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A correct classification of fungi, including yeasts, is of prime importance to understand fungal biodiversity and to communicate about this diversity. Fungal genera are mainly defined based on phenotypic characteristics and the results of single or multigene-based phylogenetic analyses. However, because yeasts often have less phenotypic characters, their classification experienced a strong move towards DNA-based data, from short ribosomal sequences to multigene phylogenies and more recently to phylogenomics. Here, we explore the usefulness of various genomics-based parameters to circumscribe fungal genera more correctly taking the yeast domain as an example. Therefore, we compared the results of a phylogenomic analysis, average amino acid identity (AAI) values, the presence of conserved signature indels (CSIs), the percentage of conserved proteins (POCP) and the presence- absence patterns of orthologs (PAPO). These genome-based metrics were used to investigate their usefulness in demarcating 13 hitherto relatively well accepted genera in Saccharomycetaceae, namely Eremothecium, Grigorovia, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces, Naumovozyma, Saccharomyces, Tetrapisispora, Torulaspora, Vanderwaltozyma, Zygosaccharomyces and Zygotorulaspora. As a result, most of these genera are supported by the genomics-based metrics, but the genera Kazachstania , Nakaseomyces and Tetrapisispora were shown to be genetically highly diverse based on the above listed analyses. Considering the results obtained for the presently recognized genera, a range of 80–92% POCP values and a range of 60–70 % AAI values might be valuable thresholds to discriminate genera in Saccharomycetaceae. Furthermore, the genus-specific genes identified in the PAPO analysis and the CSIs were found to be useful as synapomorphies to characterize and define genera in Saccharomycetaceae. Our results indicate that the combined monophyly-based phylogenomic analysis together with genomic relatedness ind

Yeasts are unicellular eukaryotic microorganisms extensively employed in various applications, notably as an alternative source of protein in feeds, owing to their nutritional benefits. Despite their potential, marine and mangrove yeast species used in the aquaculture industry have received little attention in the Philippines. Pichia kudriavzevii (A2B R1 ISO 3), sourced from bark samples, was selected and mass-produced due to its high protein content and amino acid profile. The dried biomass of P. kudriavzevii was incorporated into the diets of Nile tilapia (Oreochromis niloticus) juveniles at varying inclusion levels (0, 1, 2, and 4 g/kg diet) and its effect on their growth performance, body composition, and liver and intestinal morphology was assessed after 40 days of feeding. The groups that received P. kudriavzevii at a concentration of 2 g/kg diet exhibited higher final body weight, percent weight gain, and specific growth rate in comparison to the other treatment groups. Whole body proximate composition did not vary among the dietary groups. Intestinal and liver histopathology also indicated no abnormalities. These findings suggest the potential of ascomycetous P. kudriavzevii as a beneficial feed additive in Nile tilapia diets, warranting further investigation into its long-term effects and broader applications in fish culture.

The bioprospection of indigenous microorganism strains with biotechnological potential represents a prominent trend. Metschnikowia yeasts exhibit diverse capabilities, such as ethanol reduction in winemaking, biocontrol potential, and lipid production. In this work, local Metschnikowia strains were isolated from different fruits by their ability to produce pulcherrimic acid, a molecule that has been linked to biocontrol activity and that binds iron giving colored colonies. Five strains were selected, each from one of five distinct sources. All of them were identified as M. pulcherrima. All five were able inhibit other yeasts and one M. pulcherrima, called M7, inhibited the growth of Aspergillus nidulans. The selected strains accumulated lipid bodies in stationary phase. Certain non-conventional yeasts like Hanseniaspora vineae are very sensitive to biomass drying, but cell extracts from M. pulcherrima added to the growth media as a source of antioxidant lipids increased their tolerance to drying. All strains isolated showed good stress tolerance (particularly to heat) and have nutrient requirements similar to a commercial M. pulcherrima strain. In addition, the M7 strain had a good growth in sugarcane and beet molasses and behaved like Saccharomyces cerevisiae in a growth medium derived from agricultural waste, a persimmon hydrolysate. Therefore, the isolation of local strains of Metschnikowia able to grow in a variety of substrates is a good source of biocontrol agents.

Progressive myoclonus epilepsy of Lafora type (LD, MIM 254780) is a fatal autosomal recessive disorder characterized by the presence of progressive neurological deterioration, myoclonus, epilepsy and polyglucosan intracellular inclusion bodies, called Lafora bodies. Lafora bodies resemble glycogen with reduced branching, suggesting an alteration in glycogen metabolism. Linkage analysis and homozygosity mapping localized EPM2A, a major gene for LD, to chromosome 6q24. EPM2A encodes a protein of 331 amino acids (named laforin) with two domains, a dual-specificity phosphatase domain and a carbohydrate binding domain. Here we show that, in addition, laforin interacts with itself and with the glycogen targeting regulatory subunit R5 of protein phosphatase 1 (PP1). R5 is the human homolog of the murine Protein Targeting to Glycogen, a protein that also acts as a molecular scaffold assembling PP1 with its substrate, glycogen synthase, at the intracellular glycogen particles. The laforin–R5 interaction was confirmed by pull-down and co-localization experiments. Full-length laforin is required for the interaction. However, a minimal central region of R5 (amino acids 116–238), including the binding sites for glycogen and for glycogen synthase, is sufficient to interact with laforin. Point-mutagenesis of the glycogen synthase-binding site completely blocked the interaction with laforin. The majority of the EPM2A missense mutations found in LD patients result in lack of phosphatase activity, absence of binding to glycogen and lack of interaction with R5. Interestingly, we have found that the LD-associated EPM2A missense mutation G240S has no effect on the phosphatase or glycogen binding activities of laforin but disrupts the interaction with R5, suggesting that binding to R5 is critical for the laforin function. These results place laforin in the context of a multiprotein complex associated with intracellular glycogen particles, reinforcing the concept that laforin is involved, Ministerio de Ciencia y Tecnología, Instituto de Salud Carlos III, Asociación Lafora España, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

Producción Científica, Two strains of Saccharomyces cerevisiae (Sc01 and Sc02) and one strain of Wickerhamomyces anomalus (Wa) were isolated from organic Verdejo spontaneous fermentations and used for the development of experimental winemaking. Sc01 and Sc02 represented 52.7% of the population of the Saccharomyces strains isolated throughout the fermentation process. W. anomalus appeared as the predominant species among the non-Saccharomyces yeasts. Wa turned out to be the strain of this species with the shortest lag phase and positive enzymatic activities, and it was selected for white wine production. Fermentations with unique inoculation of S. cerevisiae strains were compared with sequential inoculation with W. anomalus. The results showed that the sequential inoculations did not affect the fermentation kinetics or physicochemical characteristics of the wines compared with the unique inoculations. However, this study identified a significant impact on the aromatic profiles of the produced wines due to the sequential inoculations. This modification resulted in a similar new aromatic profile in both sequential inoculations, demonstrating common characteristics related to the contribution of W. anomalus. In general, the sequential fermentations were mainly characterized by lower levels of acetate esters and an increase in ethyl acetate levels, whereas lower levels of ethyl octanoate and ethyl dec-9-enoate were detected. Propan-1-ol and butan-1-ol showed an increase in the sequential fermentations, while 4-methylpentan-1-ol and 2-phenylethanol were found in lower concentrations. These results highlight the great influence that the presence of specific strains of native non-Saccharomyces yeasts exerts on the characteristics of elaborate wines., Universidad de Valladolid (UVa) y Bodegas Belondrade S. L. - (grant 061/100671)

Floral nectar is frequently colonised by microbes. However, nectar microbial communities are typically species-poor and dominated by few cosmopolitan genera. One hypothesis is that nectar constituents may act as environmental filters. We tested how five non-sugar nectar compounds as well as elevated sugar impacted the growth of 12 fungal and bacterial species isolated from nectar, pollinators, and the environment. We hypothesised that nectar isolated microbes would have the least growth suppression. Additionally, to test if nectar compounds could affect the outcome of competition between microbes, we grew a subset of microbes in co-culture across a subset of treatments. We found that some compounds such as H2 O2 suppressed microbial growth across many but not all microbes tested. Other compounds were more specialised in the microbes they impacted. As hypothesised, the nectar specialist yeast Metschnikowia reukaufii was unaffected by most nectar compounds assayed. However, many non-nectar specialist microbes remained unaffected by nectar compounds thought to reduce microbial growth. Our results show that nectar chemistry can influence microbial communities but that microbe-specific responses to nectar compounds are common. Nectar chemistry also affected the outcome of species interactions among microbial taxa, suggesting that non-sugar compounds can affect microbial community assembly in flowers.

In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications., CEI Campus Moncloa, UCM-UPM (Madrid, Spain)-PICATA predoctoral fellowship, Ministerio de Educación, Cultura y Deporte (España)-FPU predoctoral fellowship, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Diplomová práce se zabývá nalezením optimálního způsobu izolace RNA z různých druhů kvasinek v maximálním možném množství a čistotě, zejména z nepatogenních kvasinek (Saccharomyces cerevisiae) a patogenních kvasinek (Candida albicans a Candida parapsilosis). V teoretické části jsou popsány základní informace o těchto kvasinkách a využití transkriptomických analýz ke studiu jejich genové regulace. Zmíněna je také charakteristika metody PCR a její použití pro transkripci RNA. Praktická část ve druhé části práce obsahuje dílčí kroky kultivace různých druhů kvasinek, izolaci RNA pomocí dvou různých izolačních metod (extrakční činidlo TRIzol-fenol chloroform a Quick-RNA Viral Kit) a elektroforetickou analýzu získané RNA., The diploma thesis deals with finding the optimal way to isolate RNA from different species of yeasts in the maximal possible amount and purity, especially from the non-pathogenic yeast (Saccharomyces cerevisiae) and pathogenic yeasts (Candida albicans and Candida parapsilosis). The theoretical section describes the basic information about these yeasts and utilization of transcriptomic analyses to study of their gene regulation. The characteristics of the PCR method, as well as its use for RNA transcription, are also mentioned. The practical section in the second part of the thesis contains partial steps of cultivation of different yeast species, RNA isolation using two different isolation methods (TRIzol reagent-phenol chloroform extraction and Quick-RNA Viral Kit), and the electrophoretic analysis of the obtained RNA., Fakulta chemicko-technologická, 1. Prezentace výsledků diplomové práce. 2. Diskuze k posudkům vedoucího a oponenta diplomové práce. 3. Studentka zodpověděla všechny dotazy a připomínky k DP., Dokončená práce s úspěšnou obhajobou

La lignocellulosa è il principale componente strutturale delle piante legnose e non, e rappresenta una delle principali fonti potenziali di materia organica rinnovabile. La lignocellulosa è composta principalmente da due polimeri di carboidrati, cellulosa ed emicellulosa e dalla lignina (un polimero aromatico). Questi polimeri complessi da un lato costituiscono spesso una biomassa residua della filiera agroalimentare, ma allo stesso tempo contengono differenti monomeri zuccherini e precursori fenolici, aventi un enorme valore biotecnologico, poiché potenzialmente possono essere convertiti in differenti prodotti ad elevato valore aggiunto. In uno scenario in cui la popolazione mondiale è in aumento insieme alla generazione di rifiuti ed inquinamento a scapito delle risorse del pianeta e del benessere umano, questo progetto mira a proporre un esempio di bioeconomia circolare e simbiosi industriale. Più in dettaglio, il progetto parte dalla valutazione quali-quantitativa delle biomasse agricole residue fino alla valorizzazione di un sottoinsieme di interesse per il nostro territorio in folati, sfruttando i lieviti come cell factory microbiche. Il folato (Vitamina B9) è una vitamina B idrosolubile con ruoli importanti nella sintesi, riparazione e metilazione degli acidi nucleici, prodotta solo dalle piante verdi e da alcuni microrganismi: per questi motivi rappresenta una componente nutritiva essenziale per l'uomo. La vitamina B9 disponibile in commercio è sintetizzata chimicamente come acido folico, non ottimale in termini di bioattività per l'uomo; la produzione di folati naturali mediante fermentazione microbica sta quindi diventando un'alternativa sostenibile e desiderabile per l'integrazione umana. Nel corso del progetto è stata acquisita ed applicata la metodologia ENEA per l'analisi dei flussi di risorse e per la creazione di possibili sinergie tra le varie aziende presenti nella regione Lombardia. Grazie a questo lavoro è stato possibile identificare l, Lignocellulose is the major structural component of woody and non-woody plants, representing a major potential source of renewable organic matter. Lignocellulose is primarily composed by two carbohydrate polymers, cellulose and hemicellulose and by lignin (an aromatic polymer). These complex polymers on the one hand often constitute a residual biomass of agro-food production chain, but at the same time they contain different sugar monomers and phenolic precursors, harbouring an enormous biotechnological value, since they can potentially be converted into different value-added products. In a scenario where the world population is increasing together with the generation of waste and pollution at the expenses of planet resources and human wellbeing, this project aims at proposing an example of circular bioeconomy and industrial symbiosis. More in detail, the project starts from the quali-quantitative evaluation of residual agricultural biomasses to the valorization of a subset of interest for our territory into folates, exploiting yeasts as microbial cell factory. Folate (Vitamin B9) is a water-soluble B vitamin with important roles in nucleic acid synthesis, repair and methylation, produced only by green plants and some microorganisms: for these reasons it represents an essential nutritional component for humans. Vitamin B9 commercially available is chemically synthetized as folic acid, suboptimal in terms of bioactivity for humans; the production of natural folates by microbial fermentation is therefore becoming a sustainable and desirable alternative for human supplementation. During the project the ENEA methodology for the analysis of resource flows and for the creation of possible synergies between the various companies present in the Lombardy region was acquired and applied. Thanks to this work it was possible to identify the main waste biomasses produced in the area in the agro-food sector and unfermented grape marcs was then selected for further studies in labo

To turn table olives into appropriate carriers of beneficial bacteria and yeasts to consumers, it is essential to have reliable methods for analysing microorganisms in biofilms. This work validates the application of a non-destructive procedure to study the lactic acid bacteria and yeasts distribution in fruits during Spanish-style green table olive fermentations. Laboratory-scale fermentations were inoculated simultaneously with three Lactiplantibacillus pentosus strains (LPG1, 119, and 13B4) and two yeasts (Wickerhamomyces anomalus Y12 and Saccharomyces cerevisiae Y30), all of them natives of table olive fermentations. Data showed that L. pentosus LPG1 and yeasts W. anomalus Y12 were quite prone to colonise olive biofilms, but only the Lactiplantibacillus strain also can penetrate the epidermis of the fruit and colonise the flesh. Applying a non-destructive treatment consisting in shelling the fruits with glass beads led to obtaining similar lactic acid bacteria and yeast recovery than the classical stomacher destructive method. However, the glass bead procedure improved the quality of the metagenomics analysis (especially when using 16 S rRNA gene-based sequencing). Results show the great utility of procedures that do not destroy the fruit for studying fermented vegetable biofilms.

Monitoring the microbiological quality of indoor air in hospital environments is a matter of comprehensive discussion due to its influence on the transmission and spread of pathogenic microorganisms. Among the artificially air-conditioned environments, hospitals are noteworthy for being specific places for the treatment and recovery of patients. In addition to problems related to patients health and professionals health, immunocompromised patients are more exposed to microorganisms present in the air currents of the refrigeration system in these environments, which can lead to consequences such as the occurrence of outbreaks. The objective of this work was to evaluate the indoor air quality in critical hospital environments of a teaching hospital in the city of Maceió, the state of Alagoas. In addition, we sought to identify the anemophilous fungal microbiota present. Air collections were taken in the rainy season, totaling, following recommendations indicated by Resolution No. 9 of the Brazilian National Health Surveillance Agency. The study was based on determining the concentration parameter of bioaerosols in indoor and outdoor air. The fungal microbiota identification was carried out by analyzing macro and microscopic characteristics for filamentous fungi and the use of molecular tools for yeasts. The most frequent species in hospital critical environments were Cladosporium cladosporioides, Penicillium piceum, Penicillium aurantiogriseum, Cladosporium herbarum and Aspergillus oryzae. In outdoor air, the most frequently found fungi were Penicillium sp., Aspergillus sp., and Cladosporium species. Candida tropicalis, C. krusei, and C. parapsilosis were identified among the yeasts in indoor and outdoor air samples. Identifying potentially pathogenic fungi in the evaluated environments points to the need for continuous monitoring of indoor air quality. Furthermore, to avoid the widespread fungal pathogens and the consequent occurrence of outbreaks, the adoption of in

Monitoring the microbiological quality of indoor air in hospital environments is a matter of comprehensive discussion due to its influence on the transmission and spread of pathogenic microorganisms. Among the artificially air-conditioned environments, hospitals are noteworthy for being specific places for the treatment and recovery of patients. In addition to problems related to patients health and professionals health, immunocompromised patients are more exposed to microorganisms present in the air currents of the refrigeration system in these environments, which can lead to consequences such as the occurrence of outbreaks. The objective of this work was to evaluate the indoor air quality in critical hospital environments of a teaching hospital in the city of Maceió, the state of Alagoas. In addition, we sought to identify the anemophilous fungal microbiota present. Air collections were taken in the rainy season, totaling, following recommendations indicated by Resolution No. 9 of the Brazilian National Health Surveillance Agency. The study was based on determining the concentration parameter of bioaerosols in indoor and outdoor air. The fungal microbiota identification was carried out by analyzing macro and microscopic characteristics for filamentous fungi and the use of molecular tools for yeasts. The most frequent species in hospital critical environments were Cladosporium cladosporioides, Penicillium piceum, Penicillium aurantiogriseum, Cladosporium herbarum and Aspergillus oryzae. In outdoor air, the most frequently found fungi were Penicillium sp., Aspergillus sp., and Cladosporium species. Candida tropicalis, C. krusei, and C. parapsilosis were identified among the yeasts in indoor and outdoor air samples. Identifying potentially pathogenic fungi in the evaluated environments points to the need for continuous monitoring of indoor air quality. Furthermore, to avoid the widespread fungal pathogens and the consequent occurrence of outbreaks, the adoption of in

Monitoring the microbiological quality of indoor air in hospital environments is a matter of comprehensive discussion due to its influence on the transmission and spread of pathogenic microorganisms. Among the artificially air-conditioned environments, hospitals are noteworthy for being specific places for the treatment and recovery of patients. In addition to problems related to patients health and professionals health, immunocompromised patients are more exposed to microorganisms present in the air currents of the refrigeration system in these environments, which can lead to consequences such as the occurrence of outbreaks. The objective of this work was to evaluate the indoor air quality in critical hospital environments of a teaching hospital in the city of Maceió, the state of Alagoas. In addition, we sought to identify the anemophilous fungal microbiota present. Air collections were taken in the rainy season, totaling, following recommendations indicated by Resolution No. 9 of the Brazilian National Health Surveillance Agency. The study was based on determining the concentration parameter of bioaerosols in indoor and outdoor air. The fungal microbiota identification was carried out by analyzing macro and microscopic characteristics for filamentous fungi and the use of molecular tools for yeasts. The most frequent species in hospital critical environments were Cladosporium cladosporioides, Penicillium piceum, Penicillium aurantiogriseum, Cladosporium herbarum and Aspergillus oryzae. In outdoor air, the most frequently found fungi were Penicillium sp., Aspergillus sp., and Cladosporium species. Candida tropicalis, C. krusei, and C. parapsilosis were identified among the yeasts in indoor and outdoor air samples. Identifying potentially pathogenic fungi in the evaluated environments points to the need for continuous monitoring of indoor air quality. Furthermore, to avoid the widespread fungal pathogens and the consequent occurrence of outbreaks, the adoption of in

Monitoring the microbiological quality of indoor air in hospital environments is a matter of comprehensive discussion due to its influence on the transmission and spread of pathogenic microorganisms. Among the artificially air-conditioned environments, hospitals are noteworthy for being specific places for the treatment and recovery of patients. In addition to problems related to patients health and professionals health, immunocompromised patients are more exposed to microorganisms present in the air currents of the refrigeration system in these environments, which can lead to consequences such as the occurrence of outbreaks. The objective of this work was to evaluate the indoor air quality in critical hospital environments of a teaching hospital in the city of Maceió, the state of Alagoas. In addition, we sought to identify the anemophilous fungal microbiota present. Air collections were taken in the rainy season, totaling, following recommendations indicated by Resolution No. 9 of the Brazilian National Health Surveillance Agency. The study was based on determining the concentration parameter of bioaerosols in indoor and outdoor air. The fungal microbiota identification was carried out by analyzing macro and microscopic characteristics for filamentous fungi and the use of molecular tools for yeasts. The most frequent species in hospital critical environments were Cladosporium cladosporioides, Penicillium piceum, Penicillium aurantiogriseum, Cladosporium herbarum and Aspergillus oryzae. In outdoor air, the most frequently found fungi were Penicillium sp., Aspergillus sp., and Cladosporium species. Candida tropicalis, C. krusei, and C. parapsilosis were identified among the yeasts in indoor and outdoor air samples. Identifying potentially pathogenic fungi in the evaluated environments points to the need for continuous monitoring of indoor air quality. Furthermore, to avoid the widespread fungal pathogens and the consequent occurrence of outbreaks, the adoption of in

The surface of microorganisms is covered with carbohydrates, which makes them unique, self-sustaining glycan probes. Lectins are able to bind to these probes, and this interaction can be exploited for selecting microorganisms or novel lectins. To examine lectin-microorganism interactions, we have previously developed an enzyme-linked lectin sorbent assay (ELLSA) with whole bacterial cells. To further test the validity of this methodology, here we compare it with flow cytometry. For this purpose, we used biotinylated recombinantly produced lectin from Musa acuminata (BanLec), this lectin’s recombinantly produced chimera with green fluorescent protein (BanLec-eGFP) and a lectin from Ricinus communis (RCA120), both biotinylated and FITC labeled. Parallel testing showed equivalent results for the two methods, in terms of the presence or absence of binding, with signal intensity yielding high Pearson correlation coefficient of 0.8 for BanLec and 0.95 for RCA120. The ELLSA method demonstrated multiple advantages, such as reliability and convenience for high-throughput analysis; it also required less lectin and yielded more consistent results. As such, ELLSA proved to be a useful tool for profiling microbial glycan structures or testing novel lectins.

Monitoring the microbiological quality of indoor air in hospital environments is a matter of comprehensive discussion due to its influence on the transmission and spread of pathogenic microorganisms. Among the artificially air-conditioned environments, hospitals are noteworthy for being specific places for the treatment and recovery of patients. In addition to problems related to patients health and professionals health, immunocompromised patients are more exposed to microorganisms present in the air currents of the refrigeration system in these environments, which can lead to consequences such as the occurrence of outbreaks. The objective of this work was to evaluate the indoor air quality in critical hospital environments of a teaching hospital in the city of Maceió, the state of Alagoas. In addition, we sought to identify the anemophilous fungal microbiota present. Air collections were taken in the rainy season, totaling, following recommendations indicated by Resolution No. 9 of the Brazilian National Health Surveillance Agency. The study was based on determining the concentration parameter of bioaerosols in indoor and outdoor air. The fungal microbiota identification was carried out by analyzing macro and microscopic characteristics for filamentous fungi and the use of molecular tools for yeasts. The most frequent species in hospital critical environments were Cladosporium cladosporioides, Penicillium piceum, Penicillium aurantiogriseum, Cladosporium herbarum and Aspergillus oryzae. In outdoor air, the most frequently found fungi were Penicillium sp., Aspergillus sp., and Cladosporium species. Candida tropicalis, C. krusei, and C. parapsilosis were identified among the yeasts in indoor and outdoor air samples. Identifying potentially pathogenic fungi in the evaluated environments points to the need for continuous monitoring of indoor air quality. Furthermore, to avoid the widespread fungal pathogens and the consequent occurrence of outbreaks, the adoption of in

The surface of microorganisms is covered with carbohydrates, which makes them unique, self-sustaining glycan probes. Lectins are able to bind to these probes, and this interaction can be exploited for selecting microorganisms or novel lectins. To examine lectin-microorganism interactions, we have previously developed an enzyme-linked lectin sorbent assay (ELLSA) with whole bacterial cells. To further test the validity of this methodology, here we compare it with flow cytometry. For this purpose, we used biotinylated recombinantly produced lectin from Musa acuminata (BanLec), this lectin’s recombinantly produced chimera with green fluorescent protein (BanLec-eGFP) and a lectin from Ricinus communis (RCA120), both biotinylated and FITC labeled. Parallel testing showed equivalent results for the two methods, in terms of the presence or absence of binding, with signal intensity yielding high Pearson correlation coefficient of 0.8 for BanLec and 0.95 for RCA120. The ELLSA method demonstrated multiple advantages, such as reliability and convenience for high-throughput analysis; it also required less lectin and yielded more consistent results. As such, ELLSA proved to be a useful tool for profiling microbial glycan structures or testing novel lectins.

This study investigates the effects of delayed sealing and repeated air ingress on the formation of primary fermentation products and other volatile organic compounds (VOC), the development of yeasts and the aerobic stability (ASTA) of maize (26.8% dry matter, DM). After packing, the silos were sealed either promptly or with a delay of 24 h, with repeated air ingress after 27, 55 and 135 days of storage. Losses of DM, fermentation pattern, including VOC, yeast numbers and aerobic stability, were determined 6 times during storage for 142 days. Yeast numbers markedly increased during the first three fermentation days, with the effect being much stronger in silage sealed with a delay than in promptly sealed silage (log10 cfu/g FM 7.27 vs. 5.88, p < 0.002). Simultaneously, the concentrations of ethanol and ethyl esters and DM losses increased. The DM losses were closely correlated with the total concentrations of alcohols and acetic acid (delay: R2 = 0.71, p < 0.001; prompt: R2 = 0.91, p < 0.001, respectively). The repeated air ingress for 24 h during storage after completion of the main fermentation phase had only a minor effect on fermentation pattern, VOC formation and DM losses. The relationship between the counts of total yeasts and lactate-assimilating yeasts (LAY) was very strong (R2 = 0.995, p < 0.001), and LAY numbers were shown to be largely responsible for aerobic instability (R2 = 0.752, p < 0.001). This trial proved the detrimental effects of air on silage fermentation with delayed sealing to be much more deleterious than repeated short-term air ingress after about one month of storage., Peer Reviewed

Plants and microbes share common metabolic pathways for producing a range of bioproducts that are potentially foundational to the future bioeconomy. However, in planta accumulation and microbial production of bioproducts have never been systematically compared on an economic basis to identify optimal routes of production. A detailed technoeconomic analysis of four exemplar compounds (4-hydroxybenzoic acid [4-HBA], catechol, muconic acid, and 2-pyrone-4,6-dicarboxylic acid [PDC]) is conducted with the highest reported yields and accumulation rates to identify economically advantaged platforms and breakeven targets for plants and microbes. The results indicate that in planta mass accumulation ranging from 0.1 to 0.3 dry weight % (dwt%) can achieve costs comparable to microbial routes operating at 40 to 55% of maximum theoretical yields. These yields and accumulation rates are sufficient to be cost competitive if the products are sold at market prices consistent with specialty chemicals ($20 to $50/kg). Prices consistent with commodity chemicals will require an order-of-magnitude-greater accumulation rate for plants and/or yields nearing theoretical maxima for microbial production platforms. This comparative analysis revealed that the demonstrated accumulation rates of 4-HBA (3.2 dwt%) and PDC (3.0 dwt%) in engineered plants vastly outperform microbial routes, even if microbial platforms were to reach theoretical maximum yields. Their recovery and sale as part of a lignocellulosic biorefinery could enable biofuel prices to be competitive with petroleum. Muconic acid and catechol, in contrast, are currently more attractive when produced microbially using a sugar feedstock. Ultimately, both platforms can play an important role in replacing fossil-derived products.

Plants and microbes share common metabolic pathways for producing a range of bioproducts that are potentially foundational to the future bioeconomy. However, in planta accumulation and microbial production of bioproducts have never been systematically compared on an economic basis to identify optimal routes of production. A detailed technoeconomic analysis of four exemplar compounds (4-hydroxybenzoic acid [4-HBA], catechol, muconic acid, and 2-pyrone-4,6-dicarboxylic acid [PDC]) is conducted with the highest reported yields and accumulation rates to identify economically advantaged platforms and breakeven targets for plants and microbes. The results indicate that in planta mass accumulation ranging from 0.1 to 0.3 dry weight % (dwt%) can achieve costs comparable to microbial routes operating at 40 to 55% of maximum theoretical yields. These yields and accumulation rates are sufficient to be cost competitive if the products are sold at market prices consistent with specialty chemicals ($20 to $50/kg). Prices consistent with commodity chemicals will require an order-of-magnitude-greater accumulation rate for plants and/or yields nearing theoretical maxima for microbial production platforms. This comparative analysis revealed that the demonstrated accumulation rates of 4-HBA (3.2 dwt%) and PDC (3.0 dwt%) in engineered plants vastly outperform microbial routes, even if microbial platforms were to reach theoretical maximum yields. Their recovery and sale as part of a lignocellulosic biorefinery could enable biofuel prices to be competitive with petroleum. Muconic acid and catechol, in contrast, are currently more attractive when produced microbially using a sugar feedstock. Ultimately, both platforms can play an important role in replacing fossil-derived products.

The article processing charge was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 491192747 and the Open Access Publication Fund of Humboldt-Universität zu Berlin., This study investigates the effects of delayed sealing and repeated air ingress on the formation of primary fermentation products and other volatile organic compounds (VOC), the development of yeasts and the aerobic stability (ASTA) of maize (26.8% dry matter, DM). After packing, the silos were sealed either promptly or with a delay of 24 h, with repeated air ingress after 27, 55 and 135 days of storage. Losses of DM, fermentation pattern, including VOC, yeast numbers and aerobic stability, were determined 6 times during storage for 142 days. Yeast numbers markedly increased during the first three fermentation days, with the effect being much stronger in silage sealed with a delay than in promptly sealed silage (log10 cfu/g FM 7.27 vs. 5.88, p < 0.002). Simultaneously, the concentrations of ethanol and ethyl esters and DM losses increased. The DM losses were closely correlated with the total concentrations of alcohols and acetic acid (delay: R2 = 0.71, p < 0.001; prompt: R2 = 0.91, p < 0.001, respectively). The repeated air ingress for 24 h during storage after completion of the main fermentation phase had only a minor effect on fermentation pattern, VOC formation and DM losses. The relationship between the counts of total yeasts and lactate-assimilating yeasts (LAY) was very strong (R2 = 0.995, p < 0.001), and LAY numbers were shown to be largely responsible for aerobic instability (R2 = 0.752, p < 0.001). This trial proved the detrimental effects of air on silage fermentation with delayed sealing to be much more deleterious than repeated short-term air ingress after about one month of storage., Peer Reviewed

Monitoring the microbiological quality of indoor air in hospital environments is a matter of comprehensive discussion due to its influence on the transmission and spread of pathogenic microorganisms. Among the artificially air-conditioned environments, hospitals are noteworthy for being specific places for the treatment and recovery of patients. In addition to problems related to patients health and professionals health, immunocompromised patients are more exposed to microorganisms present in the air currents of the refrigeration system in these environments, which can lead to consequences such as the occurrence of outbreaks. The objective of this work was to evaluate the indoor air quality in critical hospital environments of a teaching hospital in the city of Maceió, the state of Alagoas. In addition, we sought to identify the anemophilous fungal microbiota present. Air collections were taken in the rainy season, totaling, following recommendations indicated by Resolution No. 9 of the Brazilian National Health Surveillance Agency. The study was based on determining the concentration parameter of bioaerosols in indoor and outdoor air. The fungal microbiota identification was carried out by analyzing macro and microscopic characteristics for filamentous fungi and the use of molecular tools for yeasts. The most frequent species in hospital critical environments were Cladosporium cladosporioides, Penicillium piceum, Penicillium aurantiogriseum, Cladosporium herbarum and Aspergillus oryzae. In outdoor air, the most frequently found fungi were Penicillium sp., Aspergillus sp., and Cladosporium species. Candida tropicalis, C. krusei, and C. parapsilosis were identified among the yeasts in indoor and outdoor air samples. Identifying potentially pathogenic fungi in the evaluated environments points to the need for continuous monitoring of indoor air quality. Furthermore, to avoid the widespread fungal pathogens and the consequent occurrence of outbreaks, the adoption of in

Kombucha is an increasingly popular functional beverage that has gained attention for its unique combination of phytochemicals, metabolites, and microbes. Previous chemical and microbial composition analyses of kombucha have mainly focused on understanding their changes during fermentation. Very limited information is available regarding nutrient profiles of final kombucha products in the market. In this study, we compared the major chemicals (tea polyphenols, caffeine), antioxidant properties, microbial and metabolomic profiles of nine commercial kombucha products using shotgun metagenomics, internal transcribed spacer sequencing, untargeted metabolomics, and targeted chemical assays. All of the nine kombucha products showed similar acidity but great differences in chemicals, metabolites, microbes, and antioxidant activities. Most kombucha products are dominated by the probiotic Bacillus coagulans or bacteria capable of fermentation including Lactobacillus nagelii, Gluconacetobacter, Gluconobacter, and Komagataeibacter species. We found that all nine kombuchas also contained varying levels of enteric bacteria including Bacteroides thetaiotamicron, Escherischia coli, Enterococcus faecalis, Bacteroides fragilis, Enterobacter cloacae complex, and Akkermansia muciniphila. The fungal composition of kombucha products was characterized by predominance of fermenting yeast including Brettanomyces species and Cyberlindnera jadinii. Kombucha varied widely in chemical content assessed by global untargeted metabolomics, with metabolomic variation being significantly associated with metagenomic profiles. Variation in tea bases, bacteria/yeast starter cultures, and duration of fermentation may all contribute to the observed large differences in the microbial and chemical profiles of final kombucha products.

SDS-PAGE showing: Lane 1 – purified banana lectin – BanLec; Lane 2 – chimera of banana lectin and enhanced green fluorescent protein BanLec-eGFP; Lane 3 – MW – molecular weight markers; Table 1. Repeatability of flow cytometric detection of different quantities of BL-eGFP binding to C. albicans; Table 2. Repeatability of ELLSA detection of different quantities of BanLec-B binding to C. albicans; Table 3. Repeatability of flow cytometric detection of different quantities of RCA120-FITC binding to L. casei DG; Table 4. Repeatability of ELLSA detection of different quantities of RCA120-B binding to L. casei DG; Table 5. Reproducibility of BanLec-B binding to 21 different microorganisms. Experiments were done in three different laboratories, with preparing new plates each time; Fig. 1 Determining linearity of ELLSA method, by measuring binding between C.albicans and different quantity of BanLec-B; Fig. 2 Determining linearity of flow cytometry, by measuring binding between C.albicans and different quantity of BanLec-eGFP; Fig. 3 Determining linearity of ELLSA method, by measuring binding between L.casei DG and different quantity of RCA120; Fig. 4 Determining linearity of flow cytometry, by measuring binding between L.casei DG and different quantity of RCA120.

Monitoring the microbiological quality of indoor air in hospital environments is a matter of comprehensive discussion due to its influence on the transmission and spread of pathogenic microorganisms. Among the artificially air-conditioned environments, hospitals are noteworthy for being specific places for the treatment and recovery of patients. In addition to problems related to patients health and professionals health, immunocompromised patients are more exposed to microorganisms present in the air currents of the refrigeration system in these environments, which can lead to consequences such as the occurrence of outbreaks. The objective of this work was to evaluate the indoor air quality in critical hospital environments of a teaching hospital in the city of Maceió, the state of Alagoas. In addition, we sought to identify the anemophilous fungal microbiota present. Air collections were taken in the rainy season, totaling, following recommendations indicated by Resolution No. 9 of the Brazilian National Health Surveillance Agency. The study was based on determining the concentration parameter of bioaerosols in indoor and outdoor air. The fungal microbiota identification was carried out by analyzing macro and microscopic characteristics for filamentous fungi and the use of molecular tools for yeasts. The most frequent species in hospital critical environments were Cladosporium cladosporioides, Penicillium piceum, Penicillium aurantiogriseum, Cladosporium herbarum and Aspergillus oryzae. In outdoor air, the most frequently found fungi were Penicillium sp., Aspergillus sp., and Cladosporium species. Candida tropicalis, C. krusei, and C. parapsilosis were identified among the yeasts in indoor and outdoor air samples. Identifying potentially pathogenic fungi in the evaluated environments points to the need for continuous monitoring of indoor air quality. Furthermore, to avoid the widespread fungal pathogens and the consequent occurrence of outbreaks, the adoption of in

Chitin synthesis has attracted scientific interest for decades as an essential part of fungal biology and for its potential as a target for antifungal therapies. While this interest remains, three decades ago, pioneering molecular studies on chitin synthesis regulation identified the major chitin synthase in yeast, Chs3, as an authentic paradigm in the field of the intracellular trafficking of integral membrane proteins. Over the years, researchers have shown how the intracellular trafficking of Chs3 recapitulates all the steps in the intracellular trafficking of integral membrane proteins, from their synthesis in the endoplasmic reticulum to their degradation in the vacuole. This trafficking includes specific mechanisms for sorting in the trans-Golgi network, regulated endocytosis, and endosomal recycling at different levels. This review summarizes the work carried out on chitin synthesis regulation, mostly focusing on Chs3 as a molecular model to study the mechanisms involved in the control of the intracellular trafficking of proteins.

In this work, a total of 72 commercial table olive packages obtained from different international markets were analysed to determine their fungal biodiversity. Viable fungal counts ranged from the detection threshold (<1.6 log CFU/g in 25% of cases) to a maximum of 5.86 log CFU/g. Assignation of fungal taxonomy was carried out through a metataxonomic analysis of the ITS region, which revealed that almost half of the total sequences obtained from all packages corresponded to the Pichia genus (44.08%), followed by Citeromyces (14.45%), Candida (8.07%), and Wickerhamomyces (6.95%). In lower proportions were also detected other genera such as Starmerella (3.60%), Saccharomyces (2.24%), Debaryomyces (2.08%), and Dekkera (2.05%). The statistical analysis allowed to link certain taxa to specific types of elaboration (lye treated, green, and black natural olives), presentation (pitted, whole, or sliced samples), and packaging material/system (glass, PET, plastic bags, and vacuum). Likewise, Zygotorulaspora genus was especially sensitive to the presence of potassium sorbate, while other genera such as Sporobolomyces, Moniliella, and Gibellulopsis were more abundant in packages treated with this preservative. Lastly, potential pathogenic fungal genera such as Alternaria, Kodamaea, Lodderomyces, Malasessia, or Aspergillus were detected in low proportions (<0.3%), although with higher representation in some individual samples. Our results contribute to improving our knowledge of the fungal population associated with this ready-to-eat fermented vegetable, providing us a strong tool to assess the safety, stability, and quality of the final product.

Introduction. Sixty-five percent of human infections are caused by bacteria or yeasts able to form biofilms. This feature makes them more resistant to antimicrobials and antifungals.Objective. To determine biofilm formation capacity of bacterial and fungal isolates by quantitative crystal violet microtiter and qualitative Congo red agar methods.Materials and methods. Brain-heart infusion, trypticase soy broth and Müeller‑Hinton culture media were used in bacterial isolates for the quantitative method; brain-heart infusion broth and Sabouraud dextrose were used for yeasts. The same culture media plus 3% Congo red and 10% dextrose were used to apply the qualitative method in agar. The proposal by Stepanovic, et al. was used as a reference method.Results. We evaluated 103 bacterial isolates and 108 yeasts isolates. We did not recommend substitute brain-heart infusion broth for trypticase soy and Müeller-Hinton broths for biofilm formation assessment in bacterial isolates using the quantitative method. Sabouraud dextrose medium, both broth and agar, can replace brain-heart infusion to assess biofilm formation in yeasts, quantitatively and qualitatively.Conclusion. The study of biofilms in the microbiology laboratory, using Congo red agar qualitative method, is a simple, fast, and inexpensive procedure that provides precise information for the diagnosis and treatment of persistent infections caused by bacteria and yeasts., Introducción. El 65 % de las infecciones humanas son producidas por bacterias o levaduras, cuya capacidad de formar biopelículas las hace más resistentes a los antimicrobianos y antifúngicos.Objetivo. Determinar la capacidad de formación de biopelículas en aislamientos bacterianos y fúngicos por medio de los métodos cuantitativo de microtitulación con cristal violeta y cualitativo de cultivo en agar con rojo Congo.Materiales y métodos. Con el método cuantitativo, se utilizaron los medios de cultivo infusión cerebro-corazón, tripticasa de soya y Müeller-Hinton para aislamientos bacterianos; para levaduras, se usaron caldo infusión cerebro-corazón y Sabouraud dextrosa. Para el método cualitativo de cultivo en agar, se utilizaron los mismos medios de cultivo más una solución con 3 % de rojo Congo y 10 % de dextrosa. Cómo método de referencia, se utilizó la propuesta de Stepanovic et al.Resultados. Se evaluaron 103 aislamientos bacterianos y 108 de levaduras. No es recomendable sustituir el caldo infusión cerebro-corazón por los caldos tripticasa de soya y Müeller-Hinton en el método cuantitativo, para evaluar la formación de biopelículas en los aislamientos bacterianos. El medio Sabouraud dextrosa, en caldo y agar, puede sustituir al de infusión de cerebro-corazón para evaluar la formación de biopelículas en levaduras, tanto por el método cuantitativo como por el cualitativo.Conclusión. El estudio de las biopelículas en el laboratorio de microbiología, a partir del método cualitativo de cultivo en agar con rojo Congo, es un procedimiento sencillo, rápido y de bajo costo, que proporciona información útil para el diagnóstico y la terapéutica de infecciones persistentes causadas por bacterias y levaduras.

This report is the first investigation of yeast biodiversity from the oligotrophic hypersaline coastal waters of the Arabian Gulf surrounding Qatar. Yeasts and yeast-like fungi, were cultured from seawater sampled at 13 coastal areas surrounding Qatar over a period of 2 years (December 2013-September 2015). Eight hundred and forty-two isolates belonging to 82 species representing two phyla viz., Ascomycota (23 genera) and Basidiomycota (16 genera) were identified by molecular sequencing. The results indicated that the coastal waters of the Qatari oligotrophic marine environment harbor a diverse pool of yeast species, most of which have been reported from terrestrial, clinical and aquatic sources in various parts of the world. Five species, i.e., Candida albicans, C. parapsilosis, C. tropicalis, Pichia kudriavzevii and Meyerozyma guilliermondii (n = 252/842; 30% isolates) are known as major opportunistic human pathogens. Fifteen species belonging to nine genera (n = 498/842; 59%) and 12 species belonging to seven genera (n = 459/842; 55%) are hydrocarbon degrading yeast and pollution indicator yeast species, respectively. Ascomycetous yeasts were predominant (66.38%; 559/842) as compared to their basidiomycetous counterparts (33.6%; 283/842). The most isolated yeast genera were Candida (28%; 236/842) (e.g., C. aaseri, C. boidinii, C. glabrata, C. intermedia, C. oleophila, C. orthopsilosis, C. palmioleophila, C. parapsilosis, C. pseudointermedia, C. rugopelliculosa, C. sake, C. tropicalis and C. zeylanoides), Rhodotorula (12.7%; 107/842), Naganishia (8.4%; 71/842), Aureobasidium (7.4%; 62/842), Pichia (7.3%; 62/842), and Debaryomyces (6.4%; 54/842). A total of eleven yeast species ( n = 38) isolated in this study are reported for the first time from the marine environment. Chemical testing demonstrated that seven out of the 13 sites had levels of total petroleum hydrocarbons (TPH) ranging from 200 to 900 µg/L, whereas 6 sites showed higher TPH levels (> 1000-210

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct ide

The adaptive immunity system in bacteria and archaea, responsible for recognizing invasive DNA sequences, is characterized by clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated proteins (Cas) and guide RNAs complementary to specific target DNA sequences are the most widely used gene-editing tools to modify organisms. Currently, the variety of CRISPR/Cas systems has increased, adapting to microorganisms such as yeasts with biotechnological potential. This review aims to facilitate understanding of the basic concepts of the CRISPR/Cas9 system and to recognize some of the varieties of this molecular tool that have been successfully implemented in different yeasts. Likewise, some of the new technologies that are revolutionizing research based on CRISPR are described, El sistema de inmunidad adaptativa en bacterias y arqueas, encargado de reconocer las secuencias de DNA invasores, se caracteriza por tener grupos de secuencias palindrómicas cortas repetidas e intercaladas de manera regular por espaciadores (CRISPR). Las proteínas asociadas a CRISPR (Cas) y los RNAs guías complementarios a secuencias específicas del DNA blanco constituyen una de las herramientas de edición genética más utilizadas para modificar a los organismos. En los últimos años se ha incrementado la variedad de los sistemas CRISPR/Cas, adaptándose a microorganismos como las levaduras, que cuentan con potencial biotecnológico. El objetivo de esta revisión es facilitar la comprensión de los conceptos básicos del sistema CRISPR/Cas9 y reconocer algunas de las variedades de esta herramienta molecular que se han implementado de manera exitosa en distintas levaduras. Asimismo, se describen varias de las nuevas tecnologías basadas en CRISPR que están revolucionando la investigación.

The surface of microorganisms is covered with carbohydrates, which makes them unique, self-sustaining glycan probes. Lectins are able to bind to these probes, and this interaction can be exploited for selecting microorganisms or novel lectins. To examine lectin-microorganism interactions, we have previously developed an enzyme-linked lectin sorbent assay (ELLSA) with whole bacterial cells. To further test the validity of this methodology, here we compare it with flow cytometry. For this purpose, we used biotinylated recombinantly produced lectin from Musa acuminata (BanLec), this lectin’s recombinantly produced chimera with green fluorescent protein (BanLec-eGFP) and a lectin from Ricinus communis (RCA120), both biotinylated and FITC labeled. Parallel testing showed equivalent results for the two methods, in terms of the presence or absence of binding, with signal intensity yielding high Pearson correlation coefficient of 0.8 for BanLec and 0.95 for RCA120. The ELLSA method demonstrated multiple advantages, such as reliability and convenience for high-throughput analysis; it also required less lectin and yielded more consistent results. As such, ELLSA proved to be a useful tool for profiling microbial glycan structures or testing novel lectins.

The adaptive immunity system in bacteria and archaea, responsible for recognizing invasive DNA sequences, is characterized by clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated proteins (Cas) and guide RNAs complementary to specific target DNA sequences are the most widely used gene-editing tools to modify organisms. Currently, the variety of CRISPR/Cas systems has increased, adapting to microorganisms such as yeasts with biotechnological potential. This review aims to facilitate understanding of the basic concepts of the CRISPR/Cas9 system and to recognize some of the varieties of this molecular tool that have been successfully implemented in different yeasts. Likewise, some of the new technologies that are revolutionizing research based on CRISPR are described, El sistema de inmunidad adaptativa en bacterias y arqueas, encargado de reconocer las secuencias de DNA invasores, se caracteriza por tener grupos de secuencias palindrómicas cortas repetidas e intercaladas de manera regular por espaciadores (CRISPR). Las proteínas asociadas a CRISPR (Cas) y los RNAs guías complementarios a secuencias específicas del DNA blanco constituyen una de las herramientas de edición genética más utilizadas para modificar a los organismos. En los últimos años se ha incrementado la variedad de los sistemas CRISPR/Cas, adaptándose a microorganismos como las levaduras, que cuentan con potencial biotecnológico. El objetivo de esta revisión es facilitar la comprensión de los conceptos básicos del sistema CRISPR/Cas9 y reconocer algunas de las variedades de esta herramienta molecular que se han implementado de manera exitosa en distintas levaduras. Asimismo, se describen varias de las nuevas tecnologías basadas en CRISPR que están revolucionando la investigación.

This study investigated the spoilage potential of yeast strains Kluyveromyces marxianus (Km1, Km2 and Km3), Pichia kudriavzevii Pk1 and Torulaspora delbrueckii Td1 grown in skyr in cold storage. Yeast strains were isolated from skyr and identified by sequencing of the 26S rRNA gene. K. marxianus yeasts were grown in skyr to high numbers, generating large amounts of volatile organic compounds (VOC) associated with off-flavours, among them were alcohols (3-methyl-1-butanol, 2-methyl-1-propanol and 1-hexanol), esters (ethyl acetate and 3-methylbutyl acetate) and aldehydes (hexanal, methylbutanal and methylpropanal). Growth of P. kudriavzevii Pk1 led to moderate increases in several alcohols and esters (mostly, 3-methyl-1-butanol and ethyl acetate), whereas only minor shifts in VOCs were associated with T. delbrueckii Td2. The levels of the key aroma compounds, diacetyl and acetoin, were significantly decreased by all K. marxianus strains and P. kudriavzevii Pk1. In contrast to the other yeast species, K. marxianus was able to utilize lactose, producing ethanol and carbon dioxide. Based on the overall results, K. marxianus was characterised by the highest spoilage potential. The study revealed the differences between the yeast species in fermentative and spoilage activities, and clarified the role of yeast metabolites for off-flavour formation and quality defects in skyr during cold storage.

This study investigated the spoilage potential of yeast strains Kluyveromyces marxianus (Km1, Km2 and Km3), Pichia kudriavzevii Pk1 and Torulaspora delbrueckii Td1 grown in skyr in cold storage. Yeast strains were isolated from skyr and identified by sequencing of the 26S rRNA gene. K. marxianus yeasts were grown in skyr to high numbers, generating large amounts of volatile organic compounds (VOC) associated with off-flavours, among them were alcohols (3-methyl-1-butanol, 2-methyl-1-propanol and 1-hexanol), esters (ethyl acetate and 3-methylbutyl acetate) and aldehydes (hexanal, methylbutanal and methylpropanal). Growth of P. kudriavzevii Pk1 led to moderate increases in several alcohols and esters (mostly, 3-methyl-1-butanol and ethyl acetate), whereas only minor shifts in VOCs were associated with T. delbrueckii Td2. The levels of the key aroma compounds, diacetyl and acetoin, were significantly decreased by all K. marxianus strains and P. kudriavzevii Pk1. In contrast to the other yeast species, K. marxianus was able to utilize lactose, producing ethanol and carbon dioxide. Based on the overall results, K. marxianus was characterised by the highest spoilage potential. The study revealed the differences between the yeast species in fermentative and spoilage activities, and clarified the role of yeast metabolites for off-flavour formation and quality defects in skyr during cold storage.

The surface of microorganisms is covered with carbohydrates, which makes them unique, self-sustaining glycan probes. Lectins are able to bind to these probes, and this interaction can be exploited for selecting microorganisms or novel lectins. To examine lectin-microorganism interactions, we have previously developed an enzyme-linked lectin sorbent assay (ELLSA) with whole bacterial cells. To further test the validity of this methodology, here we compare it with flow cytometry. For this purpose, we used biotinylated recombinantly produced lectin from Musa acuminata (BanLec), this lectin’s recombinantly produced chimera with green fluorescent protein (BanLec-eGFP) and a lectin from Ricinus communis (RCA120), both biotinylated and FITC labeled. Parallel testing showed equivalent results for the two methods, in terms of the presence or absence of binding, with signal intensity yielding high Pearson correlation coefficient of 0.8 for BanLec and 0.95 for RCA120. The ELLSA method demonstrated multiple advantages, such as reliability and convenience for high-throughput analysis; it also required less lectin and yielded more consistent results. As such, ELLSA proved to be a useful tool for profiling microbial glycan structures or testing novel lectins.

Background and Aims Torulaspora delbrueckii is being used increasingly as a starter for alcoholic fermentation (AF) because of its chemical modulation of wine. Previous studies on this yeast in a natural must have shown a different Oenococcus oeni population by the end of MLF. In this study we aim to evaluate this aspect in a defined O. oeni strain consortium in a sterile grape must during winemaking. Methods and Results Before commencing AF with either S. cerevisiae or both T. delbrueckii and S. cerevisiae, the must was inoculated with a defined population of O. oeni strains. The use of T. delbrueckii determined the bacterial population at the end of MLF. Also, the inoculation of a selected strain after AF produced wines with different chemical composition to those fermented with the initial bacterial community. Conclusions Different yeast inoculation strategies modulate the O. oeni population, and this has an impact on the chemical composition of the wines. Moreover, the inoculation of a small O. oeni population in must leads to a process similar to spontaneous MLF. Significance of the Study Torulaspora delbrueckii can be used as a tool to modulate the O. oeni population and enhance the aromas related to MLF.

Oleogenic yeasts are characterized by the ability to accumulate increased amounts of lipids under certain conditions. These microbial lipids differ in their fatty acid composition, which allows them to be widely used in the biotechnology industry. The work focuses on the influence of various stress factors in the cultivation process, such as reduced temperature or nutritional stress through the use of various waste substrates, together with manipulating the ratio of carbon and nitrogen sources in the medium. The ability of yeast to produce significant amounts of unsaturated fatty acids was also demonstrated in the work. The most suitable substrate for lipid production was a medium containing glycerol, where the amount of accumulated lipids in the yeast M. pulcherrima 1232 was up to 36%. In our work, the crude animal fat was used for the production of high-value lipids, which to the best of our knowledge is a new result.

Arsenic (As) is quite an abundant metalloid, with ancient origin and ubiquitous distribution, which represents a severe environmental risk and a global problem for public health. Microbial exposure to As compounds in the environment has happened since the beginning of time. Selective pressure has induced the evolution of various genetic systems conferring useful capacities in many microorganisms to detoxify and even use arsenic, as an energy source. This review summarizes the microbial impact of the As biogeochemical cycle. Moreover, the poorly known adverse effects of this element on eukaryotic microbes, as well as the As uptake and detoxification mechanisms developed by yeast and protists, are discussed. Finally, an outlook of As microbial remediation makes evident the knowledge gaps and the necessity of new approaches to mitigate this environmental challenge., Ministerio de Economía, Comercio y Empresa (España), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The study highlights the fungal diversity of the traditional Algerian date’s product “Btana” prepared with direct Btana method (DBM) and undirect Btana method (UBM). Btana fungal populations were analyzed through 28S metabarcoding. Data treatment resulted in 122,997 reads representing three Phyla in which 76% reads (46 OTUs) belong to Ascomycota phylum. Zygosaccharomycesrouxii was the most prevailed species accounting for 35.40% of the total population. Similarity percentage analysis revealed a low level of resemblance in species in each of the two Btana types (DBM: 17.26%, UBM: 16.87). According to HPLC analysis, lactate was detected in nine samples within a range of 0.87-23.06 g/100g. Culture plating and subsequent D1/D2 domain of 28s DNA analysis showed the prevalence of Z. rouxii. Fermentation of non-renewed date medium revealed a high ethanol production (21.31 ± 2.89 g/100g) by Lachanceathermotolerans and 5.87 g/100g of lactates by Kluyveromycesdelphensis. Enzymatic assay revealed a high esterase (C4) and naphtol-AS-BI-phosphohydrolase activity by L. thermotolerans, K. delphensis, and Pichiasubpelliculosa, while a high level of α-fucosidase was recorded for L. thermotolerans and P. subpelliculosa. The current results demonstrated that the traditional date product Btana is a promising source for yeasts useful in production of value-added products like bioethanol and lactic acid using low-income date cultivars., SCOPUS: ar.j, info:eu-repo/semantics/published

The extensive use of synthetic fertilizers and pesticides has negative consequences in terms of soil microbial biodiversity and environmental contamination. Faced with this growing concern, a proposed alternative agricultural method is the use of microorganisms as biofertilizers. Many works have been focused on bacteria, but the limited literature on yeasts and their potential ability to safely promote plant growth is gaining particular attention in recent years. Thus, the objective of this review is to highlight the application of yeasts as biological agents in different sectors of sustainable agricultural practices through direct or indirect mechanisms of action. Direct mechanisms include the ability of yeasts to provide soluble nutrients to plants, produce organic acids and phytohormones (indole-3-acetic acid). Indirect mechanisms involve the ability for yeasts to act as biocontrol agents through their high antifungal activity and lower insecticidal and herbicidal activity, and as soil bioremediating agents. They also act as protective agents against extreme environmental factors by activating defense mechanisms. It is evident that all the aspects that yeasts offer could be useful in the creation of quality biofertilizers and biopesticides. Hence, extensive research on yeasts could be promising and potentially provide an environmentally friendly solution to the increased crop production that will be required with a growing population

In view of the growing concern about the impact of synthetic fungicides on human health and the environment, several government bodies have decided to ban them. As a result, a great number of studies have been carried out in recent decades with the aim of finding a biological alternative to inhibit the growth of fungal pathogens. In order to avoid the large losses of fruit and vegetables that these pathogens cause every year, the biological alternative's efficacy should be the same as that of a chemical pesticide. In this review, the main studies discussed concern Saccharomyces and non-Saccharomyces yeasts as potential antagonists against phytopathogenic fungi of the genera Penicillium and Aspergillus and the species Botrytis cinerea on table grapes, wine grapes, and raisins.