Reciprocating biofilters known as ReCip is a viable technology to manage nutrients, mainly nitrogen, problems at livestock operations such as swine farms and dairies. Past studies have demonstrated that ReCip is more adept at total nitrogen (TN) removal than traditional subsurface flow wetland systems. The traditional substrate used to attach biomass was rock aggregate; this media may be hard to obtain for some agricultural projects, so alternate substrates are tested and compared with the rock aggregate. The purpose of the study was twofold: first, different biofilm media were tested and compared in terms of treatment performance and, second, the long-term performance of a ReCip in continuous operation for 3 years was characterized. Four, 2.67 square meter ReCip systems with different treatment media – rock aggregate, recycled concrete aggregate (RCA), vertical-flow plastic media, and walnut shells – were operated at a 2-day THRT over the course of a 16 week study. The TN removal efficiencies for rock aggregate, RCA, plastic media, and walnut shell media were 43%, 53%, 25%, and 69% respectively. Surface based mass TN removal rates for the same media were 103, 128, 172, and 276 kg/ha-d respectively. A 134.2 square meter ReCip with rock aggregate media was running concurrently with the smaller ReCip systems. This ReCip was constructed ant operated since January 2010. TN removal efficiency and mass removal rate were 44% and 105 kg/ha-day. These values were close to results from the smaller rock media system.

Compared to conventional nitrification and denitrification, anaerobic ammonium oxidation (anammox) is a more energy saving and cost effective process for biological nitrogen removal (BNR). To date, the anammox process has been applied widely and designed mainly to treat sidestream wastewaters. However, only 15%-20% of the influent domestic sewage nitrogen loading is present in the sidestream, while the bulk of it still needs to be removed from the mainstream. Research efforts thus have shifted from sidestream to mainstream applications of anammox, including the application of anammox bioreactors at low temperature, low influent ammonium strength, and under the presence of organic carbon (characteristic of municipal mainstream wastewaters). In this dissertation research, the applicability of anammox process in lab-scale and full-scale mainstream systems have been studied. The overall goals of this dissertation research were (1) to develop an effective strategy to enrich an anammox moving bed biofilm reactor (MBBR) under low influent nitrogenous substrate concentration and ambient temperature (23 Cº), and link microbial ecology to the process performance of the enriched anammox MBBR; (2) to explore the catabolism and anabolism of anammox bacteria in a mainstream MBBR before and after dosing of organic carbon; (3) to extend the strategy of mainstream anammox enrichment under ambient temperature (23 Cº) to low temperature (15 Cº) , and link microbial ecology to the process performance; (4) to evaluate the microbial community structure, kinetics and performance during startup and long-term operation of a full-scale mainstream anammox process; (5) to investigate the reliability of the new enriched mainstream anammox MBBR under the imposition of additional wet weather flow; (6) to develop a reliable and sensitive mothed of hydrazine determination in anammox reactor. First, an anammox MBBR was successfully enriched under low nitrogenous substrate and ambient temperature. It needs to be addressed that, even with the limited fraction of Candidatus “Kuenenia stuttgartiensis” in the coming inoculum from the sidestream MBBR, Candidatus “Kuenenia stuttgartiensis” was effectively enriched in the biofilm biomass of the mainstream MBBR. Moreover, the enhanced activity of Candidatus “Kuenenia stuttgartiensis” was demonstrated through this whole time series experiments, and achieved the most competitive level among all functional groups. Therefore, the importance and necessity of bioaugmentation are addressed during the enrichment of mainstream anammox process. Second, successful enrichment of a mainstream anammox moving bed biofilm reactor was accomplished at low nitrogenous substrate and low temperature. 16S amplicon sequencing was employed to investigate the microbial ecology of the biomass in the biofilm and suspension. Results showed the dominance of Candidatus "Kuenenia" related anammox bacteria in the biofilm of mainstream reactor, though Nitrospira spp. related nitrite oxidizing bacteria were still detected in a limited fraction. These results are crucial to show the effective enrichment of anammox reactor by bioaugmentation even under low temperature, especially in a practical way. Third, the performance, kinetics and microbial ecology were studied before, during and after the imposition of additional organic carbon. The dosing of organic carbon resulted in a reversible negative impact on both the activity of AMX and the reactor performance. Stable isotope probe and 16S amplicon sequencing were applied to investigate the metabolism of functional groups. The results showed anammox bacteria are not capable of assimilating acetate, while the community assimilating 13C-labeled acetate was mainly assigned to denitrifiers. Presence of denitrifiers were observed in the mainstream MBBR and stayed inactive without sufficient organic carbon. In sum, these results demonstrate that the mainstream anammox process as tested was resilient to a short period imposition of organic carbon. Fourth, the performance and microbial ecology of the ambient-temperature mainstream anammox were investigated under wet weather condition. Based on the full recovery of reactor performance as well as the stable microbial ecology, the applicability of the mainstream MBBR under wet weather conditions was demonstrated. Fifth, real-time polymerase chain reaction was applied to evaluate the startup and operation of two parallel sidestream DEMONTM systems as well as the initiation of the mainstream anammox process through bioaugmentation. Results provided the evidence that anammox bacteria was the most abundant functional group in two parallel DEMONTM systems, showing the successful startup in the sidestream. Furthermore, anammox bacteria were selectively retained in the mainstream with high bioaugmentation rates from the sidestream. These results are critical to demonstrate the significance of bioaugmentation in the startup of mainstream anammox system even in full-scale wastewater water treatment plant. Finally, a sensitive and reliable spectrophotometric method was proposed to measure hydrazine concentration in anammox reactor. The concentration of hydrazine could be precisely determined in the presence of nitrite, when a certain amount of sulfamic acid is introduced. In sum, the application and characterization of anaerobic ammonium oxidation (anammox) process to treat sidestream and mainstream wastewaters in both lab-scale and full-scale was investigated in detail. From a practical perspective, the knowledge gained can lead to a better design and operation of engineered nitrogen removal process.

Membrane biofilm reactors (MBfR) utilize membrane fibers for bubble-less transfer of gas by diffusion and provide a surface for biofilm development. Nitrogen removal was attempted using MBfR in various configurations - nitrification, denitrification and consecutive nitrification and denitrification. Effects of loading rate and dissolved oxygen on nitrification performance were primarily investigated in a stand-alone nitrifying MBfR. Specific nitrification rate increased linearly with specific loading rate, up to the load of 3.5 g N/m²d. Beyond that load, substrate diffusion limitation inhibited further increase of specific nitrification rate. 100% oxygen utilization was achievable under limited oxygen supply condition. Effects of mineral precipitation, dissolved oxygen and temperature on hydrogenotrophic denitrification were investigated in a stand-alone denitrifying MBfR. Mineral precipitation, caused by intended pH control, caused the deterioration of denitrification performance by inhibiting the diffusion of hydrogen and nitrate. Operating reactor in various dissolved oxygen conditions showed that the denitrification performance was not affected by dissolved oxygen in MBfR. Optimum temperature of the hydrogenotrophic denitrification system was around 28°C. Total nitrogen removal in a two-step MBfR system incorporating sequential nitrification and hydrogen-driven autotrophic denitrification was investigated in order to achieve nitrogen removal by autotrophic bacteria alone. Long-term stable operation, which proved difficult in previous studies due to excessive biofilm accumulation in autotrophic denitrification systems, was attempted by biofilm control. Nitrification performance was very stable throughout the experimental periods over 200 days. Performance of autotrophic denitrification was maintained stably throughout the experimental periods, however biofilm control by nitrogen sparging was required for process stability. Biofilm thickness was also stably maintained at an average of 270 µm by the gas sparging biofilm control. According to the cost analysis of denitrifying MBfR, hydrogenotrophic denitrification can be an economical tertiary treatment option compared to conventional denitrifying filter although its economic feasibility highly depends on the cost of hydrogen gas. Although this study was conducted in a lab-scale, the findings from this study can be a valuable stepping stone for larger scale application and open the door for system modifications in future.

Membrane biofilm reactors (MBfR) utilize membrane fibers for bubble-less transfer of gas by diffusion and provide a surface for biofilm development. Nitrogen removal was attempted using MBfR in various configurations - nitrification, denitrification and consecutive nitrification and denitrification. Effects of loading rate and dissolved oxygen on nitrification performance were primarily investigated in a stand-alone nitrifying MBfR. Specific nitrification rate increased linearly with specific loading rate, up to the load of 3.5 g N/m²d. Beyond that load, substrate diffusion limitation inhibited further increase of specific nitrification rate. 100% oxygen utilization was achievable under limited oxygen supply condition. Effects of mineral precipitation, dissolved oxygen and temperature on hydrogenotrophic denitrification were investigated in a stand-alone denitrifying MBfR. Mineral precipitation, caused by intended pH control, caused the deterioration of denitrification performance by inhibiting the diffusion of hydrogen and nitrate. Operating reactor in various dissolved oxygen conditions showed that the denitrification performance was not affected by dissolved oxygen in MBfR. Optimum temperature of the hydrogenotrophic denitrification system was around 28°C. Total nitrogen removal in a two-step MBfR system incorporating sequential nitrification and hydrogen-driven autotrophic denitrification was investigated in order to achieve nitrogen removal by autotrophic bacteria alone. Long-term stable operation, which proved difficult in previous studies due to excessive biofilm accumulation in autotrophic denitrification systems, was attempted by biofilm control. Nitrification performance was very stable throughout the experimental periods over 200 days. Performance of autotrophic denitrification was maintained stably throughout the experimental periods, however biofilm control by nitrogen sparging was required for process stability. Biofilm thickness was also stably maintained at an average of 270 µm by the gas sparging biofilm control. According to the cost analysis of denitrifying MBfR, hydrogenotrophic denitrification can be an economical tertiary treatment option compared to conventional denitrifying filter although its economic feasibility highly depends on the cost of hydrogen gas. Although this study was conducted in a lab-scale, the findings from this study can be a valuable stepping stone for larger scale application and open the door for system modifications in future.

Biological nutrient removal (BNR) from wastewater, and specifically nitrogen removal, is a growing concern to wastewater dischargers such as municipalities. Excess nutrients in effluent can create problems such as eutrophication, toxicity to aquatic life, and dissolved oxygen depletion in receiving waters. BNR systems have been installed in many locations with success, but their operation presents operational and financial demands greater than conventional biological treatment. Nitrogen removal is typically performed in sequential autotrophic nitrification and denitrification, which increases needed energy input, operational complexity, and therefore cost. Simultaneous nitrification-denitrification (SNdN) achieved in a single system has also been successfully implemented, however operational parameters that compromise between ideals for aerobic nitrification and anoxic denitrification result in decreased reaction rates and removal efficiencies. The application of a product that could potentially enhance SNdN reaction rates and removal efficiencies through bioaugmentation could help ease operational and financial strains. In contrast to common sequential processes, some heterotrophic Bacillus bacteria have demonstrated SNdN (Kim et al., 2005), (Zhang et al., 2011). However, their application outside of laboratory setting has yet to be established. Aqua™ is a proprietary bioaugmentation product composed of specific Bacillus strains developed by BiOWiSH® Technologies with the intent of improving aerobic, heterotrophic SNdN rates and removal efficiencies. Screening and bench-scale experiments were performed in flasks at 35° C on orbital shakers operated at a range of speeds. Primary wastewater and minimal media were used for the experiment, and inoculation was performed with both specific Bacillus strains and Aqua™. Rapid total ammonia nitrogen (TAN) removal was observed in initial screening experiments with Aqua™ in sterile wastewater. Bacillus pumilus was identified as the fastest growing organism of the Aqua™ assemblage with the greatest TAN removal 1st order rate constant (0.32/ hr.), decreasing TAN 96% within 10 hours from an initial 48.5 ppm. The orbital shaker speed that maximized TAN removal was 100 rpm, with reduction 47% and 88% more effective than both the upper (150 rpm) and lower (50 rpm) bound tested speeds, respectively. Visible floc growth centered in flasks, along with optical density data indicated cell growth and the possibility the system could support SNdN. Carbon amendments to minimal media were then evaluated, and sodium succinate improved TAN reduction by 53% compared to dextrose amended systems. This was likely because dextrose metabolism requires glycolysis to produce pyruvate for utilization in the TCA cycle for energy production; while succinate avoids glycolysis and thus is more easily utilized. In another experiment, flasks with supplemental trace minerals had a 59% higher TAN removal than the controls. Additions of supplemental vitamin solution or yeast extract improved TAN removal by 18% and 38%, respectively. Two 10-day experiments assessed Aqua™ performance in municipal primary clarifier effluent. Nitrogen balance and optical density data showed that Aqua™ dosing at 10 ppm had no effect on nitrogen removal. The second 10-day experiment increased Aqua™ dosing to 50 ppm and evaluated product activation through incubation in growth media prior to inoculation. Nitrogen balance analysis showed no effect from Aqua™ on nitrogen removal during the second 10-day experiment as well. Systems amended with dextrose saw an initial rapid TAN first order removal rate (0.25/ hr.). However, difference between control and inoculated flasks was negligible showing no effect from Aqua™. A lack of total nitrogen losses and a lack of nitrate presence during initial rapid TAN losses confirmed these losses were by assimilation into organic nitrogen. The above experiments suggest that initial success in TAN removal during screening experiments resulted from lack of competition with other microorganisms, the high 1500 ppm dose of Aqua™, and amended dextrose.

published_or_final_version / Botany / Master / Master of Philosophy

published_or_final_version / abstract / Civil Engineering / Master / Master of Philosophy

Dissertation submitted in compliance with the requirements for the Master's Degree in Technology: Biotechnology, Technikon Natal, 2001. / Up until now extensive work has been done to develop kinetic models and related software that can be used successfully to simulate and design nitrification denitrification (ND) and nitrification denitrification biological excess phosphorus removal (NDBEPR) systems for efficient nitrogen removal. The denitrification kinetics of these systems have primarily been determined and attributed to the ordinary heterotrophic bacteria, now also known as the OHO fraction, otherwise not involved in biological excess phosphorus removal. However, denitrification kinetics determined for ND systems have been found to vary considerably at times when applied to NDBEPR systems because of varying OHO active fraction estimates and the unexplained occurrence of anoxic phosphorus removal and anysuccess achieved to date has been some what fortuitous. Ultimately variations in process performance and kinetics are attributable to inadequate control and lack of understanding of the ecological, physiological and biochemical activities of constituent microorganisms. There is growing concern and movement towards a better understanding of the microbial community within activated sludge in order to gain optimal control of the process. / M

Nitrite (NO2-) substrate under certain conditions can cause failure of N-removal processes relying on anaerobic ammonium oxidizing (anammox) bacteria. Detoxification of NO2- can potentially be achieved by using exogenous nitrate (NO3-). In this work, continuous experiments in bioreactors with anammox bacteria closely related to “Candidatus Brocadia caroliniensis” were conducted to evaluate the effectiveness of short NO3- additions to reverse NO2- toxicity. The results show that a timely NO3- addition immediately after a NO2- stress event completely reversed the NO2- inhibition. This reversal occurs without NO3- being metabolized as evidence by lack of any 30N2 formation from 15N-NO3-. The maximum recovery rate was observed with 5 mM NO3- added for 3 days; however, slower but significant recovery was also observed with 5 mM NO3- for 1 day or 2 mM NO3- for 3 days. Without NO3- addition, long-term NO2- inhibition of anammox biomass resulted in irreversible damage of the cells. These results suggest that a short duration dose of NO3- to an anammox bioreactor can rapidly restore the activity of NO2--stressed anammox cells. On the basis of the results, a hypothesis about the detoxification mechanism related to narK genes in anammox bacteria is proposed and discussed.

Many rural communities in Manitoba use wastewater lagoons to treat sewage, but the nutrient removal process is not fully understood. This thesis’ purpose is to improve understanding of chemical and biological nutrient removal mechanisms of wastewater lagoon treatment and compare two different stabilization ponds – one aerated and one facultative. Samples were collected from stabilization ponds and analysed for a pond average. The facultative lagoon achieved overall ammonia-N removals similar to those of the aerated lagoon, and lower orthophosphate removals. Nitrogen appears to be removed by ammonia volatilization; and assimilation into biomass. Phosphorus appears to be removed by assimilation into biomass; and precipitation at alkaline pH. There appears to be nitrogen limiting conditions in the secondary cells of both stabilization systems based on nitrogen-phosphorus ratios. There does not appear to be any significant advantage between aerated or facultative lagoons; they will meet their ammonia limits, but will require additional phosphorus treatment. / October 2016