Project topics and materials
Get Complete Project Material File(s) Now! »
Literature Review
Nitrification
Nitrification involves the chemical transformation of ammonia (NH4+/NH3) to nitrate (NO3-) by two separate reactions performed by nitrifiers: ammonia (NH4+/NH3) to nitrite (NO2-), and nitrite (NO2-) to nitrate (NO3-). Ammonia oxidizing bacteria (AOB) are primarily responsible for the first step in nitrification, termed nitritation: the oxidation of ammonia to nitrite. AOB are considered chemolitho-autotrophs and obligate aerobes, and are dominated in wastewater by the two geneses Nitrososomas and Nitrosospira (Kowalchuk & Stephen, 2001). AOB use two enzymes in the ammonia oxidation; ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO), with hydroxylamine formed as an intermediate (Kowalchuk & Stephen, 2001).
The conversion of nitrite (NO2-) to nitrate (NO3-), termed nitratation, is performed by nitrite oxidizing bacteria (NOB). NOB are also chemiolitho-autotrophs, and typically grow in the same environments as AOB. Predominate wastewater NOB species include the genus Nitrobacter and the genus Nitrospira (Grady et al., 2011). NOB use an enzyme called nitrite oxidoreductase (NXR) for nitrite oxidation. NOB activity usually depends on AOB activity for nitrite production, making it useful to combine the AOB and NOB stoichiometry for total nitrification.
Total nitrification involves the transfer of 8 electrons from NH4+-N(-III) to NO3–N(+V) using oxygen as the electron acceptor. The oxygen required is 4.24 mg O2/mg NH4+-N removed, of that 3.22 mg O2 is used by AOB (~76%) and 1.11 by NOB (~25) (Grady et al., 2011). Biomass is not generally a concern when designing for nitrification because of the low yield, especially when compared to heterotrophic biomass yield. A large amount of alkalinity is consumed during nitrification: 6.708 mg HCO3-/mg NH4+-N removed, or 7.14 mg CaCO3/mg NH4+-N removed (Grady et al., 2011). Nitrifiers have an optimal pH range from 7-8, and since they consume alkalinity there may be a need for alkalinity addition in order to stay in the optimal pH range. Free nitrous acid (FNA) inhibits both AOB and NOB, but AOB have shown to have a higher tolerance. NOB inhibition is observed at 0.023 mg HNO2-N/L, while AOB inhibition has been observed over the ranges of 0.40-2.81 mg HNO2-N/L ((Vadivelu, Yuan, Fux, & Keller, 2006), (Nan et al., 2019)).
The Monod equation can be used to predict growth rates for AOB and NOB, assuming that ammonia is the limiting nutrient for AOB and nitrite for NOB. For AOB, û=0.014-0.092/hr with the typical assumed value of 0.032/hr, and for NOB û= 0.021-0.042/hr. Both AOB and NOB have been observed as the faster growing nitrifier in different studies (Blackburne, Vadivelu, Yuan, & Keller, 2007; Grady et al., 2011). It is also important to note that growth rates for denitrifying bacteria (0.13/hr) are much higher than nitrifying bacteria, which can make nitrification the limiting factor in nitrogen removal systems when growth is an important factor, like in start-up systems. The Ks for AOB = 0.5-1.0 mg/L NH4+-N and the Ks for NOB = ~1.3 mg/L NO2- -N, which favors AOB growth at lower substrate concentrations (González-Cabaleiro, Curtis, & Ofiţeru, 2019). Since nitrifiers are not inhibited by oxygen like denitrifiers, oxygen can be used in the Monod equation as an additional limiting substrate. ? = ?̂( ?1 ??1+?1)( ?2 ??2+?2) (1)
For AOB S1=NH4+ and S2=DO, and for NOB S1=NO2- and S2=DO. The Ks2, also known as KO, is the oxygen half saturation coefficient, usually between 0.74-0.99 mg/L DO for AOB (Grady et al., 2011). The KO for NOB in literature is 1.4-1.75 mg/L DO (Grady et al., 2011). There has been recent research to suggest that the KO values for AOB and NOB are more complex than previously thought, and might be dependent on the dominate autotrophic species present in a system (Blackburne et al., 2007; Regmi et al., 2014).
Denitrification
Denitrification refers to the reduction of nitrate (NO3-) to nitrogen gas (N2) through a series of intermediates. Heterotrophic organisms are responsible for denitrification in wastewater, and they do so in anoxic environments using organic carbon as the electron donor and nitrite/nitrate as the election acceptor. The release of N2 gas from denitrification effectively removes nitrogen from the treated water.
Heterotrophs responsible for denitrification are known as denitrifiers. Most known denitrifiers are chemoorgano-heterotrophs, gaining their electron supply from organic carbon sources (Huijie Lu, Chandran, & Stensel, 2014). Most are also facilitative anaerobes, using nitrate as an electron acceptor in the absence of oxygen (Huijie Lu et al., 2014). Different nitrogen reductases catalyze each step of the denitrification pathway in order for denitrifiers to use each form of oxidized nitrogen as an electron acceptor in their metabolic energy-generating reactions (in order: nitrate reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Marques et al.), and nitrous oxide reductase (Nos)) (Miao & Liu, 2018).
Heterotrophic denitrification of domestic wastewater consumes hydrogen ions, and therefore produces alkalinity. The total alkalinity produced is 3.354 mg HCO3-/NO3-N removed, or 3.57 mg CaCO3/NO3-N removed. When heterotrophic denitrification is combined with nitrification, the overall alkalinity consumed is reduced by 50% because of the alkalinity produced in denitrification. When nitrification and denitrification are combined in the same system the total alkalinity loss is approximately 3.57 mg CaCO3/N-removed. It takes 2.86 g COD/g N to reduce nitrate to nitrogen gas, and when considering biomass production and assimilation it can take around 5-6 g COD/ g N (Grady et al., 2011). Considering the average COD/N of municipal wastewater is 7-12, and considering carbon consumption in other metabolic cycles, external carbon addition might be necessary for denitrification.
Optimal pH needed for denitrification is between 7-9 and the optimal temperature is between 2030°C (Huijie Lu et al., 2014). Anoxic conditions required for proper heterotrophic denitrification are usually between <0.2-0.5 mg/L DO. Denitrification can be inhibited over the required DO conditions because oxygen will be available as the preferred electron accepter over nitrate (Huijie Lu et al., 2014).
The Monod equation can be used to predict growth rates for heterotrophic bacteria growing anoxically with COD as the limiting nutrient; the û= 0.13/hr and the Ks= 76 mg/L COD (Grady et al., 2011). Heterotrophic bacteria also grow similarly under aerobic conditions, with the values û= 0.14/hr and the Ks= 67 mg/L COD (Grady et al., 2011). Anoxic heterotrophic growth can also be inhibited by low nitrate concentrations or high oxygen concentrations. To consider multiple inhibition and/or limiting factors for growth, the multiple Monod equation is used (Equation 2 below).
? = ?̂( ?? ??+??)( ???3 ???3+???3)( ??? ???+??) (2)
In this instance, Ks = COD and KNO3 is the half-saturation coefficient for nitrate, and KIO is the inhibition coefficient for oxygen. Using this equation, the values commonly accepted in most models are KNO3=0.1-0.2 mg/L of NO3-N and KIO=0.2-2.0 mg/L DO (Grady et al., 2011). Since nitrate is a limiting substrate, a minimum of 0.1-0.2 mg/L NO3-N is needed, but since oxygen is an inhibitor there can be no more than 0.2-2 mg/L DO in order for denitrification to occur.
Anammox
The 1995 discovery of anaerobic ammonium oxidation (anammox) has been making great advances in BNR (Mulder, Vandegraaf, Robertson, & Kuenen, 1995). Anammox is a bacterium that thrives in anaerobic environments and is able to convert ammonia to nitrogen gas using nitrite as an electron acceptor, bypassing nitrification and denitrification. Since its discovery, anammox has been widely studied in wastewater processes because of its potential to lower aeration costs, efficiently remove nitrogen, lower sludge yields, and decrease overall operational costs (Wang et al., 2018). ??4 + +1.32??2 − +0.066???3 − +0.13? + → 1.02?2 + 0.26??3 − +0.066??2?0.5?0.15 + 2.03?2? (3)
Anammox has been successfully implemented in side-stream treatments with high free ammonia (FA) concentrations, such as landfill leachate and sludge digestion liquid (Wang et al., 2018). Anammox thrive at a temperature above 20°C, which can be implemented in side-stream treatment much easier than in mainstream processes (O’Shaughnessy, 2016). Another reason why side-stream anammox technologies have been successful is because the slow growth rate of anammox, û= 0.0027/hr, requires a long SRT which is easier to implement in the side-stream. Anammox have been successfully retained in attached growth systems such as biofilters, but suspended growth systems (flocs or granules) need an SRT of 30-45 days in order to prevent wash out (O’Shaughnessy, 2016). The success of suspended growth systems largely depends on good settling to accurately control the SRT, which can be enhanced by cyclones separating out biomass mechanically based on density.
Currently, most applications of anammox in municipal wastewater treatment are treating small portions of flow in the side-stream. Implementing side-stream anammox processes does increase the overall efficiency of a BNR wastewater plant by decreasing the overall carbon, DO, and tank size requirements for nitrogen removal, but efficiency could be further increased if it could be implemented in the mainstream. As previously discussed, SRT, FA, and temperature are limiting factors in the mainstream. Another major factor is NOB out-selection, meaning the ability to promote AOB species over NOB species so there is enough nitrite produced for anammox but not too much consumed by NOB.As shown in Equation 15, anammox need ammonia and nitrite inputs but not nitrate, which NOB produces by oxidizing nitrite.
Short-cut nitrogen removal
Advances are being made to improve the traditional nitrification/denitrification pathways for nitrogen removal in order to improve efficiency and cost. If aeration requirements, tank sizes, and alkalinity requirements decrease, then overall cost will decrease. If less carbon is required for nitrogen removal then carbon can be diverted and used for other BNR processes or for energy recovery, increasing the overall energy efficiency of the system. Several promising short-cut pathways are being explored and developed
Nitritation/denitritation- nitrite shunt
Since nitrite is an intermediate in nitrification and denitrification, the nitrite shunt process aims to shorten the nitrification/denitrification pathway by jumping to denitritation (reduction of nitrite to nitrogen gas) after ammonia oxidation to nitrite.
??4 + +1.5?2 +2???3 − → ??2 − +3?2? +2??2 (4)
??2 − +0.5??3?? +0.5??2 → 0.5?2 + ???3 − +0.5?2? (5)
By skipping nitrite oxidation and nitrate denitrification, an overall electron transfer of six electrons is needed instead of eight. This pathway can reduce oxygen requirements by 25% and carbon requirements by 40% (Grady et al., 2011). A 40% reduction in biomass is also possible, due to the decreased use of heterotrophic bacteria that have a much higher biomass yield than autotrophic bacteria (Hellinga, Schellen, Mulder, van Loosdrecht, & Heijnen, 1998). NOB outselection is required in order for nitritation/denitritation to occur.
Deammonification: partial nitration and anammox
Deammonification refers to the combination of partial nitration, where approximately half of present ammonia is oxidized to nitrite by AOB, and anammox oxidation of ammonia to nitrogen gas using nitrite. Deammonification theoretically requires no carbon, which could greatly improve the cost and total efficiency of BNR if all carbon can be diverted before deammonification (Hellinga et al., 1998). Alkalinity consumption can potentially be reduced from the reduction of AOB activity required, and also because of the small production of alkalinity by anammox. This pathway saves even more aeration costs than the nitrite shunt, and up to 63% less than conventional nitrification (Nifong et al., 2013). Biomass production can be reduced by 80% due to elimination of heterotrophic bacteria. Nearly 100% of the total carbon can be diverted before the deammonification step to be used for other BNR processes or for energy recovery, creating potential for energy-neutral WWTPs.
Aeration is required in order to achieve partial nitritation. Oxygen requirements for AOB make combining the two deammonification steps in the same reactor an issue since anammox can be inhibited at certain DO concentrations. Some promising studies have shown no significant decrease in anammox activity at DO concentrations of 2-3 mg/L O2 (Siegrist, Salzgeber, Eugster, & Joss, 2008) and other studies show that AOB can be successfully implemented with anammox at low DO of 0.1-0.2 on granules and biofilms (Huynh et al., 2019). Deammonification can occur in single-stage processes or in two-stage systems. Single-stage systems have been successful in side-stream implementation and have shown promise in efficiency, cost reduction, and easier operation and maintenance. Examples of single-step side-stream processes already used in WWTPs are DEMON, AnitaMox, and ANAMMOX (Nifong et al., 2013; B. Wett, 2006).
Side-stream technologies only treat a portion of the wastewater treated at full-scale plants, usually high ammonia streams like digester effluent, but if the process could be modified to perform in more dilute ammonia influent conditions then the benefit of the process would increase exponentially. Mainstream deammonification is a challenge because of dilute ammonia influent conditions, anammox retention issues, and NOB out-selection challenges (See chapter 2.5 for NOB out-selection challenges). Not only do higher COD/N mainstream influent conditions take away the benefits of anammox enhancement and NOB out-selection, but too much COD is a problem for heterotrophic consumption of nitrite. Mainstream deammonification has not been successful implemented in full-scale systems but continues to be a focus of research.
Partial denitrification + anammox
Partial denitrification (PdN) combined with anammox works by using partial denitrification of nitrate to nitrite by heterotrophic bacteria to supply nitrite to anammox. This requires an external carbon addition source for the PdN. This mechanism acts as a nitrite source for anammox, and can improve the overall anammox efficiency since anammox produces nitrate (see Equation 6). When combining PdN with anammox, nitrate production can be eliminated (see Equation 6) improving the TIN efficiency of anammox from 75-80% (O’Shaughnessy, 2016) to >95% (Wu, Li, Zhao, Liang, & Peng, 2018). PdN can improve the overall COD/TIN efficiency if nitrite production from supplemental carbon is reliable so less carbon is needed for conventional nitrification/denitrification.
??4 − +1.06??2 − +0.021?6?12?6 → 1.02?2 +0.066??2?0.5?0.15 +0.066???3 − + 2.03?2? (6)
NOB out-selection
The suppression of NOB over AOB would greatly enhance the conditions necessary for anammox by providing a nitrite supply. Since AOB and NOB grow in the same environments under similar conditions, promoting one species over the other is difficult to do. NOB outselection has been successfully implemented in side-stream treatment because of high free ammonia (FA) concentrations usually present in side-stream treatment (usually exceeding 100 mg NH3). High FA inhibits NOB between 0.1-1 mgN/L, but does not inhibit AOB until 10-150 mgN/L (Anthonisen, Loehr, Prakasam, & Srinath, 1976). Side-stream applications contain higher FA amounts because of their higher temperatures and pH.
Applying NOB out-selection is more difficult to achieve in mainstream processes mostly due more dilute ammonia concentrations and lower temperatures and pH. Success has been shown for NOB out-selection in the mainstream by seeding AOB from the side-stream, controlling a short aerobic SRT, and controlling DO concentrations. Side-stream processes can produce AOBrich biomass which can be seeded back into the mainstream to bioaugment AOB and increase AOB/NOB (Al-Omari et al., 2015). Bioaugmenting AOB can decrease SRT requirements for the same nitrogen removal, due to increased AOB activity (Al-Omari et al., 2015). Aerobic SRT control works to flush out NOB, which are shown in practice to have slightly slower growth rates than AOB at certain DO concentrations (Regmi et al., 2014). Since there is only a small difference in growth rates the aerobic SRT control needs to be strictly monitored (Regmi et al., 2014). Aerobic SRT is also directly related to DO control strategies.
Table of Contents
Technical Abstract
General Audience Abstract
Acknowledgements
List of Figures
List of Tables
Abbreviations
1.Introduction
1.1 Project motivation
1.2 Research objectives
2. Literature Review
2.1 Nitrification
2.2 Denitrification
2.3 Anammox
2.4 Short-cut nitrogen removal
2.5 NOB out-selection
2.6 Phosphorus removal
2.7 Post-anoxic denitrification in EBPR systems
3. Methodology
3.1 A-stage
3.2 B-stage
3.3 A-stage WAS fermenter
3.4 Sidestream biological phosphorus remover (SBPR)
3.5 Data analysis
3.6 Sampling
3.7 AOB/NOB activity measurement
3.8 PAO/dPAO activity measurement
3.9 ISCD activity measurement
3.10 Profile measurement
3.11 Central Environmental Laboratory (CEL) sampling
3.12 EA_ANX and EA_EICD tests
3.13 Statistical analysis
4. Results and Discussion:
4.1 Determining the cause of the nitrite accumulation
4.2 Decreased fermentate addition / SBPR HRT maximization experiment
4.3 Dosing experimental setup- operational phase 3
4.4 Operational phase 3 & result phase 3
5. Conclusions
References
GET THE COMPLETE PROJECT
The Investigation of Nitrite Accumulation and Biological Phosphorus Removal in an Intermittently Aerated Process Combining Shortcut Nitrogen Removal and Sidestream Biological Phosphorus Removal
