Uncertainties in determining microbial biomass C using the chloroform fumigation–extraction method

We tested the accuracy of the chloroform fumigation–extraction method, which is commonly used to determine soil biomass C concentrations. Accurate and precise determination of total microbial biomass is important in order to characterize soil properties and to develop predictive metal transport models for soils. Two natural soils, and individual soil components, including silica sand, montmorillonite, kaolinite, a humic acid, and Bacillus subtilis bacterial cells, were fumigated for 24 h. Following the fumigation, C from fumigated and unfumigated samples was extracted using a 0.5 M K2SO4 solution. The difference between the C content in the fumigated and unfumigated samples ideally represents C due to biomass because the fumigation procedure should lyse cells and release biomass C. We observed increased 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 unfumigated samples of silica sand and of humic acid, confirming that the fumigation process does not introduce additional organic C to samples of these soil components. However, the fumigated clay samples both showed increased C release relative to the unfumigated controls, indicating that significant concentrations of the fumigant, chloroform, adsorbed onto the clay minerals studied here. Additionally, we found significant chloroform remaining in the extracts from two fumigated natural soils. Attempts to remove the chloroform from the soils or soil components prior to extraction by increasing the evacuation time, or to remove chloroform in the extracts by sparging them vigorously with nitrogen gas, both failed. This research reveals that chloroform gas may adsorb significantly to clays and the clay fraction of natural soils. Thus, the fumigation–extraction method must be corrected to account for the added chloroform C and accurately assess the concentration of biomass C in soils that contain significant concentrations of clays.

Research Highlights
► Chloroform adsorbs strongly to clay surfaces over 24 h
► Adsorbed chloroform can be interpreted as biomass in the fumigation–extraction method
► The fumigation–extraction method should be corrected for chloroform adsorption