Search

The application of biotechnologies in the mining sector has intensified over the last 30 years, driven by the increasing demand for metals associated with the rise in energy costs and the awareness for environmentally responsible mining practices. Acidophilic prokaryotes play an important role in biohydrometallurgy, facilitating the solubilisation and recovery of base metals from ores and waste materials. The potential of novel acidophiles of the phylum Firmicutes for applications in biohydrometallurgical processes is examined in this thesis. Eight strains of extremely acidophilic bacteria were studied and shown to belong to the proposed novel genus “Acidibacillus”. These had been isolated previously from several distinct global locations and were shown to be obligately heterotrophic bacteria with potential to carry out tasks critical to biomining such as regenerating ferric iron (by catalysing the dissimilatory oxidation of ferrous iron), generating sulfuric acid (by the oxidation of zero-valent sulfur and tetrathionate; two strains only), and removing potentially inhibitory dissolved organic carbon. These isolates also demonstrated the ability to catalyse the dissimilatory reduction of ferric iron in anaerobic conditions. Results obtained during this study provide the basis for future research to assess their potential roles in microbial consortia applied in the bio-processing of metal ores. A novel obligately anaerobic acidophilic Firmicute (strain I2511) isolated from sediment obtained from an abandoned copper mine, was characterised in terms of its phylogeny and physiology. This isolate formed a separated clade within the Firmicutes, and was considered to represent a novel candidate genus. It also displayed a unique set of physiological traits, distinct from currently validated species of acidophilic Firmicutes. The isolate was an obligate anaerobe that grew via zero-valent sulfur (ZVS) respiration, generating H2S over a wide pH range (1.8 - 5.0), and also catalysed the dissimilatory reduction of ferric iron. Strains of acidophilic sulfatereducing bacteria (aSRB), also Firmicutes, were shown to reduce ZVS at pH as low as 3. These aSRB, together with isolate I2511, populated a novel variant of a low pH sulfidogenic bioreactor. The “hybrid sulfidogenic bioreactor” (HSB) operated using both sulfate and ZVS as electron acceptors, and glycerol as electron donor. The bioreactor successfully remediated and recovered zinc from circum-neutral pH mine-impacted waters with distinct chemical composition collected from two abandoned lead/zinc mines in the U.K. The microbial consortium used in this system proved to be robust, in which the HSB generated H2S consistently under a wide pH range (2 – 7). Experiments demonstrated that H2S could also be generated abiotically in a non-inoculated low pH reactor, by the chemical reaction of ZVS and zero-valent iron to form iron sulfide, and the consequent acid dissolution of the latter. Operational costs and the advantages of biogenic and abiotic generation of H2S for recovery of transition metals from mine waters are discussed.

Effective treatment of wastewater is an important process in reducing the environmental impact of industry and human activity. Although conventional water treatment systems can adequately remove the principle components of waste (i.e. substances that can be represented the majority of biological and chemical oxygen demand) several materials are poorly or slowly removed. Tertiary treatment polishing processes are therefore required to remove these contaminants to ensure complete wastewater treatment. This thesis reports investigations made using film reactors that are used to remove recalcitrant materials such as ammonia- nitrogen and endocrine disrupters that although present in low concentrations, if left untreated can have a strong impact on the environment. Film reactors potentially offer several process advantages over conventional activated sludge treatments systems as they allow very long residence time and contact with high concentrations of fixed microbes with the low concentrations of pollutants so enhancing kinetic performance and efficiency of the process. Two reactor configurations, a partial fixed bed (PFBR) and moving bed biofilm reactors (MBBR) were investigated. A thirty liter reactor with a working volume of 16 liters was constructed and contained fixed microbial films on either free suspended or fixed beds plastic packing (K2 AnoxKaldnes). The investigation of ammonia-nitrogen oxidation showed that after a suitable acclimation period (2 weeks) that ammonia was oxidise rapidly reducing the feed concentrations of 35 mg/L to < 2 mg/L in the effluent. To assess the performance for ammonia-nitrogen removal the reactors operated for long periods (up to 3 months) with continuous feed using the reactor in either PFBR or MBBR modes in addition of 17alpha-ethynylestradiol (EE2) and mestranol (MeEE2), the endocrine disrupting compounds commonly found in municipal wastewater. These substances is derived from a synthetic hormones if found in the natural environment can reduced the productivity of the fish as this can cause feminization in aquatic organisms with disastrous consequences on fish populations. The MBBR and PFBR systems were used to investigate the co-metabolism of ammonia-nitrogen, 17alpha-ethynylestradiol (EE2) and mestranol from model waste water feed containing 35 mg/L of ammonia-nitrogen and 100 mug/L of 17alpha-ethynylestradiol (EE2) and mestranol (MeEE2). A kinetic analysis of the systems were made and for the PFBR reactor, the specific growth rate, mumax of 7.092 d-1 with saturation constants, Ks of 1.574 mg/L. The kinetic analysis for the MBBR system was 6.329 d-1 for the mumax with the K.S of 0.652 mg/L. When the PFBR was used removal of EE2 represents 70% MeEE2 was removed. MBBR were shown to be more effective and efficient in removing ammonia-nitrogen reducing the levels under good conditions to > 2 mg/L while the PFBR could also achieve 2 mg/L. The MBBR system was also more competent in the removal of 17alpha-ethynylestradiol (EE2) and mestranol compared to PFBR. This work demonstrates that there are considerable advantages to using thin film reactors as polishing step for the tertiary treatment of waste waters when to compared to other processes in reducing the inorganic pollutants as endocrine disrupting compounds. The significance of these results is discussed in this context.

The following thesis presents a computational framework that can capture inherently non-linear and emergent biocomplex phenomena. The main motivation behind the investigations undertaken was the absence of a suitable platform that can simulate, both the continuous features as well as the discrete, interaction-based dynamics of a given biological system, or in short, dynamic reciprocity. In order to determine the most powerful approach to achieve this, the efficacy of two modelling paradigms, transport phenomena as well as agent-based, was evaluated and eventually combined. Computational Fluid Dynamics (CFD) was utilised to investigate optimal boundary conditions, in terms of meeting cellular glucose consumption requirements and exposure to physiologically relevant shear fields, that would support mesenchymal stem cell growth in a 3-dimensional culture maintained in a commercially available bioreactor. In addition to validating the default bioreactor configuration and operational parameter ranges as suitable towards sustaining stem cell growth, the investigation underscored the effectiveness of CFD as a design tool. However, due to the homogeneity assumption, an untenable assumption for most biological systems, CFD often encounters difficulties in simulating the interaction-reliant evolution of cellular systems. Therefore, the efficacy of the agent-based approach was evaluated by simulating a morphogenetic event: development of in vitro osteogenic nodule. The novel model replicated most aspects observed in vitro, which included: spatial arrangement of relevant players inside the nodule, interaction-based development of the osteogenic nodules, and the dependence of nodule growth on its size. The model was subsequently applied to interrogate the various competing hypotheses on this process and identify the one that best captures transformation of osteoblasts into osteocytes, a subject of great conjecture. The results from this investigation annulled one of the competing hypotheses, which purported the slow-down in the rate of matrix deposition by certain osteoblasts, and also suggested the acquisition of polarity to be a non-random event. The agent-based model, however, due to being inherently computationally expensive, cannot be recommended to model bulk phenomena. Therefore, the two approaches were integrated to create a modelling platform that was utilised to capture dynamic reciprocity in a bioreactor. As a part of this investigation, an amended definition of dynamic reciprocity and its computational analogue, dynamic assimilation, were proposed. The multi-paradigm platform was validated by conducting melanoma chemotaxis under foetal bovine serum gradient. Due to its CFD and agent-based modalities, the platform can be employed as both a design optimisation as well as hypothesis testing tool.

Cancer remains a leading cause of death worldwide, with no signs of slowing down. In 2023 alone, the projected number of deaths from cancer is 600,000, with projected incidences at almost 2 million. Mortality rates have improved recently, but a cancer diagnosis still burdens patients and their families. This burden weighs on patients not only financially but also emotionally. For example, the average out-of-pocket patient cost is more than $2,000 and can be even higher for more aggressive types of cancer. Additionally, 2 in 3 cancer patients experience significant psychological stress due to their diagnosis. As such, cancer research is an increasingly important field that will only become more needed as birthrates rise and the population continues to live longer. The current in vitro cancer models generally rely on 2D monolayer cell growth conformation that lacks the sophistication required to represent physiological tissues accurately. As such, many therapeutic screening studies that employ these models fail to translate well to clinical trials. Furthermore, the xenografted nude mouse model, the most commonly used in vivo animal model, lacks many critical immune components to represent drug response and survival rates correctly. Also, as anticancer strategies are moving away from cytotoxic styles of drugs and toward inhibitory and immunotherapeutic drugs, the use cases for the nude mouse model have diminished greatly. Thus, the need for novel 3D physiologically accurate in vitro cancer research models is apparent. Bioreactors, specifically flow-perfusion bioreactors, are uniquely poised to fill this void as exceptionally useful tools for cell culture that can create 3D macro-scale cultures for tissue engineering applications. They offer unique cell culture monitoring facilities through modular sensors, particularly in the liquid phase, which leaves a gap for creating an analytical sensor for gas phase volatile metabolite detection. As such, the abovementioned deficiencies with current 2D in vitro models and the lack of proper gas phase metabolite analysis pose the guiding questions for this manuscript: 1) “How can we use novel mid-IR laser spectrometry to monitor cell cultures in bioreactors?” and 2) “In what ways does hypoxia affect the proliferation and oxygen uptake rates of cancer cells in 3D in vitro flow-perfusion bioreactor models?”. These questions will be answered by modifying the previously established flow-perfusion bioreactor system to incorporate 3D collagen hydrogel scaffolds as a more physiologically representative in vitro solid tumor model. The verification of successful cell culture was determined using cell growth and viability. Furthermore, cell culture within the solid tumor model was monitored using a novel Mid-IR laser spectrometer to detect acetaldehyde, a gas-phase metabolite, as a potential biomarker for cancer diagnosis. Further exploring the novel 3D in vitro solid tumor model illuminated the need to add hypoxia, a critical component of solid tumor pathophysiology, to create a better biomimetic model. The hypoxic component was added by directing the flow of the bioreactor such that media flowed through each chamber without reoxygenation in a series way, thus lowering the oxygen saturation stepwise. The following chapters of this investigation will focus on answering each of these questions in succession.

Environmental concerns are becoming increasingly relevant during a period of hemorrhaging ecosystem goods and services. Restoring these would result in positive outcomes for public health and economic benefit. This thesis seeks to address two environmental concerns: (1) accurate soil mapping and (2) treatment of nitrogen to affect water quality change.The current method of soil mapping, SSURGO (USDA‐NRCS Soil survey), is often erroneous and misleading. Two studies in this dissertation are conducted to evaluate the potential that different resolution digital elevation models (DEMs) have to distribute soil characteristics successfully. These studies are conducted in southwest Virginia and western Vermont. The aforementioned studies evaluated 36 and 59 soil samples, respectively. Spatial characteristics, including slope, catchment area, and topographic wetness, are derived from several DEMs. In chapter 2, these characteristics are spatially compared, and we found that small resolution rasters result in narrow flow paths relative to coarser rasters. In chapter 3, we isolate the analysis to focus on resolution size, instead of a mix of both resolution size and generation method. This is done by recursively coarsening small rasters, deriving spatial attributes from said rasters and evaluating their potential to fit the soil characteristics of interest. Here we found that slopes generated from resolutions smaller than 11m were poor predictors of soil characteristics. Both chapters are finished by proposing and evaluating a soil map. Proposed regressions beat SSURGO in all investigated properties. Furthermore, proposed maps consistently beat out uninformed smallest resolution derived maps.Chesapeake bay water quality managers are struggling to achieve targets for nitrogen loading. This is in part due to the widespread presence of legacy nitrogen. Legacy nitrogen is an emerging issue, and springs exporting high levels of nitrogen are not uncommon in northern Virginia. This thesis explores, in part, a novel concept of treating large loads of nitrogen exported from a spring with a bioreactor. Bioreactors are a young science that most typically pair carbon heavy subterranean receptacles to agricultural drainage. This provides a location for nitrogen fixing bacteria to consume nitrate/nitrite, turning these into inert nitrogen gas. A spring fed bioreactor is studied for 10 months, and bioreactor conditions including influent and effluent nitrogen concentrations, bioreactor flow, and temperature are collected. A model driven by first order reaction equations is found to be most accurate with inputs of temperature and bioreactor age. The resulting marginal effects of these inputs were consistent with previously reported studies.

Nitrate and phosphate present in agricultural subsurface drainage water can impact surface water quality thus impacting the environment and human health by causing eutrophication and Methemoglobinemia in young infant. Biological denitrification and phosphate adsorption filters are treatments options that can help reduce nitrate and phosphate concentration from agricultural subsurface drainage before it can be discharged in surface water. The first objective of this dissertation was to determine the Michaelis-Menten kinetics model parameters for nitrate removal in bioreactors using the fresh, aged, and composted woodchips. The composted woodchips achieved a denitrification rate 2.4 times higher than the fresh woodchips which was in term achieved 2.9 times higher denitrification rate than the aged woodchips. Denitrification rates decreased at 5ºC compared to 22ºC for composted woodchips and fresh woodchips. The composted still performed better at low temperatures compared to fresh woodchips. The second objective was to determine the impact of different pairing configurations of woodchips and steel chips on nitrate and phosphate removal capacity and how the pairing configurations affect the DOC and iron leaching from the reactors. Three different reactors pairing configurations were evaluated in this project including woodchips followed by steel chips, steel chips followed by woodchips, and mixed woodchips/steel chips. The effect of low temperature (5℃) on phosphate removal capacity was studied. There was no statistical difference in nitrate and phosphate removed between all three reactors configurations. Three different reactors pairing configurations were evaluated in this project including woodchips followed by steel chips, steel chips followed by woodchips, and mixed woodchips/steel chips. The effect of low temperature (5℃) on phosphate removal capacity was studied. There was no statistical difference in nitrate and phosphate removed between all three reactors configurations.

The research in this dissertation is broadly concerned with the measurement, modeling, and management of nitrogen transport and transformation in agricultural watersheds. Our objective in the first study was to quantify nitrogen losses under different cover crop treatments to provide insights into the environmental benefits of cover crops over their lifetime. We measured nitrous oxide emissions, nitrate leaching, and plant nitrogen demand in the early spring and continued nitrous oxide measurements after tillage. We found the environmental benefit of cover crops depends on the type and period of analysis. Legumes increased nitrous oxide emissions to the atmosphere post-tillage over the non-legumes. Neither legume nor non-legume, nor a mixture of the two, was effective at reducing nitrous oxide during the growth phase relative to fallow conditions. In the second study, we examined the scale-dependency of several nitrogen cycle processes in an earth system model with previously identified scale issues in its hydrologic cycle and recognized difficulty in simulating gaseous and hydrologic-driven nitrogen losses. We hypothesized that observed discrepancies in these soil nitrogen processes across grid-scales are explained by issues in hydrologic scale-dependence. The recommended spin-up procedure generated different soil hydraulic properties for a model resolution, resulting in different drainage and runoff rates. We confirmed that the choice in grid scale affected model estimates of soil moisture, nitrification, nitrate leaching, and to a lesser degree, denitrification. In the final study, we present an exploration of flow variability and denitrifying bioreactor performance that aim to improve management of nitrogen in tile drainage water. Using synthetic data modeled after observed flow distributions in an existing bioreactor in central New York, we found that a record with a few high flow events generated greater nitrate export than a record with frequent low flow events. The finding supports the general design practice of building the reactor with appropriate size and flow control to maintain a low flow rate.

The use of woodchip denitrifying bioreactors holds promise as a simple, efficient and cost-effective system to reduce nitrate loads in agricultural runoff through stimulation of microbial denitrification. While bioreactor performance at colder temperatures and under varying hydraulic residence time (HRT) has been investigated, the correlation to functional microbial communities has not been studied in great detail. In this study, we quantified denitrifying functional gene copy numbers throughout the course of a three-month study within mesoscale [1.83m x 0.3m x 0.61m] denitrification bioreactors that were operated at three temperature regimes and two HRT. We found that with increasing temperature and HRT there was a significant increase in percent nitrate removed. Temperature and HRT had little effect on nirK and nosZ clade I gene copy numbers throughout the length of the bioreactors, however, they had a significant effect on 16S rRNA and nosZ clade II gene copy numbers at the inflow locations of the bioreactors. Utilizing a hydraulic flow model developed for the denitrifying bioreactors in this study, a decrease in nitrate concentration along the length of the reactors was calculated. We correlated a decrease in 16S rRNA, nirK and nosZ clade II gene copy numbers to the decrease in nitrate concentrations predicted by the hydraulic flow model. Our results suggest that at temperatures of 14.5°C and 12-hr HRT, denitrifying bioreactor microbial communities are limited by variables other than temperature and HRT. It is suggested that carbon availability is the most likely limiting factor, indicating a need to further investigate the role of both denitrifying and decomposing communities in denitrifying bioreactors. These findings contribute to a better understanding of the microbial functional communities in denitrifying wood chip bioreactors, allowing us to optimize the design and performance of these reactors in agricultural midwestern states.

Heart failure affects 1-2 % of the population in industrialized nations. Its progression, advanced heart failure is an acute, potentially fatal condition. If a patients heart pumping function is insufficient, their only option of survival becomes either one of few available heart transplants or a mechanical circulatory support system. The latter, termed Ventricular Assist Devices (VAD) are mobile electromechanical pumps that can compensate for insufficient heart pump function. Since their invention in the 1960 they have continuously evolved, now representing a viable permanent alternative for patients ineligible for heart transplantation. However, the contact of blood with the artificial device material neccessitates an aggressive anticoagulation regime to prevent thrombosis. This causes bleeding and stroke, two major complications that persist in current generations of these devices. Housed within the umbrella of the Zurich Heart Consortium, the hybrid membrane project envisions to solve this problem by proposing a new type of VAD: A pulsatile device akin to a diaphragm pump featuring an endothelial cell (EC) layer on the entire interior, providing optimal haemocompatiblity. The most demanding challenge of this project is the maintenance of a stable endothelium on core part of the device, the cyclically deforming elastomer membrane actuating the blood. The ECs will thus be subjected to superphysiological wall shear stress up to 10 Pa while simultaneously experiencing the substrate deformation of around 10 %. From this ambitious goal derive two key challenges which are addressed within this thesis: (1) the establishment of an in vitro laboratory test system capable of replicating the stated conditions and (2) an surface-based solution capable of sustaining the EC on the elastomer membrane in this environment. In collaboration with other groups from the hybrid membrane track we successfully established such a bioreactor system. We showed that we could image an endothelialized elastomer membrane and expose it to flow. A computational fluid dynamics simulation revealed that shear patterns which are highest for the region where the flow hits the membrane as it is inflated into the channel. Actuating the membrane at 1 Hz to an apex displacement of about 8 %, we found a good correlation for the alignment of the cells with the simulation as the maximum shear was just above 5 Pa. Raising the flow to generate values of 10 Pa of maximum shear led to the eroding of cells from this zone. These results serve as a good validation of our system and showed that we could replicate the mode of failure for the new VAD that needs to be overcome by tailored engineering solutions. We then designed and fabricated a honeycomb-like surface whose wells could shelter up to 2 ECs, were found to increase the cell density (by 20 %) and proliferation (by 40 %) and would reduce the WSS at the bottom of the well by up to 60 % (as shown by CFD simulations). We subsequently evaluated endothelialized substrates bearing this topography in both a flow only system featuring cyclic olefin copolymer as the substrate as well as in the above described dynamic bioreactor test system featuring PDMS as a substrate. We found that after exposure to 8 Pa and 10 Pa for 18 h there were above 40 % more cells on the structured substrates and also the connectivity was significantly better. We also found 90% and 40% reductions of the nuclear localizations of the inflammatory markers V-CAM and I-CAM when comparring structured samples to flat controls. For combined loading of about 10 Pa of apex WSS and 5 % and 10 % of WD we found density ratios of about 2 fold % for structured vs. unstructured membranes. These results demonstrate that the introduction of the honeycomb topography is very promising with respects to EC retention under superphysiological loading conditions. Our two achievements represent important contributions towards the development of the envisioned novel VAD and might potentially see clinical integration in the near future.

Tendon and ligament tissue engineering aims to provide an alternative tissue graft for patients with acute injuries that require surgical intervention. Alternatives are necessary due to limited availability of allografts and autografts or other shortcomings in current biomaterial technologies. Tissue engineering accomplishes this through the combination of three key components: a cell source, scaffold and mechanical or chemical stimulation. While mechanical stimulation is the gold-standard for achieving tenogenic differentiation in progenitor cells, this work discusses many different methods of stimulation a differentiative response. Additionally, a great amount of recent work has been done in the field of biomaterials, and discovering new materials that can be combined with cells and stimulation techniques to achieve a more suitable graft. Additionally, further work is needed in optimizing the mechanostimulation regimen utilized in cultures. This work proposes a study in which shorter stimulation times are incorporated within more frequent rest periods to allow cells time to adapt and overcome refractory periods. Additionally, a new method of designing and producing bioreactors is proposed using fused deposition modeling, the most common 3D printing method, that allows for easy changing and rapid production of replacement parts. Finally, new protocols were developed to isolate infection sources in bioreactor cultures.

Hydraulic fracturing (hydro-fracking, HF) is widely used to extract oil, shale gas and coal bed methane. This exploration of energy reservoirs causes major challenges for water consumption and management, owing to the consumption of large volume of fresh waters and the generation of flow-back wastewater streams. Hence, there is a need for efficient and cost-effective flow-back wastewater treatment technologies. This flow-back water typically contains high levels of dissolved solids, including chloride and bromide salts, heavy metals, and hydrocarbons from natural sources as well as chemical additives from various stages of the HF process. In general, treatment of water from oil and gas exploration activities (hereafter referred to as OGWW) has occurred through either admixture to normal wastewater inputs or post-treated wastewater. However, to date, the impacts of such inputs, and in particular, the effects of high total dissolved solids (TDS) levels on secondary wastewater treatment have not been ascertained. The elevated TDS levels are of particular concern because conventional wastewater treatment is generally not effective at their removal. The effect of high salinity on the characteristics and performance of activated sludge in a bench-scale bioreactors was studied. The bioreactors were subject to salinity shocks by adding NaCl up to 50 g/L d to a synthetic municipal wastewater mimicking a medium strength influent. Relevant performance parameters were measured including mixed liquor suspended solids (MLSS), total dissolved solids (TDS), Nitrate, Ammonia, and Total Organic Carbon. In a second stage of the project, the efficiency of the bioreactors were studied to evaluate the treatment of actual hydraulic fracturing wastewater under aerobic conditions. The results indicate that increasing TDS concentrations resulted in lower COD removal rates and removal efficiencies. Additionally, high saline concentrations caused DO limitations so that nitrate was used up as electron donor.

Many food processers use salt, resulting in high strength, high salt content wastewater. The goal of this study was to determine the impact of salt concentration on the treatment of turkey slaughterhouse wastewater using sand/gravel bioreactors. Six unsaturated sand/gravel columns were intermittently dose treating the wastewater in a single pass. Turkey processing wastewater served as the control and 3 g/L and 6 g/L of table salt were added to wastewater for treatment is duplicate laboratory columns. BOD5 and NH3-N removal was measured during the 74-day experiment. The BOD5 removal achieved and maintained over 99% after day 21 at all salt levels. The NH3-N removal achieved over 99% removal after day 32. The conductivity of the effluent matched the influent indicating that the treatment system did not remove salt. It was concluded from this study that sand/gravel bioreactors were able to treat high strength, high salinity (up to 0.6%) turkey slaughterhouse wastewater.The biofilm developed on the sand/gravel surface is the functional part of wastewater treatment. In this study, ATP was measured to investigate the impact of salt on the biomass in the treatment system. Sand/gravel samples were collected from each layer of the columns in the wastewater control and wastewater plus 6 g/L salt. The results showed that salt had an initial inhibition on the biomass in the system. System performance decreased as the biomass declined. After the biomass reached a steady level, the treatment performance was maintained at high level. The study also found that the coarse sand layer was the most active layer treating wastewater.

Advancements in the field of biotechnology over the past few decades have provided solutions to many research problems and have paved the way to new scientific discoveries. From development of different applications of microbial fuel cells and discovery and characterization of new classes of microorganisms to progress in whole-genome sequencing and annotation methods, the world of biotechnology is expanding every day. Currently, the world is dependent on fossil fuels to meet its energy demand but for future, we have to invent new strategies to fulfill global energy needs. One strategy is the use of biofuels produced by genetically engineered microbes. The dissimilatory metal reducing bacteria like Shewanella oneidensis utilize metals including Fe(III), Mn(IV), and U(VI) as terminal electron acceptors for anaerobic respiration and possess the ability of extracellular electron transfer. This ability of extracellular electron transfer can be useful to many biotechnological applications, however there are three major technological challenges that must be addressed: scalability, control over operation process, and defined biological pathways. The dissimilatory metal reducing bacterium (DMRB) Shewanella oneidensis MR-1 is used as a model organism for studies in microbial fuel cells and bioelectrochemical reactors. In this study that follow, aspects related to increasing control of bioelectrochemical reactors were addressed. Work was done to improve the coulombic efficiency of S. oneidensis in reactors by improving design of anoxic bioreactors. The new reactors were found to have < 3 ppm oxygen and showed no planktonic growth. The current density was also higher in new reactors (23 µA/cm2) compared to 10 µA/cm2 in old bioreactors. The coulombic efficiency of S. oneidensis in new reactors was measured to be 86.4 ± 10.37%, a vast improvement over alternative electrochemical systems. Hydrogen metabolism in S. oneidensis biofilms was also studied in these new better controlled reactors, and the role of hydrogenases in electron transfer from S. oneidensis to electrodes in a bioreactor was studied. It was found that deletion of the hydrogenases in S. oneidensis bioreactors funneled the electron transfer to electrodes and improved the coulombic efficiency by 30% indicating their role in extracellular electron transfer. In summary, the improved bioelectrochemical system described herein will be useful in studying phenotypes of various mutants of S. oneidensis and other metal reducing bacteria under anaerobic conditions on electrodes of defined redox potential. This well-defined and robust bioelectrochemical system will aid the study of extracellular electron transfer and may provide a platform for the design of microbial fuel cells and the production of alternative fuels.

Biofilms grown on free-floating attachment surfaces, as in integrated fixed film activated sludg (IFAS) and moving bed bioreactors (MBBRs) are commonly used in wastewater treatment, but little is known about how media geometry affects the biofilms and process performance. The objective of this study was to compare nitrification and growth of biofilms grown on different length media in bench-scale MBBRs. The carriers were cut from high density polyethylene tubing with one media type one-third the length of the other, but with inner and outer diameter dimensions identical. Each bioreactor was continuously operated with coarse bubble aeration and provided with a high ammonium loading to promote greatly active nitrifying communities. Biomass measurements were taken regularly to observe growth. A series of variable velocity gradient (G) batch tests was executed to determine the effect of mixing on mass transfer through the biofilms of each media type. High ammonium and variable pH batch tests were also conducted to assess inhibition effects on nitrifiers. Greater biomass was consistently measured on the longer media despite both media types having similar ammonia uptake during continuous operation. Lower G values consistently produced greater ammonia utilization in the short media biofilm than in the long media biofilm. However, at mid to high range G values, ammonia consumption was similar between both biofilms. Ends of media typically had greater biomass and greater nitrate production than middle sections, while ammonia consumption was similar along carrier length. Abrupt changes in ammonium concentration and pH produced significantly greater inhibition effects in the short media biofilm than in the long media biofilm, suggesting greater protection in the biofilm grown on the longer media.

Bioreactor systems used for tissue engineering applications are an essential component of understanding the development of new tissues and studying the biochemical interactions between cells and their environment. A bioreactor is typically designed to mimic physiological, environmental, and mechanical stimuli that occur in vivo, and bioreactors are generally created for a specific application, such as for studying 3-dimensional tissues or dynamic fluid flow in 1-dimensional cell monolayers.The leading cause of death in the United States is coronary artery disease, which is treated with bypass graft surgery using a left internal mammary artery or human saphenous vein as the graft. Since human saphenous vein grafts often fail, investigating vascular function as a whole will help to understand more about the method of graft failure. A bioreactor system to study vascular function was successfully developed using the application of endothelial cells under shear stress in a microfluidic slide. The temperature control and diffusion rate of CO2 were recorded inside the bioreactor to confirm the system could stay within a temperature range of 37ºC +/- 0.5ºC and a CO2 concentration between 56,000 ppm and 45,000 ppm. Also, a physiological level of shear stress was determined to be feasible with the peristaltic pump. The performance characteristics of the bioreactor were analyzed, and the apparatus was determined to be successful in generating physiological relevant conditions. Then, human umbilical vein endothelial cells were exposed to both static conditions and venous shear stress conditions for up to four days in an IBIDI® microfluidic chamber. The cell morphology, alignment, and elongation were also evaluated. The cells stayed viable during the duration of all of the dynamic flow experiments, and the cells showed evidence of cell division. The cells were also more aligned and elongated towards the direction of flow for the 48 and 72 hour flow experiments compared to the 48 and 72 hour static experiments (P-value < 0.05). The 96 hour flow experiment cells were also more aligned than the cells exposed to static conditions (P-value < 0.05). The 48 hour, 72 hour, and 96 hour dynamic flow experiments had a statistically significant difference in cell alignment compared to the 24 hour flow test, and the 72 hour dynamic flow experiment also had a statistically significant difference in cell alignment compared to the 48 and 96 hour flow experiments (P-value < 0.05). The 72 hour flow experiment was more elongated than the 24, 48, and 96 hour flow experiments (P-value < 0.05). Overall, the lab setup and bioreactor system yielded desirable results and provided a system that was fully capable of studying endothelial cells under venous shear stress conditions for studies up to 4 days.

Woodchip and corn cob filled bioreactors have been shown repeatedly to be effective at reducing nitrate pollution from agricultural fields by supporting denitrifying bacteria. Little attention has been paid, however, to other microorganisms that may also proliferate in these environments. Of particular concern are sulfate reducing bacteria and other organisms known to convert mercury to highly toxic methylmercury, since supporting such organisms could lead to increased levels of methylmercury in downstream rivers and lakes. To investigate this concern, we conducted two studies. We measured total and methylmercury concentrations and related parameters in upflow column bioreactors filled with either woodchips or corn cobs. The temperature of the water pumped into the column bioreactors was in the range of 1.8�C to 18.6�C to simulate cooler autumn and winter weather. There was no significant mercury methylation detected at these temperatures. The concentration of methylmercury flowing out of the column bioreactors filled with corn cobs showed greater variability than that of woodchip filled bioreactors. The second study was set up to pump water with low concentrations of nitrate into four edge-of-field woodchip bioreactors to produce a residence time of 24 hours or more. We measured total and methylmercury concentrations along with other indicators of water chemistry and biological activity. While the conditions monitored in the bioreactors were consistent with documented conditions that support mercury methylating bacteria, we did not find evidence of methylmercury being generated within the bioreactors. These studies indicate that the production of methylmercury is not likely in Minnesota edge-of-field woodchip bioreactors or in woodchip or corn cob bioreactors where water temperatures are below 18.6�C and nitrates are not completely reduced.

Today´s rising demand for more reliable and affordable alternatives to organ transplant has led to a growing market for in vitro tissue culture systems. The main objective of tissue engineering as a whole is to employ human tissue equivalents for commercial use. The state-of-the-art processes for producing these so called tissue models are still very expensive, difficult to produce and time-consuming. To engineer tissues, in vitro, a three-dimensional support structure commonly termed scaffold is needed. Stem cells are then added to the scaffold. In order for tissue to materialize, the scaffold and the cells must be incubated (cultured) at a temperature of 37°C, 5.0% CO2, and 95% air concentration with relative humidity of 95- 99%, for a tissue engineered constructs to be viable. This requires a sterile environment, in which the tissue to be assemble. This is commonly accomplished using a petri dish inside an incubator. However, the tissue constructed is not of clinical application quality. Therefore, bioreactors come into play by exhorting physiochemical stimuli to further enhance the tissue engineered, in essence mimicking in vivo conditions and thus procuring quality construct for clinical use. Therefore, tissue engineering needs a paradigm shift in order to generate clinically useful products. The main objective of this line of research is to design, build and test Second Generation Incubator (SGI) systems that can simulate in vivo conditions under standard culture frames and sustain cell life to produce a viable tissue engineered construct. The SGI apparatus offers many features such as; ease of use, customizability, compatibility, portability and low cost, which current commercially available incubators and bioreactors lack. A two week experiment was conducted using adult human mesenchymal stem cells in conjunction with the gelatin scaffolds to form a viable bone tissue engineered construct. The results showed great thermal, gas and humidity regulation and with no adverse signs of contamination. Osteogenetic markers showed lesser osteogenetic levels in that of the SGI apparatus. Advisor: Huihui Xu

Growing algae as a source for bio-fuels has become an area of interest due to concerns about global warming and the reliability and ecology of the production of fossil fuels. Dried algae harvested from a pilot water quality improvement technology at the Rockaway Wastewater Treatment Facility in New York were examined as a source of carbohydrates and lipids for the production of bio-fuels in bio-reactors. The length of storage time, storage conditions, sugar and lipid extraction processes, and fuel production were studied. The results show that if the algae is stored dry (0.015 g/g algae even after a year in storage. The types of algae harvested has an effect on the amounts of sugars and lipids extracted, so two different methods to identify, monitor and quantify algae grown in both open and closed systems were evaluated. Capillary electrophoresis single strand conformational polymorphism (CE-SSCP) was able to identify known algae samples in an environmental system by "fingerprint" comparison, but may be most useful as a fast, accurate method of monitoring changes in the species in closed systems. We also examined and found capillary electrophoresis single base extension (CE-SBE) to be an extremely fast and accurate method to quantify the algae DNA of Chlorella vulgaris and Spirulina platensis in a closed system photo-bioreactor. A primer was designed that allowed the accurate correlation of the algae DNA amounts with the area under the curve in an electropherogram. This primer also distinguished between and quantified each species. CE-SBE demonstrated great potential for quantification of algae with difficult morphologies, and algae grown in a co-culture photo-bioreactor.

Animal cell culture is a widely used technology for producing recombinant proteins. The ability to make post-translational modifications and secrete the active forms of the protein into the culture medium represents major advantages over other processes. The growing market demand for pharmaceuticals has created a need for increased production capacity; however, achieving productivity gains in both the upstream stage and downstream processes can subject cells to aggressive environments such as those involving hydrodynamic stresses. Although numerous studies have explored the consequences of cell damage due to hydrodynamic stress, there has been a lack of understanding of the mechanism of such damage at a cellular level. Cell damage can also influence biomedical applications. Cells manipulated in instruments such as diagnosis and analysis devices can experience hydrodynamic forces.The level of cell damage is influenced by the hydrodynamic conditions in the bioprocess or biomedical equipment as well as the cell line sensitivity. To evaluate and compare cell sensitivity among different cell lines, a flow contraction device, previously designed by our group was used. Cells were exposed to well defined and controlled hydrodynamic forces and cell damage was estimated as a function of energy dissipation rate (EDR). EDR is a scalar value that represents the rate of dissipation of kinetic energy per unit of mass or volume. Using this methodology we found human cell lines highly sensitive to hydrodynamic forces.Hydrodynamic evaluations were performed in ten different bioreactor configurations Impeller-Sparger. The best configurations were chosen based on kLa response surface model for testing in cell culture experiments. The configurations chosen were used to evaluate the expression of stress proteins under moderate hydrodynamic stress in bioreactors as well as cell cycle profile and its relationship to recombinant protein production. The results suggest that for a clonal cell line evaluated G1 phase of the cell cycle may be more conducive to producing the recombinant protein. In addition, a relationship between hydrodynamic stress and expression of stress proteins was observed. The type of stress protein and the level of expression seem to be dependent on cell type and differences could even be observed between clones of the same cell line.Cell damage was also evaluated in a fluorescent activated cell sorter (FACS) models Vantage and Aria. Cells can be exposed to very high hydrodynamic forces when flowing through channels and nozzle in the sorting process. Results indicate that not only are cells damaged in a flow cytometer, but that this damage can vary from cell line to cell line as well as from specific conditions/type of flow cytometer and flow conditions. In addition, studies were conducted to evaluate cell growth behavior after stress as well as the effect of sorting on cell cycle. Extended growth lag phase was observed in cells exposed to hydrodynamic stress, and the sensitivity of any specific cell line can be a function of the growth phase of the cell

Overfishing of natural fisheries is a global issue that is becoming more urgent as the human population increases exponentially. According to the Food and Agriculture Organization of the United Nations, over 70% of the world's seafood species are fully exploited or depleted. This high demand for seafood protein is not going away; and, in fact, an astonishing one out of five people in this world depend on this source of protein. Traditional aquaculture practices use pond and flow-through systems which are often responsible for discharging pollutants into the environment. Furthermore, aquacultural feeds often contain high levels of fish protein, so the demand on wild fisheries is not completely eased. Even though traditional aquaculture has these drawbacks, there is a significant movement towards more sustainable practices. For example, implementing recirculating aquaculture systems (RAS) maximizes the reuse of culture water which decreases water demand and minimizes the levels of pollutants being discharged to the environment. And, alternative proteins (e.g., soy bean) are replacing the fish and seafood proteins in aquaculture diets. Accordingly, the research described in this dissertation focused on maximizing the reuse of freshwater fish effluent to culture marine shrimp. More specifically, by using suspended-growth biological reactors to treat a tilapia effluent waste stream and to generate microbial flocs that could be used to support shrimp culture. This RAS technology will decrease water consumption by increasing the amount of recycled water and will also improve effluent water quality. The biomass generated in the bioreactors could be used to feed shrimp with an alternative source of protein. Treating fish effluent to be reused to culture shrimp while producing this alternative feed, could significantly decrease operational costs and make these operations more sustainable. Understanding which ions are critical for the survival and normal growth of marine shrimp in freshwater effluents is essential. It is also very important to understand how to convert an effluent's organic matter into food for shrimp. Results from studies revealed that the marine shrimp, Litopenaeus vannamei, can be raised in freshwater effluent when supplemented with specific ions and wet microbial flocs fed directly to shrimp can enhance growth in shrimp fed a restricted ration of commercial feed. The treatability of the tilapia effluent using suspended-growth, biological reactors and nutritional analysis of the generated biomass were also reported. Carbon supplementation enhanced reactor performance and microbial floc generation. These microbial flocs also proved to be a superior feed ingredient when dried and incorporated into a pellet feed.

A stable mixed culture, deposited as ATCC 55644, previously shown to degrade petroleum hydrocarbons at relatively high concentrations was used as the source of inoculum. This culture was grown in Stanier’s minimal media, either in the presence of different concentrations of naphthalene, nitrobenzene and toluene (NNT) or naphthalene and toluene (NT) as the sole source of C and/or N. Results showed that the majority of the strains isolated from the mixed culture were able to grow in the presence of NNT or NT. A total of 20 different isolates were isolated from the mixed culture. Individual isolates were grown in Stanier’s minimal medium containing a single hydrocarbon as the source of carbon or carbon and nitrogen. Only one strain was found to grow solely in the presence of nitrobenzene as the source of C and N. Most of the other isolates were able to grow in the presence of naphthalene, toluene, acenaphthene, anthracene, fluoranthene and phenanthrene, n-dodecane, hexadecane, n-pentadecane, n-tetradecane, and n-octadecane. Planktonic and immobilized cells of the controlled mixed culture (ATCC 55644) were grown in separate Sequential Batch Reactors (SBR) using Stanier's media, to which naphthalene, nitrobenzene and toluene were added as the sole source of C and/or N. Biodegradation was determined by measuring the residual hydrocarbon in the SBR and the amount of trapped volatile organic carbon (VOC) and the evolved CO2. Gas chromatography data showed that immobilized cells were able to degrade NNT faster than the planktonic cells. This observation was confirmed by CO2 evolution. Over time the loading of hydrocarbon was significantly increased from a starting level of 400 ppm (Naphthalene), 100 ppm (Nitrobenzene), and 500 ppm (toluene), to a final level of 3000 ppm (Naphthalene), 400 ppm (Nitrobenzene), and 1600 ppm (toluene). While increasing nutrient loading, the frequency of re-feeding with hydrocarbons was changed from an initial re-feeding every 60 hrs to a final re-feeding frequency of 18 hrs. The experiments clearly showed that the attached, mixed microbial community was able to effectively and rapidly degrade high concentrations of hydrocarbons. This demonstrated the practical advantages of employing attached, mixed microbial cultures in a SBR.

This study focuses on the optimisation of submerged hollow fibre membrane performance by analysing the role of air sparging on the reduction of membrane fouling. In submerged hollow fibre membranes, rising bubbles have been shown to induce shear, liquid movement and fibre displacement. The interaction between fibre movement induced by bubbling and the microfiltration performance was assessed for various parameters (fibre tightness, fibre length, fibre diameter, air flowrate, nozzle size, and feed concentration). A model feed of yeast suspension and a series of isolated fibres were used. The fibre movement was assessed by monitoring the displacement using video recording. Bubble population parameters were also measured. The results suggest that bubbleinduced fibre movement plays an important role in controlling membrane fouling. Investigations of the critical flux at various operating conditions also supported these conclusions. Since energy consumption for aeration is a major contributor to the cost in submerged membranes, the potential to minimise the aeration cost has been tested by implementing intermittent aeration and different nozzle sizes. It was found that an optimum condition associated with a low fouling rate could be reached by combining various aeration intermittencies and nozzle sizes. An attempt to suppress fouling without aeration was made by incorporating vibrations into a submerged hollow fibre membrane system. The effects of vibration frequency, type of yeast (washed and unwashed) on the filtration performance were observed. The impact of coagulant addition on filtration enhancement was also analysed. The performance of microfiltration was evaluated based on its critical flux value. The findings in this preliminary study indicated potential fouling control by applying vibrations to submerged membranes. A semi-empirical model was developed to predict the filtration behaviour by taking into account the bubble-induced shear and fibre movement. The predicted critical flux values suggested that membrane fouling appears to be more prominent at low air flowrate, with tight fibres, and higher feed concentrations. The model fits the experimental data with discrepancies from approximately 0.3% to 20%. The predicted filtration profiles at different operating modes demonstrate the importance of bubble-induced shear and fibre movement in the improvement of filtration performance.

A common problem observed in large-scale cell cultivation is reduced culture performance compared with small-scale processes due to the existence of concentration gradients caused by poor mixing. Small-scale simulations using microbial cell suspensions have shown that circulation of cells through concentration gradients of oxygen, pH and glucose can result in reduction of cell growth and product formation similar to the effects observed in large-scale bioreactors. This study was aimed at using scale-down studies to investigate poor mixing in large-scale bioreactors used for suspended plant and animal cell culture. Two plant cell suspensions and a hybridoma cell line were used in this work. The range of oxygen transfer coefficients achieved in the hybridoma and plant suspensions were about 50−20 h-1 and 12−6 h-1, respectively. One-vessel simulation was developed to induce fluctuations of dissolved oxygen tension in a 2-L bioreactor using intermittent sparging of air and nitrogen. The effect of dissolved oxygen fluctuations on the cells was examined by comparing the performance of the cultures with those operated at constant dissolved oxygen tension. In the hybridoma suspension culture, only slight effects on cell growth were observed at circulation times above 300 s. No effect on the specific glucose uptake rate or antibody production was observed at the circulation times tested. Analysis of gene expression for selected hypoxia-related genes also suggested that the overall effect was limited. In plant cell suspensions, the specific growth rates and biomass yields on total sugar in the cultures under fluctuating dissolved oxygen tension were essentially the same as those at constant dissolved oxygen tension for both transgenic Nicotiana tabacum and Thalictrum minus. Under fluctuating dissolved oxygen tension, no effect on antibody accumulation was observed in transgenic N. tabacum suspensions, but a decrease in berberine accumulation was observed in T. minus. From the results, it can be concluded that only minimal effects due to the development of concentration gradients would be expected in large-scale bioreactors used for the cultivation of the hybridoma and plant cell suspensions tested in this work.

Petroleum and derivate fuels contain noticeable concentrations of Monocyclic Aromatic Hydrocarbons (MAHs) and Polycyclic Aromatic Hydrocarbons (PAHs) in their composition. MAHs and PAHs exhibit toxic carcinogenic properties and have been classified as priority pollutants by the U.S. EPA. Among these MAHs is the group forming the mixture known as BTEX, namely Benzene, Toluene, Ethyl Benzene, and Xylene. Methyl tert-Butyl Ether (MTBE) is a fuel oxygenate used extensively to reduce gasoline emissions. These compounds are frequently found in groundwater and surface water, as a consequence of leakages from storage tanks, spills at production wells, refineries, etc. PAHs have been detected as the main constituents in coal tar found contaminating water and soil at former Manufactured Gas Plant sites. Toxic PAHs include Naphthalene, 2-Methylnapthalene, and Acenaphthene. Biodegradation of these contaminants under aerobic conditions has been proven effective for the removal of these contaminants from water in the past.The objective of this study is to demonstrate that real groundwater from Millville, NJ, that has been contaminated with PAHs emanating from a former manufactured gas plant (MGP) and leakage from an oxygenated gasoline storage tank can be treated to obtain final concentrations of 5 µg/L or lower for each contaminant. Targeted contaminants include: MTBE, tert-Butyl Alcohol (TBA), Benzene, Toluene, Ethyl Benzene, p-Xylene, Naphthalene, 2-Methylnaphthalene, Acenaphthylene, Acenaphthene, and Carbazole.Two porous pot reactors were each inoculated using two different mixed bacterial cultures and then operated for a ten-month period in a continuous mode. The influent consisted of two streams: i) the contaminated groundwater buffered with sodium carbonate and ii) an acidified nutrient solution. The total influent flow was maintained at 9.44 L/day for Reactor 1 and 4.55 L/day for Reactor 2. The hydraulic retention time (HRT) was calculated as 0.64 and 1.32 days, respectively.In all cases more than 99% removal of all the investigated contaminants was achieved. All contaminants were measured at average concentrations below 1 µg/L in the reactor effluents, except in the cases of toluene (2.67 µg/L) and MTBE (2.80 µg/L) in Reactor 1. BTEX compounds exhibited the best removal efficiencies, in both reactors (>99.99%), in spite of their higher concentrations in the influent. MTBE removal rate was measured above 99.8%.This study showed that aerobic biodegradation in an ex-situ pump and treat reactor can be successfully used to remove the targeted PAHs, BTEX, MTBE and TBA in the groundwater. The final concentrations of all contaminants were below the current regulations for drinking water. The performance of both reactors did not present significant differences, although Reactor 2 performed better.

Macromolecular components, including protein and polysaccharides, are viewed as one type of major foulants in the complex feed membrane filtration systems such as membrane bioreactor (MBR). In this thesis, the mechanisms of macromolecular fouling including protein and polysaccharide in the complex feed solution are explored by using Bovine serum albumin (BSA) and alginate as model solution. During the filtration of BSA and washed yeast with 0.22 ¥ìm PVDF membrane, it was found that the critical flux of mixture solution was controlled by washed yeast concentration while the existence of BSA significantly changed the cake reversibility of much larger particles. The fouling mechanisms of alginate, as a model polysaccharide solution, were investigated both in dead end and crossflow membrane filtration. In the dead end experiments, it was found that the cake model appears to fit the entire range of the ultrafiltration data while the consecutive standard pore blocking model and cake model are more applicable to microfiltration membranes. The alginate was featured with high specific cake resistance and low compressibility despite some variations between different membranes. The specific cake resistance ( c ) is similar to c of BSA and actual extracellular polymer substance (EPS) in MBR systems reported in the literature, and higher than that of many colloidal particles. In a system contained alginate-particles mixture, it was found that the existence of alginate dramatically increased the cake specific resistance and decreased the cake compressibility. The fouling mechanism of alginate was also studied using long term cross flow filtration under subcritical flux. A two-stage TMP profile similar to that typically observed in MBR was obtained, confirming the important role of EPS during membrane fouling in MBR. In addition to adsorption, trace deposition of alginate also contributed to the initial slow TMP increase during the subcritical filtration. TMP increase during the long-term filtration was found not only due to the increase of the amount of deposition, but also the increase of specific cake resistance. A combined standard pore blocking and cake filtration model, using a critical pore size for the transition time determination, was developed and fit the experimental results well.

The purpose of this research was to develop the hollow fiber membrane (HFM) bioreactor system for treatment of chlorinated solvents in waste mixtures. This new technology employs a hollow fiber membrane reactor to separate chlorinated solvents from water or air with subsequent cometabolic biodegradation using a mutant methanotrophic microorganism, Methylosinus trichosporium OB3b PP358. Research objectives included demonstrating successful performance of the HFM bioreactor system for the treatment of trichloroethylene (TCE) contaminated water and air, increasing process efficiency for cost competitiveness with conventional technologies, extending HFM bioreactor studies to chlorinated solvent mixtures, and developing a system design strategy. HFM bioreactor demonstration experiments evaluated various process configurations, flow rates, and influent TCE concentrations. In TCE viii contaminated water experiments, mass transfer coefficients as large as 2.7×10-2 cm/min were estimated and the system was able to sustain an average first-order degradation rate constant of 0.57 L mg TSS-1 d-1 for 500 hours. While configuring the system with chlorinated solvents flowing through the HFM lumen and microorganisms flowing through the HFM shell was preferred for aqueous experiments, air treatment experiments required switching the flows because water vapor mass transfer resulted in water clogged fiber lumen. Following the demonstration experiments, shorter HFM hydraulic residence times between 0.16 and 0.57 minutes were investigated. A tradeoff between providing sufficient oxygenation and methanol stripping in the chemostat was observed. A new process decision variable, specific transformation was defined and values as large as 38.5 µg TCE/mg TSS were sustainable for TCE treatment. In HFM bioreactor experiments with a mixture of TCE and chloroform (CF), large TCE first-order degradation rate constants between 0.75 L mg TSS-1 d-1 and 1.08 L mg TSS-1 d-1 indicated that the addition of CF did not affect TCE degradation. CF mass transfer coefficients were approximately 10% less than TCE coefficients and the average CF degradation rate constant was 0.32 that of TCE. Computer models of mass transfer and biodegradation in the system were constructed and validated with experimental data. A modeling analysis was then conducted on the important decision variables and parameters and the results were used to develop a system design strategy.