Chloroform Fumigation Incubation And Extraction Methods

Microbial biomass is measured to give an indication of the response of soil microbiota to management, environmental change, site disturbance, and soil pollution. Two different approaches are both based on CO2 evolution. The chloroform fumigation incubation (CFI) method (Jenkinson and Powlson, 1976) exposes moist soil to ethanol-free chloroform for 24 h to kill the indigenous microorganisms. After removal of the fumigant, a flush of mineralized CO2 and NH4 is released during a 10-day incubation. This flush is caused by soil microorganisms that have survived the fumigation (as spores or cysts) and use cell lysates as an available C and energy source. The released CO2 is trapped in an alkaline solution and is quantified by titration. Alternatively, the CO2 that accumulates in the head-space of the sample containers is measured by gas chromatography. An assay of nonfumigated soil serves as a control. The amount of microbial biomass C is calculated as biomass C = (FC - UfC)/KC, where biomass C is the amount of carbon trapped in the microbial biomass, FC is the CO2 produced by the fumigated soil, UfC is the CO2 produced by the nonfu-migated soil sample, and KC is the fraction of the biomass C mineralized to CO2. The KC value is a constant representative of the cell utilization efficiency of the fumigation procedure. This efficiency is considered to be about 40-45% for many soils (e.g., a constant of 0.41-0.45). Deviations of this range are found for subsurface and tropical soils. The KC factor of soil samples can be estimated by measuring the 14CO2 release of soil microorganisms isolated from different soils that use radiolabeled bacterial cells as substrates.

The chloroform fumigation extraction (CFE) method involves the extraction and quantification of microbial constituents (C, N, S, and P) immediately following CHCl3 fumigation of the soil (Brookes et al., 1985). The efficiency of soil microbial biomass extraction has to be taken into account. A Kec factor of 0.45 is recommended for agricultural soils (Joergensen, 1996), and the Kec factor of soils from other environments (subsurface soils, peat soils, etc.) should be experimentally derived. The CFE method can be applied to a wide range of soils. Soils containing large amounts of living roots require a preextraction procedure of roots because these cells are also affected by the fumigation procedure.

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