1. Closed chamber incubation with alkali CO2 traps
1.1 Materials and Reagents
1 Incubation jars with gastight lids (Mason or Kilner types; Figure 45.1)
2. Glass vials (20-50 ml) for the alkali alkali solution and water
3. M NaOH solution
4. 0.5 M HCl solution
5. Phenolphthalein soiution
6. 1 M BaCl2
7. Pipettes
8. Burette or automatic titrator
9. Magnetic stirrer (optional)
10. Incubator or controlled environment room (optional)
FIGURE 45.1. Closed incubation vessel with NaOH traps for CO2
1.2 Procedure
Weigh 100-150 g (dry weight equivalent) into jars and record the weight of each jar plus soil without its lid. Place one vial containing 10 ml of 1 M MaOH and one vial containing watcr into eath jar and seal them with the lids (Figure 45.1). Incubate the jars in the dark and at the desired temperature. The CO2 can be assayed at intervals of 3-10 days typically. For each mole of CO2 trapped in the NaOH. 2 moles of NaOH will be converted to Na2CO2 (Equation 45.1). Therefore, the total CO2 produced is twice the depletion of NaOH in the trap. Remove the vials of water and NaOH and tben backtitrate the excess NaOH with HCl (Equation 45.2) using phenolphthalein as an indicator having removed dissolved CO2 and carbonates by precipitation with the addition of 2 ml BaCl2
2NaOH + CO2 → Na2CO3 + H2O (45.1)
NaOH + HCl → NaCl + H2O (45.2)
For example, example, if 5 ml of 0.5 M HCI was required to backtirate the excess NaOH in alkali trap trap that originally contained 10 ml of 1.0 M NaOH after precipitating the carbonates with BaCl2 then the CO2 content of the traps would be calculated as
CO2 in trap = 0.5 x (((VNaOH x CNaOH)/1000) – ((VHCI x CHCI)/1000)) (45.3)
Where VNaOH is the initial volume of NaOH (mL), CNaOH is the initial molar concentration of NaOH, VHCI is the volume of HCI used in the the titration (mL), and CHCI is the molar concentration of HCI used in titration.
So, CO2 in the trap = 0.5 x [((10 x 1.0)/1000) – ((5 x 0.5)/1000)]
= 0.00375 mol C
Where the incubation involved 100 g of dry weight equivalent soil and an incubation time of 48 h, C mineralization rate would be calculated as
C mineralization rate = CO2 in the trap /(soil mass in g x incubation time in h)
= 0.00375/(100 x 48)
= 0.00000078 mol C g-1 soil h-1 or 0.78 µmol C g-1 soil h-1 (45.4)
If the incubation is to be continued, wipe any continued, wipe any condensation from the inside of the jar and the lid, weigh the jars, and correct for any weight by addition of water. Then put frsh NaOH and water vials in the jars, reseal them, and continue the incubation.
1.3 Comments
The method given here is very general and may be adapted to address a wide range of specific research questions. Among other factors, the amount of soil, the temperature and moisture conditions, the concentration and amount of NaOH, and the incubation time can all be adjusted to suit particular applications. It is, however, important to be sure that the headspace in the jars is large enough to avoid the risk of anaerobiosis during long-term incubations. Typically, 100-150 g soil in a 1000 mL vessel is suitable for 3-4 days incubation intervals. It is also important to ensure that the amount of NaOH is adequate to trap all the CO2 produced. If the amount of CO2 produced. is small, reducing the NaOH concentration will increase the sensitivity of the assay. Carbonic anhydrase can be analyte to catalyze the dissolution of CO2 in water and allow titration between the pH endpoints, 8.3 to 3.7 (Underwood 1961). An automatic titrator and a magnetic stirrer can be used to help improve the precision of the titration. However, these are not essential as the assays can be carried out satisfactorily using manual equipment provided the operator is careful and skilful.
Commonly used protocols that employ closed chamber incubations to measure soil biological activity and to quantify the amount of readily mineralizable C in soil are given below. Closed chamber techniques involving alkali traps for measuring CO2 production in the field have also been described by Anderson (1982) and Zibilske (1994)