Ultrafiltration (UF) processes are often used as pretreatment before more retentive/costly processes, such as nanofiltration and reverse osmosis. This study shows the results of low-biofouling nanocomposite membranes, loaded with casein-coated silver nanoparticles (casein-Ag-NPs). Membranes were cast and imbedded with Ag-NPs using two approaches, physical blending of Ag-NPs in the dope solution (PAg-NP/CA membranes) and chemical attachment of Ag-NPs to cast membranes (CAg-NP/CA membranes), to determine their biofouling control properties. The functionalization of Ag-NPs onto the CA membranes was achieved via attachment with functionalized thiol groups with the use of glycidyl methacrylate (GMA) and cysteamine chemistries. The immobilization chemistry successfully prevented leaching of silver nanoparticles during cross-flow studies. Pseudomonas fluorescens Migula in brackish water was used for short-term dead-end filtration, where CA and CAg-NP/CA membranes displayed lower flux declines as compared to PAg-NP/CA membranes. In subsequent long-term biofouling studies, also with Pseudomonas fluorescens Migula in brackish water with addition of sodium acetate, chemically-attached Ag-NPs led to a significant reduction in the accumulation of bacterial cells, likely due to the more dispersed nanoparticles across the surface. Therefore, a method was developed to chemically immobilize Ag-NPs to membranes without losing Ag-NP’s antimicrobial properties.

La presente investigación se realizó con el fin de evaluar el uso de biocontroladores en la prevención de patógenos causantes de camping off y en el desarrollo de plantines de tomate, aplicados en un sustrato obtenido de material orgánico bioprocesado.

4 ABSTRACT (English) This thesis is based on three published research papers studying bacterial REP (repetitive extragenic palindrome) elements. REP elements are one of the best-characterized groups of bacterial DNA repeats, distributed mostly in gammaproteobacteria, including enterobacteria. They are present in noncoding parts of host genomes, usually occurring in hundreds of copies. REPs are typically aggregated in higher order repeats. In the Gram-negative model Escherichia coli, interactions of several proteins important for cell's physiology with REPs were described, indicating significant role for these elements for host cells. The first work (Nunvar et al. 2010) presents the discovery of a protein class, related to IS200/IS605 transposases. These proteins, termed RAYTs (REP-associated tyrosine transposases), contain characteristic motifs in their amino acid sequences, which are absent in canonical IS200/IS605 transposases. Another attribute of RAYTs is the arrangement of their encoding genes. These are single copy genes, always flanked at both termini by at least two REPs in inverted orientation. Based on the similarity between the REP-rayt-REP unit and insertion sequences of the IS200/IS605 family, between RAYTs and tyrosine transposases and between REPs and subterminal sequences of the IS200/IS605...

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In life science practices, cell culturing is one of the most common procedures in biological experiments involving the use of microscopes. Cell cultures are typically viewed with a microscope to monitor changes after adjusting the environment for the cells or to intermittently supervise for cellular behavior. However, it is currently not possible to continuously monitor cellular behavior due to the absence of solutions for wireless control of a microscope to image cell cultures while maintaining the environment conducive to cell growth. This thesis provides a miniaturized, cost-effective and versatile microscope down-scaled enough to operate inside incubators. This versatility encompasses the ability to switch between bright-field mode and fluorescence mode, capture images in sequences, save said sequential images to a micro SD-card as well as enabling downloading of individual images to a local file manager. All these features are done through a user interface set up by either a local WiFi or access point from the microscope, making the operation possible through wireless connection. In addition, this thesis is a continuation of a previous thesis that was completed in the spring of 2023. The purpose of this thesis is to improve on the result of the main research question of the previous work. / Inom livsvetenskapliga metoder är cellodling en av de vanligaste procedurerna i biologiska experiment som involverar användning av mikroskop. Cellkulturer ses vanligtvis med ett mikroskop för att bevaka förändringar efter att ha ändrat miljön för cellerna eller för att intermittent övervaka cellulärt beteende. Men det är för närvarande inte möjligt att kontinuerligt övervaka cellulärt beteende på grund av avsaknaden av lösningar för trådlös kontroll av ett mikroskop för att avbilda cellkulturer samtidigt som miljön som främjar celltillväxt bibehålls. Detta examensarbete tillhandahåller ett miniatyriserat, kostnadseffektivt och mångfasetterat mikroskop, som är tillräckligt nedskalad för att fungera i inkubatorer. Denna mångsidighet omfattar möjligheten att växla mellan ljusfältsläge och fluorescensläge, ta bilder i sekvenser, spara nämnda sekventiella bilder på ett micro SD-kort samt ladda ner enskilda bilder till en lokal filhanterare. Alla dessa funktioner görs via ett användargränssnitt som ställs in av antingen ett lokalt WiFi eller accesspunkt från mikroskopet, vilket gör operationen möjlig via trådlös anslutning. Dessutom är detta examensarbete en fortsättning på ett tidigare examensarbete som avslutades våren 2023. Syftet med detta examensarbete är att förbättra resultatet på den huvudsakliga forskningsfrågan i det tidigare arbetet.

Aeromonas salmonicida subsp. salmonicida est une bactérie à Gram négatif responsable de la furonculose, une infection opportuniste des salmonidés d'élevage représentant en moyenne de 30 à 60 % des diagnostics annuels posés au Québec chez l'Omble de fontaine aquicole (Salvelinus fontinalis). Les méthodes de traitement actuelles contre la furonculose reposent essentiellement sur l'antibiothérapie et l'immunoprophylaxie. Cependant, l'usage intensif d'antibiotiques en aquaculture contribue à l'émergence de souches pathogènes d'A. salmonicida antibiorésistantes (certaines résistant à jusqu'à 8 antibiotiques). De plus, les effets secondaires associés à l'antibiothérapie et la vaccination peuvent causer des altérations maladaptives des interactions hôte-microbiote, en défaveur des bactéries résidentes de l'hôte qui contribuent à la résistance aux infections. C'est pourquoi l'administration de symbiontes bactériens endogènes à des hôtes sensibles en tant que traitement probiotique semble être une solution prometteuse. L'objectif de ce projet de maîtrise était d'initier la recherche et le développement d'un traitement à base de probiotiques endogènes pour prévenir et traiter la furonculose chez l'Omble de fontaine. Dans le cadre de ce projet, des souches de Pseudomonas spp. endogènes de l'Omble de fontaine ont été découvertes sur la base de leur effet d'antagonisme marqué et inductible vis-à-vis A. salmonicida. L'analyse bio-informatique du génome de ces souches a permis de prédire qu'elles appartiennent à l'espèce P. fluorescens, qu'elles sont a priori non pathogènes et qu'elles ne portent aucun gène horizontalement transférable de résistance à des antibiotiques homologués au Canada pour usage en aquaculture. Ces souches seraient donc des candidats probiotiques prometteurs pour prévenir et traiter la furonculose chez l'Omble de fontaine. / Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium causing furunculosis, an opportunistic disease of farmed salmonids that accounts for 30 to 60% of yearly diagnoses emitted for the brook charr (Salvelinus fontinalis) in the Quebec province, Canada. Current treatment methods against furunculosis include antibiotherapy and immunoprophylaxis. However, the intensive use of antimicrobials in aquaculture contributes to the emergence of resistant A. salmonicida strains (some of which can resist up to 8 antimicrobials). Moreover, the adverse effects of antibiotherapy and vaccination may cause maladaptive changes in host-microbiota interactions, in disfavour of resident bacteria that contribute to enhance disease resistance. Therefore, using bacterial symbionts as a probiotic treatment appears to be a promising solution to treat furunculosis. The purpose of this Master's project was to pioneer research and development of an endogenous probiotic-based treatment strategy for brook charr against furunculosis. In this project, endogenous Pseudomonas spp. strains from brook charr were identified on the basis of their strong and inducible in vitro antagonistic effect against A. salmonicida. Bioinformatic analyses of those strains' genome sequence predicted that: (i) they belong to the P. fluorescens species group, (ii) they are non-pathogenic a priori, and (iii) they harbor no horizontally-transferable genes that confer resistance to antibiotics approved for use in aquaculture in Canada. These P. fluorescens strains therefore are promising probiotic candidates for in vivo trials on live brook charr.

Das Ziel dieser Arbeit war die Entwicklung eine Gerätes, welches die Überwachung von mehreren hundert Mikrobioreaktoren ermöglicht. Dabei sollte es, neben der Reduzierung von Kosten und Arbeitskraft, die konventionellen Pipettiermethoden in der zeitliche Auflösung übertreffen. Weiterhin soll es über eine Gradientenerzeugung verfügen, um sehr feine Variationen der Zusammensetzung des verwendeten Mediums zu ermöglichen. Dafür wurde ein Analysator für fluoreszierenden Tropfen (Fluorescent Droplet Analyser) entwickelt, mit dem ein segmentierter Fluss von mehreren hundert Tropfen erzeugt werden kann und für viele Tage gemessen werden kann. Für die Messung wurde der Analysator mit einer flexiblen Fluoreszenzoptik ausgestattet um unterschiedliche Fluoreszenzfarbstoffe oder Molekühle detektieren zu können. Mehrere Experimente wurden durchgeführt, welche das Potentials des Gerätes zeigen. Einzelne Zellen des Pantoffeltierchen (Paramecium tetraurelia) konnten in einzelnen Tropfen eingeschlossen werden und mit einem metabolischen Farbstoffe ihre Stoffwechselaktivität gemessen werden. Ebenfalls wurden viele Experimente mit Pseudomonas fluorescens und E.coli YFP durchgeführt. Durch die flexible Fluoreszenzoptik konnte das Wachstum beider Arten in eine Experiment gemessen werden.

Das Ziel dieser Arbeit war die Entwicklung eine Gerätes, welches die Überwachung von mehreren hundert Mikrobioreaktoren ermöglicht. Dabei sollte es, neben der Reduzierung von Kosten und Arbeitskraft, die konventionellen Pipettiermethoden in der zeitliche Auflösung übertreffen. Weiterhin soll es über eine Gradientenerzeugung verfügen, um sehr feine Variationen der Zusammensetzung des verwendeten Mediums zu ermöglichen. Dafür wurde ein Analysator für fluoreszierenden Tropfen (Fluorescent Droplet Analyser) entwickelt, mit dem ein segmentierter Fluss von mehreren hundert Tropfen erzeugt werden kann und für viele Tage gemessen werden kann. Für die Messung wurde der Analysator mit einer flexiblen Fluoreszenzoptik ausgestattet um unterschiedliche Fluoreszenzfarbstoffe oder Molekühle detektieren zu können. Mehrere Experimente wurden durchgeführt, welche das Potentials des Gerätes zeigen. Einzelne Zellen des Pantoffeltierchen (Paramecium tetraurelia) konnten in einzelnen Tropfen eingeschlossen werden und mit einem metabolischen Farbstoffe ihre Stoffwechselaktivität gemessen werden. Ebenfalls wurden viele Experimente mit Pseudomonas fluorescens und E.coli YFP durchgeführt. Durch die flexible Fluoreszenzoptik konnte das Wachstum beider Arten in eine Experiment gemessen werden.:1 Introduction 1 1.1 Motivation 1 1.2 Scope of this thesis 2 1.3 State of the art 3 2 Fundamentals 2.1 Millifluidics vs. microfluidics 5 2.1.1 Droplet based microfluidics 6 2.1.2 Surfactant in droplet-based millifluidics 7 2.2 The model of microorganism growth 8 2.3 The fluorescence based detection 9 3 Materials & Methods 3.1 The culture of the microorganisms and preparation 11 3.1.1 The Paramecium tetraurelia 11 3.1.2 Pseudomonas fluorescens 12 3.1.3 Escherichia coli 12 3.1.4 The bacteria culture 13 3.2 The Poisson distribution . 15 3.3 The composition of the emulsion 16 3.4 The fluidic components 17 3.5 Modules of the Fluorescent Droplet Analyser 18 3.5.1 The optocoupled relay card 18 3.5.2 The used fluidic pumps 19 3.5.3 The photonic elements of the FDA 19 3.5.4 The light sources 21 3.5.5 The optical fibres 23 3.5.6 The used detectors 23 3.6 The operating Software 24 4 The Fluorescent Droplet Analyser 4.1 The fluidic network for droplet generation and shuttling 26 4.1.1 Generation of a droplet sequence 28 4.1.2 Measuring a droplet sequence 30 4.2 The electric schematics of the FDA 31 4.3 The light source, guidance, and detection 32 4.3.1 Preparation of the fibres 33 4.3.2 The detection setup 35 4.4 The developed LabView program for operating the FDA 37 4.4.1 The automated measurement process 40 4.4.2 The peak mode 41 4.4.3 The time mode 42 4.4.4 The combined mode 42 4.4.5 The dynamic threshold 42 4.5 The developed VI program for analysing the data of the FDA 44 4.6 Flow characteristics of the FDA 46 4.6.1 Basic flow characteristics 46 4.6.2 Characteristics for shuttling of a droplet sequence 47 4.6.3 Influence of the hydrodynamic resistance 48 5 Monitoring of Paramecium tetraurelia 5.1 Introduction 51 5.2 Generation of a droplet sequence for Paramecium tetraurelia 53 5.3 Metabolic dynamics in droplets 54 5.4 Ecotoxicity assays 58 6 Monitoring of bacteria 6.1 Introduction 63 6.2 Generation of a droplet sequence for the bacteria experiments 64 6.3 Cell number calibration of the FDA 65 6.4 Monitoring of the bacteria growth 68 6.5 Investigation of the of the fluorescence signal 70 6.6 One bacteria cell in a droplet 71 6.7 The determination of the minimum inhibitory concentration 74 6.8 Long-term monitoring 80 6.9 Sectioning of the droplet sequence 80 6.10 Multicolor fluorescence detection 83

De senaste årtiondena har en ökning av naturligt organiskt material (NOM) observerats i ytvatten i norra Europa och Nordamerika. NOM i vattnet innebär utmaningar för vattenverk som använder ytvatten i dricksvattenproduktionen då det kan ge upphov till lukt, smak och färg och svårigheter i driften. För att åtgärda och förebygga problemen behövs mer kunskap om NOM samt hur kvantiteten och sammansättningen förändras i olika reningsprocesser. Vid Hofors vattenverk används råvatten från sjön Hyen i dricksvattenproduktionen. Driftstörningar upplevs i perioder då råvattnets färgtal (mått på halten NOM) är högt. Därför undersöktes det hur väl den nuvarande reningsprocessen vid Hofors vattenverk fungerar med avseende på avskiljning av NOM samt vilken typ av NOM som avskiljs. Resultaten jämfördes med pilotförsök med direktfällning på ultrafiltermembran för att se om detta kan ge bättre avskiljning. Olika analysmetoder, absorbans, fluorescens och mätning av löst organiskt kol (DOC), användes för att avgöra kvantitet och karaktär på NOM i vattenproverna. Även eventuell förekomst av trend i råvattnets färgtal undersöktes. Likt i många andra ytvatten har färgtalet i Hyen har ökat de senaste åren (1997-2015). Råvattnet är svårfällt och innehåller en blandning av olika NOM-fraktioner. Huvudsakligen avskiljs humuslikt, alloktont NOM i Hofors vattenverk. Avskiljningen var störst i första reningssteget, direktfällning på sandfilter. Förändringen i efterföljande reningssteg, kolfilter samt desinfektions- och pH-höjande steg, var dock svår att bestämma till följd av mycket små förändringar och motsägande analysresultat. Tre ultrafiltermembranförsök gav liknande avskiljningsresultat som sandfilter, men i två av fallen är det troligt att igensättning av membranen spelade in. Det krävs ytterligare försök för att säkert uttala sig om huruvida ultrafiltermembran är en teknik som är lämplig att använda vid Hofors vattenverk i framtiden för avskiljning av NOM. / In recent decades, an increase in natural organic matter (NOM) has been observed in surface waters in Northern Europe and North America. NOM in the water poses challenges for water treatment plants (WTP) using surface water in the drinking water production. It can cause odor, taste and color of the water and cause difficulties in the treatment process. To prevent all these problems, more information about NOM and how the quantity and composition changes in various treatment processes is needed. At Hofors WTP, raw water from the lake Hyen is used in the production of drinking water. Disruptions in running the process have been experienced in periods when the raw water color (measure of the NOM content) has been high. In this thesis, it was investigated how well the current treatment process at Hofors WTP works with respect to the separation of NOM and what type of NOM that is separated. The results were compared with pilot tests with direct filtration using an ultrafilter membrane to see if this can provide better separation. Various analytical methods as absorbance, fluorescence and measurement of dissolved organic carbon (DOC) were used to quantify and determine the composition of NOM in the water samples. It was also examined whether there is a trend in the raw water color. As in many other surface waters the color of the water has increased in Hyen in recent years (1997-2015). The raw water contains a mixture of different NOM fractions and the character indicates difficulties in removing DOC by coagulation and flocculation. It is primarily the humic like, allochtonous NOM that is separated in the treatment process with the greatest separation in the first treatment step, direct filtration with sand filter. What happens in the subsequent treatment steps, carbon filter, disinfection and pH-raising step, was however unclear due to small changes and inconsistent analysis results. Three experiments with ultrafilter membrane yielded similar results as sand filters, but in two of the cases it is likely that clogging of the membranes contributed. Further membrane pilot tests are needed to be sure whether ultrafilter membrane is a is suitable technique to use at Hofors WTP in the future for separation of NOM.

Pseudomonas is a diverse genus of Gram-negative bacteria that includes pathogens of plants, insects, and humans as well as environmental strains with no known pathogenicity. Pseudomonas fluorescens itself encompasses a heterologous group of bacteria that are prevalent in soil and on foliar and root surfaces of plants. Some strains of P. fluorescens suppress plant diseases and the genomic sequences of many biological control strains are now available. I used a combination of bioinformatic and phylogenetic analyses along with mutagenesis and biological assays to identify and compare the TonB-dependent outer-membrane proteins (TBDPs) of ten plant-associated strains of P. fluorescens and related species. TBDPs are common in Gram-negative bacteria, functioning in the uptake of ferric-siderophore complexes and other substrates into the cell. I identified 14 to 45 TBDRs in each strain of P. fluorescens or P. chlororaphis. Collectively, the ten strains have 317 TBDPs, which were grouped into 84 types based upon sequence similarity and phylogeny. As many as 13 TBDPs are unique to a single strain and some show evidence of horizontal gene transfer. Putative functions in the uptake of diverse groups of microbial siderophores, sulfur-esters, and other substrates were assigned to 28 of these TBDP types based on similarity to characterized orthologs from other Pseudomonas species. Redundancy of TBDP function was evident in certain strains of P. fluorescens, especially Pf-5, which has three TBDPs for ferrichrome/ferrioxamine uptake, two for ferric-citrate uptake and three for heme uptake. Five TBDP types are present in all ten strains, and putative functions in heme, ferrichrome, cobalamin, and copper/zinc uptake were assigned to four of the conserved TBDPs. The fluorescent pseudomonads are characterized by the production of pyoverdine siderophores, which are responsible for the diffusible UV fluorescence of these bacteria. Each of the ten plant-associated strains of P. fluorescens or P. chlororaphis has three to six TBDPs with putative roles in ferric-pyoverdine uptake (Fpv). To confirm the roles of the six Fpv outer membrane proteins in P. fluorescens Pf-5, I introduced deletions into each of the six fpv genes in this strain and evaluated the mutants and the parental strain for heterologous pyoverdine uptake. I identified at least one ferric-pyoverdine that was taken up by each of the six Fpv outer-membrane proteins of Pf-5. By comparing the ferric-pyoverdine uptake assay results to a phylogenetic analysis of the Fpv outer-membrane proteins, I observed that phylogenetically-related Fpv outer-membrane proteins take up structurally-related pyoverdines. I then expanded the phylogenetic analysis to include nine other strains within the P. fluorescens group, and identified five additional types of Fpv outer-membrane proteins. Using the characterized Fpv outer-membrane proteins of Pf-5 as a reference, pyoverdine substrates were predicted for many of the Fpv outer-membrane proteins in the nine other strains. Redundancy of Fpv function was evident in Pf-5, as some pyoverdines were recognized by more than one Fpv. It is apparent that heterologous pyoverdine recognition is a conserved feature, giving these ten strains flexibility in acquiring iron from the environment. Overall, the TBDPs of the P. fluorescens group are a functionally diverse set of structurally-related proteins present in high numbers in many strains. While putative functions have been assigned to a subset of the proteins, the functions of most TBDPs remain unknown, providing targets for further investigations into nutrient uptake by P. fluorescens spp.. The work presented here provides a template for future studies using a combination of bioinformatic, phylogenetic, and molecular genetic approaches to predict and analyze the function of these TBDPs. / Graduation date: 2012