COMPETITION BETWEEN NITRATE AND NITRITE REDUCTION IN DENITRIFICATION BY PSEUDOMONAS-FLUORESCENS

A pure culture of Pseudomonas fluorescens was used as a model system to study the kinetics of denitrification. An exponentially growing culture was harvested and resuspended in an anoxic acetate solution buffered with K/Na phosphate at pH values of 6.6, 7.0, 7.4, and 7.8. The temperature was kept at 28 degrees C in all assays. Nitrate pulses of approximately 0.2 mg NIL caused nitrite to accumulate due to a faster rate of nitrate reduction over nitrite reduction. The rate of nitrate reduction was observed to depend on its concentration as predicted by the Michaelis-Menten equation. At nonlimiting nitrate concentrations, nitrite reduction was described by the same equation. Otherwise, nitrite reduction also depended on nitrate concentration. Consequently, nitrate and nitrite reductions compete with each other for the oxidation of common electron donors. A kinetic model for nitrate competitive inhibition of nitrite reduction is proposed. The model was used to interpret the nitrate and nitrite profiles observed at the four pH values: the optimum pH value was 7.0 in both cases; the affinity for nitrate was constant (K-m NO3 = 0.04 mg N/L); the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (K-m NO2 = 0.06 mg N/L), but it decreased sharply for the pH value of 7.8. Although the ratio between the two maximum reduction rates (V-max NO2/ V-max NO3) is constant, nitrite accumulation depends on the medium pH value. Therefore, the regulation mechanism that shifts the electron flow between the two terminal reductases is readily reversible and does not change their relative maximum reduction rates. (C) 1995 John Wiley & Sons, Inc.