Добірка наукової літератури з теми "Spores and Biofilms"

Viability of Bacillus globigii spores in chlorinated polyvinyl chloride (PVC) pipe loop systems were examined under oligotrophic conditions. Three 2.5 cm × 10 m pipe loops having poised free chlorine concentrations of 0.0, 0.5, and 1.0 mg/L were seeded with 3.0 × 108B. globigii spores each and viability was assessed over a 21 day period in both the recirculating waters and within the biofilms associated with pipe wall surfaces. After 10 min of exposure, viable spores were found to be associated within the pipe biofilms. In the untreated pipe loop spore counts remained statistically consistent in both the bulk water and biofilm until 1.0 mg/L free chlorine was introduced, then spores were completely inactivated in less than seven days. Spores within the pipe loop poised at 0.5 mg/L free chlorine showed a 7.6-log10 inactivation in the bulk water phase, but only a 2.7-log10 inactivation was observed within the biofilm after 14 days of treatment. Complete inactivation was observed in the 1.0 mg/L free chlorine system in both the biofilm and the bulk water phase in less than 10 min. These data demonstrated that B. globigii spores were readily incorporated into PVC pipe biofilms, which decreased spore inactivation nearly five orders of magnitude under moderate free chlorine concentrations.

ABSTRACT Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.

Spore formation by a Bacillus strain (Bacillus subtilis SpoIVFB-GFP) engineered with a green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVF) that fluoresces during sporulation was observed. Biofilms of B. subtilis SpoIVFB-GFP containing ca. 8 log CFU/ml vegetative cells and spores below the lower detection limit (i.e., <1 log CFU/ml) were allowed to develop on glass wool (37°C). These biofilms were subsequently exposed to nutrient limitation to stimulate spore formation, which was monitored for fluorescence by confocal scanning laser microscopy. Sporulation in corresponding planktonic cells was also monitored for comparative purposes. Planktonic B. subtilis SpoIVFB-GFP cells began fluorescing after 5 h, while B. subtilis SpoIVFB-GFP biofilm cells began fluorescing after 30 h. Results suggested that an existing biofilm of vegetative B. subtilis cells may be stimulated to form spores when exposed to conditions of nutrient limitation. From a practical point of view, it may be suggested that a window of time does exist before sporulation occurs in attached Bacillus biofilms highlighting the need for shorter operating runs between cleaning and sanitation of food-processing equipment surfaces.

Clostridium perfringens is a major human pathogen that causes gastroenteritis via enterotoxin production and has the ability to form spores and biofilms for environmental persistence and disease transmission. This study aimed to compare the disinfectant and environmental resistance properties of C. perfringens vegetative cells and spores in planktonic and sessile conditions, and to examine the nucleotide polymorphisms and transcription under sessile conditions in C. perfringens strains isolated from meat. The sporulation rate of sessile C. perfringens TYJAM-D-66 (cpe+) was approximately 19% at day 5, while those of CMM-C-80 (cpe−) and SDE-B-202 (cpe+) were only 0.26% and 0.67%, respectively, at day 7. When exposed to aerobic conditions for 36 h, TYJAM-D-66, CMM-C-80, and SDE-B-202 vegetative cells showed 1.70 log, 5.36 log, and 5.67 log reductions, respectively. After treatment with sodium hypochlorite, the survival rates of TYJAM-D-66 vegetative cells (53.6%) and spores (82.3%) in biofilms were higher than those of planktonic cells (9.23%). Biofilm- and spore-related genes showed different expression within TYJAM-D-66 (–4.66~113.5), CMM-C-80 (–3.02~2.49), and SDE-B-202 (–5.07~2.73). Our results indicate the resistance of sessile cells and spores of C. perfringens upon exposure to stress conditions after biofilm formation.

This study aimed to trace the dynamics of biofilm formation by vegetative cells and endospores of Bacillus cereus DL5 and Bacillus subtilis 168. Counts of B. cereus DL5 and B. subtilis 168 vegetative cells and spores either attached to glass wool or, correspondingly, planktonic cells were determined by standard plate-counting methods. Results from this study highlighted the biofilm-forming potential of both spores and vegetative cells of two different Bacillus species. It was shown that once Bacillus spores had attached to a surface, the spores germinated under favorable (B. cereus DL5) and even unfavorable (B. subtilis 168) nutrient conditions, resulting in biofilms containing both spores and vegetative populations. Furthermore, it was suggested that vegetative B. cereus DL5 cells exhibited a low propensity for spore formation in attached and planktonic growth forms in nutrient-limited growth medium. By contrast, vegetative B. subtilis 168 cells readily formed spores in planktonic and attached microcosms when exposed to nutrient-limited growth conditions. Sporulation in attached Bacillus populations is an important practical consideration for many food industries, such as dairy processing, where bacilli are routinely isolated from populations attached to processing-equipment surfaces.

Fungi biofilms have been found growing on spacecraft surfaces such as windows, piping, cables, etc. The contamination of these surfaces with fungi, although undesirable, is highly difficult to avoid. While several biofilm forming species, including Penicillium rubens, have been identified in spacecraft, the effect of microgravity on fungal biofilm formation is unknown. This study sent seven material surfaces (Stainless Steel 316, Aluminum Alloy, Titanium Alloy, Carbon Fiber, Quartz, Silicone, and Nanograss) inoculated with spores of P. rubens to the International Space Station and allowed biofilms to form for 10, 15, and 20 days to understand the effects of microgravity on biofilm morphology and growth. In general, microgravity did not induce changes in the shape of biofilms, nor did it affect growth in terms of biomass, thickness, and surface area coverage. However, microgravity increased or decreased biofilm formation in some cases, and this was incubation-time- and material-dependent. Nanograss was the material with significantly less biofilm formation, both in microgravity and on Earth, and it could potentially be interfering with hyphal adhesion and/or spore germination. Additionally, a decrease in biofilm formation at 20 days, potentially due to nutrient depletion, was seen in some space and Earth samples and was material-dependent.

Bioactive natural products have become a hot spot for oral disease treatments. At the present study, LongZhang Gargle was investigated for its effects on single-species biofilms of Candida albicans and dual-species biofilms of Candida albicans and Streptococcus mutans. Two different models of single and dual-species biofilms were grown in YNBB medium under appropriate conditions. Biofilm biomass, biofilm architecture, and cell activity in biofilms were assessed using Crystal Violet Staining, MTT, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Significant reductions of biofilm biomass and fungus activity were obtained when treated with LongZhang Gargle at 2% ( P < 0.05 ), 4% ( P < 0.05 ), and 8% ( P < 0.05 ) in single-species biofilms of C. albicans, and at 4% ( P < 0.05 ) and 8% ( P < 0.05 ) in double-species biofilms. Suppression of density, thickness, and the proportion of hyphae and fungal spores were obtained under SEM and CLSM. In conclusion, LongZhang Gargle affects single and dual-species biofilms by inhibiting biofilm biomass, cell activity, and formation of hyphae, but it does not affect the production of Extracellular polysaccharides (EPS). We speculate that LongZhang Gargle would be a promising natural drug, which can be used in treatment against C. albicans and S. mutans in oral diseases.

ABSTRACTThe anaerobic sporeformerClostridium difficileis the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the morphotype responsible for transmission, infection, and persistence, and the outermost exosporium layer is likely to play a major role in spore-host interactions during recurrent infections, contributing to the persistence of the spore in the host. A recent study (M. Pizarro-Guajardo, P. Calderón-Romero, P. Castro-Córdova, P. Mora-Uribe, and D. Paredes-Sabja, Appl Environ Microbiol 82:2202–2209, 2016,http://dx.doi.org/10.1128/AEM.03410-15) demonstrated by transmission electron microscopy the presence of two ultrastructural morphotypes of the exosporium layer in spores formed from the same sporulating culture. However, whether these distinct morphotypes appeared due to purification techniques and whether they appeared during biofilm development remain unclear. In this communication, we demonstrate through transmission electron microscopy that these two exosporium morphotypes are formed under sporulation conditions and are also present in spores formed during biofilm development. In summary, this work provides definitive evidence that in a population of sporulating cells, spores with a thick outermost exosporium layer and spores with a thin outermost exosporium layer are formed.IMPORTANCEClostridium difficilespores are recognized as the morphotype of persistence and transmission ofC. difficileinfections. Spores ofC. difficileare intrinsically resistant to all known antibiotic therapies. Development of spore-based removal strategies requires a detailed knowledge of the spore surface for proper antigen selection. In this context, in this work we provide definitive evidence that two types of spores, those with a thick outermost exosporium layer and those with a thin outermost exosporium layer, are formed in the sameC. difficilesporulating culture or during biofilm development.

This study evaluated the adhesion and biofilm formation of Alicyclobacillus acidoterrestris on industrial orange juice processing equipment and the bactericidal efficacy of peracetic acid, sodium hypochlorite, and quaternary ammonia after biofilm formation. The efficacy of these sanitizers against the spores of this microorganism was also evaluated. Stainless steel and nylon surfaces exhibited higher cell adhesion levels than did polyvinyl chloride surfaces. Peracetic acid was the most effective in removing biofilms from all surfaces (P &lt; 0.05) and also reduced bacterial counts by 3 log CFU/cm2 on the surface of polyvinyl chloride, but the other sanitizers also reduced the bacterial counts by 2 log CFU/cm2. Quaternary ammonia exhibited the optimal minimum sporicidal concentration, preventing spore germination after only 15 s of contact at a concentration of 82 ppm. The flow cytometry results indicated that the spores and cells had low incidences of plasma membrane lysis after treatment with sanitizer, suggesting that lysis is not the principal mode of action for these sanitizers on A. acidoterrestris.

The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA.

Bacteria are often thought as single cell organisms, however they can develop into morphologically complex multicellular communities composed of different subpopulations of specialized cell types. Biofilm is an example, in which bacteria organize for protection from harmful conditions in the host and to create nutrient-rich areas. In the last years biofilm have been show to comprise an important aspect of microbial persistence in the human gut. Endospore-formers, although thought not to be major constituents of the microbiota in the human intestine, cause several intestinal diseases, usually associated with antibiotic use. Whether these bacteria persist in the intestine in biofilms or as endospores is not totally elucidated since both, biofilms and endospores, are able to resist to antimicrobial agents. Most likely sporulation and biofilm formation are tightly linked processes. For some endosporeformers, spore differentiation is induced by a sub-population of cells within the biofilm. In this work we tackled the link between bacterial biofilms and endosporulation in Bacillus subtilis. We showed that endospores produced in biofilms have higher resistance to UV radiation. We revealed that a gene, remA, conserved among endosporeformers and essential for biofilm formation is expressed during sporulation. remA is expressed in the forespore soon after asymmetric division and in the mother cell after engulfment completion. GerE represses remA expression in the mother cell at late stages of sporulation. Consequently, we found components of the biofilm matrix, TasA and BslA, on the coat of endospores produced in biofilms. We suggest that components of the biofilm matrix may be part of mature endospores. We hypothesize that some of the structural proteins that confer integrity to the matrix biofilm, as TasA, may have a role as a scaffold for the assembly of the endospore surface layers.
A percepção instalada é a de que as bactérias são organismos unicelulares. No entanto, estes organismos são capazes de se organizarem em comunidades multicelulares complexas compostas de subpopulações de células diferenciadas. Os biofilmes são um exemplo deste tipo de organização. Os biofilmes conferem protecção contra as condições desfavoráveis encontradas no hospedeiro, ao mesmo tempo que criam nichos ricos em nutrientes facilitando a implantação da população. Nos últimos anos foi demonstrado que a persistência microbiana no trato gastrointestinal humano se deve em larga medida à formação de biofilmes. Algumas bactérias que podem ser encontradas no trato gastrointestinal humano são ainda capazes de diferenciar um tipo celular altamente resistente a insultos químicos e físicos, o esporo. Nestes casos, não é claro se são os biofilmes ou os endoesporos os principais responsáveis pela persistência destes organismos, já que ambos são resistentes aos antibióticos. Neste trabalho exploramos a ligação genética entre a formação de biofilmes e a esporulação em Bacillus subtilis. Mostramos que os endoesporos produzidos em biofilmes exibem maior resistência aos UV. Mostramos que um gene, remA, conservado em bactérias formadoras de endoesporos e essencial para a formação de biofilmes é expresso durante a esporulação. remA é expresso no pré-esporo após a divisão assimétrica e na célula mãe após o envolvimento do pré-esporo. GerE reprime a expressão de remA na célula mãe em estádios tardios de desenvolvimento. Consequentemente, encontramos componentes da matriz do biofilme no manto de endoesporos maduros. Algumas das proteínas estruturais que conferem integridade à matriz do biofilme, como TasA, poderão servir como base para a montagem das camadas superficiais do esporo.

Le but de cette thèse est de développer des expériences pour comprendre la physique de la motilité dans deux systèmes microbiens, vivant dans le domaine du faible nombre de Reynolds, c’est-à-dire lorsque les forces visqueuses l’emportent sur les forces d’inertie. La première partie de la thèse traite de la croissance de biofilms bactériens sur une surface solide. Les biofilms bactériens sont des communautés de cellules étroitement empilées dans une matrice polymère. Du point de vue physique, ces colonies se comportent comme des gels et la matrice polymérique crée des flux osmotiques qui permettent aux biofilms de se développer et de se déplacer sur une surface en tant que communauté. Ici, je développe une expérience pour explorer la motilité collective des biofilms au contact de gradients externes de pression osmotique. Pour produire des gradients osmotiques stables dans des gels d’agar, je développe une configuration sur mesure au moyen de techniques de millifluidique. Les biofilms répondent au gradient externe en développant une forme asymétrique, conforme aux attentes. La deuxième partie de la thèse aborde le mécanisme de décharge des spores chez les basidiomycètes à phylum fongique. Chez ces espèces, une goutte coalesce avec la spore, ce qui entraîne son écoulement à des accélérations énormes. Cette catapulte à tension superficielle atteint son efficacité maximale lorsque la taille de la goutte est comparable à celle de la spore. J’étudie les morphologies de plusieurs champignons branchiaux, où les spores sont conditionnées à la surface de branchies complexes. Je trouve que pour ces espèces, si les spores sont emballées au maximum comme il est généralement supposé, la taille de la goutte qui coalesce avec la spore doit être bien contrôlée. Cela pose la question de comment est-ce que le champignon peut contrôler un processus purement extracellulaire, face aux fluctuations environnementales
The aim of this thesis is to develop experiments to understand the physics of motility in two microbial systems, living in the realm of low Reynolds number, i.e. when viscous forces dominate over inertial forces. The first part of the thesis discusses the growth of bacterial biofilms over a solid surface. Bacterial biofilms are communities of cells closely packed together inside a polymeric matrix. From the physical viewpoint, these colonies behave as gels and the polymeric matrix creates osmotic fluxes that enable biofilms to grow and move on a surface as a community. Here I develop an experiment to explore biofilm collective motility in contact with external gradients of osmotic pressure. To produce stable osmotic gradients in agar gels, I develop a custom-made setup through millifluidics. Biofilms respond to the external gradient by developing an asymmetric shape, consistent with the expectations. The second part of the thesis discusses the spore discharge mechanism in the fungal phylum Basidiomycetes. In these species, a drop coalesces with the spore, which results in spore discharge at enormous accelerations. This surface tension catapult reaches its maximum efficiency when the size of the drop is comparable to that of the spore. I study morphologies of several gilled mushrooms, where spores are packaged at the surface of complex shaped gills. I find that for those species, drop size must be precisely controlled. This poses the question of how mushrooms may regulate a process that occurs extracellularly, despite fluctuating physical conditions

Il existe de nombreuses technologies de décontamination, néanmoins les spores et les biofilms bactériens demeurent une préoccupation majeure dans de nombreux domaines, tels que le secteur hospitalier, alimentaire et de la biodéfense car elles sont résistantes. Une mousse novatrice contenant un biocide (l’hypochlorite de sodium ou le peroxyde d’hydrogène) et un agent stabilisant (le Xanthane) a été étudiée pour répondre à ce besoin. Cette mousse a la capacité d’être mise en oeuvre de différentes façons sur le terrain par : pulvérisation au sol ; talochage et pulvérisation sur les murs ; remplissage de pièces entières (murs et sols). Le travail de thèse est d’évaluer les modes d’action biocide de ces mousses sur les spores et les biofilms. Afin d’étudier le mode d’action de ces mousses des protocoles expérimentaux ont été mis au point sur les spores et les biofilms suivant leurs futures mises en oeuvre (horizontale, verticale et remplissage) et suivant différents facteurs environnementaux pouvant influencer leur efficacité de décontamination (températures, salissures, matériaux, …). L’ensemble de ce travail de thèse a permis de distinguer l’intérêt de la mousse au Xanthane contenant NaOCl 5% par rapport à celle H2O2 12% pour répondre aux besoins spécifiques de la décontamination des agents de la menace biologique. Cette mousse permet une décontamination rapide de 7 logs de spores en 30 minutes pour chacune des trois voies de mise en oeuvre à 20°C. De plus, elle permet la destruction de biofilms contenant 107 de bactéries/cm² en 1 heure maximum sur un support horizontal et par remplissage. Cette mousse NaOCl est suffisamment mature pour pouvoir réaliser un futur transfert industriel
Several decontamination technologies exist, however bacterial spores and biofilms remain a concern in a lot of fields, like hospital, alimentary and military. A new foam containing a biocide (sodium hypochlorite or hydrogen peroxide) and a stabilizing agent (Xanthan) has been studied to answer this problematic. This foam can be used in different ways on the field following contaminations: grounds’ spraying, walls’ covering and spraying, full pieces’ filling (walls and ground). The goal of this thesis is to evaluate the biocide efficiency of these foams on spores and biofilms. We optimized experimental protocols in order to study mechanisms of foams’ action on spores and biofilms based on theirs future applications (horizontal, vertical and filling) and depending on different environmental factors which may impact foam decontamination efficiencies (materials, temperatures, soil, …). This thesis work enabled to highlight the Xanthan foam containing 5% NaOCl from the one including 12% H2O2 in military sector. This foam allows a rapid decontamination, about 7 logs of spores in 30 minutes, for each of the three ways of use at 20°C. Moreover, the destruction of biofilms containing 107 logs of bacteria/cm² was achieved in 1 hour on a horizontal support by filling. This NaOCl foam is ready to be used for industrials

Les contaminants tels que les spores / biofilms sont problématiques dans nombreux secteurs de l'industrie alimentaire. En effet, même après les procédures d'hygiène, ces biofilm/spores pouvaient être retrouvés sur toutes les surfaces en contact direct ou non direct avec les aliments (Bénézech & Faille, 2018). Les risques associés aux biofilms peuvent être contrôlés soit en limitant le nombre des cellules adhérentes, soit en facilitant l'élimination des bactéries adhérentes. Le nettoyage en place (CIP) est une pratique de nettoyage courante et est largement utilisée dans les industries alimentaires ; cependant, il reste à un certain niveau une procédure de forte consommation d'eau. De plus, certaines études ont présenté quelques espèces bactériennes qui ont survécu même après le CIP et peuvent-être une source probable de contamination du produit.D'un autre côté, un fluide biphasique tel que la mousse peut exercer la même contrainte de cisaillement en paroi générée par un fluide monophasique, avec moins d'eau consommée. La mousse avec ses propriétés telles que le cisaillement peut être un paramètre clé pour un nettoyage mécanique des systèmes fermés tels que les tuyaux à faible consommation d'eau.Dans cette étude, nous avons étudié l'effet de l'écoulement de mousse dans les tuyaux et comparé son efficacité avec des conditions du type CIP standard sur le détachement des spores et des biofilms. La première approche consistait à travailler avec différents régimes d'écoulement de mousse (1D, 2D, 3D tout en augmentant la vitesse de 2 à 6 cm.s-1) ayant différentes qualités de mousse (quantité d'air: 50%, 60%, 70%) sur différentes espèces de micro-organismes où l'encrassement a été effectué soit en utilisant des spores de B. amyloliquefaciens 98/7 ou B. cereus 98/4 qui montrent une différence par leur caractère hydrophobe / hydrophile. Quant à P. fluorescens pf1, il a été utilisé comme un bon formateur de biofilm (biofilm de 24 heures) largement utilisé dans l'industrie alimentaire. L'encrassement a été réalisé à la position verticale ou à l'horizontale induisant des biofilms avec différentes structures. Les résultats du nettoyage de la mousse ont été comparés aux résultats des conditions de type CIP (la même action mécanique moyenne et la même concentration de tensioactif).La deuxième approche consistait à soumettre le flux de mousse à différentes singularités (réduction progressive de l'expansion soudaine - coudes) tout en travaillant avec un régime d'écoulement de mousse (1D 50%) et une espèce (B. amyloliquefaiciens 98/7 spores) pour mettre en évidence tout changement dans le flux de mousse sur l’efficacité de nettoyage.La troisième approche portait sur le travail avec une espèce (B. amyloliquefaciens 98/7) considérée comme un bon « outil microbien », en produisant la mousse à partir des différents surfactants (SDS, Capstone® FS 30, Ammonyx® LO). Ces derniers diffèrent par leurs propriétés chimiques (non ionique, anionique et zwitterion) et donc permettant de produire des mousses avec des propriétés physiques différentes en termes de taille, de nombre et de répartition des bulles, et de profil d'écoulement.Par rapport aux précédents travaux sur la caractérisation de mousse en écoulement, il a été possible de mettre en évidence le rôle potentiel de la variation de la contrainte de cisaillement en parois parallèlement à la variation d'épaisseur du film liquide au niveau des parois avec le passage des bulles, sur l'efficacité de nettoyage. De plus, selon des travaux antérieurs, l’existence des forces capillaires exercées sous les débits les plus faibles, la nature hydrophile / hydrophobe des spores et la structure du biofilm pourraient expliquer au moins en partie l'efficacité surprenante de l'élimination des spores par la mousse
Contaminants such as spores/biofilms are problematic in many food industry sectors. Indeed even after hygiene procedures, biofilms/spores could be found on every surface that is in direct contact or not with food (Bénézech & Faille, 2018). Risks associated with microorganisms can be controlled either by limiting the number of adherent cells or by facilitating the removal of adherent bacteria. Even though Cleaning in Place (CIP) is widely used and it is a common cleaning practice in food industries; however, it remains at some level a high- water consumption procedure. In addition, some studies, presented some bacterial species that still survived even after CIP and maybe a probable source of product contamination. On the other hand, a double phase fluid such as foam can impose the same wall shear stress with less water being consumed. Foam with its properties such as shearing can be key a parameter for a mechanical cleaning of closed systems such as pipes with a lower consumption of water.In this study we investigated the effect of flowing foam in pipes and compared its efficiency with standard CIP like conditions on the detachment of spores and biofilms. The first approach was working with different foam flow regimes (1D, 2D, 3D while increasing the velocity from 2 to 6 cm s-1) having different foam qualities (amount of air: 50%, 60%, 70%) on different species of microorganisms where fouling was performed either by using spores of B. amyloliquefaciens 98/7 or B. cereus 98/4 that shows a difference by their hydrophobic/hydrophilic character. As for P. fluorescens pf1 it was used as a good biofilm former (24 hrs. biofilm) widely encountered in the food industry. Fouling was performed either vertically or horizontally inducing biofilms with different structures. Results from foam cleaning were compared with CIP like conditions results (the same mean mechanical action, and the same concentration of surfactant). The second approach was subjecting foam flow to different singularities (sudden expansion gradual reduction – bends) while working with one foam flow regime (1D 50%) and one species (B. amyloliquefaciens 98/7 spores) to highlight any changes in the foam flow cleaning efficiency. The third approach was working also with one species (B. amyloliquefaciens 98/7) considered as a good “microbial tool” producing foam from the use of different surfactants (SDS, Capstone® FS 30, Ammonyx® LO) that differs by their chemical properties ( nonionic, anionic and zwitterion) thus producing different foams having different physical properties in terms of bubbles size , number and repartition, and flow pattern. Comparing to previous related works on foam flow characterization, it was possible to highlight the potential role on the cleaning efficiency of the Wall Shear Stress variations in parallel to the liquid film thickness variation at the wall with the bubbles' passage. In addition, according to previous work, the possible capillary forces exerted under the lowest flow rates and considering the hydrophilic/hydrophobic nature of the spores, in addition to biofilm structure would explain at least partly the surprising efficiency in the spores' removal by foam

Les contaminants tels que les spores / biofilms sont problématiques dans nombreux secteurs de l'industrie alimentaire. En effet, même après les procédures d'hygiène, ces biofilm/spores pouvaient être retrouvés sur toutes les surfaces en contact direct ou non direct avec les aliments (Bénézech & Faille, 2018). Les risques associés aux biofilms peuvent être contrôlés soit en limitant le nombre des cellules adhérentes, soit en facilitant l'élimination des bactéries adhérentes. Le nettoyage en place (CIP) est une pratique de nettoyage courante et est largement utilisée dans les industries alimentaires ; cependant, il reste à un certain niveau une procédure de forte consommation d'eau. De plus, certaines études ont présenté quelques espèces bactériennes qui ont survécu même après le CIP et peuvent-être une source probable de contamination du produit.D'un autre côté, un fluide biphasique tel que la mousse peut exercer la même contrainte de cisaillement en paroi générée par un fluide monophasique, avec moins d'eau consommée. La mousse avec ses propriétés telles que le cisaillement peut être un paramètre clé pour un nettoyage mécanique des systèmes fermés tels que les tuyaux à faible consommation d'eau.Dans cette étude, nous avons étudié l'effet de l'écoulement de mousse dans les tuyaux et comparé son efficacité avec des conditions du type CIP standard sur le détachement des spores et des biofilms. La première approche consistait à travailler avec différents régimes d'écoulement de mousse (1D, 2D, 3D tout en augmentant la vitesse de 2 à 6 cm.s-1) ayant différentes qualités de mousse (quantité d'air: 50%, 60%, 70%) sur différentes espèces de micro-organismes où l'encrassement a été effectué soit en utilisant des spores de B. amyloliquefaciens 98/7 ou B. cereus 98/4 qui montrent une différence par leur caractère hydrophobe / hydrophile. Quant à P. fluorescens pf1, il a été utilisé comme un bon formateur de biofilm (biofilm de 24 heures) largement utilisé dans l'industrie alimentaire. L'encrassement a été réalisé à la position verticale ou à l'horizontale induisant des biofilms avec différentes structures. Les résultats du nettoyage de la mousse ont été comparés aux résultats des conditions de type CIP (la même action mécanique moyenne et la même concentration de tensioactif).La deuxième approche consistait à soumettre le flux de mousse à différentes singularités (réduction progressive de l'expansion soudaine - coudes) tout en travaillant avec un régime d'écoulement de mousse (1D 50%) et une espèce (B. amyloliquefaiciens 98/7 spores) pour mettre en évidence tout changement dans le flux de mousse sur l’efficacité de nettoyage.La troisième approche portait sur le travail avec une espèce (B. amyloliquefaciens 98/7) considérée comme un bon « outil microbien », en produisant la mousse à partir des différents surfactants (SDS, Capstone® FS 30, Ammonyx® LO). Ces derniers diffèrent par leurs propriétés chimiques (non ionique, anionique et zwitterion) et donc permettant de produire des mousses avec des propriétés physiques différentes en termes de taille, de nombre et de répartition des bulles, et de profil d'écoulement.Par rapport aux précédents travaux sur la caractérisation de mousse en écoulement, il a été possible de mettre en évidence le rôle potentiel de la variation de la contrainte de cisaillement en parois parallèlement à la variation d'épaisseur du film liquide au niveau des parois avec le passage des bulles, sur l'efficacité de nettoyage. De plus, selon des travaux antérieurs, l’existence des forces capillaires exercées sous les débits les plus faibles, la nature hydrophile / hydrophobe des spores et la structure du biofilm pourraient expliquer au moins en partie l'efficacité surprenante de l'élimination des spores par la mousse
Contaminants such as spores/biofilms are problematic in many food industry sectors. Indeed even after hygiene procedures, biofilms/spores could be found on every surface that is in direct contact or not with food (Bénézech & Faille, 2018). Risks associated with microorganisms can be controlled either by limiting the number of adherent cells or by facilitating the removal of adherent bacteria. Even though Cleaning in Place (CIP) is widely used and it is a common cleaning practice in food industries; however, it remains at some level a high- water consumption procedure. In addition, some studies, presented some bacterial species that still survived even after CIP and maybe a probable source of product contamination. On the other hand, a double phase fluid such as foam can impose the same wall shear stress with less water being consumed. Foam with its properties such as shearing can be key a parameter for a mechanical cleaning of closed systems such as pipes with a lower consumption of water.In this study we investigated the effect of flowing foam in pipes and compared its efficiency with standard CIP like conditions on the detachment of spores and biofilms. The first approach was working with different foam flow regimes (1D, 2D, 3D while increasing the velocity from 2 to 6 cm s-1) having different foam qualities (amount of air: 50%, 60%, 70%) on different species of microorganisms where fouling was performed either by using spores of B. amyloliquefaciens 98/7 or B. cereus 98/4 that shows a difference by their hydrophobic/hydrophilic character. As for P. fluorescens pf1 it was used as a good biofilm former (24 hrs. biofilm) widely encountered in the food industry. Fouling was performed either vertically or horizontally inducing biofilms with different structures. Results from foam cleaning were compared with CIP like conditions results (the same mean mechanical action, and the same concentration of surfactant). The second approach was subjecting foam flow to different singularities (sudden expansion gradual reduction – bends) while working with one foam flow regime (1D 50%) and one species (B. amyloliquefaciens 98/7 spores) to highlight any changes in the foam flow cleaning efficiency. The third approach was working also with one species (B. amyloliquefaciens 98/7) considered as a good “microbial tool” producing foam from the use of different surfactants (SDS, Capstone® FS 30, Ammonyx® LO) that differs by their chemical properties ( nonionic, anionic and zwitterion) thus producing different foams having different physical properties in terms of bubbles size , number and repartition, and flow pattern. Comparing to previous related works on foam flow characterization, it was possible to highlight the potential role on the cleaning efficiency of the Wall Shear Stress variations in parallel to the liquid film thickness variation at the wall with the bubbles' passage. In addition, according to previous work, the possible capillary forces exerted under the lowest flow rates and considering the hydrophilic/hydrophobic nature of the spores, in addition to biofilm structure would explain at least partly the surprising efficiency in the spores' removal by foam

La caractérisation expérimentale et numérique du comportement rhéologique d'une mousse aqueuse s'écoulant à l'intérieur d'un tuyau horizontal avec et sans singularités (demi-expansion soudaine, et chicane) a été étudiée. Différentes conditions d'écoulement ont été étudiées en faisant varier les qualités de mousse (55-85%), et trois nombres de Reynolds (32, 65, et 97). Les mesures de la pression, de la répartition de la vitesse locale et de la contrariante et de l'épaisseur des films liquides au niveau de la paroi à l'aide respectivement de capteurs de pression, de la PIV, de la polarographie et de la conductimétrie ont montré une réorganisation de la mousse en aval du changement de géométrie, avec un film liquide plus épais au fond du conduit, des bulles de plus grande taille au sommet, ainsi qu'une plus grande fraction de vide de la mousse augmentant de la partie inférieure à la partie supérieure de la section du conduit. En outre, la mousse présenterait un caractère visco-élastique comparable à celui d'un liquide monophasique non newtonien. Des simulations de dynamique des fluides ont été entreprises pour prédire ce comportement rhéologique de la mousse, les deux modèles Herschel-Bulkley et Bingham ont été testés en tenant compte de la présence d'un film liquide sous-jacent au fond du canal. La comparaison entre les résultats expérimentaux et numériques a montré que, quelle que soit la qualité de la mousse, le modèle de Herschel-Bulkley pouvait décrire avec précision le comportement rhéologique de la mousse dans les différentes conditions d'écoulement analysées.Le deuxième objectif était d'étudier la capacité d'un écoulement de mousse humide (qualité de 50%) à nettoyer des surfaces en acier inoxydable contaminées par des micro-organismes. Pour cela, deux types de contamination ont été étudiés, des gouttelettes contenant des spores de Bacillus subtilis (soit hydrophiles Bs PY79 ou hydrophobes Bs PY79 spsA), et des biofilms produits par trois souches de bactéries (Escherichia coli SS2, Bacillus cereus 98/4, et Pseudomonas fluorescens Pf1). Différentes conditions d'écoulement ont été réalisées en faisant varier les contraintes de cisaillement pariétale (2.2 - 13.2 Pa), et la taille des bulles (0.18-0.34 mm) dans un conduit sans changement géométrique, afin d'identifier les mécanismes de libération de la contamination et ainsi mieux contrôler et optimiser le processus de nettoyage. Les résultats montrent que, par rapport au NEP conventionnel, le flux de mousse a éliminé efficacement les spores ainsi que les biofilms. De plus, la combinaison d'une contrainte de cisaillement pariétale élevée et de bulles de petite taille (<0,2 mm) s'est avérée prometteuse pour améliorer l'efficacité du nettoyage des spores. D'autre part, une nette amélioration de l'élimination des biofilms a été observée en augmentant la contrainte de cisaillement pariétale. La caractérisation de la mousse et des phénomènes d'interface a indiqué que des mécanismes tels que la fluctuation des contraintes de cisaillement locales, ou de l'épaisseur du film liquide entre les bulles et la paroi induite par le passage des bulles, l'imbibition de la mousse et le balayage de la contamination dans le film liquide, pourraient participer largement aux mécanismes d'élimination. Enfin, l'étude d'analyse du cycle de vie a démontré que le nettoyage à la mousse peut être une technique appropriée pour réduire la consommation d'eau et d'énergie (7 et 8 fois moins) présentant moins d'impacts environnementaux que les procédés CIP, avec environ 70%. Enfin, le nettoyage à la mousse peut être une méthode alternative, qui peut améliorer l'efficacité et réduire l'impact environnemental.D'autres activités menées au cours de la période de doctorat liées à la conception hygiénique sont présentées en soulignant le rôle des contaminants (spores et biofilms), du matériau (autre que l'acier inoxydable) et de la géométrie (conduits ou conception plus complexe) dans le contrôle de l'hygiène
In this research, experimental and numerical characterization of the rheological behavior of an aqueous foam flowing inside a horizontal pipe with and without singularities (presence of half-sudden expansion, and a fence) were investigated. Different conditions of foam flow were studied by varying the foam qualities (from 55% to 85%), and three Reynolds numbers (32, 65, and 97). Measurements of the pressure measurements, and at the wall the local velocity repartition and the thickness of the liquid films using respectively pressure sensors, Particle Image Velocimetry, and a conductimetry technique shown a reorganization of the foam downstream the geometry change, with a thicker liquid film at the duct bottom, larger bubble sizes at the top, as well as a larger foam void fraction increased from the bottom to the top part of the duct section. In addition, foam would present a visco-elastic character comparable to a non-Newtonian monophasic liquid. Computational Fluid Dynamics simulations were undertaken to predict this rheological behavior of the foam, the two models Herschel-Bulkley and Bingham were tested taken into account the presence of an underlying liquid film at the bottom of the channel . Comparison between experimental and numerical results showed that regardless of the foam quality, Herschel-Bulkley model could accurately describe the rheological behaviour of the aqueous foam under the different flow conditions analysed.The second target was to investigate the ability of a wet foam flow (quality of 50%) to clean stainless-steel surfaces contaminated by microorganisms. For this purpose, two different contamination patterns were studied, droplets containing Bacillus subtilis spores (either hydrophilic Bs PY79 or hydrophobic Bs PY79 spsA), and biofilms produced by three bacteria strains encountered in food industry production plants (Escherichia coli SS2, Bacillus cereus 98/4, and Pseudomonas fluorescens Pf1). Different flow conditions were performed by varying the wall shear stresses (2.2 - 13.2 Pa), and bubble sizes (0.18-0.34 mm) in a straight duct with no geometrical changes, in order to identify the mechanisms of contamination release and thus better control and optimize the foam cleaning process. Results show that compared to conventional cleaning-in-place, foam flow effectively removed Bs spores as well as Bc-98/4, Ec-SS2, and Pf1 biofilms. Moreover, the combination of high shear stress at the wall and small bubble sizes (<0.2 mm) showed promise for improving the cleaning efficiency of spores. On the other hand, a clear improvement of the biofilm removal was observed when increasing the mean wall shear stress. The characterization of the foam and the interface phenomenons (using polarography, conductimetry, and bubble size analysis methods) indicated that mechanisms such as fluctuation in local wall shear stresses, or in the liquid film thickness between the bubbles and the steel wall induced by bubble passage, foam imbibition, and sweeping of the contamination within the liquid film could participate largely to the removal mechanisms. Finally, the life cycle assessment study demonstrated that foam flow cleaning could be a suitable technique to reduce water and energy consumption (7 and 8 times less, respectively) presenting less environmental impacts than CIP processes, with about 70%. Lastly, foam flow cleaning can be an alternative method, which can improve efficiency and reduce environmental impact. Additional activities conducted during the PhD period related to hygienic design are presented highlighting the role of the contaminants (spores and biofilms), the material (other than stainless steel) and the geometry (ducts or more complex design) in hygiene monitoring

Orientador: Mirna Lucia Gigante
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
Made available in DSpace on 2018-08-30T23:01:21Z (GMT). No. of bitstreams: 1 Ribeiro_MariaCeciliaEnes_D.pdf: 17038739 bytes, checksum: 54c8350faac4b14e773283e6e871aa78 (MD5) Previous issue date: 2015
Resumo: O objetivo geral deste trabalho foi avaliar o efeito de diferentes matrizes na adesão e formação de biofilme em aço inoxidável por Bacillus cereus, bem como avaliar a eficiência dos procedimentos de higienização no controle de biofilmes de esporos desse micro-organismo. Nas duas primeiras etapas, avaliou-se a capacidade de adesão e formação de biofilme por B. cereus em aço inoxidável, com e sem prévio condicionamento da superfície, utilizando-se água, leite UHT desnatado e integral como matrizes e quatro diferentes tipos de inóculos, pool de células vegetativas de B. cereus isolados da indústria láctea, pool de esporos de B. cereus isolados da indústria láctea, células vegetativas da cepa de B. cereus ATCC 14579 e esporos da cepa de B. cereus ATCC 14579. Na terceira etapa do trabalho avaliou-se a influência da matriz condicionante (água e leite UHT integral), do meio de inoculação do pool de esporos de B. cereus (água e leite UHT integral) e do tempo de exposição (5 min (0,08h), 10, 24, 48 e 72 horas) sobre a adesão e formação de biofilme por B. cereus em aço inoxidável. Na quarta etapa, avaliou-se a eficiência de nove procedimentos de higienização na remoção dos biofilmes formados pelo pool de esporos de B. cereus em aço inoxidável. Todos os experimentos foram repetidos três vezes e os dados estatisticamente avaliados. A hidrofobicidade e o potencial zeta das superfícies dos esporos também foram avaliados. Os resultados das duas primeiras etapas indicaram que o pool de esporos de B. cereus isolados de indústria láctea apresentou a maior capacidade de adesão e formação de biofilme em aço inoxidável quando comparado aos outros tipos de inóculos, em todas as condições avaliadas. O maior grau de adesão de esporos de B. cereus (4,93 log UFC/cm2) foi observado ao se utilizar leite integral como matriz condicionante do aço inoxidável. Entretanto, comparando-se todas as matrizes, a menor adesão (3,01 log UFC/cm2) foi observada quando o pool de esporos de B cereus foi veiculado no leite integral sem prévio condicionamento da superfície. Na terceira etapa do trabalho observou-se que a adesão e formação de biofilme pelo pool de esporos de B. cereus foi maior quando inoculados em água, independente das matrizes de condicionamento. A adesão de B. cereus aumentou 1,02 e 0,3 log UFC/cm2 ao longo do tempo de exposição, quando o pool de esporos de B. cereus foi inoculado em água e leite integral, respectivamente. O biofilme de esporos veiculados na água apresentou maior resistência aos procedimentos de higienização. A sanitização com hipoclorito de sódio foi mais eficiente na remoção dos biofilmes quando comparada ao ácido peracético. O pool de esporos de B. cereus isolados da indústria láctea foi altamente hidrofóbico e apresentou carga negativa em uma ampla faixa de pH, com ponto isoelétrico de aproximadamente 3,0. Os esporos de B. cereus isolados da indústria láctea apresentaram maior capacidade de adesão ao aço inoxidável quando comparados aos outros inóculos avaliados, o que pode estar relacionado à alta hidrofobicidade e a baixa carga de superfície dos esporos
Abstract: The aim of this study was to evaluate the effect of different matrices on the adhesion and biofilm formation by Bacillus cereus on stainless steel, and to evaluate the effectiveness of sanitation procedures for controlling biofilm from spores of this microorganism. The first two parts were carried out in order to evaluate the adhesion and biofilm formation by B. cereus on stainless steel, with and without previous conditioning of the surface, using water, skim and whole UHT milk as matrices and four different types of inocula: a pool of B. cereus vegetative cells isolated from dairy industry, a pool of B. cereus spores isolated from dairy industry, vegetative cells of B. cereus ATCC 14579, and spores of B. cereus ATCC 14579. The third part of the study evaluated the effect of the conditioning matrix (water and whole UHT milk), the inoculation medium of pool of B. cereus spores (water and whole UHT milk) and exposure time (5 min (0.08h), 10, 24, 48 and 72 hours) on the adhesion and biofilm formation by B. cereus on stainless steel. In the fourth part, the effect of nine sanitation procedures on the removal of B. cereus spores biofilm was evaluated. All experiments were repeated three times and data were statistically evaluated. Hydrophobicity and zeta potential from spore¿s surface were also evaluated. Regarding the results to the first and second parts, the pool of B. cereus spores isolated from dairy industry had the highest ability of adhesion on stainless steel when compared to the other inocula, for all tested conditions. After stainless steel surface conditioning with whole milk, B. cereus spores showed the highest adhesion (4.93 log CFU/cm2). However, lower adhesion (3.01 log CFU/cm2) was observed when B. cereus spores were delivered in whole milk as compared to the other matrices, without previous conditioning of the surface. The results of the third part indicated that the adhesion and biofilm formation by the pool of B. cereus spores was higher when they were inoculated in water, regardless of the conditioning matrix. B. cereus spores adhesion increased by 1.02 and 0.3 log CFU/cm2 over exposure time, when the pool of B. cereus spores was inoculated into water and whole milk, respectively. Biofilm of B. cereus spores inoculated in water showed the highest resistance against all tested sanitation procedures. Sodium hypochlorite was the most effective sanitizer for removing all biofilms when compared to the peracetic acid. The pool of B. cereus spores isolated from dairy industry was highly hydrophobic and showed a negative charge at a wide pH range, with an isoelectric point of about 3.0. B. cereus spores isolated from dairy industry showed the highest ability to adhere on stainless steel when compared to the other inocula, which is possibly related to its higher hydrophobicity and lower spore surface charge
Doutorado
Tecnologia de Alimentos
Doutora em Tecnologia de Alimentos

Durante la mia attività di tesi mi sono focalizzata sulla caratterizzazione funzionale di una sorgente plasma surface Dielectric Barrier Discharge (sDBD) impiegata per l’inattivazione di spore batteriche di Geobacillus stearothermophilus, e per l’eradicazione di biofilm batterici di Staphylococcus aureus e Pseudomonas aeruginosa. Durante la prima attività è stata compiuta la caratterizzazione elettrica della sorgente sDBD e sono state condotte analisi di temperatura all’interno del volume di trattamento della sorgente. In seguito sono stati eseguiti test biologici per dimostrare l’efficacia del plasma nell’inattivazione di spore. I risultati ottenuti dimostrano come aumentando la potenza impiegata per generare la scarica di plasma aumenta anche il potere sterilizzante della sorgente e come il valore massimo di temperatura raggiunto sia rimasto al di sotto del valore di soglia. Nella seconda fase i biofilm di S. aureus e P. aeruginosa sono stati trattati tramite sorgente sDBD e l’efficacia del trattamento è stata valutata calcolando la Log Reduction. In seguito la morfologia dei biofilm è stata analizzata tramite il microscopio a scansione elettronica (SEM). I risultati mostrano come il trattamento di biofilm con sorgente plasma sDBD sia efficace sia per ceppi batterici gram positivi sia gram negativi. Le immagini al SEM hanno confermato l’effetto battericida dal plasma.

Biofilm research is growing rapidly due to the widespread existence of biofilms in pathogens and their resistance to a variety of antimicrobial therapies. World Health Organization in 2017 categorised pathogens into three categories based on their AMR [Antimicrobial resistance] and severity of infection viz. critical, high and medium. Acinetobacter baumannii, Pseudomonas aeruginosa and organisms belonging to Enterobacteriaceae family are top priority pathogens- ‘critical’, amongst which the majority of them are reported to cause the infection due to biofilm formation. As antibiotic resistance has increased tremendously in the last few years, the current research is concentrated on the development of effective approaches to inhibit biofilm formation by bacteria. Anti-biofilm activity is mediated by a spectrum of molecules obtained from plants, mammals, fungi, microbes, and marine sponges. The chapter gives a comprehensive idea about natural bioactives from plant and other sources that act as anti-biofilm agents. Clinical validation of these bioactives will aid the medical field with alternate preventive and treatment methods against pathogenic biofilms.

Stress has become the health epidemic of the twenty-first century; its burden we faced is due to social and professional pressure (spouse death, marital separation and loss of job). If stress is supposed to help an organism to go through daily issue and particular emotional issues, it can be a source of health damage, when sustained. Negative effects of stress can go from change in life style to cardiovascular events and cognitive disorders. Periodontal health can also be affected. During chronic stress, T helper 2 (Th2) lymphocytes are responsible for promoting the destruction of periodontal tissue. Wrong stress management is characterized by changes in life style like poor oral hygiene, smoking, diet modification and parafunctions. This contributes to increase dental biofilm and worsen periodontitis. The most clinically relevant effects are wound healing and response to periodontal treatment. Suppression of immune response components during stress prevents the elimination of damaged tissues as well as any new tissue buildup. Moreover, peripheral vasoconstriction and low oxygen resulting from excessive catecholamine during stress compromise wound healing and response to periodontal treatment. This highlights the importance of knowledge about psychological state of patients with periodontitis and the need for coping strategies’ improvement in an emotionally stressed patient to expect good response to treatment.

The study of the spatial-temporal and paleoecological structures of the paleocenoses of the Bolshaya Kosyu reef showed that the sphinctozoic sponges Amblysiphonelloides reticulata Rigby & Potter, 1986 and Corymbospongia sp. starting from the late Ordovician could settle in sublittoral environments with active hydrodynamics in close connection with cyanobacteria, which played a leading role in the functioning of the trophic structure of this ecosystem. Fossilized biofilms and glycocalyx, tubular and worm-like bacteriomorphs found on the surface of sponges are evidence of active participation of microbial organisms in the life of sphinctozoal sponges.

Concerns about energy efficiency and contaminants of emerging concern (CECs) in wastewater treatment plants (WWTP) lead development of new and alternative processes. Conventional activated sludge systems have a high energy consumption and footprint. Alternative processes are nowadays implemented to reduce them. In this study, we present the results of Biofungus project. Influent wastewater is treated under real conditions and continuous operation in a two-step Moving Bed Biofilm Reactor (MBBR) pilot plant based on Mucor fungus. Several strains and spontaneous mutants of the Mucor fungus were investigated. Those showing improved growth performance, wastewater resistance and nitrate consumption rate were isolated in laboratory and used at the pilot plant. Moreover, the in-situ growth at the pilot plant of the fungus from spores is implemented at the plant as a parallel process. The obtained effluent water meets regulations requirements, showing a high COD and suspended solids (SS) removal (87% and 94% on average respectively) and total nitrogen removal of 35% on average. The Biofungus pilot plant treatment works analogous to a conventional activated sludge process. One of the difficulties observed is the retention of the fungi during the process. The volume of carriers used at the MBBR is between 25-30% of the aeration tank. They allow the fungi to grow attached to them avoiding the dilution of its concentration by flotation and loss from this tank. This stage is followed by a settling tank, where the biomass is either recirculated to the aeration tank or purged to a sludge thickener tank. The process is a two-stage process with aerobic and anoxic reactors. The Mucor fungus specialized in the nitrate consumption is dosed into the anoxic reactor after a different Mucor string has consumed the DQO at the MBBR. Both stages are connected by a primary settling tank and a secondary decanter clarifies water after anoxic stage. After this secondary decanter the treated water is obtained. The retention time on the plant is between 6 and 10 hours and the treated volume is 3,6m3/day. The Mucor fungus has proved able to eliminate high ammonia and nitrate concentrations in sort periods of time, resulting on consumption rates of 1,6 mgN-NO3/h. Finally, its resistance to CECs has been evaluated under a wide range of contaminants including drugs, pesticides, herbicides, fungicides and hormones. Almost no toxicity to contaminant concentrations as high as 20 mg/l was observed. The water treatment performance has been also tested with a combination of CECs at 200µg/l and no influence has been observed after a week over the effluent water quality.