Microbial biomass C by chloroform fumigation and extraction
Garcia-Pausas, J.1; Casals P.1
1 Forest Science and Technology Centre of Catalonia (CTFC)
Last updated: 25/09/2027
Recommended citation:
Garcia-Pausas, J.; Casals P.. 2026. Microbial biomass C by chloroform fumigation and extraction. In: Pere Casals, Davide Ascoli, Lena Vilà-Vilardell, Imma Oliveras, Madoka Krick (eds.) 2026. Methods handbook for characterizing fire use behavior, assessing fire effects, and monitoring soil and vegetation responses at the stand level. FIRE-ADAPT project (number 101086416), deliverable D1.1
Indicator
Ecosystem responses: Soil responses
Brief introduction
The microbial biomass is a key component of the soil organic matter, since the soil microbes (i.e. bacteria and fungi) mineralise the soil organic matter, while releasing nutrients that can be used by plants. The method aims to assess the size of the microbial biomass, by determining the extractable C flush after chloroform fumigation of soil samples. This is useful to compare soil treatments (perturbations, management systems, etc), but comparisons among soils spanning a wide pH range should be avoided (Haney et al, 2001).
Method specifications
Method type
Soft
Hard
Method timing
Before
fire
During
fire
After fire
(short-term)
After fire
(mid-term)
After fire
(long-term)
Field skills
Low
Medium
Hard
Lab skills
Low
Medium
Hard
Material cost
Low
Medium
Hard
Time cost
Low
Medium
Hard
Calculation expertise
Low
Medium
Hard
Equipment
- Petri dishes of about 3 – 5 cm diameter
- Vacuum glass desiccator
- Vacuum pump
- Ethanol-free chloroform
- 0.5M K₂SO₄
- Filter papers
- TOC analyser for liquid samples
- Fume cupboard to avoid inhalation of chloroform vapours
Experimental design
It can be performed on the same samples used for organic C assessment in the mineral soil. The analysis must be done on fresh samples, which can be stored at 4ºC for only a few days before the analyses. To assess the short-term effect of fire, use only the uppermost mineral soil (no more than 5 cm depth). The fire effects at deeper layers may emerge in the mid- to long-term through changes in litter input quantity and/or quality).
Methodology
The method consists of quantifying the C flush after chloroform fumigation. The full lab procedure can be found at Vance et al (1987) or Horwath and Paul (1994).
Place two subsamples of about 10 g of fresh soil in two small petri dishes and note the exact weight: one subsample will be extracted after fumigation and the other without fumigation.
Store them at 4ºC till be fumigated (fumigated subsample) or to be extracted as non-fumigated subsamples.
A third subsample will be used to determine the soil moisture (60ºC – 80ºC, 48 h).
One subsample of each pair of samples (fumigate subsample) is placed in a dessicator, together with a glass containing ethanol-free chloroform and some water to avoid desiccation. Vacuum should be applied for a few minutes to create a chloroform atmosphere. Leave it overnight. Use a fume cupboard to avoid inhalation of chloroform vapours.
In the fume cupboard, take out the glass with the chloroform and evacuate chloroform from the samples by creating the vacuum and re-introducing air at least three times.
Extract C with 20 ml of 0.5M K₂SO₄ from fumigated and non-fumigated subsamples.
Analyse organic C from both extracts with a TOC analyser or by dichromate oxidation 1
1 In soils with carbonates, where we expect to have dissolved inorganic C in the extracts, organic C must be analysed by dichromate oxidation.
Calculations
Calculate the organic C content in the fumigated and unfumigated extracts of each sample, in mgC g-1 dry soil. Multiply the C in the extracts (mgC/L) by the volume of extractant (L) and divide by the dry mass of soil used in the extraction (g).
Calculate the microbial biomass C (MBC) as follows:
\mathrm{MBC}=\frac{\mathrm{C_F}-\mathrm{C}_{\mathrm{NF}}}{k_{\mathrm{EC}}}
2 When organic C is analysed by dichromate oxidation, Joergensen (1996) recommends a kEC factor of 0.38.
Relation with other methods
Mineral soil can be sampled using the method by Garcia-Pausas & Casals Organic carbon stocks in topsoils mineral layers.
Data templates
NA
Further information
Haney RL, Franzluebbers AJ, Hons FM, Hossner LR, Zuberer DA. 2001. Molar concentration of K2SO4 and soil pH affect estimation of extractable C with chloroform fumigation-extraction. Soil Biol. Biochem 33: 1501-1507
Horwath WR, Paul EA. 1994. Chapter 36: Microbial Biomass. In: Weaver RW, Angle JS, Bottomley PS (eds) Methods of soil analysis. Part 2. Microbiological and biochemical properties. SSSA Book Series: 5, Madison, Wis., pp. 753-773
Joergensen RG. 1996. The fumigation-extraction method to estimate soil microbial biomass: calibration of the kFE value. Soil Biol. Biochem 28(1): 25-31
Vance ED, Brookes PC, Jenkinson DS. 1987. An extraction method for measuring soil microbial biomass C. Soil Biol. Biochem. 19(6): 703-707