Phenyllactic acid produced by Geotrichum candidum reduces Fusarium sporotrichioides and F. langsethiae rowth and T-2 Toxin Concentration

Fusarium sporotrichioides and F. langsethiae are present in barley crops. Their toxic metabolites, mainly T-2 toxin, affect the quality and safety of raw material and final products such as beer. Therefore, it is crucial to reduce Fusarium spp. proliferation and T-2 toxin contamination during the brewing process. The addition of Geotrichum candidum has been previously demonstrated to reduce the proliferation of Fusarium spp. and the production of toxic metabolites, but the mechanism of action is still not known. Thus, this study focuses on the elucidation of the interaction mechanism between G. candidum and Fusarium spp. in order to improve this bioprocess. First, over a period of 168 h, the co-culture kinetics showed an almost 90% reduction in T-2 toxin concentration, starting at 24 h. Second, sequential cultures lead to a reduction in Fusarium growth and T-2 toxin concentration. Simultaneously, it was demonstrated that G. candidum produces phenyllactic acid (PLA) at the early stages of growth, which could potentially be responsible for the reduction in Fusarium growth and T-2 toxin concentration. To prove the PLA effect, F. sporotrichioides and F. langsethiae were cultivated in PLA supplemented medium. The expected results were achieved with 0.3 g/L of PLA. These promising results contribute to a better understanding of the bioprocess, allowing its optimization at an up-scaled industrial level

Mixed culture fermentation using Torulaspora delbrueckii and Saccharomyces cerevisiae with direct and indirect contact: impact of anaerobic growth factors

The role of the initial concentration of anaerobic growth factors (AGF) on interactions between Torulaspora delbrueckii and Saccharomyces cerevisiae was investigated in strict anaerobiosis. Experiments were performed in a synthetic grape must medium in a membrane bioreactor, a special tool designed for studying direct and indirect interactions between microorganisms. In pure culture fermentations, increased AGF concentration had no impact on S. cerevisiae behaviour, whereas it induced an extension of T. delbrueckii latency. Surprisingly, T. delbrueckii used only 75 to 80% of the consumed sugar to produce biomass, glycerol and ethanol. Physical separation influenced the population dynamics of co-fermentations. S.cerevisiae dominated the co-cultures having a single dose of AGF as its presence indirectly induced a decrease in numbers of living T. delbrueckii cells and physical contact with T. delbrueckii stimulated S.cerevisiae growth. Increasing the AGF initial concentration completely upset this domination: S. cerevisiae growth was not stimulated and T. delbrueckii living cells did not decrease. Yeasts incorporate exogenous AGFs, which probably impact their response to competing yeasts. The increase in AGF might have induced changes in the lipid composition of the T. delbrueckii membrane, which would hinder its interaction with S. cerevisiae antimicrobial peptides. The initial concentration of anaerobic growth factors influenced co-culture fermentation population dynamics tremendously, thus highlighting a new way to monitor population evolution and eventually wine organoleptic properties

Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation

Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces

Micropollutants removal in tertiary moving bed biofilm reactors (MBBRs): Contribution of the biofilm and suspended biomass

The performance of tertiary moving bed biofilm reactors (MBBRs) was evaluated in terms of micropollutants (MPs) removal from secondary-treated municipal wastewater. After stepwise establishment of a mature biofilm, monitored by scanning electron and confocal microscopies, abiotic and biotic removals of MPs were deeply studied. Since no MPs reduction was observed by the both photodegradation and volatilization, abiotic removal of MPs was ascribed to the sorption onto the biomass. Target MPs i.e. Naproxen, Diclofenac, 17ß-Estradiol and 4n-Nonylphenol, arranged in the ascending order of hydrophobicity, abiotically declined up to 2.8%, 4%, 9.5% and 15%, respectively. MPs sorption onto the suspended biomass was found around two times more than the biofilm, in line with MPs’ higher sorption kinetic constants (ksor) found for the suspended biomass. When comparing abiotic and biotic aspects, we found that biotic removal outperformed its counterpart for all compounds as Diclofenac, Naproxen, 17ß-Estradiol and 4n-Nonylphenol were biodegraded by 72.8, 80.6, 84.7 and 84.4%, respectively. The effect of the changes in organic loading rates (OLRs) was investigated on the pseudo-first order degradation constants (kbiol), revealing the dominant biodegradation mechanism of co-metabolism for the removal of Diclofenac, Naproxen, and 4n-Nonylphenol., while 17ß-Estradiol obeyed the biodegradation mechanism of competitive inhibition. Biotic removals and kbiol values of all MPs were also seen higher in the biofilm as compared to the suspended biomass. To draw a conclusion, a quite high removal of recalcitrant MPs is achievable in tertiary MBBRs, making them a promising technology that supports both pathways of co-metabolism and competitive inhibition, next to the abiotic attenuation of MPs

Understanding Kombucha Tea Fermentation: A Review

Kombucha is a beverage of probable Manchurian origins obtained from fermented tea by a microbial consortium composed of several bacteria and yeasts. This mixed consortium forms a powerful symbiosis capable of inhibiting the growth of potentially contaminating bacteria. The fermentation process also leads to the formation of a polymeric cellulose pellicle due to the activity of certain strains of Acetobacter sp. The tea fermentation process by the microbial consortium was able to show an increase in certain biological activities which have been already studied; however, little information is available on the characterization of its active components and their evolution during fermentation. Studies have also reported that the use of infusions from other plants may be a promising alternative

Kombucha fermentation of African mustard (Brassica tournefortii) leaves: Chemical composition and bioactivity

Brassica tournefortii is an edible vegetable formerly consumed by North African populations. Nowadays, this plant has been neglected and is less used. The present study aims to give an extra nutraceutical value to B. tournefortii using a 2-wk kombucha fermentation process. At the end of incubation, fermented and unfermented (control) B. tournefortii aqueous extracts were successively fractionated with ethyl acetate (EtOAc) and n-butanol to measure their chemical composition and bioactivity. Results showed that kombucha fermentation significantly increased total phenolic content, with the highest amounts in the EtOAc fraction. The antioxidant potential of B. tournefortii leaves was improved by fermentation of EtOAc extracts and conversely lowered in aqueous ones. Anti-acetylcholinesterase activity was increased with fermentation to reach ∼8-fold higher value in B. tournefortii EtOAc and aqueous extracts relative to unfermented samples. Kombucha fermentation was found to reduce cytotoxicity and xanthine oxidase inhibitory effects of B. tournefortii leaves. The findings suggested that fermentation is a promising, simple and safe bioprocess that could improve the food proprieties of less-used edible plan

Impact of fermentation conditions on the production of bioactive compounds with anticancer, anti-inflammatory and antioxidant properties in kombucha tea extracts

The production of natural bioactive compounds through the fermentation of plants has increased in recent years. The biological activities of the extracts obtained from the fermentation of black tea with the kombucha consortium were evaluated. To improve the productivity of these compounds two different vessel geometries were used and successive extractions with solvents of increasing polarity were performed. Forty-seven compounds were identified by GC–MS, including several organic acids and phenolic compounds. Total phenolic content, pH value, and antioxidant, antiproliferation and anti-inflammatory activities were measured after 21 days of fermentation. A higher surface/height (s/h) ratio seemed to enhance the anti-inflammatory activity of kombucha tea, resulting in IC50 value of 9.0 ± 0.1 μg/mL compared to 24.3 ± 0.2 μg/mL with the lowest ratio. Regarding the anticancer activity, the highest inhibition percentage of 55.3% at 50 μg/mL against the HCT-116 human colon carcinoma cell line was obtained with the ethyl acetate extract after 21 days of fermentation compared to the value of 8% obtained with the same extraction solvent using the non-fermented black tea. These results showed that fermentation may improve the biological activities of the tea and that the production of bioactive compounds can vary depending on the fermentation condition

Metabolome-microbiome signatures in the fermented beverage, Kombucha

Kombucha is a fermented tea. Here we investigate the fermentation kinetics, metabolite production, microbiome and potential health promoting properties of three different kombucha consortia. Shotgun metagenomic sequencing revealed several dominant bacterial genera such as Komagataeibacter, Gluconacetobacter and Gluconobacter. Brettanomyces and Schizosaccharomyces were the most dominant yeasts identified. Species distribution reflected different patterns of sugar consumption, with S. pombe being present in samples with the highest sugar conversion. Liquid-liquid extractions were performed with organic solvents in order to obtain dried extracts, which were later characterized. HPLC-DAD and GC-MS analysis revealed differences in the production of organic acids, sugars, alcohols and phenolic compounds, where the presence of caffeine, propanoic acid and 2,3 butanediol differ greatly across the three kombuchas. Metabolomic analysis exhibited a link between the microbiota and the production of bioactive compounds in kombucha fermentation. In vitro assays were carried out in order to evaluate potential health-promoting features of the fermented teas, with notable outcomes including antioxidant ability against DPPH radical and against the 15-lipoxygenase enzyme, indicating a potential anti-inflammatory activity. These investigations considerably enhance our understanding of the relationship between the microbiota and metabolites as well as health promoting potential of kombucha and have the potential for the development of future generations of kombucha products in which these relationships are optimized

Physicochemical properties of bacterial cellulose obtained from different Kombucha fermentation conditions

The production of bacterial cellulose has been limited due to its high cost and low productivity. Alternative low‐cost sources of this biopolymer of high purity and biocompatibility are needed in order to benefit from its enormous potential. Kombucha tea is a trend functional beverage whose production is growing exponentially worldwide, and the bacteria present in this fermented beverage belonging to the genus Komagataeibacter are capable of producing a crystalline biofilm with interesting properties. Obtaining bacterial cellulose from Kombucha tea has already been studied, however several fermentation conditions are being optimized in order to scale‐up its production. In this study, we characterized the bacterial cellulose produced from three different Kombucha fermentation conditions. The scanning electron microscopy images revealed the crystalline structure of the biofilms. The energy‐dispersive x‐ray analysis exhibited the chemical composition of the crystals. The thermogravimetric analysis showed a rate of degradation between 490 and 560°C and the differential scanning calorimetry confirmed the presence of crystalline and amorphous regions in the bacterial cellulose samples. The results suggested that crystalline cellulose could be obtained by varying the fermentation conditions of Kombucha tea

Development of devices and methods for the organization and the localisation of micro-organisms in biological complex structures

Ce projet concerne l’analyse de l’organisation de populations microbiennes au sein de structures complexescomme des dépôts ou des biofilms.Si différentes méthodes sont couramment utilisées pour étudier la structure globale ou l’organisation localed’agrégats microbiens, peu d’entre elles, permettent de réaliser simultanément ces deux analyses et nécessitentsouvent des étapes de préparation qui pénalisent un approche in-vivo et en dynamique.La stratégie proposée repose sur la mise en oeuvre de micro-organismes modèles autofluorescents (levures etbactéries) qui peuvent sans aucun traitement être directement observés en microscopie. La similitude ducomportement physiologique de ces micro-organismes avec celui des souches sauvages a été démontrée. Lesconditions d’acquisition des images en microscopie confocale ont été optimisées. Des dispositifs spécifiques ontété conçus pour générer des dépôts ou des biofilms dans des conditions de contraintes physico-mécaniques etbiochimiques maîtrisées afin d’analyser simultanément leurs caractéristiques et les performances du bioprocédé.Ainsi les dépôts ont pu être observés in-vivo et in-situ grâce à une cellule de filtration équipée d’une fenêtred’observation. Le développement d’un biofilm mixte composé de levures et bactéries modèles autofluorescentes,dans un réacteur continu spécifique, a également été analysé par microscopie confocale.Le traitement et les analyses des images acquises au cours des expériences ont été effectués et ont permisd‘étudier la structure globale des agrégats biologiques et l’organisation tridimensionnelle des micro-organismesdans ces structures, en mettant par exemple en évidence une répartition hétérogène de deux populationsmicrobiennes dans des dépôts de filtration ou en comparant la capacité de deux espèces microbiennes à formerdes biofilms en étudiant in-vivo la dynamique de croissance de chacune des espèces.Cette étude a en outre permis de démontrer la pertinence de la méthode proposée, de définir ses limites et sonchamp d’applicationThe aim of this project deals with the analysis of both the local localization and organization of microbialpopulations in complex structures such as deposits or biofilms. Different methods are currently used to study theglobal structure or the local organization of biological aggregates but only few ones allow a combined approachand require ex-vivo analyses.The proposed strategy uses home-designed model auto-fluorescent microorganisms (yeasts and bacteria) whichcan be observed directly by microscopy without any dying treatment. Same kinetic behaviours between the wildstrains and their recombinant ones were demonstrated. The confocal microscopy conditions were optimised.Specific devices were developed to generate deposits or biofilms under controlled and known hydrodynamic orbiochemical environment conditions to analyse their structure characteristics linked to the bioprocessperformances.Based on the proposed strategy, microbial deposits modifications due to pressure constraints were observed invivo in a specifically designed flow cell equipped with a microscope glass coverslip. A mixed biofilm composedby our auto-fluorescent yeasts and bacteria was carried out in a specific bioreactor allowing the sampling ofbiofilms during their development to be analysed by confocal microscopy. Both studies have shown specificorganisations between yeasts and bacteria mainly depending on their size and on the environment conditions(pressure or dilution rate).These studies of both local and global structure of biological aggregates and 3D-organisation of themicroorganisms within theses structures demonstrated the relevance of the proposed strategy defining the limitsof the method and proposing various perspectives for further characterizations and application