Microbial community analysis: a kinetic approach to constructing potential C source utilization patterns

The analysis of multiple substrate metabolism by assemblages of bacterial strains may be used to differentiate inocula from environmental samples. Biolog plates, 96-well microtiter plates containing nutrients, a single carbon test substrate in each well and a tetrazolium redox dye to monitor substrate oxidation, have been used for this purpose. One of the difficulties faced by users of this technique is determining which substrates have been metabolized. Reliance on single-time-point absorbance data for each well is problematic due to variably non-linear rates of color development for each well. Previous efforts to use color-normalized single plate readings have been successful in discriminating between environmental sample types, but substrate-use contributions to sample classifications vary depending on the duration of the plate incubation. We present a model based on the logistic equation for density-dependent population growth providing a good (low χ2) fit to the sigmoidal kinetics of color development data. The kinetic parameters generated by the model can be used as surrogates for single-time-point data in constructing carbon source utilization patterns, and contribution of substrate use to sample classification does not depend on incubation time. This technique obviates the need to choose the time following inoculation to read the plate absorbance data and also provides two kinetic parameters that are invariant with respect to inoculum density. We provide a comparison of community potential substrate use analyses using single-time-point microplate data and parameters from our kinetic model.