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No. of bitstreams: 1 Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2015-11-19 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nanotechnology is a science of nano-sized systems, comprised in a range of 10-9 m, which proposes to create a new organizational structure able to show different behaviors and properties of materials currently known and available. Through integration with biotechnology, the term "nanobiotechnology" may be used to define the use of microorganisms in studies concerning medical, pharmaceutical and cosmetics industries, among others. By virtue of their biotechnological potential for synthesizing antibacterial compounds, antifungal, antitumor, and antiparasitic compounds, beyond other substances of industrial interest, filamentous bacteria (actinomycetes) has potential for the development of drugs through biological synthesis of silver nanoparticles (AgNPS). This study aimed to evaluate the potential of AgNPs synthesis with antimicrobial activity against pathogenic bacteria and opportunistic fungi using as source of biomolecules species of actinomycetes. Ten samples of actinomycetes were selected from the collection Collection of Culture-DPUA and were subcultured and authenticated in ISP-2A (malt extract-yeast extract agar), based on morphological characteristics. The identification at the species level was performed by the technique of PCR (Polymerase Chain Reaction). For biogenic synthesis AgNPs, actinomycetes underwent submerged fermentation. The recovered crude extract and biosynthesized nanoparticles were used to determine the antimicrobial activity by agar diffusion method. The AgNPs that showed significant antagonistic action have been used to determine the MIC (Minimum Inhibitory Concentration), Cytotoxicity and characterized by different techniques, such as UV-Vis, DLS (Dynamic Light Scattering), XRD (diffraction X-ray) FTIR (Infrared Spectroscopy Fourier Transform), ICP-OES (Optical Emission Spectroscopy by Inductively Coupled Plasma) and TEM (Transmission Electron Microscopy). From these actinomycetes, 100% viable expressed morphological characteristics of Streptomyces. For the test of PCR the following species were identified Streptomyces parvulus (40%), Streptomyces bullii (10%), Streptomyces seoulensis (10%), Streptomyces owasiensis (10%), Streptomyces malasyensis (20%), Streptomyces lavendulae (10%). By the method agar block antimicrobial activity was confirmed and 80% of the bacteria inhibited S. aureus; 30% M. smegmatis and 40% the Candidas (albicans, atlantica and valderwaltii). Only 10% of actinomycetes inhibited the growth of E. coli. When tested across the crude extract to microorganisms test, 50% of the samples inhibited S. aureus; 30% M. smegmatis and 20% proved to be effective against C. albicans, C. atlantica and C. valderwaltii. In these tests the halo diameters varied on average 12 to 29 mm. As for the AgNPs, 100% inhibited the growth of S. aureus and E. coli with halos ranging from 12 to 34 mm. Data indicating the broad spectrum of these bioactive compounds. The AgNPs Streptomyces DPUA 1549, Streptomyces DPUA 1747 and Streptomyces DPUA 1748 showed antagonistic action for greater efficiency. Promising AgNPs showed spherical shape and sizes ranging from 1-40 nm. However, nanoparticles produced by Streptomyces sp. DPUA 1549 were the most efficient with MIC against S. aureus at low concentrations (1.95 μM). Through the images displayed in AFM was observed structural damage on cell membrane of S. aureus. In toxicity tests the AgNPs reduced the proliferation of NIH-3T3 cells. In addition, the cytotoxic activity of this nanomaterial is time dependent. These scientific evidences show the potential application of nanoparticles biosinthesyzed by Streptomyces DPUA 1549 on preparation of products for use in medical, cosmetic and pharmaceutical industries / A Nanotecnologia é uma ciência de sistemas de tamanho nano, compreendida em uma escala de 10-9 m, que propõe criar uma nova organização estrutural capaz de apresentar comportamentos e propriedades diferentes dos materiais atualmente conhecidos e disponibilizados. Por meio da integração com a biotecnologia, o termo “nanobiotecnologia” pode ser empregado para definir o uso de micro-organismos em estudos voltados as indústrias médica, farmacêutica, cosmética, dentre outras. Em virtude do seu potencial biotecnológico por sintetizarem compostos antibacterianos, antifúngicos, antitumorais, antiparasitários, além de outras subtâncias de interesse industrial, as bactérias filamentosas (actinomicetos) apresentam potencial para o desenvolvimento de medicamentos por meio da síntese biológica de Nanopartículas de Prata (AgNPs). Este estudo teve como objetivo avaliar o potencial de síntese de AgNPs com atividade antimicrobiana sobre bactérias patogênicas e fungos oportunistas, utilizando como fonte biomoléculas de espécies de actinomicetos. Dez amostras de actinobactérias foram selecionadas do acervo da Coleção de Cultura-DPUA e foram subcultivados e autenticados em ISP-2A (extrato de levedura-extrato de malte, agar), com base nas características morfológicas. A identificação em nível de espécie foi realizada pela técnica da PCR (Reação em Cadeia da Polimerase). Para síntese biogênica de AgNPs, os actinomicetos foram submetidos a fermentação submersa. O extrato bruto recuperado e as nanopartículas biossintetizadas foram utilizados na determinação da atividade antimicrobiana pelo método de difusão em ágar. As AgNPs que apresentaram ação antagônica significativa foram utilizadas para determinação do MIC (Concentração Inibitória Mínima), Citotoxicidade e caracterizadas por diferentes técnicas, como: espectroscopia de UV-Vis, DLS (Espalhamento Dinâmico de Luz), DRX (Difratometria de Raios X), FTIR (Espectroscopia de Infravermelho com Transformada de Fourier), ICP-OES (Espectroscopia Óptica de Emissão por Plasma Indutivamente Acoplado) e TEM (Microscopia Eletrônica de Transmissão). Dos actinomicetos avaliados, 100% expressaram as características morfológicas viáveis de Streptomyces. Pelo teste da PCR foram identificadas as seguintes espécies, Streptomyces parvulus (40%), Streptomyces bullii (10%), Streptomyces seoulensis (10%), Streptomyces owasiensis (10%), Streptomyces malasyensis (20%), Streptomyces lavendulae (10%). Pelo método bloco de gelose a atividade antimicrobiana foi confirmada e, 80% das bactérias inibiram S. aureus; 30% M. smegmatis e 40% as Candidas (albicans, atlantica e valderwaltii). Apenas 10% dos actinomicetos inibiram o crescimento de E. coli. Quando testado o extrato bruto frente aos micro-organismos teste, 50% das amostras inibiram S. aureus; 30% M. smegmatis e 20% mostrou-se eficiente contra C. albicans, C. atlantica e C. valderwaltii. Nestes testes o diâmetro do halo variou, em média, de 12 e 29 mm. Já para as AgNPs, 100% inibiram o crescimento de S. aureus e E. coli, com halos variando de 12 a 34 mm. Dados que indicam o amplo espectro destes compostos bioativos. As AgNPs de Streptomyces DPUA 1549, Streptomyces DPUA 1747 e Streptomyces DPUA 1748 apresentaram ação antagônica de maior eficiência. As AgNPs promissoras apresentaram forma esférica e tamanhos variando entre 1-40 nm. Contudo, nanopartículas produzidas por Streptomyces sp. DPUA 1549 foram as mais eficientes, apresentando MIC frente a S. aureus, em baixas concentrações (1,95 μM). Por meio das imagens visualizadas em AFM foi possível observar os danos estruturais da membrana celular de S. aureus. Nos testes de toxicidade, as AgNPs reduziram a proliferação de células NIH-3T3. Além disso, a atividade citotóxica deste nanomaterial é dependente do tempo. Estas evidências científicas demonstram a potencial aplicação de nanopartículas biossintetizadas por Streptomyces sp. DPUA 1549 na elaboração de produtos para utilização nas indústrias médicas, cosméticas e farmacêuticas

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<p>Sedan december 2003 har Kalmar läns slakteris (KLS) nya reningsverk varit i drift. Entreprenör för det nya reningsverket samt driftansvariga under det två första åren är Läckeby Water Group. Verket är av SBR-typ (Sekventiell Biologisk Rening) med biologisk kväverening och kemisk fällning av fosfor med hjälp av järnklorid. Från slakteriet leds avloppsvattnet genom en 2 km lång ledning ner till reningsanläggningen. Verket hade under 2004 problem med höga halter fosfor i utgående vatten, flertalet mekaniska haverier samt två perioder av slamsvällning. Slamsvällningen orsakades av filamentösa (trådformiga) bakterier, första gången av Thiothrix spp. och andra gången av Typ 021N. Syftet med examensarbetet var att finna orsaken till den senare slamsvällningen samt att söka förebyggande åtgärder mot Typ 021N. Examensarbetet utfördes genom litteraturstudier, laboratorieförsök, fullskaleförsök, genomgång av driftsdata samt mikroskopering av aktivt slam vid verket.</p><p>Utifrån litteraturstudier konstaterades att filamentösa bakterier kan gynnas under perioder av låga syrehalter samt av låg näringstillförsel eftersom dessa bakterier har en högre tillväxthastighet vid låga substratkoncentrationer än flockbildande bakterier. Specifikt för Typ 021N är att dessa har möjlighet att utnyttja reducerat svavel som energikälla samt gynnas vid tillgång på korta lättnedbrytbara kolföreningar. Laboratorieförsök visade inte entydigt att låga fosfor eller syrehalter gynnade de filamentösa bakterierna. Inverkan av FeCl3, Ecofloc, PAXXL60, NaOCl och H2O2 studerades under korttids laboratorieförsök och effekten utvärderades i mikroskop. I några fall hämmades filamenten men aldrig utan att även påverka övriga mikroorganismer negativt. PAX-XL60 hämmade filamentförkomsten mest och påverkade andra organismer förhållandevis lite. Tillsats av PAX i filamenthämmande och flockbildande syfte utfördes därefter i fullskala. Effekten av tidigare tillsatser av NaOCl och H2O2 i filamenthämmande syfte studerades och visade sig ha givit varierande resultat. NaOCl visade sig effektivt bekämpa filamentösa bakterier i processen då inblandning skedde under rätt förutsättningar.</p><p>Processdata för våren 2004 jämfördes med data från en period under hösten, vilken följdes av en slamsvällning. Perioderna visade stora skillnader m.a.p. syrehalt, temperatur, dosering av järnklorid och organisk belastning. En on-line mätning i inkommande vatten visade på mycket höga halter av svavelväte. Svavelväte bildas under anaeroba förhållanden t.ex. i stillastående avloppsvatten. Orsaker till slamsvällningen i september-oktober 2004 tros vara höga halter av svavelväte, perioder med låga syrehalter, höga vattentemperaturer samt tillgång på lättnedbrytbart organiskt material. Svavelvätet kan förslagsvis elimineras genom en tidsstyrd dosering av CaNO3 i inkommande ledning. Noggrann övervakning av syre samt tillgång på syre måste garanteras i processen. Det inkommande vattnets mikroflora kan förändras genom installation av en aerob selektor för att gynna de flockformande bakterierna. För att sänka fosforhalterna i utgående vatten samt att inte riskera fosforbrist i processen har en tillfällig efterfällning med extra tillsats av FeCl3 och polymer installerats.</p> / <p>In December 2003 the new plant treating slaughterhouse wastewater from KLS was taken into operation. Läckeby Water Group was entrepreneur and responsible for the maintenance during the following two years. The treatment plant is of SBR-type and has biological nitrate removal and chemical precipitation of phosphate with iron chloride. The wastewater from the slaughterhouse passes a 2 km long pipeline before entering the treatment plant. During 2004, the plant had problems with high levels of phosphorous in the effluent, several mechanical problems and two occasions of sludge bulking caused by filamentous bacteria. The first incident was caused by Thiothrix spp. and the second by Type 021N. The aim with the thesis was to find causes for the latest period of sludge bulking as well as investigate preparatory actions against Type 021N. The thesis included literature studies, laboratory and full-scale tests, evaluation of prior process data and continuous microscopic analysis of the activated sludge at the plant.</p><p>The literature study showed that filamentous bacteria are favoured by low oxygen and low nutrient concentrations due to their possibly higher growth rate during low substrate concentrations. Type 021N, specifically, can use reduced sulphides as energy source and benefits from an excess of low molecular substrates. Laboratory experiments did not verify that the filamentous bacteria were favoured by low oxygen concentration or low phosphate levels. The effect of FeCl3, Ecofloc, PAX-XL60, NaOCl and H2O2 added to a bulking sludge was evaluated by microscopic analysis. No chemical was found to suppress the filamentous bacteria without also affecting the floc-forming bacteria negatively. PAX-XL60 showed the largest negative effects on filamentous bacteria and only a minor impact on other microorganisms. Full-scale tests with PAX were thereafter performed in order to suppress filamentous bacteria as well as flocculate particulate solids. The effect of earlier additions of NaOCl and H2O2 into the process gave varied results. NaOCl was efficient against filamentous bacteria when addition was made during correct circumstances.</p><p>Process data from two separate periods during 2004 was compared. One period was followed by good effluent values and another period by a sludge bulking period. Large differences between the two periods were seen in oxygen conditions, temperature, FeCl3 dosage and organic load. Measurements on influent wastewater showed high levels of hydrogen sulphide, which can be produced during anaerobe conditions i.e. in stagnant sewage pipes. Likely causes for the sludge bulking in September-October 2004 were high levels of hydrogen sulphide in the influent, periods of insufficient oxygen concentrations, high water temperatures and access to easy degradable substrate. The hydrogen sulphide can be eliminated through time-controlled dosage of CaNO3 in influent pipeline. Sufficient oxygen levels must be guaranteed in the process. The microbiological fauna in influent can be changed by installation of an aerobe selector to benefit floc-forming bacteria. To lower the phosphorous levels in effluent water and not risk phosphorous deficiency in the process a post-precipitation have been installed. The post-precipitation include extra dosage of FeCl3 and polymer and a drum screen to minimize suspended solids.</p>

Sedan december 2003 har Kalmar läns slakteris (KLS) nya reningsverk varit i drift. Entreprenör för det nya reningsverket samt driftansvariga under det två första åren är Läckeby Water Group. Verket är av SBR-typ (Sekventiell Biologisk Rening) med biologisk kväverening och kemisk fällning av fosfor med hjälp av järnklorid. Från slakteriet leds avloppsvattnet genom en 2 km lång ledning ner till reningsanläggningen. Verket hade under 2004 problem med höga halter fosfor i utgående vatten, flertalet mekaniska haverier samt två perioder av slamsvällning. Slamsvällningen orsakades av filamentösa (trådformiga) bakterier, första gången av Thiothrix spp. och andra gången av Typ 021N. Syftet med examensarbetet var att finna orsaken till den senare slamsvällningen samt att söka förebyggande åtgärder mot Typ 021N. Examensarbetet utfördes genom litteraturstudier, laboratorieförsök, fullskaleförsök, genomgång av driftsdata samt mikroskopering av aktivt slam vid verket. Utifrån litteraturstudier konstaterades att filamentösa bakterier kan gynnas under perioder av låga syrehalter samt av låg näringstillförsel eftersom dessa bakterier har en högre tillväxthastighet vid låga substratkoncentrationer än flockbildande bakterier. Specifikt för Typ 021N är att dessa har möjlighet att utnyttja reducerat svavel som energikälla samt gynnas vid tillgång på korta lättnedbrytbara kolföreningar. Laboratorieförsök visade inte entydigt att låga fosfor eller syrehalter gynnade de filamentösa bakterierna. Inverkan av FeCl3, Ecofloc, PAXXL60, NaOCl och H2O2 studerades under korttids laboratorieförsök och effekten utvärderades i mikroskop. I några fall hämmades filamenten men aldrig utan att även påverka övriga mikroorganismer negativt. PAX-XL60 hämmade filamentförkomsten mest och påverkade andra organismer förhållandevis lite. Tillsats av PAX i filamenthämmande och flockbildande syfte utfördes därefter i fullskala. Effekten av tidigare tillsatser av NaOCl och H2O2 i filamenthämmande syfte studerades och visade sig ha givit varierande resultat. NaOCl visade sig effektivt bekämpa filamentösa bakterier i processen då inblandning skedde under rätt förutsättningar. Processdata för våren 2004 jämfördes med data från en period under hösten, vilken följdes av en slamsvällning. Perioderna visade stora skillnader m.a.p. syrehalt, temperatur, dosering av järnklorid och organisk belastning. En on-line mätning i inkommande vatten visade på mycket höga halter av svavelväte. Svavelväte bildas under anaeroba förhållanden t.ex. i stillastående avloppsvatten. Orsaker till slamsvällningen i september-oktober 2004 tros vara höga halter av svavelväte, perioder med låga syrehalter, höga vattentemperaturer samt tillgång på lättnedbrytbart organiskt material. Svavelvätet kan förslagsvis elimineras genom en tidsstyrd dosering av CaNO3 i inkommande ledning. Noggrann övervakning av syre samt tillgång på syre måste garanteras i processen. Det inkommande vattnets mikroflora kan förändras genom installation av en aerob selektor för att gynna de flockformande bakterierna. För att sänka fosforhalterna i utgående vatten samt att inte riskera fosforbrist i processen har en tillfällig efterfällning med extra tillsats av FeCl3 och polymer installerats. / In December 2003 the new plant treating slaughterhouse wastewater from KLS was taken into operation. Läckeby Water Group was entrepreneur and responsible for the maintenance during the following two years. The treatment plant is of SBR-type and has biological nitrate removal and chemical precipitation of phosphate with iron chloride. The wastewater from the slaughterhouse passes a 2 km long pipeline before entering the treatment plant. During 2004, the plant had problems with high levels of phosphorous in the effluent, several mechanical problems and two occasions of sludge bulking caused by filamentous bacteria. The first incident was caused by Thiothrix spp. and the second by Type 021N. The aim with the thesis was to find causes for the latest period of sludge bulking as well as investigate preparatory actions against Type 021N. The thesis included literature studies, laboratory and full-scale tests, evaluation of prior process data and continuous microscopic analysis of the activated sludge at the plant. The literature study showed that filamentous bacteria are favoured by low oxygen and low nutrient concentrations due to their possibly higher growth rate during low substrate concentrations. Type 021N, specifically, can use reduced sulphides as energy source and benefits from an excess of low molecular substrates. Laboratory experiments did not verify that the filamentous bacteria were favoured by low oxygen concentration or low phosphate levels. The effect of FeCl3, Ecofloc, PAX-XL60, NaOCl and H2O2 added to a bulking sludge was evaluated by microscopic analysis. No chemical was found to suppress the filamentous bacteria without also affecting the floc-forming bacteria negatively. PAX-XL60 showed the largest negative effects on filamentous bacteria and only a minor impact on other microorganisms. Full-scale tests with PAX were thereafter performed in order to suppress filamentous bacteria as well as flocculate particulate solids. The effect of earlier additions of NaOCl and H2O2 into the process gave varied results. NaOCl was efficient against filamentous bacteria when addition was made during correct circumstances. Process data from two separate periods during 2004 was compared. One period was followed by good effluent values and another period by a sludge bulking period. Large differences between the two periods were seen in oxygen conditions, temperature, FeCl3 dosage and organic load. Measurements on influent wastewater showed high levels of hydrogen sulphide, which can be produced during anaerobe conditions i.e. in stagnant sewage pipes. Likely causes for the sludge bulking in September-October 2004 were high levels of hydrogen sulphide in the influent, periods of insufficient oxygen concentrations, high water temperatures and access to easy degradable substrate. The hydrogen sulphide can be eliminated through time-controlled dosage of CaNO3 in influent pipeline. Sufficient oxygen levels must be guaranteed in the process. The microbiological fauna in influent can be changed by installation of an aerobe selector to benefit floc-forming bacteria. To lower the phosphorous levels in effluent water and not risk phosphorous deficiency in the process a post-precipitation have been installed. The post-precipitation include extra dosage of FeCl3 and polymer and a drum screen to minimize suspended solids.

<p>Slamsvällning orsakar stora problem i avloppsreningsverk med biologisk rening i aktivt slamprocesser. Slamsvällning orsakas av filamentösa (trådformiga) bakterier, som inverkar negativt på slammets sedimenteringsegenskaper.</p><p>Himmerfjärdens vattenreningsverk har drabbats av detta problem som leder till ett stabilt lager av slam på ytan av sedimenteringsbassängen som inte sedimenterar.</p><p>För att lösa detta problem behandlades returslammet från sedimenteringsbassängen med ozon för att minska mängden filamentösa bakterier i returslamflödet. Ozon är en starkt oxiderande gas, som är väl användbar för icke-specifik bekämpning av slamsvällning. När ozon kommer i kontakt med den filamentösa bakteriens cellvägg penetreras det in i cellen, varvid cellen lyserar.</p><p>Ozonbehandlingen resulterade i en förminskning av antalet filamentösa bakterier. Ozonbehandling av returslam förbättrade sedimenteringsegenskaperna hos svällande slam utan att påverka andra viktiga mikrobiologiska processer t.ex. nitrifikation.</p> / <p>Bulking sludge causes major problems in wastewater treatment plants that deal with biological nutrient removal in activated sludge processes. Bulking sludge is caused by filamentous bacteria, which have a negative impact on the sludge settling properties.</p><p>Himmerfjärden wastewater treatment plant suffers from this type of problem with bulking sludge which creates a stable layer at the surface that does not settle in the clarifier.</p><p>In order to solve this problem, on site generated ozone was used to decrease the amount of filamentous bacteria in the return activated sludge flow. Ozone is a strong oxidant is suitable for non-specific bulking control. It stresses the filamentous bacteria causing inactivation through cell wall disintegration.</p><p>The ozone treatment resulted in decreased abundance of filamentous bacteria. Ozone treatment of the recycled activated sludge improves the settling properties of bulking sludge, without interfering with other important microbiological processes e.g. nitrification.</p>

Slamsvällning orsakar stora problem i avloppsreningsverk med biologisk rening i aktivt slamprocesser. Slamsvällning orsakas av filamentösa (trådformiga) bakterier, som inverkar negativt på slammets sedimenteringsegenskaper. Himmerfjärdens vattenreningsverk har drabbats av detta problem som leder till ett stabilt lager av slam på ytan av sedimenteringsbassängen som inte sedimenterar. För att lösa detta problem behandlades returslammet från sedimenteringsbassängen med ozon för att minska mängden filamentösa bakterier i returslamflödet. Ozon är en starkt oxiderande gas, som är väl användbar för icke-specifik bekämpning av slamsvällning. När ozon kommer i kontakt med den filamentösa bakteriens cellvägg penetreras det in i cellen, varvid cellen lyserar. Ozonbehandlingen resulterade i en förminskning av antalet filamentösa bakterier. Ozonbehandling av returslam förbättrade sedimenteringsegenskaperna hos svällande slam utan att påverka andra viktiga mikrobiologiska processer t.ex. nitrifikation. / Bulking sludge causes major problems in wastewater treatment plants that deal with biological nutrient removal in activated sludge processes. Bulking sludge is caused by filamentous bacteria, which have a negative impact on the sludge settling properties. Himmerfjärden wastewater treatment plant suffers from this type of problem with bulking sludge which creates a stable layer at the surface that does not settle in the clarifier. In order to solve this problem, on site generated ozone was used to decrease the amount of filamentous bacteria in the return activated sludge flow. Ozone is a strong oxidant is suitable for non-specific bulking control. It stresses the filamentous bacteria causing inactivation through cell wall disintegration. The ozone treatment resulted in decreased abundance of filamentous bacteria. Ozone treatment of the recycled activated sludge improves the settling properties of bulking sludge, without interfering with other important microbiological processes e.g. nitrification.

Disease has been identified as a major problem in the aquaculture industry for the welfare of the fish stocked as well as for its economic impact. The number of diseases affecting cultured fish has increased significantly during recent years with the emergence of several conditions that have added to the overall impact of disease on the industry. Frequently, a lack of scientific knowledge about these diseases is compounded by an absence of effective treatment and control strategies. This has been the case with rainbow trout gastroenteritis (RTGE), an emerging disease of rainbow trout (Oncorhynchus mykiss Walbaum). This study investigated several aspects related to its aetiology and control. A retrospective survey of UK rainbow trout farmers was undertaken to ascertain the extent and severity of RTGE in the UK as well as to identify RTGE risk factors at the site level. Participants in this study accounted for over 85% of UK rainbow trout production in 2004. It was found that the total number of RTGE-affected sites had risen from 2 in the year 2000 to 7 in 2005. The disease was only reported from sites producing more than 200 tonnes of trout/year for the table market. Analysis of risk factors associated with RTGE at the site level showed that this syndrome was associated with large tonnage and rapid production of rainbow trout for the table market. The data collected during this study enabled the identification of those sites that were most likely to present with RTGE the following year and this information was used to study the epidemiology of RTGE at the unit level. A prospective longitudinal study was undertaken in 12 RTGE-affected UK sites. It described in detail the impact, presentation, current control strategies and spread pattern of RTGE within affected UK sites. The risk factors associated with RTGE presence and severity were also investigated. Data were collected for each productive unit (i.e. cage, pond, raceway or tank) on the mortalities, fish origin, site management and environmental factors. RTGE was identified using a case definition based on gross pathological lesions. Analysis of these data revealed that RTGE behaved in an infectious manner. This conclusion was supported by the presence of a pattern typical of a propagating epidemic within affected units. Also, the risk of an unaffected unit becoming RTGE positive was increased if it had received fish from or was contiguous to a RTGE-affected unit. The presentation also suggested an incubation period of 20-25 days. Risk factor analysis identified management and environmental risk factors for RTGE, including high feed input and stressful events, which could be used to generate a list of control strategies. A study of the histopathological and ultrastructural presentation of RTGE was conducted. The location of segmented filamentous bacteria (SFB) and pathological changes found in affected fish were examined. Pyloric caeca were the digestive organ where SFB were found more frequently and in higher numbers, suggesting that this was the best location to detect SFB in RTGE-affected trout. Scanning and transmission electron microscopy revealed a previously undescribed interaction of SFB with the mucosa of distal intestine and pyloric caeca and this included the presence of attachment sites and SFB engulfment by enterocytes, as previously described in other host species. The SFB were not always adjacent to the pathological changes observed in the digestive tract of RTGE-affected trout. Such changes included cytoskeletal damage and osmotic imbalance of enterocytes, with frequent detachment. These observations suggested that if SFB are indeed the cause of RTGE their pathogenesis must involve the production of extracellular products. Analysis of the gross presentation and blood biochemistry in RTGE-affected fish was used to examine the patho-physiologic mechanisms of RTGE. To enable identification of positive RTGE cases for this study, a case definition was created from the information available on RTGE gross presentation in the literature. This case definition was assessed in a sample including 152 fish cases and 152 fish controls from 11 RTGE-affected UK sites, matched by unit of origin. The analysis of these fish using bacteriology, packed cell volume (PCV) and histopathology revealed that RTGE occurred simultaneously with other parasitic and bacterial diseases in a percentage of fish identified with this case definition. With the information gained after analysing the gross presentation, RTGE-affected fish without concurrent disease were selected for the study of the pathogenesis, which included blood biochemical analyses. These analyses revealed a severe osmotic imbalance, and a reduced albumin/globulin ratio suggesting selective loss of albumin, typical for a protein losing enteropathy. The role of the SFB “Candidatus arthromitus” in the aetiology of RTGE was assessed using a newly developed “C. arthromitus”-specific polymerase chain reaction assay (PCR) in conjunction with histological detection. This technique was applied to eight different groups of trout, including an RTGE-affected group and seven negative control groups. This analysis was conducted on DNA extracted from paraffin wax-embedded tissues as well as fresh intestinal contents. The results revealed the presence of “C. arthromitus” DNA in apparently healthy fish from sites where RTGE had never been reported. Additionally, SFB were observed histologically in two trout from an RTGE-free hatchery. These findings do not permit the exclusion of “C. arthromitus” as the aetiological agent for RTGE, although they suggest that the presence of these organisms in the digestive system of healthy trout is not sufficient to cause clinical disease, and therefore other factors are necessary. In conclusion, this study has used a multidisciplinary approach to the study of RTGE which has generated scientific information related to the epidemiology, pathogenesis and aetiology of this syndrome. The results of this project have suggested priority areas where further work is required, including experimental transmission of RTGE, field assessment of the control strategies proposed and further investigation into the aetiology of RTGE.

Abstract Flocculation plays a critical role in the activated sludge process, where flocs are removed by settling and where unsatisfactory flocculation is resulting in poor effluent quality. Control and operation of the process is also challenging as it is sensitive to external and internal disturbances. Furthermore, stricter environmental demands are also being placed on wastewater treatment and discharge quality thus solutions are needed to improve the current systems. A novel optical monitoring method employing a tube flow and a CCD camera was developed to characterize the flocs and filaments of the sludge, and the method was tested on samples from full-scale activated sludge plants. An online device operating on the same principle was also developed and this was tested over a period of eight months at municipal wastewater treatment plant. Optical monitoring was employed in the laboratory to study the breakage of activated sludge flocs. Based on the image analysis data, in the industrial plant the major breakage process was large-scale fragmentation. In the two municipal plants, it was surface erosion. The flocs had more filaments and were more irregular in shape in the industrial plant, which could be the reason for the large-scale fragmentation. The effect of floc morphology on the effluent clarity of the activated sludge process was studied in the industrial and municipal activated sludge plants by optical monitoring over periods of three months and eight months, respectively. The changes in floc morphology took place slowly in both plants. Four major factors that correlated with the purification results were the size and shape of the flocs and the quantities of small particles and filaments. The image analysis results suggested that the settling problem that occurred during the test periods in the industrial plant was caused by dispersed growth, whereas that in the municipal plant was caused by filamentous bulking. In conclusion, it is possible to use the developed method online in order to analyse the state of flocculation. Thus the method could be useful when developing online monitoring applications for quantifying floc characteristics and for diagnosing the causes of settling problems in the wastewater treatment plants. / Tiivistelmä Aktiivilieteprosessissa flokkulaatiolla on merkittävä rooli, sillä muodostuneet flokit poistetaan prosessista laskeutuksen avulla. Siten huono flokkulaatio johtaa puhdistetun jäteveden kiintoainemäärän lisääntymiseen. Prosessin säätö ja operointi on kuitenkin hankalaa, sillä aktiivilieteprosessi on herkkä ulkoisille ja sisäisille häiriöille. Jätevedenpuhdistukseen liittyvät ympäristövaatimukset ja päästöehdot vesistöihin ovat myös tiukentuneet, joten uusia menetelmiä tarvitaan parantamaan nykyisiä prosesseja. Tässä työssä kehitettiin uusi, optinen kuvantamismenetelmä karakterisoimaan flokkeja ja rihmoja. Menetelmä hyödyntää putkivirtausta ja CCD-kameraa ja sitä testattiin aktiivilietelaitosten näytteillä. Lisäksi kehitettiin samaa periaatetta noudattava online-laitteisto, jota testattiin kahdeksan kuukauden ajan. Optista kuvantamista testattiin laboratoriossa flokkien hajoamistutkimuksessa. Kuva-analyysitulosten perusteella kahden kunnallisen aktiivilietelaitoksen flokit hajosivat pintaeroosioon perustuvan mallin mukaan ja teollisen aktiivilietelaitoksen flokit hajosivat fragmentaatiomallin mukaan. Teollisen aktiivilietelaitoksen flokeissa oli enemmän rihmoja ja ne olivat epäsäännöllisemmän muotoisia, mikä voi olla syynä flokkien fragmentaatioon. Flokkien morfologian vaikutus jäteveden puhdistustuloksiin tutkittiin teollisessa (kolmen kuukauden ajan) ja kunnallisessa (kahdeksan kuukauden ajan) aktiivilietelaitoksessa optisella kuvantamismenetelmällä. Molemmissa laitoksessa muutokset flokkien morfologiassa tapahtuivat hitaasti. Neljä tärkeintä tekijää, jotka korreloivat puhdistustulosten kanssa, olivat flokkien koko ja muoto sekä pienten partikkelien ja rihmojen määrä. Kuva-analyysitulosten perusteella laskeutumisongelma teollisessa jätevesilaitoksessa johtui flokinmuodostajabakteerien liian pienestä määrästä ja kunnallisessa jätevesilaitoksessa rihmamaisten bakteerien liikakasvusta. Yhteenvetona voidaan todeta, että kehitettyä menetelmää on mahdollista käyttää online-mittarina sekä sen avulla voidaan arvioida flokkulaation tilannetta. Siten menetelmää on mahdollista hyödyntää flokkien ominaisuuksien karakterisoinnissa ja arvioidessa jätevedenkäsittelylaitoksen laskeutumisongelmien aiheuttajaa.

Les métaux lourds et les radionucléides sont présents dans différents écosystèmes du monde à cause de contaminations naturelles ou des activités anthropiques. L’utilisation de micro-organismes pour restaurer ces écosystèmes pollués, processus connu sous le nom de bioremédiation, suscite beaucoup d’intérêt, spécialement aux pH proches de la neutralité. Les minéraux de fer qui encroûtent les bactéries neutrophiles du fer, notamment les Oxydes de Fer Biogéniques (BIOS en anglais), ont une structure très faiblement cristalline, qui en plus de leur grande surface et réactivité font d’eux d’excellents supports pour le piégeage de polluants inorganiques. Dans cette thèse nous avons étudié les différents mécanismes de piégeage de l’uranium uranium par la bactérie neutrophile Sphaerotilus natans, choisie comme modèle bactérien de micro-organismes du fer capables de filamenter en formant des gaines. S. natans peut croître sous forme de cellules individuelles ou formant des filaments. Ces derniers ont été utilisés pour étudier la biosorption d’U(VI) et sa sorption sur les BIOS. De plus, la sorption d’U(VI) sur les analogues abiotiques de ces minéraux de fer a été testée. Afin d’utiliser les filaments de S. natans pour piéger l’U(VI), il était nécessaire d’identifier les facteurs induisant la filamentation de S. natans. L’influence de l’oxygène a été établie en utilisant des techniques de biologie moléculaire et nos résultats ont démontré que tandis qu’en condition d’oxygène saturé elle croît sous forme de cellules individuelles, une diminution modérée d’oxygène à ~ 3 mg O2.L-1 la fait croître sous la forme désirée, des filaments de S. natans.Les BIOS attachés aux filaments de S. natans ainsi que ses analogues abiotiques ont été analysés pas XAS au seuil K du Fe. Les deux matériaux identifiés sont des phosphates de fer(III) amorphes avec une faible proportion de fer(II), qui présentent une réactivité élevée pour le piégeage de polluants inorganiques. L’EXAFS au seuil LIII de l’U a montré la même structure pour les couches O, tandis que celles P, Fe et C étaient différentes en fonction des sorbants. Une étude intégrée qui combine des techniques expérimentales avec des calculs de spéciation a permis de décrire les isothermes d’adsorption de l’U(VI) en utilisant un modèle de complexation de surface. Ces résultats suggèrent que les groupes phosphoryles et carboxyles sont les groupes fonctionnels principaux pour la biosorption d’U(VI) par des filaments de S. natans. Les résultats de cette thèse vont aider à comprendre les processus contrôlant l’immobilisation de l’U(VI), soit par la biosorption sur S. natans, la sorption sur les BIOS ou la sorption sur les phosphates de fer, et en conséquence le devenir de l’U en conditions neutres / Heavy metals and radionuclides are present in some ecosystems worldwide due to natural contaminations or anthropogenic activities. The use of microorganisms to restore those polluted ecosystems, a process known as bioremediation, is of increasing interest, especially under near-neutral pH conditions. Iron minerals encrusting neutrophilic iron-related bacteria, especially Bacteriogenic Iron Oxides (BIOS), have a poorly crystalline structure, which in addition to their large surface area and reactivity make them excellent scavengers for inorganic pollutants. In this PhD work we studied the different mechanisms of uranium scavenging by the neutrophilic bacterium Sphaerotilus natans, chosen as a model bacterium for iron-related sheath-forming filamentous microorganisms. S. natans can grow as single cells and filaments. The latter were used to investigate U(VI) biosorption and U(VI) sorption onto BIOS. In addition, uranium sorption onto the abiotic analogues of such iron minerals was assessed. In order to use S. natans filaments for U(VI) scavenging, it was necessary to identify factors inducing S. natans filamentation. The influence of oxygen was ascertained by using molecular biology techniques and our results revealed that while saturated oxygen conditions resulted in single cell growth, a moderate oxygen depletion to ~ 3 mg O2.L-1 led to the desired filamentous growth of S. natans. BIOS attached to S. natans filaments as well as the abiotic analogues were analysed by XAS at Fe K-edge. Both materials were identified as amorphous iron(III) phosphates with a small component of Fe(II), with a high reactivity towards scavenging of inorganic pollutants. In addition, EXAFS at the U LIII-edge revealed a common structure for the O shells, while those for P, Fe and C were different for each sorbent. An integrated approach combining experimental techniques and speciation calculations made it possible to describe U(VI) adsorption isotherms by using a surface complexation model. These results suggested the role of phosphoryl and carboxyl groups as the main functional groups involved in the U(VI) biosorption by S. natans. The results of this PhD work will help to better understand the processes governing U(VI) immobilization, either by S. natans biosorption, sorption onto BIOS or sorption onto iron phosphates, an thus the fate of uranium in near-neutral pH environments