Nitric Oxide Reductase-Targeted Real-Time PCR Quantification of Denitrifier Populations in Soil

Abstract

ABSTRACT The quantification of denitrifying bacteria is a component in the further understanding of denitrification processes in the environment. Real-time PCR primers were designed to target two segments of the denitrifier population ( cnorB P [ Pseudomonas mandelii and closely related strains] and cnorB B [ Bosea , Bradyrhizobium , and Ensifer spp.]) in agricultural soils based on functional cnorB (nitric oxide reductase) gene sequences. Total population numbers were measured using 16S rRNA gene real-time PCR. Two soil microcosm experiments were conducted. Experiment 1 examined the response of the indigenous soil microbial population to the addition of 500 mg/kg glucose-C daily over 7 days in soil microcosms. Changes in the total population were correlated ( r = 0.83) between 16S rRNA gene copy numbers and microbial biomass carbon estimates. Members of the cnorB P population of denitrifiers showed typical r-strategy by being able to increase their proportion in the total population from starting levels of <0.1% to around 2.4% after a daily addition of 500 mg/kg glucose-C. The cnorB B guild was not able to increase its relative percentage of the total population in response to the addition of glucose-C, instead increasing copy numbers only in proportion with the total population measured by 16S rRNA genes. Experiment 2 measured population dynamics in soil after the addition of various amounts of glucose-C (0 to 500 mg/kg) and incubation under denitrifying conditions. cnorB P populations increased proportionally with the amount of glucose-C added (from 0 to 500 mg/kg). In soil microcosms, denitrification rates, respiration, and cnorB P population densities increased significantly with increasing rates of glucose addition. cnorB B guild densities did not increase significantly under denitrifying conditions in response to increasing C additions.