Many industrial activities discharge sulphate- and metal-containing wastewaters, including the manufacture of pulp and paper, mining and mineral processing, and petrochemical industries. Acid mine drainage (AMD) is an example of such sulphate- and metal-containing waste streams. Formation of AMD is generally the result of uncontrolled oxidation of the sulphide minerals present in the terrain in which the drainage flows with concomitant leaching of the metals. Acid mine drainage (AMD) and other sulphate- and metal-containing waste streams are amenable to active biological treatment. Anaerobic reduction of sulphate, reaction of produced sulphide with metal ions present in the waste stream, and biooxidation of excess sulphide are three main sub-processes involved in the active biotreatment of AMD. Anaerobic reduction of sulphate can be achieved in continuous stirred tank bioreactors with freely suspended cells or in immobilized cell bioreactors. The application of freely suspended cells in a continuous system dictates a high residence time to prevent cell wash-out, unless a biomass recycle stream is used. In an immobilized cell system biomass residence time becomes uncoupled from the hydraulic residence time, thus operation of bioreactor at shorter residence times becomes possible. In the present work, kinetics of anaerobic sulphate reduction was studied in continuous immobilized cell packed-bed bioreactors. Effects of carrier matrix, concentration of sulphate in the feed and sulphate volumetric loading rate on the performance of the bioreactor were investigated. The bioreactor performance, in terms of sulphate reduction rate, was dependent on the nature of the carrier matrix, specifically the total surface area which was provided by the matrix for the establishment of biofilm. Among the three tested carrier matrices, sand displayed the superior performance and the maximum volumetric reduction rate of 1.7 g/L-h was achieved at the shortest residence time of 0.5 h. This volumetric reduction rate was 40 and 8 folds faster than the volumetric reduction rates obtained with glass beads (0.04 g/L-h; residence time: 28.6 h) and foam BSP (0.2 g/L-h; residence time: 5.3 h), respectively. Further kinetic studies with sand as a carrier matrix indicated that the extent of volumetric reduction rate was dependent on the feed sulphate concentration and volumetric loading rate. At a constant feed sulphate concentration, increases in volumetric loading rate caused the volumetric reduction rate to pass through a maximum, while increases in feed sulphate concentrations from 1.0 g/L to 5.0 g/L led to lower volumetric reduction rates. The maximum volumetric reduction rates achieved in the bioreactors fed with initial sulphate concentration of 1.0, 2.5 and 5.0 g/L were 1.71, 0.82 and 0.68 g/L-h, respectively. The coupling of lactate utilization to sulphate reduction was observed in all experimental runs and the rates calculated based on the experimental data were in close agreement with calculated theoretical rates, using the stoichiometry of the reactions involved. The maximum volumetric reduction rates achieved in the immobilized cell bioreactors were significantly faster than those reported for freely suspended cells employed in the stirred tank bioreactors.

Many industrial activities discharge sulphate- and metal-containing wastewaters, including the manufacture of pulp and paper, mining and mineral processing, and petrochemical industries. Acid mine drainage (AMD) is an example of such sulphate- and metal-containing waste streams. Formation of AMD is generally the result of uncontrolled oxidation of the sulphide minerals present in the terrain in which the drainage flows with concomitant leaching of the metals. Acid mine drainage (AMD) and other sulphate- and metal-containing waste streams are amenable to active biological treatment. Anaerobic reduction of sulphate, reaction of produced sulphide with metal ions present in the waste stream, and biooxidation of excess sulphide are three main sub-processes involved in the active biotreatment of AMD. Anaerobic reduction of sulphate can be achieved in continuous stirred tank bioreactors with freely suspended cells or in immobilized cell bioreactors. The application of freely suspended cells in a continuous system dictates a high residence time to prevent cell wash-out, unless a biomass recycle stream is used. In an immobilized cell system biomass residence time becomes uncoupled from the hydraulic residence time, thus operation of bioreactor at shorter residence times becomes possible. In the present work, kinetics of anaerobic sulphate reduction was studied in continuous immobilized cell packed-bed bioreactors. Effects of carrier matrix, concentration of sulphate in the feed and sulphate volumetric loading rate on the performance of the bioreactor were investigated. The bioreactor performance, in terms of sulphate reduction rate, was dependent on the nature of the carrier matrix, specifically the total surface area which was provided by the matrix for the establishment of biofilm. Among the three tested carrier matrices, sand displayed the superior performance and the maximum volumetric reduction rate of 1.7 g/L-h was achieved at the shortest residence time of 0.5 h. This volumetric reduction rate was 40 and 8 folds faster than the volumetric reduction rates obtained with glass beads (0.04 g/L-h; residence time: 28.6 h) and foam BSP (0.2 g/L-h; residence time: 5.3 h), respectively. Further kinetic studies with sand as a carrier matrix indicated that the extent of volumetric reduction rate was dependent on the feed sulphate concentration and volumetric loading rate. At a constant feed sulphate concentration, increases in volumetric loading rate caused the volumetric reduction rate to pass through a maximum, while increases in feed sulphate concentrations from 1.0 g/L to 5.0 g/L led to lower volumetric reduction rates. The maximum volumetric reduction rates achieved in the bioreactors fed with initial sulphate concentration of 1.0, 2.5 and 5.0 g/L were 1.71, 0.82 and 0.68 g/L-h, respectively. The coupling of lactate utilization to sulphate reduction was observed in all experimental runs and the rates calculated based on the experimental data were in close agreement with calculated theoretical rates, using the stoichiometry of the reactions involved. The maximum volumetric reduction rates achieved in the immobilized cell bioreactors were significantly faster than those reported for freely suspended cells employed in the stirred tank bioreactors.

Cartilage tissue engineering is an emerging treatment option for osteoarthritis and trauma related joint injuries. A continuing challenge for cartilage tissue engineering is increasing construct extracellular matrix production and material properties. Shear stress and oxygen tension play an important role in tissue engineering of cartilage. In this select stimulatory conditions using combinations of shear stress and oxygen tension have been used to enhance the construct extracellular matrix deposition and material properties. Additionally, a perfusion concentric cylinder bioreactor has been developed to incorporate multiple fluid flow regimes through the construct. This thesis attempts to elucidate the effect of shear stress and biochemical conditions on cartilage development in vitro to provide functional tissue engineered constructs.

Articular cartilage has a limited ability to heal due to its avascular, aneural, and alymphatic nature. Currently, there is a need for alternative therapies for diseases that affect articular cartilage such as osteoarthritis. Recently, it has been shown that tissue constructs, which resemble cartilage in structure and function, can be cultured in vitro in a cell-polymer bioreactor system. Bioreactors provide a three dimensional environment that promotes cell proliferation and matrix production. The primary objective of this study is to accurately simulate fluid mechanics using the lattice Boltzmann method for application to a cell-polymer bioreactor system. Lattice Boltzmann (LB) is a flexible computation technique that will allow for the simulation of a moving construct under various bioreactor conditions. The method predicts macroscopic hydrodynamics by considering virtual particle interactions. Derived from the Lattice Gas Automata, lattice Boltzmann allows for mass transfer, complex geometries, and particle dynamics. A primary goal is to characterize the accuracy of the LB implementation and eventually the shear stresses felt by a tissue construct in this dynamic environment. This information is important since recent studies show that chondrocytic function may depend on the mechanical stimuli produced by fluid flow. Hence, shear stress may affect the final mechanical properties of tissue constructs. In this study, numerical simulations are done first in 2D and then extended to 3D to test the LB implementation. Simulations of the rotating wall vessel (RWV) bioreactor are then undertaken. The results are benchmarked against computations done with a commercial CFD package, FLUENT, and compared with analytic solutions and experimental data.

Estrogens are a group of endocrine disrupting compounds capable of causing abnormalities in the reproductive systems of the wildlife. Wastewater is a major source of environmental estrogens, in part due to incomplete removal of estrogens in biological wastewater treatment processes. This dissertation investigated factors affecting estrogen biodegradation in bioreactors. Specifically, research efforts were placed on characterization of several bacterial estrogen degraders (model strains: Aminobacter strains KC6 and KC7, and a Sphingomonas strain KC8) and examination of the effects of operating parameters on estrogen removal and estrogen-degrading microbial community structure. Sphingomonas strain KC8 can use 17beta-estradiol as a sole carbon source, suggesting that estrogen degradation by KC8 is a growth-linked, metabolic reaction; however, estrogen degradation by strains KC6 and KC7 might be a non-growth linked, cometabolic reaction. One important finding was that strain KC8 can also degrade and further utilize testosterone as a growth substrate. Strain KC8 was characterized in terms of its utilization kinetics toward estrogens and testosterone with the results that showed relatively smaller kinetic parameters than the typical values for heterotrophs in activated sludge. Strain KC8 can also grow on other organic constituents (glucose, succinate, and acetate). Strain KC8 retained its ability to degrade both 17beta-estradiol and estrone (after 15 d of growth on a complex nutrient medium without 17beta-estradiol). Effective removals (>98.7 %) of 17beta-estradiol with no significant differences were observed in sequencing batch reactors (SBRs) under three solid retention times (SRTs of 5, 10, 20 d). The population ratios of known estrogen degraders (strains KC8 and ammonia-oxidizing bacteria (AOB)) and amoA gene (associated with ammonia oxidation) to total bacteria decreased as SRT increased in SBRs. These observations correspond to the decreasing percentages of 17 beta-estradiol biodegraded in SBR when SRT increased from 5 to 20 d, when the sorption of 17 beta-estradiol onto biomass was considered. Real-time terminal restriction fragment length polymorphism showed that more ribotypes were observed in SBR-20d than SBR-5d. The species evenness (E) in microbial community structures in SBRs was not affected by SRT. However, diversity indices (Shannon-Weaver diversity index (H) and the reciprocal of Simpson?s index (1/D)) suggest that longer SRTs might lead to a more diverse microbial community structure.

published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

Not available / text

Determining how biological cells respond to external factors in the environment can aid in understanding disease progression, lead to rational design strategies for tissue engineering, and contribute to understanding fundamental mechanisms of cellular function. Dynamic mechanical forces exist in vivo and are known to alter cellular response to other stimuli. However, identifying the roles multiple external factors play in regulating cell fate and function is currently impractical, as experimental techniques to mechanically stimulate cells in culture are severely limited in throughput. Hence, determining cell response to combinations of mechanical and biological factors is technically limited. In this thesis, microfabricated systems were designed, implemented and characterized to screen for the effects of mechanical stimulation in a high-throughput manner. Realizing these systems required the development of a fabrication process for precisely-aligned multilayer microstructures, and the development of a method to integrate non-traditional and clinically-relevant biomaterials into the microfabrication process. Three microfabricated platforms were developed for this application. First, an array was designed for experiments with high mechanical throughput, in which cells cultured on a surface experience a range of cyclic, uniform, equibiaxial strains. Using this array, a novel time- and strain-dependent mechanism regulating nuclear β-catenin accumulation in valve interstitial cells was identified. Second, a simpler system was designed to screen for the effects of combinatorially manipulated mechanobiological parameters on the pathological differentiation of valve interstitial cells. The results demonstrate functional heterogeneity between cells isolated from different regions of the heart valve leaflet. Last, a microfabricated platform was developed for high-throughput mechanical stimulation of cells encapsulated in a three-dimensional biomaterial, enabling the study of mechanical forces on cells in a more physiologically relevant microenvironment. Overall, these studies identified novel biological phenomena as a result of designing higher-throughput systems for the mechanical stimulation of cells.

Determining how biological cells respond to external factors in the environment can aid in understanding disease progression, lead to rational design strategies for tissue engineering, and contribute to understanding fundamental mechanisms of cellular function. Dynamic mechanical forces exist in vivo and are known to alter cellular response to other stimuli. However, identifying the roles multiple external factors play in regulating cell fate and function is currently impractical, as experimental techniques to mechanically stimulate cells in culture are severely limited in throughput. Hence, determining cell response to combinations of mechanical and biological factors is technically limited. In this thesis, microfabricated systems were designed, implemented and characterized to screen for the effects of mechanical stimulation in a high-throughput manner. Realizing these systems required the development of a fabrication process for precisely-aligned multilayer microstructures, and the development of a method to integrate non-traditional and clinically-relevant biomaterials into the microfabrication process. Three microfabricated platforms were developed for this application. First, an array was designed for experiments with high mechanical throughput, in which cells cultured on a surface experience a range of cyclic, uniform, equibiaxial strains. Using this array, a novel time- and strain-dependent mechanism regulating nuclear β-catenin accumulation in valve interstitial cells was identified. Second, a simpler system was designed to screen for the effects of combinatorially manipulated mechanobiological parameters on the pathological differentiation of valve interstitial cells. The results demonstrate functional heterogeneity between cells isolated from different regions of the heart valve leaflet. Last, a microfabricated platform was developed for high-throughput mechanical stimulation of cells encapsulated in a three-dimensional biomaterial, enabling the study of mechanical forces on cells in a more physiologically relevant microenvironment. Overall, these studies identified novel biological phenomena as a result of designing higher-throughput systems for the mechanical stimulation of cells.

North American petroleum refineries use landfarming to dispose of hydrocarbon-containing wastes for bioremediation by indigenous soil microorganisms. In this study, we isolated PAH-degrading bacteria from landfarm soil by enrichment with hydrocarbon-containing effluent. One isolate, Arthrobacter sp. UG50, was capable of using phenanthrene and anthracene as sole carbon sources. The strain degraded phenanthrene (200 mg/L) within 24 h in pure culture at high cell density (10e8 cells/mL). Anthracene (50 mg/L) was slowly degraded, with 29% degraded within 21 days. The strain could not use naphthalene, pyrene, chrysene or benzo(a)pyrene as sole carbon sources, but could degrade pyrene (50 mg/L) cometabolically when phenanthrene was provided. Anthracene degradation (50 mg/L) was enhanced by phenanthrene, with 100% degraded within 6 days. The addition of strain UG50 to petroleum sludge in baffled flasks increased total hydrocarbon degradation and degradation of low concentrations of fluorene, phenanthrene, pyrene and chrysene compared to flasks with limited aeration or containing sludge alone. / NOVA Chemicals and the Ontario Centres of Excellence