<p class=MsoNormal><span style="mso-spacerun: yes">          </span>Stable isotopic analysis of nitrate (¹⁵N/¹⁴N and ¹⁸O/¹⁶O) was used to trace nitrate sources and cycling under undisturbed conditions and following harvest at the Turkey Lakes Watershed (TLW), located near Sault Ste. Marie, Ontario, Canada. <span style="mso-spacerun: yes">  </span> <p class=MsoNormal><span style="mso-spacerun: yes">    </span><span style="mso-spacerun: yes">      </span>Bulk precipitation collected biweekly at the TLW from 1995 to 2000 had nitrate isotope values that ranged from +42. 4 to +80. 4&permil; for <span style='font-family:Symbol'>d</span>¹⁸O and -6. 3 to +2. 8&permil; for <span style='font-family:Symbol'>d</span>¹⁵N. <span style="mso-spacerun: yes">  </span>An incubation experiment indicated that the isotopic composition of atmospheric nitrate was not compromised by collection methods whereby unfiltered bulk precipitation samples remain in the collector for up to two weeks. <span style="mso-spacerun: yes">  </span> <p class=MsoNormal><span style="mso-spacerun: yes">          </span>The first direct measurement of the isotopic composition of microbial nitrate produced <i>in situ</i> was obtained by eliminating precipitation inputs to three forest floor lysimeters and subsequently watering the area with a nitrate-free solution. <span style="mso-spacerun: yes">  </span>Microbial nitrate had <span style='font-family:Symbol'>d</span>¹⁸O values that ranged from +3. 1 to +10. 1&permil; with a mean value of +5. 2&permil;, only slightly higher than values predicted based on the <span style='font-family:Symbol'>d</span>¹⁸O-H₂O of the watering solution used. <span style="mso-spacerun: yes">  </span><span style='font-family:Symbol'>d</span>¹⁸O values of soil O₂ (+23. 2 to +24. 1&permil;) down to a depth of 55cm were not significantly different from atmospheric O₂ (+23. 5&permil;) and therefore respiratory enrichment of soil O₂ did not affect the <span style='font-family:Symbol'>d</span>¹⁸O values of microbial nitrate produced at the TLW. <span style="mso-spacerun: yes">  </span> <p class=MsoNormal><span style="mso-spacerun: yes">          </span>Nitrate export from two undisturbed first-order stream basins was dominated by microbial nitrate, with the contribution of atmospheric nitrate peaking at about 30% during snowmelt. <span style="mso-spacerun: yes">  </span>Clear-cutting of catchment 31 in 1997 resulted in elevated nitrate concentrations, reaching levels that exceeded the drinking water limit of 10 mg N/L. <span style="mso-spacerun: yes">  </span>Isotopic analysis indicated that the source of this nitrate was predominantly chemolithoautotrophic nitrification. <span style="mso-spacerun: yes">  </span>The <span style='font-family:Symbol'>d</span>¹⁸O values of microbial nitrate in stream 31 progressively increased during the post-harvest period due to an increase in the proportion of nitrification that occurred in the summer months. <span style="mso-spacerun: yes">  </span>Despite drastic alteration of nitrogen cycling in the catchment by the harvest, <span style='font-family:Symbol'>d</span>¹⁵N-nitrate values in shallow groundwater did not change from the pre-harvest. <span style="mso-spacerun: yes">   </span>Denitrification and plant uptake of nitrate in a small forested swamp in catchment 31 attenuated 65 to 100% of surface water nitrate inputs following harvest, reducing catchment-scale nitrate export by 35 to 80%.

This study explores the use of N2O and its isotopic composition to investigate nitrogen processes in groundwater aquifers. Groundwater sampling was undertaken in 2008-2009 at two septic system sites (Long Point site and Lake Joseph site) and two agricultural sites (Strathroy site and Woodstock site). All of these four sites have been studied previously, and denitrification zones were identified by using NO3- isotopes. Extremely broad ranges of N2O-N concentrations are present at septic system sites (1 to 1071 μg/L at Long Point and 0.1 to 106 μg/L at Lake Joseph). N2O concentrations at the agricultural sites show lower levels and narrower ranges (0.1 to 3.3 μg/L at Strathroy and 14.6 to 40.5 μg/L at Woodstock site). However, N2O-N concentrations at four sites except Strathroy are higher than the atmospheric equilibrium values (0.27 to 0.37 μg/L at 8 to 17°C) as well as N2O-N values in surface water (0.2 to 1.2 μg/L, Grand River). This provides indication of N2O production in subsurface in both septic system sites and agricultural sites. Using reported enrichment factors and measured ranges for NH4+ and NO3- isotopic values, ranges were calculated for the isotopic composition expected for N2O produced by nitrification and denitrification. At Long Point site, δ15N-N2O and δ18O-N2O ranging from -43.9 to +24.9 ‰ and +20.6 to +89.4 ‰ indicates that nitrification is mainly responsible for N2O accumulation in both proximal shallow and deep zones while some N2O at the bottom of the aquifer is presumably produced from denitrification. After N2O is produced in the plume core, δ15N and δ18O in N2O reveal that N2O is further consumed to N2. Also, N2O isotopic values cleanly show seasonal N2O production shifted from mostly nitrification in early season to primarily denitrification in late season. At Lake Joseph, δ15N-N2O and δ18O-N2O varying from -4.4 to -43.2 ‰ and +24.7 to +56.7 ‰ reveal that nitrification N2O was mainly present in aerobic zone whereas denitrification zone was found in downgradient anaerobic area. At Strathroy site, δ15N-N2O (+1.7 to -29.7 ‰) and δ18O-N2O (+33 to +65 ‰) show that N2O in shallow aquifer (< 5m depth) is presumably derived from atmosphere and nitrification whereas in deep aquifer (>5m depth), N2O formation occurs during denitrification. At Woodstock site, δ15N-N2O (-4.7 to -15.9 ‰) and δ18O-N2O (+30.7 to +37.1 ‰) at Woodstock provide indication of N2O production from a mixing of denitrification N2O and tropospheric N2O. N2O isotopic signatures are highly useful to identify N2O sources which include nitrification, denitrification, and dissolution of atmospheric N2O at both septic system sites and agricultural sites. Further, at Lake Joseph site and Woodstock site, denitrification evidence from NO3- concentration/isotopes is lacking but N2O isotopes suggest the occurrence of denitrification. At Long Point site, N2O isotopes indicated N2O production was by denitrification occurred early in the season; however, no NO3- isotopic enrichment was t that depth until in late season. These sites provide evidence that N2O is an early and sensitive indicator of denitrification in groundwater at both septic system and agricultural sites. Additionally, N2O isotopes are valuable for detecting N2O consumption whereas NO3- isotopes cannot provide insight into this process.

The biologically-mediated process of nitrification can occur in chloraminated drinking water distribution systems. In this process, ammonia is oxidized to nitrite by ammonia-oxidizing bacteria (AOB) and archaea (AOA). In complete nitrification, nitrite is further converted to nitrate by nitrite-oxidizers; however, bacterial mediation of this step is less critical as a chemical-oxidation pathway also exists. The initial conversion of ammonia to nitrite is also more critical due to its role in the degradation of the disinfectant residual. Nitrification is affected by factors such as the concentrations of ammonia and total chlorine, the pH of the drinking water, and the temperature. The key consequence of distribution system nitrification is an accelerated decay of the disinfectant residual; it can also lead to increases in nitrite and nitrate, and a potential proliferation of heterotrophic bacteria. The goal of this thesis is to enhance understanding of distribution system nitrification; one aspect to this goal is the evaluation of models for nitrification. The approach followed in this study was to collect water samples from two full-scale distribution systems in Southern Ontario. In the first phase, a sampling campaign was conducted at sites in these systems, with water samples being analyzed for parameters considered relevant to nitrification, such as the concentrations of nitrogen species affected by nitrification, the disinfectant residual, and the levels of ammonia-oxidizing microorganisms. In the second phase, batch tests were conducted with water from these same distribution systems. In the course of the field sampling campaign some indications of nitrification were detected, but there were no severe nitrification episodes as indicated by major losses of the disinfectant or prolonged elevations in nitrite levels. On some occasions at some sites there were small rises in nitrite above baseline levels; moderate declines in total chlorine residual were also seen. Nitrifying microorganisms were present in most samples, as detected by both culture-based and molecular methods (PCR). The latter was able to distinguish AOA from AOB; both were detected in the systems included in this study, with AOB gene counts outnumbering those of AOA at most sites. Using Spearman non-parametric correlations, significant correlations were found between some parameters relevant to nitrification. Notably, AOB were found to be positively correlated with heterotrophic plate counts (HPC), reinforcing the latter's role as a useful indicator of microbial regrowth conditions in a distribution system. Also of interest is the negative correlation between total chlorine residual and levels of microorganisms, reminding drinking water professionals of the value of maintaining a stable disinfectant residual. Batch testing investigations compared total chlorine decay curves between inhibited and uninhibited samples to provide insight into the microbial contribution to disinfectant decay. Four types of decay curves were identified, with qualitative differences in the microbial contribution to the disinfectant residual decay. Liquid chromatography with organic carbon detection (LC-OCD) was applied to investigate changes in the character of the dissolved organic carbon over the course of the batch tests. Based on the results of this study, it is recommended to evaluate the results of nitrification batch tests based on a visual identification of the curve type and calculation of the decay rates and critical threshold residual (CTR), rather than relying on the microbial decay factor alone to express the results. An application of this work was in making comparisons to some models for nitrification proposed in the literature. The ultimate goal of these models is to provide drinking water system operators with a prediction of when nitrification episodes will occur so that action may be taken to avert them. The models considered in this study differ in their degree of complexity and in whether they are based on mechanistic considerations. The differences in the underlying principles and data required for analysis make these models suitable for different applications. The results of this evaluation support the use of the model of Fleming et al. (2005) in full-scale distribution systems and the use of the model by Yang et al. (2008) for research applications, while the other models considered can still offer some useful insights. The results of this research can be applied to monitoring and operational practices in chloraminated distribution systems where nitrification is a potential concern. The correlations between parameters that have significance to distribution system nitrification that were found in this study, along with the modelling and batch testing evaluated in this work, can provide insight into predicting conditions favourable to nitrification and avoiding or averting nitrification episodes.

Pulse crops have been long associated with biological dinitrogen fixation and therefore improve the sustainability of cropping systems when included in rotation. However, studies indicate there may be additional benefits of including pulse crops in rotation. To quantify these potential benefits, soil processes and properties related to nitrogen (N) and carbon (C) cycling were examined in five crop rotations with and without field pea (<i>Pisum sativum</i> L.) in Scott, Saskatchewan. Gross mineralization and nitrification rates were determined using the 15N isotope dilution technique in intact soil cores. To estimate the proportion of nitrous oxide (N2O) emissions derived from nitrification related processes rather than denitrification processes tracer techniques using 15N were used. Field incubations were performed in 2008 at seeding (May 13), anthesis (July 8) and just after harvest (October 8). Mean mineralization and nitrification rates were not significantly different among rotations on any date and there was no significant difference in mean N2O emissions among rotations. From labeled 15NO3- cores, it was determined that nitrification-related processes were the major contributors to N2O emissions. There was no difference among the rotations in microbial biomass carbon (MB-C) or microbial biomass N (MB-N) with the exception of MB-C in the continuous field pea (FP) and the canola (<i>Brassica napus</i> L.)-wheat (<i>Triticum aestivum</i> L.)-field pea (CNL-W-FP) rotation at anthesis. There was no effect of rotation on dissolved organic carbon (DOC) and only seasonal differences were observed with DOC levels being lower before seeding than at anthesis and post-harvest. Based on the results obtained from a single growing season, our results show that N benefits of including field pea in rotation, beyond dinitrigen fixation, were not detectable and that the immediate N benefit of including field pea in rotation may be due simply to the direct effects of biological dinitrogen (N2) fixation. However, there have been reports of pulse crop benefits to succeeding crops in rotation. As a result, we investigated both the quantity and quality of crop residues, which can have an impact on soil properties and processes. Plants enriched with isotopic tracers can be used to trace crop residue decomposition to various C pools but only if the tracer is homogeneously distributed throughout the plant. In order to determine if repeat-pulse labeling could be used to trace crop residue decomposition, this method was followed using 13CO2 to enrich plant material of field pea and canola plants in a controlled environment. The distribution of 13C throughout the plant parts (roots, stem, leaves, and pod) and biochemical fractions [acid detergent fiber (ADF) and acid detergent lignin (ADL)] were determined. It was found that 13C was not homogeneously distributed throughout the plant parts or biochemical fractions. The pod fraction in particular was much less enriched in comparison to the other fractions. The ADL fraction was less enriched than the ADF fraction. Because of the heterogeneity of the label throughout the plant, modifications of the method are needed and 13C distribution through out the plant needs to be assessed before the repeat-pulse method can be used to trace C residue through various C pools. Nevertheless, root contributions to below-ground C were successfully determined from the enriched root material and the resulting enriched soil. It was found that canola contributed more above- and below-ground residues than field pea, however canola was also higher in ADF and ADL fractions indicating a more recalcitrant residue. Research should continue to better define the impact of pulse crop residues on C and N cycling and subsequent crops in rotation.

Ammonia, a colorless gas with a characteristic pungent odor, is produced by various industrial and agricultural activities. Emissions of ammonia into the atmosphere not only cause a nuisance in the vicinity of the sources, but also have various environmental effects, such as eutrophication and acidification of terrestrial and aquatic ecosystems, and visibility problems resulting from the formation of aerosols. The traditional treatment of ammonia emissions is based on physical and/or chemical processes, both of which are expensive and produce secondary pollutants. Biological methods are effective and economical for biodegradable odorants and VOC contaminants. This study used fixed-film bioreactors, a biofilter and a biotrickling filter, to remove and oxidize gas-born ammonia. Firstly, a pilot-scale biofilter consisted of two columns (40 cmW ¡Ñ 40 cmL ¡Ñ 70 cmH acrylic column) arranged in series. A medium consisting solely of fern chips, on which biofilms were cultivated, was used as a packing material. The biofilter was tested continuously for 110 days, measuring the removal efficiency, empty bed residence time (EBRT), removal capacity, pressure drop, moisture content and pH. Most of ammonia was eliminated in the first biofiltration column and the removal efficiency increased with the increase in EBRT. Complete removal of the influent ammonia (20-120 ppm) was obtained with an ammonia loading as high as 5.4 g N kg-1 dry media d-1 during the experiment. The Michaelis-Menten equation was tested to be adequate for modeling the ammonia elimination kinetics in the biofilter and the maximum removal rate (Vm) and the half-saturation constant (Ks) were estimated to be 28.2 g N kg-1 dry media d-1 and 129 ppm, respectively. Secondly, a pilot-scale reactor, consisting of a set of two-stage-in-series biotrickling filters, an influent gas supply system and a liquid recirculation system, was utilized to treat ammonia in an air stream. Each stage of the biotrickling filter was constructed from a 20 cm ¡Ñ 200 cm (inner diameter ¡Ñ height) acrylic column packed with cokes (average diameter = 3.0 cm, specific area = 150 m2/m3) of 125 cm height. Experimental results indicate that a time of 30 days is required for development of biofilms for nitrification of the absorbed ammonia from the gas. Long-term (187 days) experimental results show that, in the conditions of EBRT (empty bed gas retention time) = 7.25 s, ¡§circulation liquid/gas¡¨ flow rate ratio = 7.7 L m-3, and liquid pH = 6.65, the level of ammonia in the influent gas was reduced from 230 to 4.0 ppm. With the volumetric ammonia loading of less than 7.37 g NH3-N m-3 hr-1, the system could achieve ammonia removal and nitrification efficiencies of 98 and 94%, respectively, without supplementary glucose as a carbon source. However, with a loading of 13.1 g NH3-N m3 h-1, both decreased gradually due to a lake of carbon source and an accumulation of ammonium and nitrite ions in the recirculation liquid.

published_or_final_version / Civil Engineering / Master / Master of Philosophy

Οι διεργασίες βιολογικής απομάκρυνσης του αζώτου μέσω της νιτροποίησης και της απονιτροποίησης βρίσκουν σήμερα ευρεία εφαρμογή στην επεξεργασία τόσο των αστικών και των βιομηχανικών υγρών αποβλήτων όσο και στην προεπεξεργασία του πόσιμου νερού. Η νιτροποίηση (βιολογική οξείδωση της αμμωνίας) υλοποιείται από δύο διαφορετικές κατηγορίες αυτότροφων βακτηριών. Η πρώτη ομάδα (νιτρωδοποιητές) μετατρέπει την αμμωνία (+4 NH) σε νιτρώδη (−2NO) και στη συνέχεια η δεύτερη ομάδα, οι νιτρικοποιητές, οξειδώνει περαιτέρω το ενδιάμεσο προϊόν (νιτρώδη) σε νιτρικά. Η απονιτροποίηση είναι η βιολογική διεργασία, η οποία ευθύνεται για την απομάκρυνση του αζώτου με τη μορφή των νιτρικών και/ή νιτρωδών από τα απόβλητα μέσω μετατροπή τους σε αέριο άζωτο. Τα τελευταία χρόνια, γίνεται σημαντική ερευνητική προσπάθεια για να παρακαμφθεί το στάδιο της νιτρικοποίησης. Είναι επιθυμητό η αμμωνία να οξειδώνεται σε νιτρώδη και μετά απευθείας να λαμβάνει χώρα η απονιτροποίηση, παρά να γίνεται πρώτα η μετατροπή σε νιτρικά στα συστήματα απομάκρυνσης αζώτου. Θεωρητικά εξοικονομείται περίπου 25% σε δέκτη ηλεκτρονίων (οξυγόνο) και 40% σε δότη ηλεκτρονίων, ενώ επίσης ο ρυθμός απονιτροποίησης αυξάνεται κατά 63% με μικρότερη παραγωγή βιομάζας, οφέλη ιδιαίτερα σημαντικά από οικονομικής πλευράς, καθώς μειώνεται αρκετά το κόστος λειτουργίας της μονάδας επεξεργασίας αποβλήτων. Η παράκαμψη αυτή συνήθως επιτυγχάνεται ρυθμίζοντας κατάλληλα τη συγκέντρωση του διαλυμένου οξυγόνου, το pH και τη θερμοκρασία. Ο σκοπός της παρούσας εργασίας ήταν η εύρεση του βέλτιστου τρόπου λειτουργίας αντιδραστήρα SBR για την απομάκρυνση του αζώτου από τα λύματα με παράκαμψη της παραγωγής των νιτρικών μέσω κατάλληλης ρύθμισης του πλήθους και της διάρκειας των αερόβιων και ανοξικών φάσεων λειτουργίας του. Η παράκαμψη της νιτρικοποίησης επιτεύχθηκε για λειτουργία του συστήματος με 12ωρο κύκλο, με 3 ζεύγη αερόβιας/ανοξικής φάσης και αναλογία φάσεων 2:3. Η μείωση της διάρκειας του κύκλου λειτουργίας σε 8 ώρες οδήγησε σε εξίσου ικανοποιητική απόδοση, ταυτόχρονα όμως επιτρέπει την επεξεργασία μεγαλύτερου όγκου αποβλήτου στο ίδιο χρονικό διάστημα. / -

This study explores the use of N2O and its isotopic composition to investigate nitrogen processes in groundwater aquifers. Groundwater sampling was undertaken in 2008-2009 at two septic system sites (Long Point site and Lake Joseph site) and two agricultural sites (Strathroy site and Woodstock site). All of these four sites have been studied previously, and denitrification zones were identified by using NO3- isotopes. Extremely broad ranges of N2O-N concentrations are present at septic system sites (1 to 1071 μg/L at Long Point and 0.1 to 106 μg/L at Lake Joseph). N2O concentrations at the agricultural sites show lower levels and narrower ranges (0.1 to 3.3 μg/L at Strathroy and 14.6 to 40.5 μg/L at Woodstock site). However, N2O-N concentrations at four sites except Strathroy are higher than the atmospheric equilibrium values (0.27 to 0.37 μg/L at 8 to 17°C) as well as N2O-N values in surface water (0.2 to 1.2 μg/L, Grand River). This provides indication of N2O production in subsurface in both septic system sites and agricultural sites. Using reported enrichment factors and measured ranges for NH4+ and NO3- isotopic values, ranges were calculated for the isotopic composition expected for N2O produced by nitrification and denitrification. At Long Point site, δ15N-N2O and δ18O-N2O ranging from -43.9 to +24.9 ‰ and +20.6 to +89.4 ‰ indicates that nitrification is mainly responsible for N2O accumulation in both proximal shallow and deep zones while some N2O at the bottom of the aquifer is presumably produced from denitrification. After N2O is produced in the plume core, δ15N and δ18O in N2O reveal that N2O is further consumed to N2. Also, N2O isotopic values cleanly show seasonal N2O production shifted from mostly nitrification in early season to primarily denitrification in late season. At Lake Joseph, δ15N-N2O and δ18O-N2O varying from -4.4 to -43.2 ‰ and +24.7 to +56.7 ‰ reveal that nitrification N2O was mainly present in aerobic zone whereas denitrification zone was found in downgradient anaerobic area. At Strathroy site, δ15N-N2O (+1.7 to -29.7 ‰) and δ18O-N2O (+33 to +65 ‰) show that N2O in shallow aquifer (< 5m depth) is presumably derived from atmosphere and nitrification whereas in deep aquifer (>5m depth), N2O formation occurs during denitrification. At Woodstock site, δ15N-N2O (-4.7 to -15.9 ‰) and δ18O-N2O (+30.7 to +37.1 ‰) at Woodstock provide indication of N2O production from a mixing of denitrification N2O and tropospheric N2O. N2O isotopic signatures are highly useful to identify N2O sources which include nitrification, denitrification, and dissolution of atmospheric N2O at both septic system sites and agricultural sites. Further, at Lake Joseph site and Woodstock site, denitrification evidence from NO3- concentration/isotopes is lacking but N2O isotopes suggest the occurrence of denitrification. At Long Point site, N2O isotopes indicated N2O production was by denitrification occurred early in the season; however, no NO3- isotopic enrichment was t that depth until in late season. These sites provide evidence that N2O is an early and sensitive indicator of denitrification in groundwater at both septic system and agricultural sites. Additionally, N2O isotopes are valuable for detecting N2O consumption whereas NO3- isotopes cannot provide insight into this process.

Agriculture management including tillage and crop residues impact the functioning of soil microbiota. Soil microbiota cycle nutrients, with greenhouse gases being a byproduct within the cycle. The main objectives of this thesis were to 1) assess tillage and corn residue impact on N-cycling soil microorganisms and N2O emissions in situ (Chapter 3); and 2) evaluate N-cycling soil microorganisms in situ relative to N2O flux during a spring thaw cycle (Chapter 4). In situ sampling addresses how changing field conditions influence soil bacterial processes. Results indicated tillage and removal of corn residue declined soil microbial abundance and increased N2O emissions. These responses were dependent on local environmental conditions; moisture, carbon and nitrogen availability. The spring thaw study highlighted N-cycling microorganisms were present and active over the spring thaw event, and delayed nosZ denitrifier activity was related to the timing of significant N2O emission events, suggesting new evidence of de novo denitrification. / Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) and Natural Science and Engineering Research Council (NSERC)

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.