Effect of nitrite concentration and pH on most probable number enumerations of non-growing Nitrobacter spp.

Abstract Enumerations of nitrite-oxidizing bacteria in soil samples by a Most Probable Number technique, often showed relatively high cell numbers at a low nitrite concentration compared with the numbers of ammonium-oxidizing bacteria. It was hypothesized that the high numbers enumerated at low nitrite concentration would represent non-growing or organotrophically growing cells of nitrite-oxidizing species. In this paper, the sensitivity of non-growing Nitrobacter species to high nitrite concentrations as well as to low pH was examined. Different Nitrobacter species were pre-cultured at 0.5 mM nitrite. Non-growing cells differing in age were enumerated at different nitrite concentrations and pH values. The incubation period lasted for 5 months at 20°C. However, during the incubation periods of the older non-growing cells, it appeared that a period of 5 months might have been too short for reaching constant numbers. Early stationary cells of all species that were studied appeared not to be affected by high nitrite concentrations or low pH. Eight- and 18-month-old non-growing cells of Nitrobacter hamburgensis were also insensitive to 5 mM nitrite. The numbers of 8- and 18-month-old resting cells of N. vulgaris were only repressed by a combination of 5 mM nitrite and a low pH. Eight-month-old non-growing cells of N. winogradskyi were sensitive to 5 mM irrespective of pH, but 18-month-old cells only to 5 mM nitrate at low pH. The numbers of 8- and 18-month-old resting cells of N. winogradskyi serotype agilis were repressed by low pH rather than high nitrite concentration. Hence, it was concluded that the large differences in numbers of nitrite-oxidizing bacteria obtained with low and high nitrite concentrations in the incubation medium, was not likely to be due to the presence of non-growing Nitrobacter species in soil samples, but rather to the existence of organotrophically growing Nitrobacter cells.