In the original chloroform fumigation method, soil samples are fumigated to lyse all microorganisms. The released carbon dioxide is mineralized. Chloroform kills microbes, but does not solubilize other organic matter found in soil. Soil scientists have determined that 24 hours of chloroform fumigation is enough to lyse all microorganisms. Captured carbon dioxide is compared to a similar unfumigated soil sample, which is the control sample. Subtracting the values of the unfumigated control sample from the fumigated soil sample gives a value for the carbon biomass.
To determine carbon or nitrogen in a soil sample, extract chloroform-fumigated soil samples with potassium sulfate (K2SO4).This has become a standard step in the analysis of chloroform-fumigated soil samples. After the extraction with the potassium sulfate solution, filter the chloroform-fumigated and unfumigated soil samples. Measure total dissolved carbon on a TIC/TOC (total inorganic carbon/total organic carbon) analyzer or other method. Assess total carbon biomass by subtracting the values for the unfumigated sample from the chloroform-fumigated sample. To determine the nitrogen content of the soil sample, subject the potassium sulfate extract to Kjeldahl digestion or a similar procedure.
The characteristics of the soil sample -- such as large amounts of non-microbial organic material and excess water content -- may require modifications to the standard chloroform fumigation method. In the standard method, you subtract the unfumigated soil sample from the chloroform-fumigated sample to measure carbon biomass. When dealing with changes or disturbances in soil, scientists often do not subtract an unfumigated control sample. Sometimes, soil scientists extend the time of chloroform fumigation incubation for more than 24 hours for optimal results. Scientists, measuring biomass in flooded rice samples, developed a chloroform fumigation protocol without using a vacuum. Chloroform fumigation under vacuum is standard. Scientists also modified the chloroform fumigation extraction method to reduce salt concentration when measuring radioactive carbon in soil samples. They used potassium chloride instead of potassium sulfate for extraction and added a dializing step.
When soils contain large amounts of organic matter, are waterlogged or contain high clay content, the chloroform fumigation extraction method may over or underestimate carbon or nitrogen content. For example, in clay soil samples, some chloroform residues cannot be removed after fumigation and remain absorbed in the clay. This increases the amount of carbon in biomass measurements, according to a 211 report in Chemical Geology. In problem soil samples, isotope labeling and other methods such as phospholipid fatty acid analysis or substrate-induced respiration may have to be used.