Aerobic respiration rates of bacteria: The influence of nutrient-limitation, cell density, and nutrient pulsing

Many environments on Earth experience nutrient limitation and as a result have non-growing bacterial populations. To better understand bacterial respiration under environmentally relevant conditions, the effect of nutrient limitation on heterotrophic bacterial respiration rates was measured. The oxygen consumption of batch cultures of Escherichia coli ZK126, Shewanella oneidensis MR-1, and Marinobacter aquaeolei VT8 was determined as they transitioned into a state of nutrient limitation. Specific oxygen consumption, QO2, (&mgr;mol O2 CFU -1day-1) was determined by measuring the rate of change of the concentration of dissolved oxygen in a subsample of the culture, normalized to the subsample's population of platable cells (CFUs). Transitioning from nutrient-excess (growth) to nutrient-limitation (stationary phase) reduced QO2 (ZK126 by 420x, MR-1 by 210x, VT8 by 490x). However, following death phase, the small persisting population (LTSP) showed QO2 levels increasing to values close to those during growth phase. After a stationary phase minimum, the fraction of cells that stained positive for respiration increased and a subpopulation of highly respiring cells emerged. The large reduction in QO2 from growth to stationary phase suggests that nutrient status is an important factor when considering environmental respiration rates. Increasing rates observed after death phase suggest that the population of respiring cells is not well represented by CFUs, rather a population of respiring but not platable cells develops during LTSP along with a subpopulation of highly respiring cells.;When the post-death QO2 data from the 200 day experiment is compared to its corresponding CFU ml-1 data, there is a systematic inverse relationship. To confirm this relationship in ZK126, a 30 day old culture was split into 10X, 1X, 1/2X, and 1/10X concentrations and analyzed for oxygen consumption, CFUs, phase contrast cells and respiring cells. The bulk solution community oxygen consumption of each new population converged over 23.2 days while the number of CFUs per split remained stable. The number of phase contrast and respiring cells changed with large and significant increases in the 1/10X split. The distribution of respiration intensity within the respiring cell fraction changed too. The 10X sample showed a decline in "high respiration" cells over the experiment while the 1/10X sample showed a general pattern of higher intensity respiration than the other splits. A combination of changes in respiring population size and distribution of respiration intensity was responsible for the convergence in community oxygen consumption.;To understand how nutrient pulsing affects bacterial evolution, clonal populations of Shewanella oneidensis MR-1 were subjected to two different 39 day nutrient pulse schedules and measured for changes in growth rate, yield, lag, and oxygen consumption. Schedule 1 populations experienced long pulses of high-nutrient media punctuated by short pulses of low-nutrient media. Schedule 2 populations experienced long pulses of low-nutrient media punctuated with short pulses of high-nutrient media. Both schedules received the same total amount of nutrients. Population density was tracked over the two schedules and patterns diverged strongly from each other but were very consistent within schedule replicates. Final populations were analyzed in high and low-nutrient media. Both schedule final population traits changed significantly from the Ancestor, but showed no significant divergence in traits based on the schedule experienced. Both populations combined demonstrate significant (p < 0.004) decrease in growth rate, increase in yield, and decrease in lag time in high-nutrients. In low nutrients, growth rate increased (p = 0.07), yield decreased (p = 0.18), and oxygen consumption increased (p = 0.08). These results suggest that pulsed populations experienced selection for slower more efficient growth in high nutrients and faster more competitive growth in low nutrients. Growth, yield, and oxygen consumption traits of the final populations support an evolutionary trade-off in low-nutrients but not high. This suggests that the MR-1 Ancestor has previously evolved in a low-nutrient environment. Nutrient pulsing quickly changed growth and efficiency characteristics of MR-1 in a short laboratory experiment, but the specific timing and duration of the nutrient pulses was not an important factor.