Fluorescent in situ hybridization (FISH) was used to quantify the ammonia-oxidizing populations within intact biofilm samples collected from a full-scale nitrifying trickling filter (NTF). Ammonia, nitrite, and nitrate concentrations were measured for aqueous samples taken in conjunction with biofilm samples at multiple filter depths. Correlation coefficients for individual sampling events, calculated by simple linear regression of FISH signal area and ammonia removal rates, ranged from 0.558 when using probe NEU23a to 0.982 when using probe Nso190. The improved correlations with Nso190 suggest that genera other than Nitrosomonas are present in this system. Percent biofilm coverage, as determined by 4Ј,6-diamidino-2phenylindole counterstaining and dry weight biomass measurements, did not change throughout the NTF. This indicates that biofilm growth is fairly uniform throughout the filter even if nitrifier growth is not. Water Environ. Res., 73, 329 (2001).

The effect of alkalinity on nitrifying biofilm activity was determined by collecting 21‐day‐old biofilm samples from the top of a full‐scale nitrifying trickling filter and evaluating bench‐scale nitrate plus nitrite generation rates at (1) various initial carbonate alkalinity concentrations and (2) with four types of available alkalinity: carbonate only, phosphate only, phosphate plus hydroxide, and phosphate plus carbonate. Initial carbonate alkalinity concentrations were varied between 308and 20 mg/L as calcium carbonate (CaCO3). Ammonia, nitrite, and nitrate concentrations were measured at time zero, 90 minutes, 180 minutes, and 270 minutes. Generation rates in grams of nitrogen per square meter per day were calculated for each time period and normalized against dry‐weight biomass. Generation rates were impaired at initial carbonate alkalinity concentrations of 40 mg/L and lower (as CaCO3) and were unaffected at concentrations of 45 mg/L and greater. For reactor runs with different alkalinity types, ammonia, nitrite, and nitrate concentrations were measured at time zero and at 375 minutes. The type of alkalinity, carbonate versus phosphate, affected nitrification rates. When the carbonate alkalinity was less than 45 mg/L, nitrification rates were impaired regardless of the total alkalinity concentration. This effect seems to be independent of pH for the range of 6.92 to 7.99 evaluated here. This suggests that in addition to neutralizing the acid generated by the nitrification process, a minimum level of carbonate alkalinity is necessary to meet the ammonia‐oxidizer's inorganic carbon requirement for cellular synthesis and growth.

Biofilm samples from a carbonaceous trickling filter were evaluated in bench scale reactors to determine their maximum potential denitrification rates. The samples were collected as intact, undisturbed biofilms by inserting clean microscope slides into the trickling filter at various locations, leaving them in place for 28 days, and then removing them and bringing them back to the laboratory. In the laboratory, they were placed into 0.6 L bench-scale reactors filled with sterilized, primary clarifier effluent that had been spiked with nitrate to a final concentration of 16-18 mg/L as N. Dissolved oxygen (D.O.) concentrations were maintained between 2 and 4 mg/L in the bulk aqueous phase to simulate conditions within the full-scale system. Nitrate loss from the reactors was monitored over a five hour period. Denitrification rates of 3.09 to 5.55 g-N/m 2 ·d were observed with no initial lag period. This suggests that the capacity for denitrification is inherent in the biofilm and that denitrification can take place in the deeper layers of the biofilm even when oxygen is present at significant concentrations in the bulk aqueous phase. There were no significant differences in denitrification rates per unit area of media (g-N/m 2 ·d) either between (a) experimental runs or (b) sampling locations over the TF. This suggests that denitrification potentials are uniform over the entire volume of the full-scale TF. For wastewater treatment plants with TFs that currently nitrify downstream, recycle flows may be used to remove nitrate in their existing upstream TFs. This approach may be used to meet less stringent permitted discharge concentrations and may allow some facilities to postpone or eliminate construction of additional unit processes for denitrification.

The effect of alkalinity on nitrifying biofilm activity was determined by collecting twenty-one day old biofilm samples from a full-scale nitrifying trickling filter and evaluating bench-scale nitrate plus nitrite generation rates at (1) various initial carbonate alkalinity concentrations and (2) with four types of available alkalinity; carbonate only, phosphate only, phosphate plus hydroxide, and phosphate plus carbonate alkalinity. Initial carbonate alkalinity concentrations were varied between 308 and 20 mg/L as CaCO 3 . Ammonia, nitrite, and nitrate concentrations were measured at time zero, 90 minutes, 180 minutes, and 270 minutes. Generation rates in g N/m 2 *day were calculated for each time period and normalized against dry weight biomass. The effect of the initial carbonate alkalinity concentration on generation rate was evaluated by twotailed ANOVA followed by LSD analysis at a 95% confidence interval. Generation rates were impaired at initial alkalinity concentrations at and below 40 mg/L as CaCO 3 and were unaffected at concentrations of 45 mg/L and above. This is consistent with Gujer and Boller's (1984) proposal and Szwerinski's et al. (1986) data that alkalinity is rate limiting below 1-2 millimoles/liter or 50-100 mg/L as CaCO 3 . For reactor runs with different alkalinity types, ammonia, nitrite, and nitrate concentrations were measured at time zero and at 375 minutes. Generation rates were calculated and normalized against dry weight biomass measurements. The effect of alkalinity type on generation rate was evaluated by two-tailed ANOVA followed by Tukey analysis. The type of alkalinity, carbonate versus phosphate, affected nitrification rates. If the carbonate alkalinity was below 45 mg/L, nitrification rates were impaired regardless of the total alkalinity. This effect seems to be independent of pH for the range of 6.92 to 7.99 evaluated here. This would suggest that in addition to neutralizing the acid generated by the nitrification process, a minimum level of carbonate alkalinity is necessary to meet the ammoniaoxidizer's inorganic carbon requirement for cellular synthesis and growth.

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