<div>Fresh produce has been the leading source of foodborne illness outbreaks in the US, surpassing typical pathogen carriers such as meat, dairy, and seafood. Among the fresh produce popular to the consumers, cantaloupe and sprouts are mostly susceptible to pathogen contaminations and outbreaks. However, it has been a challenge to address the key factor in the contamination - the biofilms formed by pathogens are highly resistant to conventional washing and cleaning procedures. For cantaloupe, the net-like and porous surface forms a barrier for washing. For sprouts, the fragile texture of seedlings prevents aggressive cleaning operation and biofilm removal.</div><div><br></div><div>In this study, innovative interventions were developed to improve microbial safety of fresh produce, using cantaloupe and alfalfa sprouts as models. For cantaloupe, abrasive brushing was designed to remove pathogen biofilm from cantaloupe. Our research found pathogens could form biofilm at cantaloupe rind surface as the residence time of pathogens increased. Biofilm formed on cantaloupe rind was imaged by cryo-scanning electron microscopy (cryo-SEM), and its resistance to sodium hypochlorite and lauroyl arginate ethyl (LAE) was confirmed. Furthermore, abrasive brushing with peroxyacetic acid (PAA) could effectively remove biofilm formed at cantaloupe rind. The efficacy of this novel cleaning technique was highly desirable, which could achieve 3 log reduction in pathogen population. Mechanism of abrasive brushing to remove biofilm at cantaloupe rind surface was also proposed. Conceivably, brushing with diatomaceous earth (DE) and PAA could be an innovative and cost-effective method to remove pathogen biofilm from cantaloupe rind.</div><div><br></div><div>For alfalfa sprouts, since most of the outbreaks are linked to the sprouting seeds, seed disinfection treatments are considered to be the most effective method to improve microbial safety of sprouts. In this study, a newly developed alginate-based, antimicrobial seed coating treatment was evaluated for its efficacy to reduce foodborne pathogens from alfalfa seeds and sprouts. The calcium alginate coating in the presence of 2.5% lactic acid (CA-LA coating) reduced foodborne pathogens inoculated on alfalfa seeds to an undetectable level on day 1 during 28 day-seed storage, while chlorine (20,000 ppm) or lactic acid (2.5%) treatment took longer time to reach the same level. With sprouts, CA-LA coating resulted in > 2.5 log reduction for pathogen cells. In contrast, log reduction was < 0.6 for either chlorine (20,000 ppm) or lactic acid (2.5%) treatment. In general, this study indicated the effect of calcium alginate coating on reducing bacterial load of alfalfa seeds and sprouts, however, the germination rate of treated seeds was compromised due to the addition of lactic acid in the seed coating. Further study is needed to select antimicrobial compounds with minimum impact on germination rate of seeds.</div><div><br></div>

Předložená dizertační práce s názvem “Rastrovací elektronová mikroskopie a její aplikace pro senzitivní vzorky” pojednává o problematice rastrovací elektronové mikroskopie v kontextu instrumentálního a metodologického vývoje vedoucího k inovativnímu řešení, které je dobře aplikovatelné zejména v mikrobiologickém výzkumu. Součástí práce je rozprava o historii a současném stavu elektronové mikroskopie (EM) jakožto vědecké zobrazovací a analytické techniky, tato část se nachází v úvodních kapitolách. Nepopiratelný přínos EM v biologických a lékařských oborech je dokazován mnoha citovanými vědeckými publikacemi. Předložená dizertační práce přináší novinky z oblasti přípravy preparátů a kryogenní rastrovací elektronové mikroskopie (cryo-SEM) vyvinuté na pracovišti Ústavu přístrojové techniky AV ČR, v.v.i. v Brně. Jedná se především o návrhy a výrobu speciálních držáků vzorků a vývoj nových metodik v oblasti přípravy mikrobiologických preparátů vedoucích k nalezení optimálních parametrů jednotlivých procesů. V experimentální části se nachází ověření metodologických postupů při studiu hydratovaných a na elektronový svazek senzitivních preparátů. Následné srovnání různých přístupů na definovaném biologickém systému z oblasti mikrobiologie přispívá k rozšíření interpretace doposud známých výsledků. Mezi zkoumanými mikrobiologickými kmeny byly biofilm-pozitivní bakterie Staphylococcus epidermidis a kvasinky jako Candida albicans a Candida parapsilosis, jež jsou považovány za klinicky významné, protože se podílejí na vzniku závažných infekcí zejména u imunokompromitovaných pacientů. Dále byl studován vliv růstu biofilmu bakterie Bacillus subtilis na biodeteriorizaci a biodegradaci poly--kaprolaktonových fólií. Vývoj v oblasti cryo-SEM byl aplikován ve výzkumu mikrobů s biotechnologickým potenciálem, jako jsou např. Cupriavidus necator a Sporobolomyces shibatanus.

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Chemotherapy of tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (M. tuberculosis), is successful against actively-growing bacilli but ineffective against dormant/persistent organisms, found mainly in a protective lipid-laden granuloma, possibly necessitating the use of lipophilic antibiotics. In vitro, these bacilli are encased in lipid-rich biofilms. In this study, the antimycobacterial activity of one such agent, clofazimine, and its nanoparticle formulation, have been investigated against Mycobacterium smegmatis (M. smegmatis), as a surrogate for M. tuberculosis, by determining the bacteriostatic and bactericidal activities of the native (NC) and spray-dried (SDC) preparations of this agent on planktonic and biofilm populations, as well as their effects on biofilm formation and its lipid compositions, specifically free mycolic acid (FM) content. Both preparations were comparable, being bacteriostatic for rapidly-proliferating bacilli, bactericidal for slow-growing, biofilm-producing sessile bacteria, but ineffective against non-replicating, biofilm-encased M. smegmatis organisms. However, similar studies in M. tuberculosis are required. / Dissertation (MSc)--University of Pretoria, 2013. / Immunology / Unrestricted

Actinobacillus succinogenes cells were grown on Poraver® support particles in a packed-bed reactor. Dilution rates (D) of 0.054–0.72 h-1 were investigated. Glucose was used as substrate. CO2 (g) was bubbled into a complex medium to satisfy the fixation requirements and maintain anaerobic conditions. At D ≥ 0.31 h-1, an initial glucose concentration of 35 g.L-1 was used; at lower dilution rates, this was increased to 60 g.L-1 in order to avoid substrate limitations. By-product formation included acetic and formic acids. A maximum productivity of 10.7 g.L-1 was obtained at D = 0.7 h-1. It was found that the system provided repeatable results at a given D. The longest steady state period was maintained for about 97 h at D = 0.31 h-1. Steady state stability was maintained for > 72 h at D < 0.31 h-1. For periods longer than 75 h, however, inhibitory acid titres resulted in a gradual decline in productivity. At higher dilution rates, long-term stability could not be maintained. The low acid titres produced significant biofilm sloughing following aggressive biofilm growth, resulting in oscillatory system behaviour. For fermentation times < 115 h, the dilution rate was secondary to the attachment area in determining the total biomass at steady state. Total biomass values were then used to determine specific rates. A clear trend was observed, with the specific glucose consumption rate, and specific acid production rates, increasing with increasing D. This was explained by assuming a maintenance-driven system at all Ds studied. A product analysis indicated that at ΔS < 15 g.L-1, pyruvate formate lyase was the preferred oxidative route. A shift to the pyruvate dehydrogenase pathway occurred at higher ΔS values, so that the highest YSS values obtained exceeded 0.85 g.g-1. A decrease in C3 by-product formation resulted in high YSS values being maintained, indicating an additional, unknown source of nicotinamide adenine dinucleotide (NADH). It is recommended that any process utilising immobilised A. succinogenes cells should operate at an intermediate D, in order to maintain long-term reactor stability, high productivities and good yields. / Dissertation (MEng)--University of Pretoria, 2013. / gm2014 / Chemical Engineering / unrestricted

Continuous fermentations were performed in a novel external-recycle, biofilm reactor using D-glucose and CO2 as carbon substrates. Corn steep liquor (CSL) and yeast extract (YE) served as nitrogen sources. In anaerobic fermentations using medium containing CSL and YE, succinic acid (SA) yields were found to be an increasing function of glucose consumption. The ratio of SA to the major by-product, acetic acid (YAASA), increased from 2.4 g g-1 at a glucose consumption of 15 g L-1, to 5.7 g g-1 at a glucose consumption of 46 g L-1. For medium containing no CSL, YAASA remained near 1.97 g g-1, exceeding this for cases where biofilm grown on CSL-containing medium was present. The ratio of formic acid to acetic acid (YAAFA), for CSL-containing medium, decreased from an equimolar value (0.77 g g-1) at a glucose consumption of 10 g L-1 to zero at 46 g L-1 glucose consumed. In contrast, YAAFA for YE-only medium remained at 0.77 g g-1. Therefore, pyruvate was metabolised solely by pyruvate-formate lyase when no CSL was present. The highest SA yield obtained on glucose, SA titre and SA productivity were 0.91 g g-1, 48.5 g L-1 and 9.4 g L-1 h-1, respectively, all for medium containing CSL. Medium that included CSL significantly outperformed medium that excluded CSL, achieving 64%, 21% and 203% greater SA titres, yields on glucose and productivities respectively. Metabolic flux analyses based on the established C3 and C4 metabolic pathways of Actinobacillus succinogenes revealed that the increase in YAASA, for CSL-containing fermentations, could not be attributed to the decrease in formate and biomass formation, and that an additional source of reducing power was present. The fraction of reducing power (NADH) unaccounted for increased with glucose consumption, suggesting that the maintenance or non-growth metabolism encountered at higher SA titres differs from the growth metabolism. It is postulated that the additional reducing power originates from an active pentose phosphate pathway in non-growing cells or from an undetected component(s) in the fermentation medium. No major metabolic flux variations were found in fermentations that excluded CSL. / Dissertation (MEng)--University of Pretoria, 2013. / gm2014 / Chemical Engineering / unrestricted

Biofilms of Actinobacillus succinogenes, grown in a biofilm reactor system, were investigated for structure and cell viability, through microscopic visualisation with a confocal scanning laser microscope (CSLM) and a scanning electron microscope (SEM). Biofilms were sampled and visualised at steady state conditions with the broth containing succinic acid titres between 15 and 21 g/L. All sampled biofilm was 6 days old. Six-day-old biofilms of A. succinogenes showed a heterogeneous biofilm architecture composed of cell micro-colony pillars which varied considerably in thickness, area and shape. Microcolony pillars consisted of a densely packed entanglement of sessile cells. Quantitative analysis revealed that the pillars were mostly large, with a mean pillar diameter of 170 m and a mean thickness of 92 m, although pillar diameter and thickness were variable as they ranged from 25 – 500 m and 30 – 300 m, respectively. In the regions close to the substratum surface, pillars were characterised by having defined borders with a network of channels ranging from 40 – 200 m in width separating them. However, towards the middle of the biofilm depth some of the pillars coalesced. For this reason low cross sectional area coverage of biofilm consistently occurred at the bottom portion of the biofilm whilst the highest coverage was in the middle portion of the biofilm. Regarding cell morphology, very large differences were observed. Planktonic cells were rod-shaped, whereas sessile cells expressed an elongated rod morphology and thus were much longer and thinner compared with planktonic cells. Planktonic cells were 1 – 2 m thick and 4 – 5 m long, while sessile cells were 0.5 – 1 m thick and 5 – 100 m long. Long sessile cells resulted in extensive tangling in microcolony pillars, which may have contributed to the structural stability of the pillars. Fibre-like connections of constant diameter were observed between cells, and between the cells and surface. The diameter of these connections was approximately 20 – 30 nm. Viability stains showed that in the bottom portion (from 0 - 20 m above the substratum surface) of the biofilm, most of the cells were dead. However, the portion of covered area attributed to living cells increased past the middle of the biofilm towards the top part of the biofilm. A high percentage of living cells was thus found towards the top part of the biofilm. Overall, 65% (with 2% standard deviation) of the entire biofilm was composed of dead cells. In this way, the results show that operation at high acid conditions comes at a cost of low overall biomass productivity due to decreased active biomass. / Dissertation (MEng)--University of Pretoria, 2017. / Chemical Engineering / MEng / Unrestricted

En 2000, la Directive Cadre sur l’Eau a exigé le retour au bon état chimique et écologique des cours d’eau. Elle a notamment classé 45 substances comme étant prioritaires pour cette évaluation (directive 2013/39/UE), dont en grande partie des pesticides. En effet, en raison de leur utilisation massive, on retrouve aujourd’hui ces contaminants dans tous les compartiments de l’environnement. Par ailleurs, au vu de sa capacité à intégrer les contaminations, le biofilm est considéré comme un excellent bioindicateur pour l’évaluation de la qualité de l’eau. Celui-ci est à la base de la chaine trophique dans les milieux aquatiques et se compose de microorganismes (microalgues, bactéries, champignons, etc…) enchâssés dans une matrice de substances polymériques extracellulaires (EPS).Cette thèse porte sur l’étude des mécanismes de transfert et de distribution des pesticides dans les biofilms en lien avec les effets toxiques associés. Ces travaux ont été réalisés avec une approche toxicocinétique par laquelle la bioaccumulation du diuron (un herbicide inhibiteur de la photosynthèse) a été suivie dans les différents compartiments du biofilm. Ce suivi a été réalisé sous plusieurs conditions abiotiques (vitesses de courant, températures et photopériodes) pour différentes durées d’exposition. En parallèle des descripteurs fonctionnels et structuraux ont été mesurés comme des activités photosynthétique (pour les communautés autotrophes) et enzymatiques (pour les communautés hétérotrophes), ainsi que la biomasse totale, la production de protéines et de polysaccharides.L’ensemble des expérimentations menées au cours de cette thèse a permis de décrire les mécanismes de sorption du diuron dans les biofilms, à savoir des processus d’absorption par les cellules et d’adsorption par la matrice EPS. Puis l’influence des différents paramètres environnementaux étudiés a pu être mise en avant. Ces travaux démontrent la pertinence de l’approche toxicocinétique-toxicodynamique pour l’étude de l’impact des pesticides sur les biofilms fluviaux. / In 2000, the Water Framework Directive required the return of rivers to good chemical and ecological status. In particular, it has classified 45 substances as priority for this assessment (Directive 2013/39/EU), including a large proportion of pesticides. Indeed, due to their massive use, these contaminants are now found in all compartments of the environment. In addition, given its ability to integrate contamination, biofilm is considered an excellent bioindicator for water quality assessment. It is at the base of the trophic chain in aquatic environments and is composed of microorganisms (microalgae, bacteria, fungi, etc...) embedded in a matrix of extracellular polymeric substances (EPS).This thesis focused on the analysis of mechanisms of pesticide transfer and distribution in biofilms related to toxic impacts. This work was carried out using a toxicokinetic approach whereby the bioaccumulation of diuron (a photosynthesis inhibitor herbicide) was monitored in the different compartments of the biofilm. This assessment was carried out under several abiotic conditions (flow velocity, temperatures and photoperiods) under varying exposure durations. In parallel, functional and structural descriptors were measured as photosynthetic (for autotrophic communities) and enzymatic (for heterotrophic communities) activities, as well as biofilm biomass and protein and polysaccharide production.All the experiments performed during this thesis made it possible to highlight the sorption mechanisms of diuron in biofilm, i.e. absorption processes by cells and adsorption phenomenon within the EPS matrix. Then the influence of the different environmental parameters studied was emphasized. This work demonstrates the relevance of the toxicokinetic-toxicodynamic approach to the study of the impact of pesticides on fluvial biofilms.

Cette thèse est consacrée à l'étude de systèmes d'équations aux dérivées partielles. En particulier nous nous intéressons à des systèmes issus de la mécanique des fluides avec contraintes qui permettent de décrire de manière continue en temps et en espace des quantités physiques telles que la densité ou la vitesse. Dans ce cadre nous construisons des modèles pour la biologie : modélisation de la croissance d'un biofilm de micro-algues et modélisation du gros intestin et de sa couche de mucus. Ces modèles sont ensuite testés numériquement à l'aide de schémas numériques spécifiquement élaborés pour ces modèles. Cette thèse est complétée par une étude numérique du modèle d'Aw-Rascle avec contrainte pour le trafic routier / This thesis is devoted to the study of partial differential equation systems. In particular, we are interested in constrained systems coming from the fluid mechanics field which allow to describe, in time and space, physical quantities such as density or speed. In this context we build models for biology: modeling of the growth of micro-algae biofilms and modeling of the large intestine and its mucus layer. These models are then tested numerically using numerical schemes specifically developed for these models. This thesis is supplemented with a numerical study of Aw-Rascle model with constraint for road traffic

Natürliche Biofilme von zwei urankontaminierten Standorten, dem ehemaligen Uranbergwerk in Königstein (Sachsen) und dem Gebiet der ehemaligen Aufstandsfläche der Gessenhalde (Thüringen), wurden in dieser Arbeit näher untersucht. An beiden Standorten konnte in den Minenwässern die hochmobile, gelöste Uranspezies Uranylsulfat (UO2SO4) als dominierend nachgewiesen werden. Aufgrund der Instabilität vieler kommerzieller Fluoreszenzfarbstoffe bei niedrigen pH-Werten war eine gezielte Anfärbung der Mikroorganismen in den sauren Biofilmen nicht möglich, ohne den pH-Wert der Biofilmproben anzuheben, was die Probenchemie maßgeblich verändert. In Kooperation mit der Firma DYOMICS (Jena, Deutschland) wurden neue, kommerziell nicht erhältliche, säurestabile Farbstoffe erstmals hinsichtlich ihrer Eignung zur Anfärbung von Mikroorganismen in sauren Biofilmen ohne Veränderung des pH-Wertes sowie der sonstigen Probenchemie getestet. Die neuen Farbstoffe DY-601XL, V07-04118, V07-04146 und DY-613 zeigten eine Eignung für solche Färbungen, da sie eine intensive Anfärbung der Mikroorganismen bei niedrigen pH-Werten unter pH 3 – 4 herbeiführen und außerhalb des Emissionsbereiches von Uran fluoreszieren. In dieser Arbeit wurde die Fähigkeit von Euglena Mutabilis-Zellen zur Bioakkumulation des Urans im pH-Wertbereich 3 – 6 in den Hintergrundmedien Natriumperchlorat (9 g/l) oder Natriumsulfat (3,48 g/l) an lebenden Zellen untersucht. Unabhängig vom Medium konnte bei sauren pH-Werten um pH 3 – 4 über 90 % des vorgelegten Urans aus den Probelösungen abgetrennt werden. Die Speziation des an den Euglena-Zellen akkumulierten Urans, wurde mittels laserinduzierter Fluoreszenzspektroskopie (LIFS) untersucht. Es zeigte sich, dass unabhängig vom Hintergrundmedium, Lebenszustand und pH-Wert eine vergleichbare neue Uranspezies an den Zellen gebildet wird. Durch den Vergleich der Daten aus den LIFS-Messungen mit Referenzwerten, konnte die gebildete Uranspezies auf eine Anbindung durch (organo)phosphatische und/oder carboxylische funktionelle Gruppen eingegrenzt werden. Mit Hilfe der zeitaufgelösten FT-IR-Spektroskopie konnte die carboxylische Anbindung des Urans an toten Zellen nachgewiesen werden. Ein Ausschluss der (organo)phosphatischen Komplexierung konnte jedoch mit dieser Methode nicht geführt werden. Untersuchungen zur Lokalisation des Urans an bzw. in den Zellen, mittels der gekoppelten CLSM/LIFS-Technik zeigten erstmals ein Indiz für die intrazelluläre Akkumulation von Uran in den lebenden Zellen. Ergänzende TEM/EDX-Messungen bestätigten die intrazelluläre Aufnahme und belegen eine Akkumulation in runden bis ovalen Zellorganellen, bei denen es sich vermutlich um Vakuolen oder Vakuolen-ähnliche Vesikel handelt. An den toten Zellen konnte mit diesen Methoden kein Uran detektiert werden. Dies lässt auf eine passive, homogen verteilte Biosorption des Urans an die verfügbaren Bindungsplätze an der Zelloberfläche der toten Biomasse schließen.