Methodology for assessing respiration and cellular incorporation of radiolabeled substrates by soil microbial communities

Abstract

A method is described for determining biodegradation kinetics of both naturally occurring and xenobiotic compounds in surface and sub-surface soil samples. The method measures both respiration and uptake into cellular biomass of¹⁴C-labeled substrates. The estimation of biomass incorporation entailed removal of cells from soil particles by washing the soil with a polyvinyl-pyrrolidone/pyrophosphate solution and H₂O₂. After separation of the cells and the soil particles by centrifugation, the cells were trapped on membrane filters for liquid scintillation counting. Mass balances were easily obtained. The technique was used to measure metabolic activity in soil profiles, including unsaturated and saturated zones. First order rate constants (K₁) were in the range of 10⁻³−10⁻² hour⁻¹ for amino acid metabolism and 10⁻⁵−10⁻⁴ hour⁻¹ for m-cresol metabolism. Saturation kinetics were observed for amino acids and m-cresol. m-Cresol K₁ values for uptake often exceeded those for respiration by greater than a factor of ten. V_max values were low (amino acids, 10¹−10² ng g⁻¹ hour⁻¹; m-cresol, 10⁻¹ ng g⁻¹ hour⁻¹), whereas K_m values were quite high (amino acids, 10³−10⁴ ng g⁻¹; m-cresol 10³−10⁵ ng g⁻¹). Saturation was not observed in many horizons even at 10⁵ ng g⁻¹ dry soil. Frequently, respiration obeyed saturation kinetics whereas uptake was first order. It is concluded that measuring only kinetics of respiration may lead to severe underestimations of biodegradation rates.