CHLOROFORM FUMIGATION DIRECT EXTRACTION (CFDE) PROTOCOL FOR MICROBIAL BIOMASS CARBON AND NITROGEN

5 May 1998, S. E. Hobbie

References:

Beck, T., R. G. Joergensen, E. Kandeler, F. Makeschin, E. Nuss, H. R. Oberholzer, and S. Scheu. 1997. An inter-laboratory comparison of ten different ways of measuring soil microbial biomass C. Soil Biol. Biochem. 29 (7):1023-1032.

Brookes, P. C., A. Landman, G. Pruden, and D. S. Jenkinson. 1985. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 17:837-842.

Fumigation and Extraction

Important if measuring microbial biomass carbon: before doing CFDE, remove the ethanol from the chloroform by running 500 ml over 25 g of basic grade 1 alumina in a syringe or column. (or purchase ethanol-free chloroform).

1. For each sample, you will require 3 subsamples:
-one subsample for determining gravimetric soil moisture (48 hours @ 100°C)
-one non-fumigated sample (10 g oven-dry equivalent) for immediate extraction with 0.5 M K₂SO₄.
-one fumigated sample (10 g oven-dry equivalent).

2. Place non-fumigated subsample in a specimen cup (e.g., Fisher 14-375-148) with 50 ml 0.5 M K₂SO₄. Place on shaker for 1 hour. After shaking, filter through pre-leached (with 0.5 M K₂SO₄) Whatman No. 1 filter paper. Freeze extract to store.

3. Place samples to be fumigated in 50 ml glass beakers. Mark beakers with pencil, as sharpee will run in chloroform. Put the beakers with soil into a vacuum dessicator. Beakers can be stacked in the dessicator by layering with chicken wire.

4. Place a 50-ml beaker or scintillation vial containing boiling chips and 20 ml of chloroform in the dessicator.

5. Evacuate until chloroform boils. Vent.

6. Repeat (5) three more times, not venting the last time. (you may have to replace the boiling chips each time to get the chloroform to boil).

7. Let sit in the dark for 3 days (darkness prevents the chloroform from breaking down). (I cover the dessicator with a garbage bag).

8. Release the vacuum and remove the chloroform.

9. Draw a vacuum and release to remove excess chloroform. Repeat three times. To ensure that you've vented all the chloroform, you may want to do the famous "sniff test" invented by Eldor Paul (it's probably not that good for your health).

10. Extract the sample in 50 ml K₂SO₄. Shake for 1 hour. Filter through pre-leached (with 0.5 M K₂SO₄) Whatman No. 1 filter paper. Freeze extract to store.

For Microbial Biomass Carbon:

1. Determine total dissolved carbon on a TIC/TOC analyzer. The difference between C in the fumigated and non-fumigated samples is the chloroform-labile C pool (EC), and is proportional to microbial biomass C (C):

C=EC/kEC

where kEC is soil-specific, but is often estimated as 0.45 (Beck et al. 1997).

For Microbial Biomass Nitrogen:

1. Digest 20 ml of extract using Kjeldahl digestion. Run digest on autoanlyzer for total N. The difference between N in the fumigated and non-fumigated samples is the chloroform-labile N pool, and is proportional to microbial biomass N (N):

N=EN/kEN

where kEN is soil-specific, but is often estimated as 0.54 (Brookes et al. 1985).