10.2 Biological Nitrogen Removal: Nitrification & Denitrification

10.2 Biological Nitrogen Removal: Nitrification & Denitrification

Nitrogen compounds in municipal wastewater discharges exert severe environmental impacts on receiving water bodies. Un-ionized ammonia ($\text{NH}_3$) is directly toxic to fish and aquatic life at concentrations as low as $0.05 - 0.20\text{ mg/L}$. The biological oxidation of ammonia consumes large quantities of dissolved oxygen (Nitrogenous Biochemical Oxygen Demand / NBOD), while nitrate ($\text{NO}_3^-\text{-N}$) in drinking water sources causes infant methemoglobinemia ("blue baby syndrome," enforced under the Safe Drinking Water Act with a Maximum Contaminant Level of $10\text{ mg/L as N}$). Furthermore, excessive nitrogen fuels marine and estuarine coastal eutrophication (such as the Gulf of Mexico hypoxic dead zone).

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Nitrogen Fractions in Wastewater

Total Nitrogen (TN) in raw municipal wastewater averages $20\text{ to }45\text{ mg/L as N}$ and consists of several distinct chemical fractions:

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|                                      WASTEWATER NITROGEN FRACTIONS                                      |
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| 1. ORGANIC NITROGEN (Org-N):                                                                            |
|    - Bound in amino acids, proteins, urea, and nucleic acids (typically 8 - 15 mg/L in raw sewage).     |
|    - Undergoes microbial enzymatic hydrolysis (ammonification) in collection sewers and treatment       |
|      basins, converting rapidly into soluble ammonium ions (NH4+).                                      |
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| 2. AMMONIA NITROGEN (NH3 / NH4+):                                                                       |
|    - Exists in chemical equilibrium: NH3 (un-ionized gas, highly toxic) + H2O <==> NH4+ (ammonium ion, |
|      non-toxic) + OH-. At standard wastewater pH (7.0 - 7.8), > 95% is in the ionized NH4+ form.        |
|    - Typically constitutes 12 - 30 mg/L in raw influent.                                                |
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| 3. TOTAL KJELDAHL NITROGEN (TKN):                                                                       |
|    - Defined analytically as: TKN = Organic Nitrogen + Ammonia Nitrogen.                               |
|    - Represents the total un-oxidized nitrogen present in the wastewater.                               |
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| 4. OXIDIZED NITROGEN (NO2- and NO3-):                                                                   |
|    - Nitrite (NO2-) and Nitrate (NO3-). Raw municipal sewage contains < 0.5 mg/L oxidized nitrogen.     |
|    - Total Nitrogen is calculated as: TN = TKN + NO2-N + NO3-N.                                         |
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Two-Step Biological Nitrification by Autotrophs

Biological nitrification is an obligate aerobic, two-stage biochemical process performed by specialized chemolithoautotrophic bacteria. Unlike heterotrophic bacteria that consume organic carbon, nitrifiers obtain cellular carbon by fixing inorganic carbon dioxide ($\text{CO}_2$) and bicarbonate alkalinity ($\text{HCO}_3^-$), deriving metabolic energy solely from the oxidation of inorganic nitrogen compounds.

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|                                 TWO-STEP BIOCHEMICAL NITRIFICATION PATHWAY                              |
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| STEP 1: Ammonia Oxidation to Nitrite (Ammonia-Oxidizing Bacteria / AOB)                                 |
| Dominant Genera: Nitrosomonas, Nitrosococcus, Nitrosospira                                              |
| Biochemical Reaction:                                                                                   |
|        2 NH4+  +  3 O2   ────────►   2 NO2-  +  4 H+  +  2 H2O  +  Energy                               |
|                                                                                                         |
| STEP 2: Nitrite Oxidation to Nitrate (Nitrite-Oxidizing Bacteria / NOB)                                 |
| Dominant Genera: Nitrobacter, Nitrospira                                                                |
| Biochemical Reaction:                                                                                   |
|        2 NO2-  +  O2     ────────►   2 NO3-  +  Energy                                                  |
|                                                                                                         |
| OVERALL COMBINED STOICHIOMETRIC REACTION:                                                               |
|        NH4+  +  2 O2  +  2 HCO3-  ──►  NO3-  +  2 CO2  +  3 H2O  +  Biomass                             |
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Stoichiometric & Environmental Requirements for Nitrification

1. Dissolved Oxygen Consumption:

   • Complete nitrification requires $4.57\text{ mg of dissolved oxygen (O}_2)$ per $1.0\text{ mg of NH}_4^+\text{-N}$ oxidized ($3.43\text{ mg O}_2$ for the $\text{AOB}$ step plus $1.14\text{ mg O}_2$ for the $\text{NOB}$ step).
   • Operators must maintain basin dissolved oxygen at $\ge 2.0\text{ mg/L}$ throughout the aeration zone. If DO drops below $1.0\text{ mg/L}$, nitrification rates decline dramatically, and nitrite accumulation ($\text{NO}_2^-$) may occur.

2. Alkalinity Consumption & Destruction:

   • The biological oxidation of ammonium releases hydrogen ions ($\text{H}^+$), neutralizing wastewater buffering capacity. Nitrification destroys $7.14\text{ mg of total alkalinity as CaCO}_3$ for every $1.0\text{ mg of NH}_4^+\text{-N}$ oxidized to nitrate.
   • If residual mixed liquor alkalinity falls below $50\text{ to }100\text{ mg/L as CaCO}_3$, mixed liquor pH will drop precipitously, immediately inhibiting nitrifying bacteria. Operators must dose supplemental alkalinity (hydrated lime, soda ash, caustic soda, or magnesium hydroxide) when influent alkalinity is insufficient.

3. pH Sensitivity:

   • Optimal biological nitrification occurs between $\text{pH } 7.5\text{ and }8.5$.
   • Nitrification rates decline sharply below $\text{pH } 7.0$, drop by over $50\%$ at $\text{pH } 6.5$, and virtually stop at $\text{pH } < 6.0$.

4. Temperature Dependency & Missouri Winter Operation:

   • Nitrifying autotrophs are exceptionally sensitive to cold wastewater. Biological activity decreases by approximately $50\%$ for every $10^\circ\text{C}$ drop in temperature ($\theta = 1.072 - 1.10$).
   • In Missouri winters, when aeration basin wastewater temperatures drop below $10^\circ\text{C}$ ($50^\circ\text{F}$), the minimum generation time of nitrifiers increases from 1 day to over 4 days. To prevent nitrifiers from being washed out in the effluent, operators must substantially increase the Mean Cell Residence Time (MCRT / Sludge Age) to $15 - 25+\text{ days}$ by reducing WAS wasting.

5. Solids Retention Time (MCRT):

   • Autotrophic nitrifiers have very slow growth rates (cell yield of only $0.10 - 0.15\text{ lb VSS / lb NH}_4^+$-N oxidized, compared to $0.6\text{ lb VSS / lb BOD}$ for heterotrophs). Minimum design MCRT is $8 - 15\text{ days}$ under warm summer conditions ($20^\circ\text{C}$).

Biological Denitrification

While nitrification converts toxic ammonia to nitrate, it does not remove nitrogen from the wastewater. Biological denitrification is a facultative heterotrophic process performed under anoxic conditions (where dissolved oxygen is absent, but chemically bound nitrate oxygen is present). Denitrifiers reduce nitrate into harmless, inert nitrogen gas ($\text{N}_2$), which bubbles out of solution into the atmosphere (which is naturally $78\%\text{ N}_2$).

$$\text{NO}_3^- \longrightarrow \text{NO}_2^- \longrightarrow \text{NO}\uparrow \longrightarrow \text{N}_2\text{O}\uparrow \longrightarrow \text{N}_2\uparrow$$

Biochemical & Environmental Requirements for Denitrification

1. Anoxic Environment:

   • Dissolved oxygen must be zero or near-zero ($\text{DO} < 0.2\text{ mg/L}$). If dissolved oxygen is present, facultative heterotrophs preferentially utilize free $\text{O}_2$ because it yields higher thermodynamic energy, immediately halting denitrification enzyme synthesis.

2. Organic Carbon Source (Electron Donor):

   • Denitrifying heterotrophs (Pseudomonas, Alcaligenes, Paracoccus denitrificans, Bacillus) require an electron donor (organic carbon) to reduce nitrate:
     • Influent BOD: The readily biodegradable soluble COD in raw or primary wastewater (most cost-effective carbon source, utilized in pre-anoxic zones).
     • Endogenous Carbon: Cellular breakdown in post-anoxic zones (slow reaction rate).
     • Supplemental Chemical Carbon: Dosed when influent wastewater is carbon-deficient ($\text{BOD}_5 : \text{TKN} < 4 : 1$). Common external carbon sources include methanol ($\text{CH}_3\text{OH}$), sodium acetate ($\text{CH}_3\text{COONa}$), glycerin, or proprietary sugar formulations.

• Stoichiometry with Methanol:

$$6\text{NO}_3^- + 5\text{CH}_3\text{OH} \longrightarrow 3\text{N}_2\uparrow + 5\text{CO}_2 + 7\text{H}_2\text{O} + 6\text{OH}^- + \text{Biomass}$$

To achieve complete nitrate reduction, operators typically dose $2.5\text{ to }3.5\text{ lbs of methanol}$ per lb of $\text{NO}_3^-\text{-N}$ reduced (accounting for dissolved oxygen scavenging).

1. Alkalinity Recovery & Oxygen Credit:
   • Alkalinity Production: Denitrification releases hydroxyl ions ($\text{OH}^-$), producing $3.57\text{ mg of total alkalinity as CaCO}_3$ for every $1.0\text{ mg of NO}_3^-\text{-N}$ reduced to $\text{N}_2$ gas. This recovers exactly $50\%$ of the alkalinity destroyed during nitrification, stabilizing mixed liquor pH.
   • Oxygen Credit: Utilizing nitrate bound oxygen saves $2.86\text{ lbs of dissolved oxygen equivalent}$ per lb of $\text{NO}_3^-\text{-N}$ reduced, reducing aeration blower power consumption by $15\%\text{ to }25\%$.

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Biological Nitrogen Removal (BNR) Process Configurations

To achieve efficient nitrification and denitrification within a continuous flow facility, several engineered tank configurations are utilized:

1. Modified Ludzack-Ettinger (MLE) Process

The Modified Ludzack-Ettinger (MLE) process is the most widely adopted BNR configuration for municipal wastewater:

• Flowsheet: An Anoxic Zone (HRT $1 - 2\text{ hours}$) is placed directly ahead of the Aerobic Aeration Basin (HRT $4 - 8\text{ hours}$), followed by a secondary clarifier.
• Internal Mixed Liquor Recycle (IMLR): A high-capacity low-head submersible axial flow pump recycles nitrified mixed liquor from the discharge end of the aerobic basin back to the influent of the anoxic basin at a rate of $200\%\text{ to }400\%$ of influent flow ($Q$).
• Mechanism: Raw wastewater provides the readily biodegradable organic carbon (BOD) needed for denitrification in the anoxic basin. The de-nitrified mixed liquor then flows into the aerobic zone where remaining BOD is removed and ammonia is completely nitrified. Return Activated Sludge (RAS at $50\% - 100\% Q$) also returns secondary clarifier nitrate to the anoxic basin.
• Performance: Achieves $70\%\text{ to }85\%$ Total Nitrogen removal, yielding effluent TN of $5 - 8\text{ mg/L}$.

$$\text{Theoretical Max TN Removal (\%)} = \left( \frac{Q_{\text{IMLR}} + Q_{\text{RAS}}}{Q + Q_{\text{IMLR}} + Q_{\text{RAS}}} \right) \times 100$$

2. 4-Stage Bardenpho Process

When effluent total nitrogen permits are stringent ($< 3.0\text{ mg/L TN}$), the 4-Stage Bardenpho configuration is used:

• Stage 1 (Primary Anoxic Zone): Receives raw influent BOD and IMLR ($300 - 400\% Q$) from Stage 2 to achieve primary denitrification ($70 - 80\%$ TN reduction).
• Stage 2 (Primary Aerobic Zone): Complete carbon oxidation and biological nitrification of ammonia to nitrate.
• Stage 3 (Secondary / Post-Anoxic Zone): Eliminates residual nitrate escaping Stage 1 via endogenous respiration or supplemental carbon addition (methanol).
• Stage 4 (Re-aeration Zone): Brief aeration ($10 - 20\text{ minutes}$) to raise DO to $4.0 - 6.0\text{ mg/L}$ and strip residual nitrogen gas bubbles from flocs, preventing "clumping / rising sludge" in secondary clarifiers.
• Performance: Achieves $> 90\% - 95\%$ Total Nitrogen removal (effluent TN $< 2 - 3\text{ mg/L}$).

3. 5-Stage Bardenpho (Phoredox) Process

Integrates an Anaerobic Selector Zone ahead of the 4-Stage Bardenpho system (Anaerobic $\rightarrow$ Pre-Anoxic $\rightarrow$ Aerobic $\rightarrow$ Post-Anoxic $\rightarrow$ Re-aeration) to achieve simultaneous Enhanced Biological Phosphorus Removal (EBPR) and Total Nitrogen Removal in a single suspended-growth system.