Tout support immergé dans l'eau est rapidement colonisé par de nombreux organismes micro- et macroscopiques. Ce phénomène séquentiel et complexe appelé biofouling est à l'origine de nombreux préjudices économiques et écologiques, notamment dans le milieu marin. L'interdiction récente de certaines substances toxiques, utilisées comme biocides dans les revêtements antifouling des coques de bateaux notamment, a relancé l'intérêt de rechercher de nouvelles molécules antifouling respectueuses de l'environnement. L'objectif de cette thèse a été de développer et d'amorcer l'étude de la représentativité d'un bio-essai permettant d'évaluer le potentiel antifouling de molécules et de revêtements sur des " biofilms " mono et plurispécifiques in vitro en microplaques grâce à l'utilisation de fluorochromes. Le choix a été fait de se focaliser sur le biofilm primaire car il est envisagé que l'élimination ou la limitation de ce dernier réduisent le biofouling. Cinq souches de bactéries marines pionnières, isolées de la Rade de Toulon et en Bretagne, ont été utilisées afin de comparer l'efficacité anti-adhésion de molécules commerciales et naturelles. Deux dérivés de synthèse de substances naturelles marines (TFA E et Z) ont présenté une activité significative associée à une absence de toxicité sur bactéries, suggérant ainsi un mode d'action anti-adhésion spécifique. En outre, les différences de sensibilité entre souches ont confirmée l'importance de réaliser le bio-essai avec un panel diversifié de bactéries.Afin de voir si les données obtenues en laboratoire reflétaient ce qui se produit dans le milieu naturel, une comparaison entre les résultats du bio-essai en microplaque appliqué à six revêtements et les biofilms qui ont colonisés ces mêmes peintures immergées un mois dans la Rade de Toulon (analysés par cytométrie de flux, microscopie et PCR-DGGE), a été effectuée. Les analyses quantitatives ont suggéré une cohérence entre les deux approches même si l'absence de revêtement d'efficacité intermédiaire et le nombre de systèmes testés limite la portée de nos conclusions

<p>When paper is manufactured, deposits often form in the machines. These deposits are slimelike and can interfere with the papermaking process. The slimelike deposits are aggregates of micro-organisms, also known as biofilm. One single type of micro-organism can form a biofilm, but most biofilms consists of a mixture of several different kinds of micro-organisms and can form on about any conceivable surface. To control the aggregates of micro-organisms a slimecide is added, a so-called biocide. To examine what kind of bacteria that is included in the biofilm and also which bacteria that is killed or not killed by the biocide, Terminal Restriction Fragment Length Polymorphism analysis (T-RFLP) can be used.</p><p> </p><p>In this report we examine biocides impact on biofilm produced in the laboratory.The biocides were first tested for possible interference with the PCR-step of the T-RFLP analysis. None of the tested ten biocides inhibited the PCR process the biofilm was formed on metal plates when these were lowered in a beaker with white water. Three different beakers were set up, one with addition of a biocide with active component 4,5-DICHLORO-1,2-DITHIOLONE from the start, one with the addition of the same biocide after three days and one with no addition at all of biocide. Samples were taken from the beakers and analyzed with T-RFLP.</p><p> </p><p>In this report, we show that biocides affect planktonic and biofilm micro-organisms differently. There are however some micro-organisms in the biofilm that does not get affected by the biocide.</p><p> </p><p>The experimental in this report is a good way of investigate the influence that biocides have on planktonic and biofilm micro-organisms, but to get even greater result the experiment should be done over a longer period of time and repeatedly.</p>

<p>Polymeric and particulate organic carbon (POM) are fundamental compounds in the global cycling of carbon, and constitute significant amounts of BOD in municipal wastewater.</p><p>The main objective of this work is to study molecular size effects on degradation dynamics in biofilm systems. Specifically, the effect of substrate molecular weight on degradation kinetics and transport dynamics, location of depolymerisation enzyme activity and depolymerisation intermediate formation dynamics are assessed. A mathematical model for biofilm degradation dynamics is presented, and used for data interpretation and simulations.</p><p>Dextran, an -1,6 Glucan, was used as model substrate during batch degradation in a Rototorque biofilm reactor, in addition to batch tests on biofilm sub samples retrieved from the Rototorque, and during pure endo- and exo-Dextranase studies. Oxygen utilisation rate (OUR) estimates and bulk phase TOC mass balances were used to evaluate the effect of variable initial molecular weight on the observed half order removal coefficient (Harremoës, 1978; Rittmann and McCarty, 1980). Size exclusion-HPLC analysis for determination of bulk phase depolymerisation intermediates, and specific enzyme assays were used to evaluate transport dynamics of polymers and location of enzyme activity in the enhanced mixed population biofilm system.</p><p>Dextran removal rate decrease with increasing Dextran molecular weight. The observed areal half order removal rate coefficient, k1/2,A, demonstrate an approximate 10-fold decrease in the 1-500 kDa range, showing negative logarithmic correlation to the initial MW of Dextran. A less distinct correlation is observed above this transition limit (1-10 MDa). Evaluation of the Thiele moduli, from one step depolymerisation modelling, suggests that the logarithmic reduction in observed removal rate is caused by combined reaction rate and transport limitations. Transport limitations dominates as the polymeric substrate size increase and hinders biofilm matrix diffusion, and the removal rate becomes a surface limited process. Removal of Dextran is biomass dependent in what appears to be a non-linear dependency on biofilm thickness. Expressed as biomass areal density (g/m2), no depolymerisation is observed for thin biofilms (0.7 g/m2), slow for medium (3.7 g/m2) and high for thicker biofilms (5.2 g/m2).</p><p>Depolymerisation intermediates accumulated in the bulk phase over the entire Dextran size range during pure Dexranase studies, with even size distributions. Final products were oligo-isomaltoses (DP 2-6). Dextran was not depolymerised by -Glucosidase nor Oligo-1,6 Glucosidase. During biofilm reactor and slide sub-sample tests, low MW Dextran intermediates (1-10 kDa) accumulated in the bulk during depolymerisation of 160 kDa Dextran at 250 and 200 mg/l initial concentrations, but were not detected during experiments with 100 mg/l initial concentrations. Intermediate range Dextran (10-100 kDa) did not accumulate in either case. At the same conditions, some assimilable range Dextran (0.2-0.9 kDa) accumulated in the bulk liquid during initial 250 and 200 mg/l batches, but was not detected during 100 mg/l initial Dextran concentrations. The extent of bulk phase accumulation seems to depend on the biofilm growth rate, where more bulk phase accumulation is observed during experiments with starved compared to more actively growing biofilms. More intermediates accumulate during low MW initial standards, compared to higher. These observations indicate that the extent of bulk phase intermediate accumulation is balanced by the rate of depolymerisation, and the substrate uptake rate (growth). Accumulation of intermediate hydrolysis products in biofilm systems is therefore dependent on the slowly biodegradable organic (SBCOD) loading rate. </p><p>Dextranase was detected in the cellular fraction of the biofilms. The enzyme activity was not detected in any other biofilm sub compartments, implying that the exogenous enzyme remains attached to the cells while working on polymers. These findings support the conceptual model of Confer and Logan (1998), implying that bulk phase intermediate accumulation observed in this study and by others, is not a result of enzymatic activity in the bulk phase, but transport of intermediates from the biofilm matrix.</p>

Polymeric and particulate organic carbon (POM) are fundamental compounds in the global cycling of carbon, and constitute significant amounts of BOD in municipal wastewater. The main objective of this work is to study molecular size effects on degradation dynamics in biofilm systems. Specifically, the effect of substrate molecular weight on degradation kinetics and transport dynamics, location of depolymerisation enzyme activity and depolymerisation intermediate formation dynamics are assessed. A mathematical model for biofilm degradation dynamics is presented, and used for data interpretation and simulations. Dextran, an -1,6 Glucan, was used as model substrate during batch degradation in a Rototorque biofilm reactor, in addition to batch tests on biofilm sub samples retrieved from the Rototorque, and during pure endo- and exo-Dextranase studies. Oxygen utilisation rate (OUR) estimates and bulk phase TOC mass balances were used to evaluate the effect of variable initial molecular weight on the observed half order removal coefficient (Harremoës, 1978; Rittmann and McCarty, 1980). Size exclusion-HPLC analysis for determination of bulk phase depolymerisation intermediates, and specific enzyme assays were used to evaluate transport dynamics of polymers and location of enzyme activity in the enhanced mixed population biofilm system. Dextran removal rate decrease with increasing Dextran molecular weight. The observed areal half order removal rate coefficient, k1/2,A, demonstrate an approximate 10-fold decrease in the 1-500 kDa range, showing negative logarithmic correlation to the initial MW of Dextran. A less distinct correlation is observed above this transition limit (1-10 MDa). Evaluation of the Thiele moduli, from one step depolymerisation modelling, suggests that the logarithmic reduction in observed removal rate is caused by combined reaction rate and transport limitations. Transport limitations dominates as the polymeric substrate size increase and hinders biofilm matrix diffusion, and the removal rate becomes a surface limited process. Removal of Dextran is biomass dependent in what appears to be a non-linear dependency on biofilm thickness. Expressed as biomass areal density (g/m2), no depolymerisation is observed for thin biofilms (0.7 g/m2), slow for medium (3.7 g/m2) and high for thicker biofilms (5.2 g/m2). Depolymerisation intermediates accumulated in the bulk phase over the entire Dextran size range during pure Dexranase studies, with even size distributions. Final products were oligo-isomaltoses (DP 2-6). Dextran was not depolymerised by -Glucosidase nor Oligo-1,6 Glucosidase. During biofilm reactor and slide sub-sample tests, low MW Dextran intermediates (1-10 kDa) accumulated in the bulk during depolymerisation of 160 kDa Dextran at 250 and 200 mg/l initial concentrations, but were not detected during experiments with 100 mg/l initial concentrations. Intermediate range Dextran (10-100 kDa) did not accumulate in either case. At the same conditions, some assimilable range Dextran (0.2-0.9 kDa) accumulated in the bulk liquid during initial 250 and 200 mg/l batches, but was not detected during 100 mg/l initial Dextran concentrations. The extent of bulk phase accumulation seems to depend on the biofilm growth rate, where more bulk phase accumulation is observed during experiments with starved compared to more actively growing biofilms. More intermediates accumulate during low MW initial standards, compared to higher. These observations indicate that the extent of bulk phase intermediate accumulation is balanced by the rate of depolymerisation, and the substrate uptake rate (growth). Accumulation of intermediate hydrolysis products in biofilm systems is therefore dependent on the slowly biodegradable organic (SBCOD) loading rate. Dextranase was detected in the cellular fraction of the biofilms. The enzyme activity was not detected in any other biofilm sub compartments, implying that the exogenous enzyme remains attached to the cells while working on polymers. These findings support the conceptual model of Confer and Logan (1998), implying that bulk phase intermediate accumulation observed in this study and by others, is not a result of enzymatic activity in the bulk phase, but transport of intermediates from the biofilm matrix.

Biofilm systems such as the moving bed biofilm reactor (MBBR) are finding increased application in wastewater treatment. One important process that governs MBBRs and yet is poorly understood is the rate of biofilm detachment. The detachment of cells from biofilm surfaces controls both the accumulation of biofilm and the quantity of biomass that is suspended in the bulk liquid phase. This changing balance of attached and suspended cells, in this thesis named the biosolids dynamics, can impact the efficacy of MBBRs. The goal of this research was to investigate how the biosolids dynamics are influenced by process changes relevant to applied wastewater treatment systems and suggest new routes to reactor design and optimization. To achieve this goal, the work addresses three separate but interconnected lines of inquiry. First, multivariate analysis (Principal Component Analysis, Partial Least Squares) was used to examine 2 years of historical data from an MBBR operating at a Canadian pulp mill in order to identify key process variables, perform process diagnostics, and act as a predictive tool. Secondly, the effect of calcium concentration on biofilm structure, microbiology and reactor performance was investigated in four laboratory-scale MBBRs operated at a range of calcium concentrations (1 to 300 mg/L Ca2+). It was found that above a threshold calcium concentration between 1-50 mg/L, MBBR biofilms were observed to be thicker with greater density, contain larger anoxic regions adjacent to the carrier substratum, have more proteinaceous EPS, and have altered microbial community structure. The results suggest an important role for calcium that should be considered in the design and operation of MBBRs. In the final line of inquiry, a diffusion-reaction biofilm model was adapted to represent the key processes of the MBBR. The model was found to simulate average trends observed in the lab-scale experiments allowing for quantification of the detachment rate. Transient periods of reactor starvation were also simulated by introducing a novel metabolic state function to account for down-regulation of metabolism as a result of starvation. This approach was found to accurately simulate starvation response when coupled with detachment expressions that were growth-dependant.

The application of membrane bioreactors (MBRs) to wastewater treatment is increasing due to their ability to operate at high biomass concentrations and to deliver effluents of high quality. The major challenges associated with the application of MBRs is fouling which can shorten the useful life of the membrane, increase in the amount of energy consumed, and the cost for membrane cleaning. The main reasons for fouling are the deposition of solids as a cake layer, pore plugging by colloidal particles, adsorption of soluble compounds and biofouling. Fouling is a particular problem for activated sludge membrane bioreactors (AS-MBRs) since this process deals with liquors having a high concentration of total solids as well as dissolved compounds such as extracellular polymeric substances (EPS). The combination of a moving bed biofilm reactor and a membrane reactor (MBBR-MR) has significant potential. It may be considered as a compact wastewater treatment process which can compensate for the drawbacks of AS-MBRs. Readily biodegradable COD is removed in the MBBR while particulate matter is separated by the membrane. To further reduce the membrane fouling the effects of adding an intermediate coagulation stage was investigated critically on membrane fouling. The present study includes an overall assessment of the performance of a combined MBBR-MR system, based on the chemical oxygen demand (COD) removal efficiency and membrane fouling mechanism. The required test runs were conducted using pilot-scale MBBR and ultra filtration membrane. The pilot MBBR had a working volume of 1.8 m3 with a 60% carrier fill fraction. The MBBR was operated with loading rate of 78 ± 21 g/m2/d (HRT of 4 h). The ultra-filtration was spiral wound and composed of polyethersulfone (PES) with a pore size of 0.03 microns. The MBBR feed was obtained from a final treated wastewater effluent in a food processing plant located in SW Ontario. In this research, ferric chloride was also employed as a coagulant and influences of different coagulant doses and permeate fluxes on membrane fouling were studied. Based on the experimental results, it was found that the combination of MBBR with membrane filtration can produce a constant high quality permeate that is appropriate for water reuse purposes. The composition analysis of permeate showed that the stream is free of suspended solids and the average COD turns to 75 ± 25 mg/l. In addition, the MBBR had a SCOD removal of 76% ± 7% which is considered as a reasonable efficiency for a single reactor. Operating the membrane without adding coagulant caused rapid fouling in a short time period and the Trans Membrane Pressure (TMP) reached the maximum allowable pressure of 10 psi. However, addition of coagulant was found to decrease the fouling of the membrane as well as increasing the filtration time. The extent of the pre-coagulation effect on membrane fouling was found to strongly depend on the dosage of the coagulant and the MBBR effluent characteristics. A coagulant dose of 400 mg/l with a permeate flux of 7.6 LMH performed the best at reducing membrane fouling. Colloidal fouling was found to be a significant fouling mechanism at low coagulant dose (e.g. 200 mg/l), while cake formation appeared to be mainly responsible for fouling at higher coagulant doses. Permeate flux was found to have a significant effect on the fouling of the membrane. The presence of colloidal matters at low fluxes and TSS at higher fluxes were responsible for fouling of the membrane by blocking the pores and formation of the cake layer on the membrane surface, respectively. Then later addition of Dissolved Air Flotation (DAF) inside the factory had a noticeable effect on wastewater characteristics and consequently on fouling of the membrane. A 22% and 31% improvement in TCOD and TSS in the wastewater was observed leading to reduction in the fouling.

Biofilm systems such as the moving bed biofilm reactor (MBBR) are finding increased application in wastewater treatment. One important process that governs MBBRs and yet is poorly understood is the rate of biofilm detachment. The detachment of cells from biofilm surfaces controls both the accumulation of biofilm and the quantity of biomass that is suspended in the bulk liquid phase. This changing balance of attached and suspended cells, in this thesis named the biosolids dynamics, can impact the efficacy of MBBRs. The goal of this research was to investigate how the biosolids dynamics are influenced by process changes relevant to applied wastewater treatment systems and suggest new routes to reactor design and optimization. To achieve this goal, the work addresses three separate but interconnected lines of inquiry. First, multivariate analysis (Principal Component Analysis, Partial Least Squares) was used to examine 2 years of historical data from an MBBR operating at a Canadian pulp mill in order to identify key process variables, perform process diagnostics, and act as a predictive tool. Secondly, the effect of calcium concentration on biofilm structure, microbiology and reactor performance was investigated in four laboratory-scale MBBRs operated at a range of calcium concentrations (1 to 300 mg/L Ca2+). It was found that above a threshold calcium concentration between 1-50 mg/L, MBBR biofilms were observed to be thicker with greater density, contain larger anoxic regions adjacent to the carrier substratum, have more proteinaceous EPS, and have altered microbial community structure. The results suggest an important role for calcium that should be considered in the design and operation of MBBRs. In the final line of inquiry, a diffusion-reaction biofilm model was adapted to represent the key processes of the MBBR. The model was found to simulate average trends observed in the lab-scale experiments allowing for quantification of the detachment rate. Transient periods of reactor starvation were also simulated by introducing a novel metabolic state function to account for down-regulation of metabolism as a result of starvation. This approach was found to accurately simulate starvation response when coupled with detachment expressions that were growth-dependant.

The objectives of this work are: 1) to agarose-stabilize fragile biofilms for quantitative structure analysis; 2) to understand the influences of LuxS on biofilm formation; 3) to improve teacher quality by preparing Georgia’s middle school science teachers to integrate inquiry-based, hands-on research modules in the classroom. Quantitative digital image analysis demonstrated the effectiveness of the agarose stabilization technique for generating reproducible measurements of three dimensional biofilm structure. The described method will also benefit researchers who transport their flow cell-cultivated biofilms to a core facility for imaging. AI-2-dependent and independent effects of LuxS on biofilm-related phenotypes were revealed, suggesting that LuxS is a versatile enzyme, possessing multiple functions in E. coli ecology that could assist E. coli in adapting to diverse conditions. Overall, the work presented in this dissertation supported the concept that quorum sensing, biofilm formation, and cell adhesion are largely related. Additionally, through this project, teachers enhanced content knowledge and confidence levels, mastered innovative teaching strategies and integrated inquiry-based, inter-disciplinary, hands-on activities in the classroom. As a result, student learning was enhanced, and teachers are better equipped to give Georgia’s students a solid foundation in the sciences.

In this dissertation I describe a model system to characterize the response of an oral bacterial pathogen, Aggregatibacter actinomycetemcomitans to the metabolic byproducts of a representative member of the oral flora, Streptococcus gordonii. A. actinomycetemcomitans is a causative agent of periodontal infections in humans. To cause infection, A. actinomycetemcomitans must overcome numerous challenges, including the host immune system and toxic metabolite production from other microbes. The most numerically dominant flora in the oral cavity are oral streptococci, which are well known for their ability to produce copious amounts of lactic acid and H₂O₂. By studying the response to H₂O₂ and lactic acid in pure and co-cultures, I have demonstrated that A. actinomycetemcomitans responds to these metabolites by several novel mechanisms that both enhance its survival in the presence of the host immune system and in the presence of the model oral streptococci S. gordonii. These studies have demonstrated that metabolites produced by normal flora can impact the survival of a single species in vivo as much as previously known virulence factors have done. In addition, I present a new method for measuring metabolite production in an attached cell population. This method is a novel application of scanning electrochemical microscopy (SECM) and I used this technique to study H₂O₂ production in the three dimensional space surrounding a multispecies biofilm in real time. In a related study I present the use of SECM to discover a novel redox chemistry phenomenon in the opportunistic pathogen Pseudomonas aeruginosa. / text

Biofuel production from microalgal biomass offers a clean and sustainable liquid fuel alternative to fossil fuels. In addition, algae cultivation is advantageous over traditional biofuel feedstocks as (i) it does not compete with food production, (ii) it potentially has a much greater areal productivity, (iii) it does not require arable land, and (iv) it can use marginal sources of water not suitable for irrigation or drinking. However, current algae cultivation technologies suffer from (i) low solar energy conversion effiencies, (ii) large thermal fluctuations which negatively affect the productivity, and (iii) large evaporative losses which make the process highly water intensive. This thesis reports a numerical study that address these key issues of planktonic as well as benthic algal photobioreactor technologies. First, radiant energy transfer in planktonic algal photobioreactors containing cells with different levels of pigmentation was studied. Chlamydomonas reinhardtii and its truncated chlorophyll antenna transformant tla1 were used as model organisms. Based on these simulations guidelines are derived for scaling the size and microorganism concentration of photobioreactors cultivating cells with different levels of pigmentation to achieve maximum photosynthetic productivity. To achieve this, the local irradiance obtained from the solution of the radiative transport equation (RTE) was coupled with the specific photosynthetic rates of the microorganisms to predict both the local and total photosynthetic rates in a photobioreactor. For irradiances less than 50 W/m2, the use of genetically modified strains with reduced pigmentation was shown to have negligible effect on increasing photobioreactor productivity. However, at irradiances up to 1000 W/m2, improvements of up to 30% were possible with cells having 63% less pigment concentration. It was determined that the ability of tla1 to transmit light deeper into the photobioreactor was the primary mechanism by which a photobioreactor using the modified strain can achieve greater productivity. Furthermore, it was determined photobioreactors using each strain have dead zones in which the local photosynthetic rate is negligible due to nearly complete light attenuation. These dead zones occur at local optical thicknesses greater than 169 and 275 in photobioreactors using the wild strain and the genetically modified strain, respectively. In addition, a thermal model of an algae biofilm photobioreactor was developed to assess the thermal fluctuations and evaporative loss rate of these novel photobioreactors under varying outdoor conditions. The model took into account air temperature, irradiance, relative humidity, and wind speed as inputs and computed the temperature and evaporative loss rate as a function of time and location in the photobioreactor. The model was run for a week-long period in each season using weather data from Memphis, TN. The range of the daily algae temperature variation was observed to be 13.2C, 12.4C, 12.8C, and 9.4C in the spring, summer, winter, and fall, respectively. Furthermore, without active cooling, the characteristic evaporative water loss from the system is approximately 6.3 L/m2-day, 7.0 L/m2-day, 4.9 L/m2-day, and 1.5 L/m2-day in the spring, summer, fall, and winter, respectively. / text