest implantation values. On the basis of this characterization, two strains have been selected which are suitable as starter cultures for MLF of Tempranillo wine. Use of these strains will ensure that MLF proceeds successfully and gives retention of the organoleptic characteristics of wines made in Castilla-La Mancha.

Genotypic and technological characterization of Leuconostoc isolates to be used as adjunct starters in Manchego cheese manufacture.

Twenty-seven Leuconostoc (Ln.) isolates from Manchego cheese were characterized by phenotypic and genotypic methods, and their technological abilities studied in order to test their potential use as dairy starter components. While phenotypic diversity was evaluated by studying the biochemical characteristics of technological interest (i.e. acidifying and aminopeptidase activities), genotypic diversity was evidenced by using Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). Additional technological abilities such as lipolytic, proteolytic and autolytic activities, salt and pH tolerance and production of dextran, flavour compounds and biogenic amines, were investigated. The marked differences among strains reflected the existing biodiversity in naturally fermented products. After statistically evaluating their performance, strains C0W2, belonging to Ln. lactis, and C16W5 and N2W5, belonging to Ln. mesenteroides subsp. dextranicum, revealed the best properties to be used in mixed dairy starter cultures. This study evidences the fact that natural environments can be considered as a proper source of useful strains, for the dairy industry.

Intraspecific genetic diversity of lactic acid bacteria from malolactic fermentation of Cencibel wines as derived from combined analysis of RAPD-PCR and PFGE patterns.

Three molecular techniques, randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), pulsed field gel electrophoresis (PFGE) and differential display-polymerase chain reaction (DD-PCR) have been used to assess the intraspecific diversity of the lactic microbiota responsible for spontaneous malolactic fermentation (MLF) in Cencibel wines made at a cellar in Castilla-La Mancha (Spain). The results indicate that RAPD-PCR and PFGE are of value in typing this microbiota. Better discrimination was achieved by RAPD-PCR. Reproducibility using DD-PCR was not good, which makes this method unsuitable. Combined numerical analysis of the patterns obtained from RAPD-PCR and PFGE allowed a better discrimination; this would therefore be a suitable tool to discriminate the diversity of bacterial communities like those found in MLF of wines. Genetic diversity data from combined numerical analysis suggest that there is considerable microbial diversity within MLF of Cencibel wines, with some genotypes coinciding in the two vinifications analysed.

Genetic diversity, dynamics, and activity of Lactobacillus community involved in traditional processing of artisanal Manchego cheese.

A total of 248 strains of predominant lactobacilli isolated during the manufacture and ripening of artisanal Manchego cheeses obtained from two dairies were obtained and the genetic diversity of 197 investigated using random amplified polymorphic DNA (RAPD-PCR). 51 isolates could not be lysed and were therefore not genotyped. Forty-two distinct RAPD patterns, grouped in six major clusters at a similarity level of 54%, were obtained. Phenotypic characterization of isolates enabled their assignment to the species L. plantarum, L. brevis, L. paracasei subsp. paracasei, L. fermentum, L. pentosus, L. acidophilus and L. curvatus. In samples from both dairies, the species L. plantarum, L. brevis and L. paracasei subsp. paracasei dominated during ripening. Three genotypes showed excellent physiological characteristics and were therefore proposed as adjunct cultures for Manchego cheese manufacture.