Sensitive and accurate methodology for measuring the kinetics of concentration-dependent hydrocarbon metabolism rates in seawater by microbial communities.

A method having sufficient sensitivity to resolve the kinetic constants for dissolved nonpolar substrate metabolism, together with the related rate constants in natural waters, is presented. The method is based on the rate of CO(2) recovery from radioactive dissolved substrate. Sensitivity is enhanced by using large seawater volumes, high-specific-activity isotopes, and by reducing background radioactivity. Before use, commercial isotopes are purified by mild alkaline hydrolysis followed by sublimation from base to remove CO(2) as well as interfering polar C-substrates. During sample analysis, chilled Tenax resin is used to remove volatile C-substrate from the nitrogen stream containing CO(2) recovered from substrate oxidation. Chromatographic evidence of purity, shown to be insufficient, is augmented by kinetic data from toluene utilization by mixed cultures and by rates in induced versus noninduced pure cultures. Accuracy is enhanced by using short (<10 h) incubation times and small hydrocarbon concentrations so that the metabolism rates in unamended natural water systems can be evaluated. Toluene metabolism rates in seawater as low as 1 pg/liter per h and at concentrations as low as 20 ng/liter have been determined.