Partial denitrification anammox (PdNA) is an emerging wastewater treatment technology with the potential to increase process capacity and save on energy and carbon. PdNA circumvents potential issues with stability of the more familiar mainstream partial nitritation anammox (PNA) process. The PdNA process can be used to effectively remove ammonia, nitrate, and nitrite from mainstream municipal waste streams. To retain slow growing anammox, some sort of retention system is needed with media being a common solution to this problem. PdNA has been successfully implemented in mainstream full-scale systems in sand filters and with moving media. The goal of this study was to assess the denitrifying capabilities, anammox treatment capacity, and effective surface area to volume of two types of fixed media. A nitrifying pilot was set up to assess the effective surface area to volume. To assess the nitrifying and anammox ammonia removal capabilities of the fixed media, a fixed media PdNA system was installed in the second anoxic zone of a full-scale municipal wastewater treatment plant. The fixed media system consisted of three modules of sheets modified to mimic a plug flow system. After accounting for the estimated nitrate removal from mixed liquor, denitrification rates normalized to media surface area were 0.52 +/- 1.9 g/m2-day in the first module, 0.62 +/- 0.91 g/m2-day for the second module, and 0.56 +/- 0.90 g/m2-day for the third module. In ex situ batch testing it was found that maximum ex-situ anammox ammonia removal rates for the / Master of Science / Urban population growth has created a two-pronged problem for wastewater treatment plants. Plants in populated areas are seeing increases in flow along with growing space restrictions that limit new infrastructure construction. Additionally, rising environmental awareness from the public has spurred regulatory agencies to impose tighter limits on the quality of water leaving plants and entering sensitive watersheds. These factors have driven a need for treatment techniques that allow plants to operate better with their existing equipment. Overall, this concept is known as process intensification. One such method that treatment plants are using to intensify wastewater treatment is the addition of plastic media into their existing tanks. This media provides additional surfaces for the microorganisms that biodegrade the pollutants in the wastewater to grow and allows waste to be treated faster in the same area. It also allows slow growing organisms to be retained in the system that would otherwise not have time to grow. Such slow-growing microbes are especially critical for the removal of ammonia, a toxic form of nitrogen that occurs in high concentrations in wastewater. The partial denitrification-anammox process is an intensification process that leverages microbial metabolisms to convert nitrate to nitrite instead of denitrifying the nitrate all the way to nitrogen gas. Plants then let more ammonia pass through the aeration zone, where ammonia is converted to nitrate. The bleed through ammonia and the nitrite generated from partial denitrification is used by microbes called anammox, which denitrify without the addition of carbon. The full denitrification process requires externally added carbon, which is energy intensive to produce and expensive, and aeration requires energy to run the aeration blowers. Bypassing the full denitrification process using PdNA results in two-fold cost and energy savings. The plastic media help slow-growing anammox bacteria attach and grow to achieve this aim. Most of the plants that use plastic media use media that is free floating in the tank. However, certain plants cannot use this floating plastic media because it can either plug up the system, or flow to the end of the treatment tank and have no way to get back to the front. In instances such as these it could be beneficial to use a type of media that is fixed in place. One potential use of fixed media that has never been tried before is with partial denitrification with anammox. This research sets out to evaluate the effectiveness of fixed media with use in a partial denitrification anammox process and compare it to a treatment tank of moving media that is present at the same plant to find out whether it may be a viable option for retrofitting plants that cannot use moving media.

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.

The research in this thesis was conducted at the Hampton Road Sanitation District's biological nutrient removal pilot, located at the Chesapeake-Elizabeth WWTP in Virginia Beach, VA. The pilot is operated in an A/B process with a high-rate, carbon-diverting A-stage, followed by a biological nitrogen removal B-stage containing four intermittently aerated CSTRs, followed by an anammox polishing MBBR. The goal of this research was to successfully combine short-cut nitrogen removal with sidestream enhanced biological nutrient removal (EBPR) in the most efficient way possible, specifically aiming to decrease cost and energy requirements, divert the most amount of carbon possible before B-stage, and to achieve low effluent nitrogen and phosphorus concentrations. A RAS fermenter (SBPR) and an A-stage WAS fermenter that feeds VFA into the SBPR (the supernatant of the fermenter is called fermentate) were implemented in order to enhance biological phosphorus removal. About 8 months after the RAS and WAS fermenter implementation, there was a 28 day consecutive period of low B-stage effluent OP <1 mg/L, with an average of 0.5 ± 0.1 mg/L OP. Following this low effluent OP period, bio-P became more unstable and there was high nitrite accumulation in the B-stage effluent for 106 days with concentrations ranging from 1.1-5.9 mg/L NO2. The nitrite accumulation was not due to NOB out-selection, confirmed by AOB and NOB maximum activity tests. It was determined that the nitrite accumulation was due to partial denitrification of nitrate to nitrite by bacteria using internally stored carbon, because profiles and activity tests showed anoxic nitrite accumulation at the end of the aerobic process. Post-anoxic denitrification using internally stored carbon compounds has been observed in other EBPR systems (Vocks, Adam, Lesjean, Gnirss, and Kraume, 2005). Fermentate addition was then halted, and nitrite accumulation and bio-P activity ceased all together, linking the fermentate addition to both bio-P activity and nitrite accumulation. Fermentate was then controlled to dose at 60% of the sCOD/OP (fermentate sCOD g/day / total OP- fermentate + influent - g/day) of the first low effluent OP period. During this fermentate dosing period where the average sCOD/OP was 15.6 ± 3.0 g/g, no nitrite accumulation was observed, but another consecutive low effluent OP period was observed with an average of 0.6 ± 0.2 mg/L OP. Linear correlation analysis shows that the highest r2 values relating the low effluent OP periods and the COD loads to the SBPR for both periods were between VFA g/day vs OP effluent mg/L, at r2=0.18 for the first low effluent OP period and r2=0.65 for the second. There were also high tCOD r2 values for the second low effluent OP period showing that COD hydrolysis in the SBPR could have impacted bio-P activity. However, the VFA r2 value was higher than any tCOD r2 value, concluding that the fermentate dosing mainly worked to enhance biological phosphorus removal by increasing the VFA load in g VFA as acetate/day. Since no nitrite was observed in a period with a lower VFA/OP dose, then the probable VFA load needed to provide enough internal storage to produce nitrite accumulation by partial denitrification is between 5-9 (g VFA as acetate/ g total OP). If sidestream EBPR systems could be studied further to promote nitrite accumulation and bio-P activity to produce low effluent OP, then short-cut nitrogen removal and EBPR could be successfully combined in an efficient way. / Master of Science / It is important to reduce nitrogen and phosphorus concentrations in wastewater treatment effluent in order to both protect the environment from eutrophication and to meet the increasingly stringent nutrient effluent discharge limits imposed by the EPA. Conventional biological nitrogen removal is achieved through nitrification and denitrification converting ammonia to nitrogen gas, where nitrogen gas is volatile and leaves the system naturally. Phosphorus removal can be achieved through either chemical addition or through biological phosphorus removal, where phosphorus is taken up in cells and removed from the system by the subsequent solids wasting of these cells. The combination of biological nitrogen and phosphorus removal can be improved to increase energy efficiency, reduce costs including aeration and chemical addition costs, increase system capacity and reduce tank sizes, and reduce biomass production, all while achieving low effluent N and P concentrations. Short-cut nitrogen removal can increase the efficiency of biological nitrogen removal. Deammonification, the combination of partial nitritation and anammox, has the potential to reduce wastewater treatment plant (WWTP) aeration costs by 63%, carbon requirements by 100%, and biomass production by 80% (Nifong, Nelson, Johnson, and B. Bott, 2013). Deammonification is the combination of partial nitritation and anammox. Anaerobic ammonia oxidation (anammox) is a useful class of bacteria that converts ammonia and nitrite straight to nitrogen gas in anaerobic conditions, which is a more direct pathway than the conventional nitrification-denitrification pathway. Anammox requires a nitrite supply, which can supplied by partial nitratation of ammonia to nitrite, performed by ammonia oxidizing bacteria (AOB) aerobically in the deammonification process. In order for partial nitratation to work, there needs to be nitrite oxidizing bacteria (NOB) out-selection so that the nitrite produced by AOB does not get oxidized to nitrate. Enhanced biological phosphorus removal (EBPR) is accomplished by the taking up and storing of orthophosphate (OP) by phosphorus accumulating organisms (PAOs). These organisms require an anaerobic carbon-storage phase followed by an aerobic growth phase where the internally stored carbon is used for growth. During the cell growth phase of PAOs in aerobic conditions, PAOs are able to take up more OP than they previously released in anaerobic conditions, creating a net OP removal from the system. There has been recent success in recycle activated sludge (McIlroy et al.) fermentation to enhance biological phosphorus removal, which works to promote hydrolysis, fermentation, and EBPR enhancement (Houweling, Dold, and Barnard, 2010). A portion of the RAS is introduced to an anaerobic zone before returning to the main process, allowing for extra VFA production and adsorption by PAOs. RAS fermentation solves the issue of carbon needed for EBPR in VFA/carbon limited systems without having to add too much additional carbon, creating a carbon efficient EBPR system. The research outlined in this study was done at the Hampton Road Sanitation District's (HRSD) pilot plant located within HRSD's Chesapeake-Elizabeth WWTP in Virginia Beach VA. The pilot is run in an A/B process that works in two separate steps: the A-stage is the first step that works to remove carbon by oxidation, and by adsorption so it can potentially be diverted, and the B-stage is the second step where biological nitrogen removal (BNR) is done. The BNR phase consists of an anaerobic selector followed by four completely stirred tank reactors (CSTRs) that are intermittently aerated to provide aerobic and anoxic phases. The pilot also has an anammox polishing step following B-stage. The nitrogen removal goal for this research was short-cut nitrogen removal via deammonification, by producing partial nitritation in B-stage and polishing with anammox. A B-stage RAS fermenter, along with an A-stage waste activated sludge (WAS) fermenter that feeds VFA into the RAS fermenter, was implemented to the existing pilot to enhance biological phosphorus removal. The overall goal of this study was to successfully combine short-cut nitrogen removal with sidestream EBPR to achieve low effluent N and P concentrations in the most energy and carbon efficient way possible. EBPR was achieved about eight months after the implementation of the RAS and WAS fermenter to the pilot. A period of B-stage effluent OP that was consistently below 1 mg/L OP was observed right before an unexpected period of high nitrite in the B-stage effluent. The high effluent nitrite lasted for 106 days and ranged from 1.1-5.9 mg/L of effluent nitrite during this time. The nitrite accumulation was unexpected because weekly maximum activity tests for AOB and NOB showed that NOB out-selection was not occurring. The first phase of this research investigates the cause of the nitrite accumulation. Based on profiles taken in the reactors in the aerobic and anoxic phases, and based on denitrification activity tests, it was determined that the nitrite accumulation was due to partial denitrification of nitrate to nitrite. Because this partial denitrification was happening in the reactor anoxic times where external should have been used up, it was determined that the source of the partial denitrification was from a bacteria using internally stored carbon during anoxic periods as the electron supply for partial denitrification. Research has showed that EBPR systems promote bacteria that are capable of storing carbon internally and keeping that carbon stored through an aerobic phase and then using that stored carbon for denitrification following an aerobic phase (Vocks et al., 2005), like observed in this research. The second phase of this research sought to link the nitrite accumulation and bio-P activity to the VFA added to the RAS fermenter. The VFA addition was decreased in phases, and with that a decrease in nitrite in the effluent was observed. The bio-P activity became more unstable after the nitrite accumulation occurred, but all bio-P activity ceased after VFA addition to the RAS fermenter ceased. It was concluded, unsurprisingly, that the VFA added to the RAS fermenter was the source of the internally stored carbon that caused the nitrite accumulation, and necessary for bio-P enhancement. The third phase of this research sought to recreate the low effluent OP period and the nitrite accumulation by controlling the VFA dose to the RAS fermenter. The average soluble chemical oxygen demand (sCOD) per OP (fermenter sCOD g/day / total OP-fermenter + influent- g/day) of the period of low effluent OP was calculated, and the dose from the WAS fermenter was controlled to meet 60% of the calculated value. The calculated dose was 13.6 gC/gP, but the actual average dose from controlling the load during this period was 15.6 ± 3.0 gC/gP. The average VFA/OP (g VFA as acetate/ g total OP) dose for the first low effluent OP period was 9.4 ± 3.6 g/g, and the average dose for the third phase of research was 5.5 ± 1.3 g/g. No nitrite accumulation occurred in this phase, but another consistent low effluent OP period did occur. From linear correlation analysis, the highest r2 values relating the low effluent OP periods and the COD loads to the RAS fermenter for both periods were between VFA g/day vs OP mg/L, at r2=0.18 for the first period and r2=0.65 for the second. This shows that effluent OP < 1 mg/L can be achieve at 5.5 or 9.4 (g VFA as acetate/ g total OP). Since no nitrite was observed in phase 3, than the probable VFA load needed to provide enough internal storage to produce nitrite accumulation by partial denitrification is probably between 5.5-9.4 (g VFA as acetate/ g total OP). This research was significant because the link between nitrite accumulation and bio-P enhancement with sidestream RAS and WAS fermentation was confirmed. Partial denitrification of nitrate to nitrite could be used as an alternative source of nitrite for anammox, instead of NOB out-selection and partial nitritation of ammonia to nitrite by AOB, in combined EBPR and short-cut nitrogen removal systems. If sidestream EBPR systems could be used to promote nitrite accumulation and bio-P activity to produce low effluent OP and nitrogen removal efficiently than short-cut nitrogen removal and EBPR could be successfully combined in an efficient way. Future work needs to be done on the organism that is capable of nitrite accumulation and if that organism can be enhanced in conjunction with EBPR organisms to promote both nitrite accumulation and low effluent OP simultaneously.

Partial denitrification/anammox (PdNA) is a biological nitrogen removal technology with significant carbon and aeration savings when compared with conventional nitrification/denitrification. Yet despite these benefits, the use of PdNA in mainstream wastewater treatment remains limited. One of the main barriers to implementation of anammox-based technologies is the slow growth rate of anammox (AMX), which results in a long startup time. To accelerate startup, the typical approach to commissioning AMX-based processes, specifically used for sidestream partial nitritation/AMX, is with biomass augmentation, which is practically unrealistic for full-scale mainstream applications. Thus, this study evaluated startup strategies for mainstream PdNA without AMX inoculation in moving bed biofilm reactors (MBBRs) with two simultaneous experiments. In one experiment, an MBBR was started using IFAS carriers with a preliminary biofilm and no external carbon dosing or AMX biomass inoculation. The feed was controlled to 20°C and included mainstream conditions of nitrite and ammonia controlled to the stoichiometric requirements for AMX growth. After only 84 days of operation, AMX activity was confirmed in the reactor with evidence of activity a few weeks before testing. In the second experiment, four reactors were started with either virgin carriers or integrated fixed-film activated sludge (IFAS) carriers with a preliminary biofilm of heterotrophs and nitrifiers. The reactors were fed mainstream levels of ammonia and nitrate with a temperature control target of 20°C and one reactor of each carrier type was dosed with carbon in the form of either glycerol or methanol. Carbon dosing was based on a feedback proportional-integrative-derivative (PID) control loop with a nitrate residual of 1-1.5 mgNO3-N/L. Of the four reactors, the preliminary biofilm carrier reactor dosed with glycerol achieved AMX activity first after 224 days of operation, but it was determined this was likely limited by synthetic feeding for the first 184 days. These results, along with other recent PdNA work, suggest that growth of AMX on biofilm carriers can be established in mainstream conditions in 50-100 days, depending on media selection and carbon source. Ultimately, this research will help utilities understand methods for starting up mainstream PdNA MBBRs from scratch and make this technology more accessible. / Master of Science / Intensification is the practice by which operational changes and new technologies are employed to reduce economic, resource, energy, and space requirements of wastewater treatment plants. One area of increasing focus involves the use of anaerobic ammonia oxidizing bacteria, or anammox (AMX), to reduce the aeration and carbon dosing needs for treating wastewater. One of the main barriers to implementation of AMX-based technologies is the slow growth rate of AMX, which results in a long startup time. To accelerate startup, the typical approach to commissioning AMX-based processes, specifically used for sidestream partial-nitritation/AMX, is with augmentation of biomass, which is practically unrealistic for full-scale mainstream applications. Thus, this study evaluated startup strategies for mainstream moving bed biofilm reactors (MBBRs) without AMX biomass inoculation in two simultaneous experiments in an AMX MBBR and a partial denitrification/AMX (PdNA) MBBR. In one experiment, idealized stoichiometric conditions for AMX growth were provided to a mainstream MBBR started with carriers from an aerobic integrated fixed-film activated sludge (IFAS) process to determine how fast AMX could potentially grow. In another experiment, different carrier types, virgin or preliminary biofilm carriers from an IFAS process, and different carbon sources, methanol and glycerol, were tested to determine the best methods for encouraging AMX attachment and growth in a PdNA process. These results, along with other recent PdNA work, suggest that growth of AMX on biofilm carriers can be established in mainstream conditions within 50-100 days, depending on media selection and carbon source. Ultimately, this research will help utilities understand methods for starting up mainstream PdNA MBBRs from scratch and make this technology more accessible.