Chloroform fumigation extraction

The chloroform fumigation extraction (CFE) is a method for determining the microbial biomass in soils .

principle

The soil sample is divided into two sub-samples. A partial sample is extracted with 0.5 mol L ⁻¹ K ₂ SO ₄ solution and the extract is filtered. The second partial sample is fumigated with chloroform in a desiccator for 24 hours . The chloroform penetrates the soil pores, kills the microorganisms and lyses the cells. The chloroform is then removed by evacuating and ventilating several times . The partial sample is then also extracted. The dissolved organic carbon (DOC) or total nitrogen is determined in the extracts .

The microbial carbon C _mic is calculated as:

${\ displaystyle C_ {mic} = {\ frac {C_ {DOC, f} -C_ {DOC, nf}} {k_ {EC}}}}$

C _{DOC, f} - DOC concentration in the extract from the fumigated partial sample (gg ⁻¹ dry matter)
C _{DOC, nf} - DOC concentration in the extract from the non-fumigated partial sample (gg ⁻¹ dry matter)
k _EC - extraction factor

The microbial nitrogen N _mic is calculated as:

${\ displaystyle N_ {mic} = {\ frac {C_ {N, f} -C_ {N, nf}} {k_ {EN}}}}$

C _{N, f} - N concentration in the extract from the fumigated partial sample (gg ⁻¹ dry matter)
C _{N, nf} - N concentration in the extract from the non-fumigated partial sample (gg ⁻¹ dry matter)
k _EN - extraction factor

Extraction factors

The extraction factors take into account that not all cells are completely lysed and some of the organic carbon or nitrogen is adsorbed on the soil or retained during filtration. The factors were determined by comparing results from the CFE method with results from the chloroform fumigation incubation . The mean values determined are k _EC = 0.45 and k _EN = 0.54. For individual soils, these values can differ significantly from the mean value. Nevertheless, the mean values are mostly used and the method is not calibrated in individual cases.

Cons and criticism

The procedure is very imprecise. Even with sieved and thus homogenized samples, coefficients of variation of 20% are not uncommon. Fine roots can lead to an overestimation of the microbial biomass and may need to be removed before the procedure. Especially in clay soils, chloroform is in some cases not completely removed by evacuation and can therefore also lead to an overestimation. Despite these disadvantages, the method is widely used because it is inexpensive and easy to carry out and possible alternatives also have disadvantages.

Alternatives

Possible alternatives are substrate-induced respiration (SIR), chloroform fumigation incubation and determination of extractable phospholipid fatty acids (PLFA). The determination of the total DNA is less suitable as the proportion of DNA in the total biomass (especially between bacteria and fungi) can vary considerably.

supporting documents

DIN ISO 14240-2: 1997 Soil quality - Determination of the microbial biomass of soils - Part 2: Fumigation extraction method

Individual evidence

↑ Joergensen, RG, 1996. The fumigation-extraction method to estimate soil microbial biomass: calibration of the k _EC value. Soil Biology and Biochemistry 28, 25-31.
↑ Joergensen, RG, Mueller, T., 1996. The fumigation-extraction method to estimate soil microbial biomass: Calibration of the k _EN value. Soil Biology and Biochemistry 28, 33-37.
↑ Alessi, DS, Walsh, DM, Fein, JB, 2011. Uncertainties in determining microbial biomass C using the chloroform fumigation extraction method. Chemical Geology 280, 58-64.
↑ DIN ISO 14240-1: 1999-10
↑ Jenkinson, DS, Powlson, DS, 1976. The effects of biocidal treatments on metabolism in soil - V. A method for measuring soil biomass. Soil Biology and Biochemistry 8, 209-213.
↑ ^a ^b Leckie, SE, Prescott, CE, Grayston, SJ, Neufeld, JD, Mohn, WW, 2004. Comparison of chloroform fumigation-extraction, phospholipid fatty acid, and DNA methods to determine microbial biomass in forest humus. Soil Biology and Biochemistry 36, 529-532.
↑ Bailey, VL, Peacock, AD, Smith, JL, Bolton Jr., H., 2002. Relationships between soil microbial biomass determined by chloroform fumigation – extraction, substrate-induced respiration, and phospholipid fatty acid analysis. Soil Biology and Biochemistry 34, 1385-1389.

[kec-1] Joergensen, RG, 1996. The fumigation-extraction method to estimate soil microbial biomass: calibration of the k _EC value. Soil Biology and Biochemistry 28, 25-31.

[ken-2] Joergensen, RG, Mueller, T., 1996. The fumigation-extraction method to estimate soil microbial biomass: Calibration of the k _EN value. Soil Biology and Biochemistry 28, 33-37.

[clay-3] Alessi, DS, Walsh, DM, Fein, JB, 2011. Uncertainties in determining microbial biomass C using the chloroform fumigation extraction method. Chemical Geology 280, 58-64.

[SIR-4] DIN ISO 14240-1: 1999-10

[CFI-5] Jenkinson, DS, Powlson, DS, 1976. The effects of biocidal treatments on metabolism in soil - V. A method for measuring soil biomass. Soil Biology and Biochemistry 8, 209-213.

[PLFA1-6] Leckie, SE, Prescott, CE, Grayston, SJ, Neufeld, JD, Mohn, WW, 2004. Comparison of chloroform fumigation-extraction, phospholipid fatty acid, and DNA methods to determine microbial biomass in forest humus. Soil Biology and Biochemistry 36, 529-532.

[PLFA2-7] Bailey, VL, Peacock, AD, Smith, JL, Bolton Jr., H., 2002. Relationships between soil microbial biomass determined by chloroform fumigation – extraction, substrate-induced respiration, and phospholipid fatty acid analysis. Soil Biology and Biochemistry 34, 1385-1389.