ABSTRACT
Nitrosomonas europaea and Nitrobacter winogradskyi (strain "Engel") were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25 degrees C and pH 8. Fitting the retentostat biomass and oxygen consumption data of N. europaea and N. winogradskyi to the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 micromol of O2 mg-1 of biomass-C x h-1 for both N. europaea and N. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster for N. winogradskyi than for N. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable. N. europaea more readily recovers from starvation than N. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 microg x ml-1) inhibition experiments with N. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514-1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.
