Microbial Biomass C, N



Sampling Frequency: minimum of once per month through the growing season (April -October)

The chloroform fumigation-incubation method (Jenkinson and Powlson 1976) is used to estimate microbial biomass C and N. Triplicate subsamples from the composite soil sample of each plot (30 g freshweight) are placed inside 50 ml glass beakers. Two treatments, control and fumigated, are used. Samples designated for fumigation are placed in a vacuum desiccator lined with moist paper towels. Acid washed chloroform is placed in a 50 ml beaker in the center of the desiccator. The desiccator is sealed, placed in a laboratory hood and evacuated, allowing the chloroform to boil for approximately 30 secs. The boiling is repeated 4 times. Samples are incubated for 24 h in the chloroform vapor-saturated atmosphere. At the end of theincubation period the desiccator is vacuum air-flushed 8 times in order to remove the chloroform. Samples are removed from the desiccator, placed in quart wide mouth canning jars and sealed with lids fitted with rubber septa for gas sampling. At the same time, unfumigated soil samples are placed in canning jars and treated in the samefashion, serving as controls. Evolved CO2 is measured using a TCD gas chromatograph equipped with a Porapak Q column. Biomass C, Bc, is derived from the relationship:

Bc = Fc/Kc

where Fc = [ (CO2-C evolved from fumigated soil during the 10 day incubation) – (CO2-C evolved from the control during the 10 d incubation)] and Kc = 0.41.

Biomass N is estimated by measuring the flush of 2 M KCl extractable NH4 at the end of the 10 d incubation period. A 10 g subsample of the fumigated and unfumigated incubated samples is taken and analyzed for NH4 and NO3-N. Microbial biomass N, Bn, is derived using the equation:

Bn = Fn/Kn

where Fn = [( N mineralized by a fumigated soil for the 10 d incubation period) – (N mineralized by the unfumigated soil)] and Kn = [-0.14 (CO2-C evolved from fumigated soil over the 10 day incubation period/N mineralized by the fumigated soil over the 10 day fumigation period) + 0.39].


Chloroform Fumigation-Incubation: Protocol for Soil Microbial Biomass C and N Determination


  • Commercial chloroform stabilized with ethanol (0.75%)

  • Anhydrous K

  • 2 M NaOH solution

  • 1 L separatory funnel

  • 100 mL glass beakers for fumigated samples

  • similar containers for control samples (need not be glass)

  • Quart canning jars

  • 20 mL polyethylene scintillation vials

  • Large glass vacuum desiccators (20L) (each desiccator will hold about 20 soil samples)

  • rotary vacuum pump with cold trap or water pump

Removal of ethanol from chloroform

In a fume hood:

  1. Wash approximately 100 mL of chloroform for each desiccator with 200 mL distilled water 3 times in a separatory funnel. Discard upper phase.

  2. Dry the distilled chloroform over anhydrous K

The ethanol-free chloroform can be stored in the dark for a few weeks.

Soil preparation

Sample collection, sieving, compositing and distribution to recipient laboratories takes 2-3 days. Samplesare stored at 4 degrees C in this period.

  1. Mix and composite soil samples by sieving to 4 mm. Do this at field moisture unless partial drying is necessary to maintain micro-aggregate stability.

  2. Determine the soil moisture content by oven drying samples of each composite.

  3. Weigh 20g (fw) samples into 100 mL beakers and label samples to be fumigated with pencil (chloroform will dissolve most markers).

  4. Add water to bring each sample to 50% of its WHC (waterholding capacity; = 19% of soil dry weight for KBS LTER site soil).

  5. Preincubate the samples for 5 days at 25 degrees C in closed canning jars with a few mL of water in the bottom (100% humidity).


In a fume hood:

  1. Line the vacuum desiccator(s) with wet paper towels.

  2. Arrange fumigation samples (c. 20) and a conical flask containing 75 mL ethanol-free chloroform and a few boiling chips in each desiccator.

  3. Connect the desiccator(s) to a vacuum manifold which includes a vacuum gauge. The pump should be protected from excessive chloroform vapor with a cold trap (-20 degrees C or better). Change the pump oil frequently.

  4. Evacuate the desiccators until the chloroform boils for 1 min.

  5. Close the desiccator valves.

  6. Incubate the samples in chloroform vapor in the desiccator at room temp (in the fume hood) for 24 h.

  7. After fumigation open the desiccators and remove the chloroform flask and paper towel.

  8. Remove the residual chloroform by evacuating to 250 Pa and venting to atmosphere 6 times. The meticulous removal of residual chloroform is essential.


  1. Check the sample weights and re-adjust moisture content to 50% water holding capacity (WHC) if necessary.

  2. Place the fumigated and control samples in canning jars with an open 20 mL scintillation vial containing 2 mL 2 M NaOH to trap CO
    ; for soils with very high organic matter content the volume or molarity of the trap can be adjusted to be equivalent to about twice the expected CO
    flush. A few mL of water in the bottom of the jar will maintain 100% humidity. In the case of preincubated control samples open the canning jars for at least 5 min to equilibrate with atmospheric CO

  3. Include several blank jars which contain everything except soil.

  4. Tightly close the jars and incubate for 10 days



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