Testing the Accuracy of the Chloroform Fumigation-Extraction Method for Determining Microbial Biomass C in Soils.

Accurate and precise determination of total microbial biomass in soils is important in order to characterize soil properties and to develop predictive metal transport models.  In recent decades, chloroform fumigation-extraction has become the most widely used method for the determination of microbial biomass C in soils, however the method has never been rigorously tested with control experiments to determine the effect of chloroform adsorption on the accuracy of the procedure. We performed control experiments, measuring biomass C in individual soil components such as sand, clay, humic acid, and bacteria, to test the accuracy of the method.  Each individual component was fumigated for 24 hours with chloroform gas, which was subsequently evacuated under vacuum.  Following the fumigation, C from fumigated and un-fumigated samples was extracted using a 0.5M K₂SO₄ solution.  The difference between the C content in the fumigated and un-fumigated samples ideally represents C due to biomass because the fumigation procedure should lyse cells and release biomass C. We observed enhanced C release upon fumigation for bacteria-only samples, confirming the ability of fumigation to lyse cells.  There was no difference in extracted C concentration between fumigated and un-fumigated samples of biomass-free silica sand and humic acid, confirming that the fumigation process does not introduce additional organic C to samples of these soil components.  However, fumigated samples of two clays, kaolinite and montmorillonite, showed enhanced C release relative to the un-fumigated controls, indicating that significant concentrations of the fumigant, chloroform, adsorbed onto these clay minerals.  Our work reveals that the fumigation-extraction method cannot be used to accurately assess the concentration of biomass C in soils that contain significant concentrations of clays. Because most soils in temperate environments do contain a significant proportion of clays, another approach must be developed to quantify concentrations of biological cells in these soils.