Robotized incubation system for monitoring gases (O 2, NO, N 2O N 2) in denitrifying cultures

Abstract

As genomic data for bacteria are unraveled at an increasing speed, there is a need for more efficient and refined techniques to characterize metabolic traits. The regulatory apparatus for denitrification, for instance, has been explored extensively for type strains, but we lack refined observations of how these and wild type denitrifiers respond metabolically to changing environmental conditions. There is a need for new “phenomic” approaches, and the present paper describes one; an automated incubation system for the study of gas kinetics in 15 parallel bacterial cultures. An autosampler with a peristaltic pump takes samples from the headspace, and replaces the sampled gas with He by reversing the pump. The sample flows through the injector of a micro GC (for determination of N 2, O 2, CH 4, CO 2, N 2O) to the inlet of a chemoluminescence NO analyzer. The linear range for NO is 0.5–10 4 ppmv (CV = 2%, detection limit 0.2 ppmv). The gas leakage of N 2 into the system is low and reproducible, allowing the quantification of N 2 production (in flasks with He + O 2 atmosphere) with a detection limit of 150–200 nmol N 2 for a single time increment. The gas loss by each sampling is taken into account, securing mass balance for all gases, thus allowing accurate estimation of electron flows to the various terminal acceptors (O 2, NO 2 -, NO, N 2O) throughout the culture's depletion of O 2 and NO x . We present some experimental results with Agrobacterium tumefaciens, Paracoccus denitrificans and denitrifying communities, demonstrating the system's potential for unraveling contrasting patterns of denitrification gene expression as a function of concentrations of O 2 and NO in the medium.