8.1 Biological Nitrogen Removal: Nitrification & Denitrification

Nitrogen Forms & Chemistry in Municipal Wastewater

Nitrogen compounds in municipal wastewater originate primarily from domestic human waste (urea, proteins, amino acids) and commercial/industrial discharges. Discharging un-treated nitrogen into receiving waters causes severe environmental degradation, including dissolved oxygen depletion in rivers and estuaries, toxic un-ionized ammonia ($NH_3$) mortality in fish, accelerated eutrophication and harmful algal blooms (HABs), and groundwater nitrate contamination that poses the risk of infant methemoglobinemia ("blue baby syndrome").

┌────────────────────────────────────────────────────────────────────────┐
│                     Nitrogen Fractions in Wastewater                   │
├───────────────────────────────┬────────────────────────────────────────┤
│ Total Nitrogen (TN)           │ Organic-N + NH3/NH4+-N + NO2--N + NO3--N│
├───────────────────────────────┼────────────────────────────────────────┤
│ Total Kjeldahl Nitrogen (TKN) │ Organic-N + Ammonia-N (NH3 + NH4+-N)   │
├───────────────────────────────┼────────────────────────────────────────┤
│ Total Inorganic Nitrogen (TIN)│ Ammonia-N + Nitrite-N + Nitrate-N      │
└───────────────────────────────┴────────────────────────────────────────┘

1. Organic Nitrogen

Organic nitrogen encompasses nitrogen bound in cellular proteins, peptides, nucleic acids, and urea ($CO(NH_2)_2$). In raw municipal influent, organic nitrogen accounts for 30% to 40% of Total Kjeldahl Nitrogen (TKN) (typically 10 to 25 mg/L as N). In collection systems and preliminary/primary treatment units, extracellular bacterial enzymes rapidly hydrolyze urea and proteins into free ammonium through ammonification / deamination:

$\text{CO(NH}_2)_2 + \text{H}_2\text{O} \xrightarrow{\text{Urease}} 2\text{NH}_3 + \text{CO}_2$

2. Ammonia-Nitrogen ($NH_3$ and $NH_4^+$)

Ammonia-nitrogen exists in aqueous solution in a dynamic chemical equilibrium between the un-ionized gas ($NH_3$, free ammonia) and the ionized ammonium ion ($NH_4^+$):

$\text{NH}_3 + \text{H}_2\text{O} \rightleftharpoons \text{NH}_4^+ + \text{OH}^- \quad (pK_a = 9.25 \text{ at } 25^\circ\text{C})$

• Toxicity & pH/Temperature Sensitivity: Un-ionized ammonia ($NH_3$) is a neutral, lipid-soluble molecule that readily diffuses across fish gill membranes, causing acute aquatic toxicity at concentrations as low as $0.05\text{ to }0.20\text{ mg/L }NH_3\text{-}N$. As wastewater pH and temperature increase, the equilibrium shifts strongly to the left, dramatically increasing the fraction of toxic un-ionized $NH_3$.
• Typical Concentrations: Raw domestic wastewater contains 20 to 45 mg/L of ammonia-nitrogen ($NH_3\text{-}N$), representing 60% to 70% of raw influent TKN.

3. Nitrite-Nitrogen ($NO_2^-$)

Nitrite is an unstable, intermediate oxidation state (+3) formed during nitrification. In healthy biological systems, nitrite is rapidly oxidized to nitrate and rarely exceeds $0.1\text{ to }0.5\text{ mg/L }NO_2^-\text{-}N$. Elevated effluent nitrite is a major operational concern because nitrite exerts a massive chemical chlorine demand: $1.0\text{ mg/L of }NO_2^-\text{-}N$ consumes approximately $5.0\text{ mg/L}$ of free chlorine ($Cl_2$).

4. Nitrate-Nitrogen ($NO_3^-$)

Nitrate is the fully oxidized end-product (+5 oxidation state) of complete nitrification. It is highly soluble, mobile in soil, and poses a major threat to drinking water aquifers. The California Primary Maximum Contaminant Level (MCL) for drinking water is $10\text{ mg/L as }NO_3^-\text{-}N$ (equivalent to $45\text{ mg/L as }NO_3^-$).

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The Biochemistry of Two-Step Biological Nitrification

Biological nitrification is a two-step aerobic, autotrophic process mediated by specialized chemolithoautotrophic bacteria. Unlike heterotrophic bacteria that derive carbon and energy from organic matter (BOD), nitrifiers utilize inorganic carbon (dissolved carbon dioxide, $CO_2$, and bicarbonate, $HCO_3^-$) to build cellular biomass, deriving metabolic energy solely from the chemical oxidation of reduced nitrogen.

                       STEP 1: Ammonia Oxidation
               (Ammonia-Oxidizing Bacteria: Nitrosomonas)
         2 NH4+ + 3 O2  ──►  2 NO2- + 4 H+ + 2 H2O + Energy
                                  │
                                  ▼
                       STEP 2: Nitrite Oxidation
                (Nitrite-Oxidizing Bacteria: Nitrobacter)
               2 NO2- + O2  ──►  2 NO3- + Energy

Step 1: Ammonia Oxidation to Nitrite (AOB)

Ammonia-Oxidizing Bacteria (AOB), predominantly belonging to the genus Nitrosomonas (along with Nitrosococcus and Nitrosospira), oxidize ammonium to nitrite:

$2\text{NH}_4^+ + 3\text{O}_2 \xrightarrow{\text{Nitrosomonas}} 2\text{NO}_2^- + 4\text{H}^+ + 2\text{H}_2\text{O} + \text{Energy}$

• Oxygen Demand: Oxidation of ammonium to nitrite consumes 3.43 lbs $O_2$ per lb $NH_4^+\text{-}N$ oxidized.
• Acid Production: Releases two hydrogen ions ($H^+$) per mole of ammonium oxidized, directly neutralizing bicarbonate alkalinity in the mixed liquor.

Step 2: Nitrite Oxidation to Nitrate (NOB)

Nitrite-Oxidizing Bacteria (NOB), primarily Nitrobacter and Nitrospira, oxidize the intermediate nitrite to nitrate:

$2\text{NO}_2^- + \text{O}_2 \xrightarrow{\text{Nitrobacter / Nitrospira}} 2\text{NO}_3^- + \text{Energy}$

• Oxygen Demand: Oxidation of nitrite to nitrate consumes 1.14 lbs $O_2$ per lb $NO_2^-\text{-}N$ oxidized.

Overall Complete Nitrification Reaction

Summing the two biological steps yields the overall biochemical stoichiometry:

$\text{NH}_4^+ + 2\text{O}_2 \rightarrow \text{NO}_3^- + 2\text{H}^+ + \text{H}_2\text{O}$

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Stoichiometric Requirements & Critical Process Control Parameters

Nitrification is exceptionally sensitive to environmental conditions, dissolved oxygen availability, pH, alkalinity, temperature, and Mean Cell Residence Time (MCRT).

┌────────────────────────────────────────────────────────────────────────┐
│               Nitrification Stoichiometry & Operating Windows          │
├──────────────────────────┬─────────────────────────────────────────────┤
│ Stoichiometric O2 Demand │ 4.57 lbs O2 per lb NH3-N oxidized           │
├──────────────────────────┼─────────────────────────────────────────────┤
│ Alkalinity Destruction   │ 7.14 lbs Alkalinity (as CaCO3) per lb NH3-N │
├──────────────────────────┼─────────────────────────────────────────────┤
│ Optimal Operating pH     │ 7.5 – 8.5 (Severe inhibition below pH 6.8)  │
├──────────────────────────┼─────────────────────────────────────────────┤
│ Minimum Aerobic DO       │ >= 2.0 mg/L in bulk aeration basin liquid   │
├──────────────────────────┼─────────────────────────────────────────────┤
│ Minimum Aerobic MCRT     │ 4–8 days (at 20°C) / 10–18 days (at 10°C)   │
└──────────────────────────┴─────────────────────────────────────────────┘

1. Oxygen Stoichiometry ($4.57\text{ lbs }O_2 / \text{lb }NH_3\text{-}N$)

• The complete biological oxidation of $1.0\text{ lb of }NH_3\text{-}N$ requires $4.57\text{ lbs of dissolved oxygen}$ ($3.43\text{ lbs }O_2$ for Step 1 + $1.14\text{ lbs }O_2$ for Step 2).
• In operational practice, taking into account the small fraction of nitrogen assimilated into heterotrophic biomass synthesis, the actual gross oxygen demand is approximately $4.2\text{ to }4.3\text{ lbs }O_2\text{ per lb }NH_3\text{-}N$.

2. Alkalinity Consumption & pH Control ($7.14\text{ lbs }CaCO_3 / \text{lb }NH_3\text{-}N$)

• During Step 1 ammonia oxidation, hydrogen ions ($H^+$) are released into the bulk liquid, neutralizing bicarbonate buffer ($HCO_3^-$):

$\text{H}^+ + \text{HCO}_3^- \rightarrow \text{CO}_2 \uparrow + \text{H}_2\text{O}$

• Nitrification destroys $7.14\text{ lbs of total alkalinity (as }CaCO_3\text{)}$ for every $1.0\text{ lb of }NH_3\text{-}N$ oxidized.
• pH Decline & Souring: If the raw wastewater lacks adequate native alkalinity, nitrification rapidly depletes the buffering capacity, causing mixed liquor pH to plummet. Nitrification rates drop by 50% at pH 6.8 and cease almost completely below pH 6.5.
• Chemical Alkalinity Addition: To sustain complete nitrification, operators must maintain a residual alkalinity of at least $50\text{ to }100\text{ mg/L as }CaCO_3$ in the aeration basin effluent by dosing supplemental alkalinity:
  • Sodium Hydroxide (Caustic Soda, $NaOH$): $0.80\text{ lb }NaOH$ per lb $CaCO_3$ equivalent (fast acting, hazard: freeze point 54°F at 50% solution).
  • Hydrated Lime ($Ca(OH)_2$): $0.74\text{ lb }Ca(OH)_2$ per lb $CaCO_3$ equivalent (inexpensive, increases sludge volume).
  • Sodium Bicarbonate ($NaHCO_3$): $1.68\text{ lb }NaHCO_3$ per lb $CaCO_3$ equivalent (safe, impossible to overdose past pH 8.3, higher chemical cost).
  • Magnesium Hydroxide ($Mg(OH)_2$): Slurry form, self-buffering up to pH 9.0.

3. Dissolved Oxygen (DO) Requirements

• Autotrophic nitrifiers reside embedded deep inside biological activated sludge flocs. To overcome mass-transfer diffusion resistance across the floc boundary layer, the bulk liquid DO concentration in the aeration basin must be maintained at $\ge 2.0\text{ mg/L}$ under peak diurnal ammonia loading.
• When bulk DO falls below $1.0\text{ mg/L}$, Nitrosomonas and Nitrobacter growth kinetics drop exponentially, leading to incomplete nitrification and elevated effluent ammonia/nitrite.

4. Mean Cell Residence Time (MCRT / Sludge Age) & Temperature Kinetics

Autotrophic nitrifiers grow substantially slower than carbonaceous heterotrophic bacteria. The maximum specific growth rate of nitrifiers ($\mu_N \approx 0.3\text{ to }0.6\text{ day}^{-1}$) is a fraction of heterotrophic rates ($\mu_H \approx 2.0\text{ to }6.0\text{ day}^{-1}$), resulting in a very low biomass yield ($Y_N \approx 0.10\text{ to }0.15\text{ lb VSS / lb }NH_3\text{-}N$).

• Washout Prevention: To prevent nitrifiers from being washed out with the Waste Activated Sludge (WAS), the aerobic MCRT ($\theta_c$) must exceed the minimum generation time:

$\theta_c^{\text{min}} = \frac{1}{\mu_N - b_N}$

• Temperature Dependence: Microbial growth kinetics are governed by the Arrhenius temperature relationship ($\mu_T = \mu_{20} \cdot \theta^{T-20}$). As wastewater temperature drops, nitrifier growth rates slow dramatically:
  • At $20^\circ\text{C}$ ($68^\circ\text{F}$): Required aerobic MCRT is 4 to 8 days.
  • At $10^\circ\text{C}$ ($50^\circ\text{F}$): Required aerobic MCRT increases to 10 to 18 days.
• Winter Process Adjustment: During cold weather operations, operators must reduce WAS wasting rates to build mixed liquor suspended solids (MLSS) inventory and increase MCRT, compensating for depressed nitrifier metabolic rates.

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The Biochemistry of Biological Denitrification

Denitrification is the biological reduction of oxidized nitrogen species (nitrate, $NO_3^-$, and nitrite, $NO_2^-$) to inert, gaseous dinitrogen ($N_2$), which vents harmlessly to the atmosphere. This process completes Total Nitrogen (TN) removal.

                               DENITRIFICATION PATHWAY
         Nitrate       Nitrite      Nitric Oxide   Nitrous Oxide   Nitrogen Gas
          NO3-   ──►    NO2-   ──►       NO    ──►     N2O    ──►      N2 (gas)
       (+5 state)    (+3 state)      (+2 state)     (+1 state)      (0 state)

1. Microbial Ecology & Anoxic Conditions

Denitrification is conducted by facultative heterotrophic bacteria widely present in activated sludge, including species of Pseudomonas, Bacillus, Paracoccus, Alcaligenes, and Rhodobacter.

• Anoxic Environment: True denitrification occurs under anoxic conditions—defined as the absence of dissolved molecular oxygen ($\text{DO} < 0.2\text{ mg/L}$) combined with the presence of bound nitrate/nitrite oxygen ($NO_x^-$).
• Respiratory Mechanism: Because these bacteria are facultative heterotrophs, they preferentially utilize dissolved oxygen for respiration ($32\text{ ATP}$ generated per mole glucose). When free dissolved oxygen is depleted, the bacteria activate nitrate reductase enzyme pathways, utilizing nitrate ($NO_3^-$) as the terminal electron acceptor in the electron transport chain ($26\text{ ATP}$ generated per mole glucose).

2. Alkalinity Recovery & Oxygen Credit

Denitrification provides two massive operational benefits that offset nitrification costs:

1. Alkalinity Recovery: Denitrification produces $3.57\text{ lbs of alkalinity as }CaCO_3$ per $1.0\text{ lb of }NO_3^-\text{-}N$ reduced. This represents an exact 50% recovery of the alkalinity destroyed during the preceding nitrification step.
2. Oxygen Credit: The bound oxygen in nitrate is utilized to oxidize carbonaceous BOD, recovering $2.86\text{ lbs of }O_2\text{ equivalent}$ per $1.0\text{ lb of }NO_3^-\text{-}N$ reduced. This reduces aeration blower electrical consumption by 15% to 25% in facilities equipped with pre-anoxic selectors.

3. Carbon Source Requirements (Electron Donors)

Heterotrophic denitrifiers require an organic carbon electron donor to reduce nitrate. Without sufficient carbon, denitrification kinetics halt.

• Endogenous Influent BOD: In raw municipal wastewater, the readily biodegradable COD (rbCOD) serves as the carbon source. Complete denitrification requires a raw wastewater $\text{COD}/\text{TKN}$ ratio $\ge 4.5\text{ to }5.0$ (or $\text{BOD}_5/\text{TKN} \ge 3.5$).
• Supplemental External Carbon Sources: In wastewater plants with weak influent BOD, strict total nitrogen limits ($TN < 3\text{ mg/L}$), or post-anoxic configurations, external carbon must be injected:

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

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Biological Nutrient Removal (BNR) Process Configurations

                      1. MODIFIED LUDZACK-ETTINGER (MLE) PROCESS

          ┌────────────────── Internal Nitrate Recycle (INR 200 - 400% Q) ─────────────────┐
          │                                                                                │
          ▼                                                                                │
Influent ────► [ PRE-ANOXIC ZONE ] ──► [ AEROBIC ZONE ] ─────────────────────────► [ SECONDARY CLARIFIER ] ──► Effluent
                     ▲                    (Nitrification)                                  │
                     │                                                                     │
                     └────────────────────── Return Activated Sludge (RAS) ────────────────┘


                      2. 4-STAGE BARDENPHO SYSTEM (DEEP TN REMOVAL)

          ┌─────── Internal Nitrate Recycle (INR 300 - 400% Q) ────────┐
          │                                                            │
          ▼                                                            │
Influent ──► [ PRE-ANOXIC ] ──► [ AEROBIC 1 ] ──► [ POST-ANOXIC ] ──► [ RE-AERATION ] ──► [ CLARIFIER ] ──► Effluent
               (Primary           (Complete         (Endogenous /      (DO Polishing /      │
             Denit. w/ BOD)     Nitrification)     Ext. Carbon)        N2 Gas Stripping)    │
                   ▲                                                                        │
                   └─────────────────────── RAS ────────────────────────────────────────────┘

1. Modified Ludzack-Ettinger (MLE) Process

The MLE process is the most widely deployed single-sludge BNR configuration for nitrogen removal:

• Pre-Anoxic Basin: Raw influent wastewater and Return Activated Sludge (RAS) blend with a high-rate Internal Nitrate Recycle (INR / Nitrified Liquor Recycle) pumped from the discharge end of the aerobic basin.
• Internal Nitrate Recycle (INR) Rate: Operates at $200%\text{ to }400%$ of forward influent flow ($2Q\text{ to }4Q$).
• Operational Advantages:
  1. Uses raw influent readily biodegradable BOD as the carbon source for denitrification, eliminating external carbon purchase.
  2. Destroys 50% to 75% of total nitrogen before the mixed liquor enters the aeration basin.
  3. Recovers 50% of the destroyed alkalinity and reduces overall aeration blower power by 15% to 20%.
• Effluent Quality: Produces effluent Total Nitrogen of $6\text{ to }10\text{ mg/L}$.

$\text{Theoretical TN Removal Fraction} = \frac{\text{INR} + \text{RAS}}{1 + \text{INR} + \text{RAS}}$

(For example, with $\text{INR} = 300%$ ($3.0$) and $\text{RAS} = 100%$ ($1.0$), theoretical removal is $\frac{3.0 + 1.0}{1 + 3.0 + 1.0} = \frac{4.0}{5.0} = 80%$).

2. 4-Stage Bardenpho System

Designed for stringent effluent Total Nitrogen limits ($\text{TN} < 3.0\text{ mg/L}$):

1. Stage 1 (Pre-Anoxic): Denitrifies the high-volume INR stream ($300%\text{–}400%$) using influent BOD.
2. Stage 2 (Aerobic 1): Complete carbonaceous BOD oxidation and full ammonia nitrification to nitrate.
3. Stage 3 (Post-Anoxic): Polishing denitrification basin that receives no raw influent BOD. Heterotrophs denitrify remaining nitrate via slow endogenous respiration or via metered supplemental carbon (methanol/glycerol).
4. Stage 4 (Re-Aeration / 2nd Aerobic): Short detention basin (10 to 30 minutes HRT) equipped with diffused aeration. Accomplishes two critical functions:
   • Elevates mixed liquor DO to $2.0\text{ to }4.0\text{ mg/L}$ to prevent anoxic conditions and phosphorus release in the secondary clarifier.
   • Strikes and strips supersaturated dissolved nitrogen gas ($N_2$) microbubbles out of solution, preventing them from floating sludge blankets in downstream clarifiers.

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Troubleshooting: Clarifier Rising Sludge from Denitrification

A classic operational problem in nitrifying activated sludge plants is rising sludge (clumping/floating sludge) in secondary clarifiers:

• Mechanism: When mixed liquor containing high nitrate ($NO_3^-$) settles in a secondary clarifier with a deep, stagnant sludge blanket, dissolved oxygen is depleted within minutes. Facultative heterotrophs in the sludge blanket switch to anoxic respiration, denitrifying nitrate into insoluble nitrogen gas ($N_2$). Microscopic $N_2$ bubbles become trapped within the biological flocs, increasing floc buoyancy until massive sheets or clumps of brown sludge float to the surface.
• Distinction from Bulking: Unlike filamentous bulking (where sludge settles slowly and has an SVI >150 mL/g due to filamentous organisms), rising sludge settles rapidly in a 30-minute settleometer test but floats to the top of the cylinder after 45 to 120 minutes with bubbles clinging to the mass.
• Corrective Operator Actions:
  1. Increase RAS Pumping Rate: Immediately increase RAS return rate to purge the clarifier sludge blanket and reduce solids detention time below the threshold where anoxia develops.
  2. Increase Aeration / Re-aeration DO: Ensure DO entering the clarifier is $\ge 2.0\text{ mg/L}$.
  3. Optimize Pre-Anoxic Denitrification: Increase INR recycle rate in the MLE/Bardenpho train to reduce nitrate loading to secondary clarifiers.