FUNDAMENTALS OF NITRIFICATION

2016 So. Cal. Water Education Seminar Orange, California – August 10, 2016

Hélène Baribeau, Ph.D., P.E. Agenda • Background • Problems caused by nitrification • Regulatory compliance • Causes and triggers: – Water quality conditions – Distribution system characteristics, and operational practices • Nitrification indicators Wolfe et al., AEM, 56:2:451, 1990 Agenda • Nitrification may be occurring in 63% of water providers that use monochloramine • 48% of utilities have experienced nitrification (2004 survey): – 25% are responding to nitrification ≥2 times per summer “If you don’t – Not all systems are monitoring think you have specifically for nitrification nitrification, you (sample numbers, frequency, haven’t looked locations, water quality parameters) hard enough!” Nitrification is

+ ‐ + • AOB: NH4 + 3/2 O2 → NO2 + H2O + 2 H ‐ ‐ NOB: NO2 + 1/2 O2 → NO3 ‐ • Incomplete nitrification: NH3 → NO2 ‐ • Complete nitrification: NH3 → NO3 • No single microorganism can oxidize to

Monochloramine decomposition AOB NOB

Monochloramine  Ammonia   Nitrate + ‐ ‐ (NH2Cl) (NH3 / NH4 ) (NO2 ) (NO3 ) Nitrification is • Two unrelated groups of : – AOB: Nitroso‐ • Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosovibrio, Nitrosolobus • Also ammonia‐oxidizing – NOB: Nitro‐ • , Nitrospina, Nitrococcus, • Limited information regarding genera/species in drinking water distribution systems, and identification depends on methods used • Wide variety of characteristics and differences: – Shape, pigments, cell wall, flagella, reproduction... Nitrification is Responsible for Regulations, Effects of Nitrification Problems High levels of nitrite (and nitrate) Decrease in disinfectant residuals SWTR, TCR  May allow bacterial growth, does not protect the DS against contaminations High HPCs, potential presence coliforms SWTR, TCR and E. coli, cell lysis consumes chloramine Decrease in pH, dissolved , and Lead and Copper Rule alkalinity  May enhance corrosion Tastes and odors, particles Customer complaints “Free chlorine burn” DBPRs Nitrification and Regulations • Regulated as Inorganic Contaminants: ‐ – Nitrate MCL: 45 mg/L NO3 (10 mg/L N) Nitrite MCL: 1 mg/L N Nitrite + nitrate MCL: 10 mg/L N – At entry points to the distribution system: • Increases in nitrite/nitrate in the distribution system?

Nitrosomonas Watson et al., Bergey’s Manual of Systematic Bacteriology, 1989 Nitrification and Regulations • Difficult to exceed MCLs due to nitrification:

• Low Cl2:NH3 ratio, high free ammonia concentration, high chloramine residual • Many other challenges encountered before (e.g., loss in disinfectants, microbial growth, …) Nitrification and Regulations • Bacteriological quality determined by the (Revised) Total Coliform Rule (TCR): • Collect x samples per week/month (based on system size and type) • Coliforms (fecal coliforms or Escherichia coli) • Chlorine residual • MCL violation if: • > y% of samples are positive for total coliforms (based on system size and type) • Repeat sample is positive for fecal coliforms (FC) or E. coli • Following a FC+ or E. coli +, a repeat sample is TC+ • Revised TCR Nitrification and Regulations • Minimum residual set by Bacteriological Quality (Surface Water Treatment Rule, SWTR): • Entry points: Disinfectant “shall not be <0.2 mg/L for >4 hours in any 24‐hour period” • Distribution system: • Disinfectant “cannot be undetectable in >5% of samples in a month for any 2 consecutive months” • “HPC 500 ≤ CFU/mL is deemed to have detectable residual disinfectant” • Maximum residual set by the Stage 1 and Stage 2 Disinfectants and Disinfection Byproduct Rules (≤ MRDLs) Nitrification and Regulations • Minimum residual • Maximum residual set by the Stage 1 and Stage 2 Disinfectants and Disinfection Byproduct Rules: • Running Annual Average (RAA) of quarterly averages

• Monochloramine MRDL: 4.0 mg/L Cl2 Free chlorine MRDL: 4.0 mg/L Cl2 Chlorine dioxide MRDL: 0.8 mg/L ClO2 • DBP MCLs (chlorine burn): • THM4: 0.080 mg/L • HAA5 (ClAA, Cl2AA, Cl3AA, BrAA, Br2AA): 0.060 mg/L CAUSES AND TRIGGERS OF NITRIFICATION Triggers of Nitrification

Distribution system Water quality characteristics, operation • Substrate (free ammonia) • Water age • Monochloramine residual • Sediments, biofilms • Chlorine‐to‐ammonia ratio (system cleanliness) • • Temperature Corrosion • • pH, alkalinity Darkness • Organic material (treatment processes) Triggers – Ammonia • Obligate chemolithotrophs:

– dioxide (CO2) as carbon source – Inorganic substrates as energy source: • AOB: Ammonia + (NH3 may be the preferred source over NH4 ) • NOB: Nitrite – Some exceptions: • Organic substrates (Nitrobacter) → Slower growth • Organic material is not a food source, but affects chloramine stability → Ammonia release → Food • (Phosphorus is needed for nitrifier growth) Triggers – Chloramine • Chloramine degradation (decay and demand)  Releases ammonia  Food for AOB • Monochloramine stability is affected by: – Formation conditions: • Monochloramine dose (faster decay at higher doses) • Chlorine‐to‐ammonia ratio (faster decay at higher ratios) • pH (faster decay at low pH) – Decay reaction (auto‐decomposition): ‐ + 3 NH2Cl → N2 + NH3 + 3 Cl + 3 H – Monochloramine demand: Triggers – Chloramine • Monochloramine demand: – Microorganisms and biofilms – Corrosion products and tubercles – Organic material – Inorganic material: • Nitrite • Bromide

Vikesland et al., Water Research, 37:7:1766, 2001 Triggers –Cl2:NH3 Ratio • Desirable: 4.5:1 to 5:1 – Less ammonia available upon chloramine degradation – But monochloramine degrades faster at higher ratios

• As monochloramine decays, the Cl2:NH3 ratio decreases Triggers – Temperature • Range: 4‐60°C Optimum: 20‐35°C (25‐30°C) • Typically: >15°C • Duel effect of temperature: – Nitrifiers growth rate increases with temperature – Chloramine decomposition increases with temperature

Vikesland et al., Water Research, 37:7:1766, 2001 Triggers –pH

• Optimum: 6.5 to 8.5: .05 ) 1

– 7.5 to 8.0 for AOB .04 7.6 to 7.8 for NOB .03 • Dual effect of pH: .02 – Affect .01

– Affect chloramine stability 0.0 (chloramines are more Specific Growth rate (h - 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 stable at higher pH) pH • pH decreases during nitrification in low‐alkalinity waters  May increase corrosion Triggers – Alkalinity, DO

• ~7.1 g alkalinity CaCO3 consumed per 1 mg NH3‐N • Dissolved oxygen (DO): – Aerobic microorganisms + – AOB: 3.2 mg O2 per mg NH4 ‐N oxidized ‐ NOB: 1.1 mg O2 per mg NO2 ‐N oxidized – Some exceptions: • Low oxygen concentrations • Facultative aerobes

Nitrococcus mobilis Watson et al., Bergey’s Manual of Systematic Bacteriology, 1989 Inhibitors of Nitrification • Metals: – Nickel, chromium, iron, aluminum, zinc, lead, manganese – Copper: • Low levels may enhance nitrifier growth (<10 μg/L) • High levels are toxic to nitrifiers (>100 μg/L) • Cyanides • Phenols, mercaptans, • Halogenated compounds: e.g., THMs, perchlorate Inhibitors of Nitrification • THMs can delay the onset of nitrification: – AOB can biodegrade THMs through cometabolism, with production of compounds that are toxic to AOB • Brominated THMs are particularly toxic to nitrifiers

Speitel et al., Journal AWWA, 103:1:69, 2011 Triggers –Water Age • Nitrification generally occurs at high water age: – Lower monochloramine residual • Distribution system characteristics: – Dead‐ends: • By design • Closed valves – Storage facilities: • Poorly cycled or mixed • Common inlet/outlet – Oversized pipes – Multiple pressure zones Triggers – Corrosion • Nitrification can occur in pipes of any materials, however… – Corrosion tubercles can harbor microorganisms – Corrosion tubercles consume disinfectants: + 2+ 3+ + ‐ ½ NH2Cl + H + Fe → Fe + ½ NH4 + ½ Cl – Corrosion control practices can affect nitrifiers because they need phosphorous to grow – Benefits of corrosion control outweigh its drawbacks

Golden State Water Company Triggers – Darkness • Nitrifiers are inhibited by light, but not fully inactivated by it • NOB may be more sensitive than AOB • Recovery from inhibition in darkness • Photo‐inhibition and recovery depend on: • Illumination intensity • Attenuation properties of water • Water circulation (exposure of microorganisms)

AWWA, Opflow 2010 INDICATORS AND MONITORING OF NITRIFICATION Nitrification Indicators • No ideal method to identify and enumerate nitrifiers: Advantages Disadvantages Culturing • Relatively easy • Underestimate counts techniques • Enumeration • Difficult to identify species possible • Long incubation time Microscopy • Faster than • Initial isolation required and serological culturing • Large diversity requires techniques techniques different antibodies Molecular • Identification • Large diversity requires methods possible different primers/probes • Fastest • Complex; not readily usable by water utilities Nitrification Indicators • Monitoring based on indicators: • Substrates: Ammonia (free), and nitrite • Products: Nitrite, and nitrate • Chloramine residual • HPCs • Temperature, pH Nitrospina gracilis, • Drawbacks of indicators: Watson et al., 1989  Indicators are the results of nitrification; They do not predict nitrification  Trends are system dependent  Trends may be due to factors other than nitrification Summary • Carried out by various bacteria that belong to different groups, different characteristics • Potential causes are numerous, including water quality, and distribution system characteristics and operations • Favorable growth conditions of nitrifiers are similar to those observed in distribution systems • Lack of simple, quantitative and rapid methods for identification and enumeration ‐ ‐ – Monitored via indicators (NH3, NO2 , NO3 , NH2Cl) Thank you!

Hélène Baribeau, Ph.D., P.E. (714) 488‐0496 [email protected]