Microbial Phosphorus - Chloroform Extraction Method
Reagents
0.5M NaHCO3 (enough for 50 samples)
In 2 l volumetric flask
- 84 g Sodium Bicarbonate
- Adjust pH to 8.5
- Bring to volume with DI water.
Keep refrigerated for a month.
0.5 M HCl
In 2 liter volumetric flask
- 85.8 ml Hydrochloric Acid
- Bring to volume with DI water.
Procedure
In 50 ml Oakridge centrifuge tubes
In duplicate
- 2 g Field fresh, sieved soil
- 40 ml DI water
- 2 Bicarb charged resin strip (2 x 6 cm; Charging: shake strips 10 minutes in 100 ml 0.5 M NaHCO3, rinse 3 times with DI water, shake dry.)
Shake @ 150 rpm for 4 hours.
Remove resin strips and wash with minimal amount of DI water back into the sample cup. The purpose for the resin strips is to remove inorganic ortho-P.
Centrifuge at high speed for 45 minutes; discard supernatant without removing the soil.
Fumigation
- Add 1 ml chloroform (CHCl3) to one tube labeled “F” for fumigated.
- Shake @ 250 rpm for 1 hour.
- Allow chloroform to evaporate overnight in the fume hood.
Store the other tube labeled “C” for control (i.e., unfumigated) at 4°C.
- Add 30 ml of 0.5 M NaHCO3 to F and C labeled tubes.
- Shake @ 150 rpm for 16 hours, centrifuge at high speed for 30 minutes.
- Pour supernatant into 15 ml tubes and centrifuge again at high speed for 10 min.
Analyze for total P on the ICP.
Calculations
- µg P/g in microbial biomass = [(Total P in ‘F’ soil) – (Total P in ‘C’ soil)]/kp
Where:
- kp = extraction efficiency (0.4) or sorption efficiency for each soil (see P sorption methods)
Lajtha K. et al. (1999) Soil Phosphorus. Standard soil methods for long-term ecological research.
Created: 8/19/09 Revised: 10/10/11