Robotized incubation system for monitoring gases (O2, NO, N2O N2) in denitrifying cultures

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 N2, O2, CH4, CO2, N2O) to the inlet of a chemoluminescence NO analyzer. The linear range for NO is 0.5–104 ppmv (CV = 2%, detection limit 0.2 ppmv). The gas leakage of N2 into the system is low and reproducible, allowing the quantification of N2 production (in flasks with He + O2 atmosphere) with a detection limit of 150–200 nmol N2 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 (O2, NO2-, NO, N2O) throughout the culture's depletion of O2 and NOx. 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 O2 and NO in the medium.