Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil

Abstract

ABSTRACT Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene ( amoA ) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 × 10 5 cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH 4 ) 2 SO 4 application. AOB populations in amended treatments increased from an initial density of approximately 4 × 10 6 cells/g of dry soil to peak values (day 7) of 35 × 10 6 and 66 × 10 6 cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 × 10 9 and 2.2 × 10 9 cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 × 10 6 to 38.0 × 10 6 cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH 4 + h −1 cell −1 and decreased with time in both microcosms and the field. Growth yields were 5.6 × 10 6 , 17.5 × 10 6 , and 1.7 × 10 6 cells/mol of NH 4 + in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.