Partial denitrification/anammox (PdNA) is an emerging biological nutrient removal (BNR) process that can be used to remove ammonia (NH3) and NOx from wastewater. This process is a combination of partial denitrification (PdN), which serves to reduce nitrate (NO3) to nitrite (NO2), and anaerobic ammonia oxidation, or anammox (AMX), which uses the nitrite as an electron acceptor to oxidize ammonia. PdNA provides significant aeration and external carbon savings when compared to the conventional nitrification/denitrification biological removal process for nitrogen but has been difficult to implement in mainstream treatment conditions due to many factors. One of these factors is the slow growth rate and startup time of anammox bacteria. This research first focused on determining the required startup time and startup optimization methods for a proposed mainstream polishing PdNA MBBR at Hampton Roads Sanitation District's James River Treatment Plant (JRTP). These two MBBRs were started with either virgin carriers or carriers coated with a preliminary biofilm and were fed secondary effluent The MBBRs were dosed with glycerol based on a feedforward carbon control approach and were not seeded with anammox at any point. Anammox activity was detected in the preliminary biofilm and virgin media MBBRs approximately 52 and 86 days after startup, respectively. Based on these results, starting up a mainstream PdNA reactor without seed is possible, and using preliminary biofilm carriers can speed up startup by approximately one month. A second experiment was conducted to determine the carbon demand and nitrogen removal capabilities of a glycerol fed PdNA MBBR and AMX MBBR in series. A nitrifying MBBR was also added to the MBBR train to test how well residual nitrite leaving the MBBRs could be polished off to limit ozone/disinfectant demand downstream. Additionally, a methanol-fed PdNA integrated fixed-film activated sludge (IFAS) reactor was also operated to determine the carbon demand and nitrogen removal capabilities for a PdNA process in an IFAS reactor. The PdNA and AMX MBBRs had average effluent TIN concentrations of 3.75 ± 1.25 and 2.81 ± 1.21 mg TIN/L, respectively, with a COD dosed per TIN removed ratio (COD/TIN) of 2.42 ± 0.77 g COD/g TIN for the entire process. The PdNA IFAS reactor had average effluent TIN concentrations of 4.07 ± 1.66 mg/L and 3.30 ± 0.96 mg/L at hydraulic retention times (HRTs) of 30 and 25 minutes. At these two HRTs, the PdNA IFAS process had a COD/TIN ratio of 1.08 ± 0.38 and 2.18 ± 0.99 g COD/g TIN, respectively. Overall, this indicated that both the PdNA MBBR and IFAS processes could reach low effluent TIN limits in mainstream conditions with low demand for COD, even with relatively low and unstable PdN efficiencies. Additionally, the nitrifying MBBR managed to keep the effluent nitrite concentration consistently below 0.5 mg/L at ammonia and nitrite influent loadings rates of 0.055 ± 0.035 and 0.379 ± 0.112 g N/m2/day. This research demonstrated that starting a PdNA process in mainstream conditions, without seed, can be accomplished within a reasonable timeframe and provides knowledge that can help engineers better understand the advantages of PdNA and design and startup mainstream polishing PdNA MBBRs and IFAS reactors. / Master of Science / As the human population continues to grow and wastewater discharge requirements continue to become more stringent, researchers and engineers have been exploring new technologies and methods to treat wastewater more efficiently. Once such method that is currently being explored is the integration of anaerobic ammonia oxidation, or anammox (AMX), bacteria with a variety of wastewater treatment technologies to remove nitrogen more efficiently from wastewater. AMX synchronously remove ammonia, which exists naturally in wastewater, and nitrite through an oxidation/reduction reaction in which the nitrogen leaves the wastewater in the form of dinitrogen gas. This process greatly reduces the amount of aeration and external carbon needed for the removal of nitrogen from wastewater compared to the commonly used method of full nitrification and denitrification, which are large operational costs at a wastewater treatment plant. While AMX have found use at full-scale plants in treating concentrated sidestreams with the use of partial nitrification (PN) to produce nitrite for the AMX, little progress has been made to integrate AMX into a full-scale mainstream treatment process where the stream is less concentrated and not ideal for consistent PN. Partial denitrification (PdN), however, has shown some promise in reliably producing nitrite in mainstream conditions for AMX usage. On top of the demand for nitrite, AMX bacteria also grow very slowly compared to most bacteria, which means these processes require relatively large amounts of time to get started. A common strategy for decreasing the startup time of AMX processes has been the addition of AMX biomass to a reactor during startup, but this is not feasible in a full-scale mainstream process due to the large amount of biomass that would be required. Therefore, other methods for startup optimization must be evaluated, which this study sought to do through two startup experiments in separate mainstream polishing moving bed biofilm reactors (MBBRs), which use plastic carriers to develop biofilms of bacteria. These two MBBRs were started with different types of carriers in them, one with carriers coated with a pre-established preliminary biofilm and one with brand-new, virgin carriers, to see what kind of effect these different types of carriers have on AMX startup time. AMX activity was detected in the preliminary biofilm and virgin media MBBRs approximately 52 and 86 days after startup, respectively, which was much quicker than expected. This indicates that starting up a mainstream PdNA reactor without seed is possible and using the preliminary biofilm carriers can speed up startup by approximately one month. After the startup experiment, one of the MBBRs was converted to a PdNA integrated fixed-film activated sludge (IFAS) reactor through the addition of activated sludge. This PdNA IFAS reactor was operated alongside a PdNA MBBR and AMX MBBR to test their nitrogen removal and carbon savings capabilities. Operation of these reactors demonstrated that both a PdNA MBBR or IFAS process are capable of consistently removing nitrogen to low levels with relatively low amounts of external carbon addition, even with inconsistent PdN. Overall, this research provided valuable insight into startup methods and design requirements of PdNA MBBRs and IFAS reactors which will make the implementation of these treatment processes more feasible.

O processo de tratamento de esgoto por lodo ativado pode ser adaptado para o recebimento de maior carga orgânica ou para a remoção de nitrogênio por meio da introdução de suportes plásticos móveis, em um processo conhecido por IFAS Integrated Fixed-Film Activated Sludge. O objetivo do projeto é avaliar comparativamente os desempenhos dos sistemas de lodo ativado e IFAS na remoção de matéria orgânica e nitrogênio de esgoto doméstico, associados à variação da idade do lodo com referência na biomassa suspensa. O efeito da adição de suportes plásticos móveis em sistemas existentes de lodo ativado é avaliado como forma de subsidiar análises de viabilidade de emprego dessa solução para a ampliação e adaptação de estações de tratamento de esgotos domésticos. A pesquisa foi desenvolvida em escala piloto, mantendo dois sistemas em funcionamento em paralelo, um representando um sistema de lodo ativado com remoção de nitrogênio e outro idêntico, exceto para a introdução dos suportes plásticos móveis. Desta forma, foi possível atribuir a diferença nos resultados à presença de biomassa aderida. Foram utilizados elementos suporte com área superficial específica de 300 m²/m³ e fração de enchimento de 50%. Ambos os sistemas foram mantidos em operação estável e eficiente, considerando a remoção de matéria orgânica. No entanto, o sistema IFAS teve melhor desempenho na remoção de nitrogênio em todas as fases experimentais, confirmando as vantagens antecipadas. Os resultados foram verificados em termos de taxas de aplicação previstas para cada porção de biomassa, de acordo com as idades do lodo estudadas. / The activated sludge wastewater treatment process can be retrofitted to either receive larger organic loads or for nitrogen removal by introducing plastic media carriers, in a process known as IFAS Integrated Fixed-Film Activated Sludge. The project aims to comparatively assess the performances of activated sludge and IFAS systems in removing organic matter and nitrogen from domestic sewage, associated to the variation of the sludge age with reference to the suspended biomass. The effect of adding plastic media carriers on existing activated sludge systems is evaluated as a subsidy for prefeasibility analysis of using this solution for the upgrading and retrofitting of municipal wastewater treatment plants. The study was developed on a pilot scale, operating two systems in parallel, representing an activated sludge system with nitrogen removal and another identical system except for the introduction of plastic media carriers. Thus, it was possible to assign the difference in results to the presence of attached biomass. Carriers were used with 300 m²/m³ specific surface area and 50% filling fraction. Both systems were kept under stable and efficient operation considering the removal of organic matter. However, the IFAS system had better performance at removing nitrogen in all experimental phases, confirming the anticipated advantages. The results were verified in terms of application rates expected for each portion of biomass in accordance with the sludge ages studied.

Submitted by YEMALL ALEXANDER MAIGUAL ENRIQUEZ (alex.feisunesp@gmail.com) on 2018-05-08T00:18:49Z No. of bitstreams: 1 TESE MBBR-ICT-7-MAY-2018.pdf: 5856438 bytes, checksum: f1c36146463af4ee146d48da7c3be308 (MD5) / Approved for entry into archive by Bruna Bacalgini null (bruna@sorocaba.unesp.br) on 2018-05-08T14:17:18Z (GMT) No. of bitstreams: 1 enriquez_yam_dr_soro.pdf: 5856438 bytes, checksum: f1c36146463af4ee146d48da7c3be308 (MD5) / Made available in DSpace on 2018-05-08T14:17:18Z (GMT). No. of bitstreams: 1 enriquez_yam_dr_soro.pdf: 5856438 bytes, checksum: f1c36146463af4ee146d48da7c3be308 (MD5) Previous issue date: 2018-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A aquicultura é uma atividade de produção de espécies hidrobiológicas sob condições controladas. Esta atividade tem evoluído nos processos produtivos, mas estão gerando águas residuárias, quando lançadas diretamente nos corpos de água constituindo riscos potenciais à saúde. O reator de leito móvel com biofilme (MBBR) é uma variação do processo de lodos ativados. É um sistema de tratamento de efluentes que contem biofilme que cresce aderida aos suportes, que se movem livremente no volume do reator. O objetivo desta pesquisa foi determinar a eficiência na remoção de material, nitrogenado no MBBR dos efluentes gerados num sistema de recirculação para aquicultura “SRA”. O sistema MBBR com circulação (SC) - e com aeração (SA) obteve remoções medias de 38,7% para Nitrogênio Total Kjedahl (NTK), 28,9% de fósforo total, 44,5% de DBO, Nitrogênio Amoniacal Total (NAT) de 40% e 38,9% para amônia não ionizada e para nitrito foi 59,3%. Os sólidos Totais (ST), Sólidos Suspensos Totais (SST) e Sólidos Voláteis Totais (SVT) tiveram remoções de 28,0%, 45,9% e 57,4% respectivamente. Durante o período avaliado houve aumento do biofilme nos dois reatores. No MBBR-SA foi verificada a presença de protozoários, microinvertebrados e estruturas filamentosas semelhantes a estreptococos. No MBBR-SC, bactérias na forma de cocos, diplococos e estreptococos. Nas analises realizadas com MEV nos lodos dos dois sistemas foi encontrado traços de silício, alumínio, zinco, fósforo, sódio e magnésio. Também foi encontrado bactérias do tipo vibrio spp, bacilus spp, além da formação de aglomerados. As analises térmicas feitas nos lodos indicaram que houve perda de umidade até os 150°C. A maior parte da massa se perde entre 220°C e 580°C, faixa onde sofrem transformação térmica as proteínas, lipídeos, carboidratos, ácidos graxos, restos de paredes celulares bacterianas. Após os 600°C existe o restante, as cinzas são compostas por carbonatos e outros minerais transformando-se em matérias cristalinos, com baixa variação na massa. / Aquaculture is an activity of production of hydrobiological species under controlled conditions. These activities have evolved in the productive processes, but are producing wastewater, when thrown directly into bodies of water constituting potential health risks. The moving bed biofilm reactor (MBBR) is a variation of the activated sludge process. It is an effluent treatment system containing biofilm growing attached to the carriers, which move freely in the reactor volume. The aim of this research was to determine the efficiency in the removal of nitrogenous compounds in MBBR from the effluents generated in a recirculating aquaculture system "RAS". The MBBR system with circulation (CS) - and with aeration (AS) obtained mean removals of 38.7% for NTK, 28.9% of total phosphorus, 44.5% of BOD, 40% TAN and 38.9% for non-ionized ammonia and for nitrite was 59.3%. Total Solids (TS), Total Suspended Solids (TSS) and Total Volatile Solids (TSV) had removals of 28.0%, 45.9% and 57.4% respectively. During the evaluated period there was an increase of biofilm in the two reactors. In the MBBR-AS the presence of protozoa, microinvertebrates and filamentous structures similar to streptococcus was verified. Also, in MBBR-CS was verified coccus, diplococcus and streptococcus bacteria. In the analyzes performed with SEM in the sludge of the two systems, traces of silicon, aluminum, zinc, phosphorus, sodium and magnesium. Vibrio and bacillus type bacteria were present and besides granule formation. Thermal analyzes indicated that there was in the sludge moisture loss until 150°C. Most of the mass is loss between 220°C and 580°C, where they undergo thermal transformation of proteins, lipids, carbohydrates, fatty acids, remains of bacterial cell walls. After the 600°C the ashes are composed of carbonates and other minerals transforming into crystalline materials, with low mass variation. / CAPES: 12616-13-3

O processo de tratamento de esgoto por lodo ativado pode ser adaptado para o recebimento de maior carga orgânica ou para a remoção de nitrogênio por meio da introdução de suportes plásticos móveis, em um processo conhecido por IFAS Integrated Fixed-Film Activated Sludge. O objetivo do projeto é avaliar comparativamente os desempenhos dos sistemas de lodo ativado e IFAS na remoção de matéria orgânica e nitrogênio de esgoto doméstico, associados à variação da idade do lodo com referência na biomassa suspensa. O efeito da adição de suportes plásticos móveis em sistemas existentes de lodo ativado é avaliado como forma de subsidiar análises de viabilidade de emprego dessa solução para a ampliação e adaptação de estações de tratamento de esgotos domésticos. A pesquisa foi desenvolvida em escala piloto, mantendo dois sistemas em funcionamento em paralelo, um representando um sistema de lodo ativado com remoção de nitrogênio e outro idêntico, exceto para a introdução dos suportes plásticos móveis. Desta forma, foi possível atribuir a diferença nos resultados à presença de biomassa aderida. Foram utilizados elementos suporte com área superficial específica de 300 m²/m³ e fração de enchimento de 50%. Ambos os sistemas foram mantidos em operação estável e eficiente, considerando a remoção de matéria orgânica. No entanto, o sistema IFAS teve melhor desempenho na remoção de nitrogênio em todas as fases experimentais, confirmando as vantagens antecipadas. Os resultados foram verificados em termos de taxas de aplicação previstas para cada porção de biomassa, de acordo com as idades do lodo estudadas. / The activated sludge wastewater treatment process can be retrofitted to either receive larger organic loads or for nitrogen removal by introducing plastic media carriers, in a process known as IFAS Integrated Fixed-Film Activated Sludge. The project aims to comparatively assess the performances of activated sludge and IFAS systems in removing organic matter and nitrogen from domestic sewage, associated to the variation of the sludge age with reference to the suspended biomass. The effect of adding plastic media carriers on existing activated sludge systems is evaluated as a subsidy for prefeasibility analysis of using this solution for the upgrading and retrofitting of municipal wastewater treatment plants. The study was developed on a pilot scale, operating two systems in parallel, representing an activated sludge system with nitrogen removal and another identical system except for the introduction of plastic media carriers. Thus, it was possible to assign the difference in results to the presence of attached biomass. Carriers were used with 300 m²/m³ specific surface area and 50% filling fraction. Both systems were kept under stable and efficient operation considering the removal of organic matter. However, the IFAS system had better performance at removing nitrogen in all experimental phases, confirming the anticipated advantages. The results were verified in terms of application rates expected for each portion of biomass in accordance with the sludge ages studied.

L'objectif de cette thèse est d'évaluer le concept d'un dispositif intégré comprenant un bioréacteur à biofilm fluidisé bio-augmenté, couplé à une membrane de nanofiltration de type polyelectrolyte multicouche, destiné à éliminer les micropolluants en traitement tertiaire des eaux usées domestiques, traitées conventionnellement. Les résultats montrent que, pour des micropolluants ciblés, chacun des procédés est efficace comme traitement tertiaire. Les mécanismes biologiques et de rétention membranaires sont explicités. Cependant des challenges restent à relever en particulier pour l'étape de bio augmentation (survie et implantation de la souche apportée) pour une exploitation de cette étape. D'autre part, des investigations plus poussées sont nécessaires à l'élaboration d'une membrane fiable et robuste. Un tel procédé couplé MBBR-NF pourra alors être entièrement justifié dans le contexte d'une élimination performante de micropolluants ciblés. Il aura toute sa place dans le panel des technologies vertes pour la préservation de l'environnement. / This thesis aims at answering whether the concept of an integrated layout comprised of a coupled "bioaugmented moving bed biofilm reactors (bMBBRs) - polyelectrolyte multilayer (PEM)-based nanofiltration (NF) membrane" can be considered as a promising technology to eliminate target MPs from conventionally-treated municipal wastewater. Results presented herein indicate that each given component of the layout is efficient in the tertiary removal of MPs. Still, several challenges ahead of the process bioaugmentation (such as the survival and maintenance of inoculated strains) must be in-depth studied to find convenient operating solutions. On the other hand, further investigations are definitely needed to achieve a robust PEM-based membrane as a long-lasting technology. Even though a coupled bMBBR-NF system for enhanced MPs removal can be experimentally justified is, however, practically questionable. "The tale of bMBBR-NF" deserves much more scientific endeavors as plenty of environmental considerations are placed in, whereby achieving a future Green technology will not be far from our expectation.

This research focuses on anoxic ammonium oxidation (anammox). The anammox process for treating high ammonium and low organic carbon wastewater can reduce operational costs to a greater extent than the conventional autotrophic/heterotrophic treatment process can. The process has been widely researched because of its potential economic benefits. However, during long-term reactor operation, sudden reductions of nitrogen removal rates have been reported; maximum nitrogen removal rates in different reactor configurations could not approach values predicted based on mathematical modeling; and the crucial stability parameter, such as nitrite, did not have defined threshold concentration. It was hypothesised that free ammonia (FA) increase is the precursor of the instability of the anammox reactor. If it is true that nitrite up to about 200 mg N/L should stimulate nitrogen removal rate inside of the anammox reactor, when FA is kept below the inhibition threshold concentration. The research presented in the thesis argues that FA plays a larger role than has been previously considered in the instability of the anammox reactor. This study found FA inhibited nitrogen removal rates (NRR) at concentrations exceeding 2 mg N/L. In the pH range 7 to 8, the decrease in anammox activity was independent of pH and related only to the concentration of FA. Nitrite concentrations of up to 200 mg N/L did not negatively affect nitrogen removal rate. This study further found that low nitrite provided stable anammox reactor performance, but that high nitrite was not necessarily the cause for reactor destabilization. During the research high nitrogen removal rate was achieved when low FA was provided. During regular reactor operation at pH 6.5, the NRR at about 6.2 g N/Ld was archived. This value was never achieved before till this study was conducted. Conducted research showed controlling FA at low level is required to approach high rates in anammox reactors. Achieving high rates in anammox reactors allow significant reduction in reactor volume which saves resources. Further studies will be required to identify the FA effect on different microbial interactions, and that may provide more in-depth understanding of the nitrite and FA effect than observations based on NRR alone.

New federal regulations with regards to ammonia in wastewater effluent discharge will require over 1000 existing wastewater treatment facilities to be upgraded. Although biological treatment is the most common and economical means of wastewater ammonia removal, nitrification rates can be completely impeded at cold temperatures. Moving bed biofilm reactors (MBBR) have shown promise as an upgrade nitrifying unit at pilot-scale and full-scale applications with respect to low temperature nitrification. MBBR technologies offfer the advantages of less space requirement, utilizing the whole tank volume, no sludge recycling, and no backwashing, over other attached growth systems. Two laboratory MBBRs were used in this study to investigate MBBR nitrification rates at 20deg.C, after long-term exposure to 1deg.C, and at the kinetic threshold temperature of 5deg.C. Furthermore, the biologically produced solids from the MBBR system 20deg.C and after long-term exposure to 1deg.C, and the Arrhenius temperature correction models used to predict nitrification rates after long-term exposure to 1deg.C. The nitrification rates at 1deg.C over a four month exposure period as compared to the rate at 20deg.C were 18.7 + 5.5% and 15.7 + 4.7% for the two reactors. The nitrification rate at 5deg.C was 66.2 + 3.9% and 64.4 + 3.7% compared to the rate measured at 20deg.C for reactors 1 and 2, respectively, and as such was identified as the kinetic temperature threshold. The quantity of solids detached from the nitrifying MBBR biocarriers was low and did not vary significantly at 20deg.C and after long-term exposure to 1deg.C. Lastly, a temperature correction model based on exposure time to cold temperatures, developed by Delatolla et al. (2009) showed a strong correlation to the calculated ammonia removal rates relative to 20deg.C following a gradual acclimatization period to cold temperatures. Biofilm morphology along with biomass viability at various depths in the biofilm were investigated using variable pressure electron scanning microscope imaging (VPSEM) and confocal laser scanning microscope (CLSM) imaging in combination with viability live/dead staining. The biofilm thickness along with the number of viable cells showed significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant changes. Hence, this study observed higher cell activities at warm temperatures and a slightly greater quantity of biomass with lower activities at cold temperatures in nitrifying MBBR biofilms. Using DNA sequencing analysis, 'Nitrosomonas' and 'Nitrosospira' (ammonia oxidizers)as well as 'Ntrospira' (nitrite oxidizer) were identified in which no population shift was observed during 20deg.C and after long-term exposure to 1deg.C. Furthermore, a number of non-nitrifiers were identified int he biofilm during warm and cold temperatures presenting the possibility that their presence may have provided some form of protection to the nitrifiers during long-term temperature exposure.

This research focuses on anoxic ammonium oxidation (anammox). The anammox process for treating high ammonium and low organic carbon wastewater can reduce operational costs to a greater extent than the conventional autotrophic/heterotrophic treatment process can. The process has been widely researched because of its potential economic benefits. However, during long-term reactor operation, sudden reductions of nitrogen removal rates have been reported; maximum nitrogen removal rates in different reactor configurations could not approach values predicted based on mathematical modeling; and the crucial stability parameter, such as nitrite, did not have defined threshold concentration. It was hypothesised that free ammonia (FA) increase is the precursor of the instability of the anammox reactor. If it is true that nitrite up to about 200 mg N/L should stimulate nitrogen removal rate inside of the anammox reactor, when FA is kept below the inhibition threshold concentration. The research presented in the thesis argues that FA plays a larger role than has been previously considered in the instability of the anammox reactor. This study found FA inhibited nitrogen removal rates (NRR) at concentrations exceeding 2 mg N/L. In the pH range 7 to 8, the decrease in anammox activity was independent of pH and related only to the concentration of FA. Nitrite concentrations of up to 200 mg N/L did not negatively affect nitrogen removal rate. This study further found that low nitrite provided stable anammox reactor performance, but that high nitrite was not necessarily the cause for reactor destabilization. During the research high nitrogen removal rate was achieved when low FA was provided. During regular reactor operation at pH 6.5, the NRR at about 6.2 g N/Ld was archived. This value was never achieved before till this study was conducted. Conducted research showed controlling FA at low level is required to approach high rates in anammox reactors. Achieving high rates in anammox reactors allow significant reduction in reactor volume which saves resources. Further studies will be required to identify the FA effect on different microbial interactions, and that may provide more in-depth understanding of the nitrite and FA effect than observations based on NRR alone.

New federal regulations with regards to ammonia in wastewater effluent discharge will require over 1000 existing wastewater treatment facilities to be upgraded. Although biological treatment is the most common and economical means of wastewater ammonia removal, nitrification rates can be completely impeded at cold temperatures. Moving bed biofilm reactors (MBBR) have shown promise as an upgrade nitrifying unit at pilot-scale and full-scale applications with respect to low temperature nitrification. MBBR technologies offfer the advantages of less space requirement, utilizing the whole tank volume, no sludge recycling, and no backwashing, over other attached growth systems. Two laboratory MBBRs were used in this study to investigate MBBR nitrification rates at 20deg.C, after long-term exposure to 1deg.C, and at the kinetic threshold temperature of 5deg.C. Furthermore, the biologically produced solids from the MBBR system 20deg.C and after long-term exposure to 1deg.C, and the Arrhenius temperature correction models used to predict nitrification rates after long-term exposure to 1deg.C. The nitrification rates at 1deg.C over a four month exposure period as compared to the rate at 20deg.C were 18.7 + 5.5% and 15.7 + 4.7% for the two reactors. The nitrification rate at 5deg.C was 66.2 + 3.9% and 64.4 + 3.7% compared to the rate measured at 20deg.C for reactors 1 and 2, respectively, and as such was identified as the kinetic temperature threshold. The quantity of solids detached from the nitrifying MBBR biocarriers was low and did not vary significantly at 20deg.C and after long-term exposure to 1deg.C. Lastly, a temperature correction model based on exposure time to cold temperatures, developed by Delatolla et al. (2009) showed a strong correlation to the calculated ammonia removal rates relative to 20deg.C following a gradual acclimatization period to cold temperatures. Biofilm morphology along with biomass viability at various depths in the biofilm were investigated using variable pressure electron scanning microscope imaging (VPSEM) and confocal laser scanning microscope (CLSM) imaging in combination with viability live/dead staining. The biofilm thickness along with the number of viable cells showed significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant changes. Hence, this study observed higher cell activities at warm temperatures and a slightly greater quantity of biomass with lower activities at cold temperatures in nitrifying MBBR biofilms. Using DNA sequencing analysis, 'Nitrosomonas' and 'Nitrosospira' (ammonia oxidizers)as well as 'Ntrospira' (nitrite oxidizer) were identified in which no population shift was observed during 20deg.C and after long-term exposure to 1deg.C. Furthermore, a number of non-nitrifiers were identified int he biofilm during warm and cold temperatures presenting the possibility that their presence may have provided some form of protection to the nitrifiers during long-term temperature exposure.

Water and wastewater treatment solutions incorporating biofilm systems are becoming increasingly popular due to more stringent regulations pertaining to drinking water and wastewater effluent discharge in Canada and in other parts of the world. As a major component of biofilm, extracellular polymeric substances (EPS) have been considered as an important factor affecting the physical and chemical properties of biofilm. Further, the selected method of EPS extraction and the methods of detecting the composition of the EPS have shown to affect the results of EPS measurements. In this research, protocols for EPS extraction and EPS composition analysis were investigated and optimized for nitrifying moving bed biofilm reactor (MBBR) biofilm. In addition, the confocal Raman microscopy (CRM) spectra of EPS in nitrifying MBBR biofilm and the protein, polysaccharide and extracellular DNA (eDNA) percent concentrations of the EPS were investigated at various operating temperatures. Further, the CRM spectra and the protein, polysaccharide and eDNA percent concentration of EPS in nitrifying MBBR biofilm along with the biofilm morphology and thickness and the viability of the embedded cells were investigated at various hydraulic retention times (HRTs). The EPS was characterized at various temperatures and HRTs in order to investigate potential correlation between the EPS components of the nitrifying biofilm and the ammonia removal kinetics. The biofilm morphology and thickness along with the bacterial viability of the biofilm were also investigated at various HRTs. Biofilm morphology images and thickness measurements were acquired using a variable pressure scanning electron microscope (VPSEM). The percentages of viable embedded cells in the biofilm were quantified using live/dead staining in combination with confocal laser microscopy (CLSM) imaging. The research demonstrates that an increase in protein content and subsequently a decrease in polysaccharides and eDNA contents in the EPS of nitrifying MBBR biofilm were observed at the lowest operational HRT and the highest temperature in this work. In particular, the EPS protein to polysaccharide (PN/PS) ratio of nitrifying MBBR systems was shown to significantly decrease below a value of 3 when the system was underloaded (observed at the highest operational temperature in this study) or hydraulically overloaded (observed at the lowest HRT in this study). As such, data obtained at lower operational temperatures, with the system no longer underloaded, and at longer HRTs, with the system no longer hydraulically overloaded, all demonstrate an EPS PN/PS ratio of approximately 3. Correlations were observed between the chemically measured EPS PN/PS ratios and the measured Raman spectra intensity ratios; supporting the concept of higher PN/PS ratios of EPS in more optimal nitrifying MBBR operations. Further, the ammonia removal kinetics and EPS response at HRT values of 0.75 and 1.0 h indicate that nitrifying MBBR systems may be optimized to operate at HRTs as low as 0.75 to 1.0 hour as opposed to conventional HRTs of 2.0 to 6.0 h.