Protocol

Author

Davey Jones

OVERVIEW

This protocol outlines the use of ¹⁴C-labelled glucose and amino acid solutions to calculate the cumulative rate of ¹⁴CO₂ production in soils. This can be used to determine the level of soil microbial activity. In particular it uses a low molecular weight carbon substrate which is ubiquitous in soil, namely glucose. Other substrates can be used as well depending upon the question being asked (e.g. organic acids, amino acids, amino sugars etc). The benefit of using glucose is that nearly every microbe living in soil can use this substrate as a carbon source. Once taken up into the microbial cell it can be used in catabolic (e.g. respiration) or anabolic (e.g. making new cell walls) processes. The assay used here assesses the role of glucose in respiration which provides a good measure of total soil microbial activity. The benefit of using ¹⁴C-labelled glucose over non-radioactively labeled glucose is that low concentrations of glucose can be used that are more representative of that occurring in soil (i.e. <10 mM). This contrasts with some of the alternative methods using non-¹⁴Cglucose which use very high glucose concentrations to ensure sufficient CO₂ is given off to allow detection. The assay described here is also extremely rapid allowing hundreds of samples to be screened. Another benefit is that the assay can be scaled down to use only mg quantities of soil (e.g. for use in rhizosphere soil samples).

BACKGROUND

Understanding the rate of C turnover in soils offers an indication of microbial activity. This is particularly important in changing climates, for example High Arctic regions, where there were concerns that increases in soil temperature are stimulating soil permafrost melting and microbial activity, thereby accelerating losses of greenhouse gases (Boddy et al. 2008).

MATERIALS/EQUIPMENT

• 60 mL polypropylene tubes
• Cold room 10∓2^∘C
• Large (1 litre) plastic beaker
• Solid NaOH (pellets)
• Deionized/distilled water
• Stir bar and stir plate
• Plastic storage bottle and cap (at least 1 L)
• 37 kBq ml^-1 14C-stock solution
• 10 mM glucose stock solution
• ¹⁴CO₂ trapping vials – 1 per sample (the 5 mL plastic vials used for the scintillation counter)
• rubber bungs
• Pipettes
• Vortexer
• Liquid beta scintillation counter

UNITS, TERMS, DEFINITIONS

Liquid beta scintillation counter

PROCEDURE

1. Weigh out 5 g of field-moist soil into labelled 60 mL polypropylene tubes. Normally these are weighed out on the day of use. Cap the tubes to prevent soil water loss and place in cold room at 10∓2^∘C.
2. Make up some 1 M NaOH in a labelled plastic bottle (i.e. 20 g of solid NaOH per 500 mL; enough for 500 samples) for the ¹⁴CO₂ traps. To do this place the NaOH in a large (1 litre) plastic beaker and add 500 mL of deionized/distilled water. Add a stir bar and place on a stir plate for a few minutes until NaOH pellets have dissolved. Do not attempt to put the small pellets of NaOH into the bottle directly as they spill easily, go everywhere and are difficult to clean up. Pour the NaOH from the beaker into the storage bottle and cap. Store at room temperature. This will keep, if capped, for many months.
3. Make up a 10 mM glucose solution (0.09 g per 50 ml).
4. To a 50 ml tube add the following:
   a. 500 μL of a 37 kBq ml^-1 14C-stock solution.
   b. 500 μL of a 10 mM glucose stock solution.
   c. 49 ml of distilled water
5. Pipette 1 mL of the NaOH into the bottom of ¹⁴CO₂ trapping vials. These are the 5 mL plastic vials used for the scintillation counter. Make sure there is no NaOH on the outside of the vial. You need one for each sample. Label in small letters (big letters interfere with light emission in the scintillation counter).
6. Add 500 μL of the radioactive solution (from step 4) to the soil samples, mix gently by shaking, gently lower a NaOH trap onto the soil surface and stopper the tubes with a rubber bung. The lowering of the NaOH trap is easiest by placing the sample tube at 45^∘ and sliding it down the side. The experiment is now running and the start time should be noted.
7. Place an additional single aliquot of 500 μL of the radioactive solution in a scintillation vial (ca. 5 mL volume) and add 4 mL of scintillation fluid and shake well. This is your standard and should be read along with your other samples.
8. Remove the NaOH traps after 0.5, 1, 3, 6, 24, 48 h and 7 d. Label them if you haven’t done so already.
9. Add 4 mL of scintillation fluid to the NaOH trap and mix well on the vortexer. The solutions should turn milky for a few seconds and then go clear.
10. Measure the ¹⁴C on a liquid beta scintillation counter.
11. Calculate the cumulative rate of ¹⁴CO₂ production. Examples can be found in Boddy et al. (2007) and Boddy et al. (2008). The data can be fitted to a double exponential kinetic mineralization model as described by Boddy et al. (2008) or by contacting Davey Jones at Bangor University (d.jones@bangor.ac.uk).

LITERATURE REFERENCES

Boddy, E; Roberts, P; Hill, PW, Jones DL (2008) Turnover of low molecular weight dissolved organic C (DOC) and microbial C exhibit different temperature sensitivities in Arctic tundra soils. SOIL BIOLOGY & BIOCHEMISTRY 40: 1557-1566

Boddy, E; Hill, PW; Farrar, J, Jones DL (2007) Fast turnover of low molecular weight components of the dissolved organic carbon pool of temperate grassland field soils. SOIL BIOLOGY & BIOCHEMISTRY 39: 827-835