The main principle of the Respicond respirometer has been described previously [7] . Briefly, it consists of water baths with inserted hermetically closed vessels for soil incubation. Each vessel includes a smaller open vessel with KOH solution and platinum electrodes inserted into the solution. Each set of electrodes is connected to a digital volt-/amperemeter and a source of constant current (Figure 1). The CO₂ released from the soil sample is absorbed by the KOH solution. The amount of absorbed CO₂ is determined by measuring changes in conductivity of the KOH solution. Data from each vessel are collected in hourly increments and analyzed using the software supplied with the Respicond VI. The chemical reaction of the absorption of CO₂ by the KOH solution is

The concentration of ions in solution decreases linearly in relation to the amount of absorbed CO₂. The amount of accumulated CO₂ during incubation N(CO₂) can be calculated using the following equation (reformulated from [7] ):

where C(t₀) was the conductance at the beginning of the incubation (time t₀) before any absorption of CO₂ by KOH, C(t₁) was the conductance at time t₁ and A was an empirically determined constant expressing the theoretical maximum amount of CO₂ absorbed and it can be calculated as A = N(CO₂) if C(t₁) = 0. This calculation assumes that the temperature of the solution is constant during the experiment. The constant A is proportional to the volume and concentration of the solution. The default value of the constant A used by the software of the Respicond VI is 219 mg CO₂ for 10 ml of 0.6 M KOH solution, corresponding to 36.5 mg CO₂ ml⁻¹ 1 M KOH. Another published value for constant A is 9.94 mg CO₂ for 5 ml of 0.05 M KOH solution [5] , corresponding to 39.8 mg CO₂ ml⁻¹ 1 M KOH. No further calibrations for other concentrations of KOH are available, although, a reliable detection of low CO₂ evolution rates (<100 µg CO₂ h⁻¹) is possible only with lower concentrations of KOH solution [7] . To meet the objective of this study, it was necessary to re-determine the value of the constant A for suitable concentrations of KOH, its dependence on the temperature and finally to develop a method to perform measurements under variable temperatures to allow for variations in experimental conditions or induced temperature treatments.

The determination of the constant A can be done using two different approaches [5] [6] :

1. Production of a defined amount of CO₂ in a closed vessel following the reaction

The emitted CO₂ is absorbed by the KOH solution, resulting in a changing conductance of the KOH solution.

2. Replacing the KOH solution by mixtures with different ratios of KOH and potassium carbonate (K₂CO₃). According to Reaction 1, this imitates the absorption of defined amounts of CO₂.

To apply the first calibration method, 10 ml 1.0 M hydrochloric acid (HCl) were added to empty vessels. After closing the vessels, sodium carbonate (Na₂CO₃) solution was injected through a septum into the HCl. Twelve injections were made, each of which with 1 ml 0.2 M Na₂CO₃ producing 8.8 mg CO₂. After each injection, the conductance of the KOH solution (10 ml, originally 0.5 M ) was measured as soon as it stabilized (approx. 30 minutes). The whole experiment was repeated five times at 20˚C.

The constant A was calculated from the linear relationship between the CO₂ evolved and expected to be absorbed by KOH (N(CO₂)), and the measured conductance after injection C(t_inj):

where C(t₀) and C(t_inj) were the conductance of KOH solution before and after injection, respectively.

For the second calibration method, it is assumed that the absorption of one mole of CO₂ converts two moles of KOH to one mole of K₂CO₃ and one mole of H₂O (see reactions above). The absorption of CO₂ could be simulated by replacing KOH with CO₂-equivalent amounts of K₂CO₃ solution. We mixed 0.5 M , 0.1 M and 0.05 M KOH with 0.25 M , 0.05 M and 0.025 M K₂CO₃, respectively, each of which at 6 different ratios of KOH to K₂CO₃: 0/10, 2/8, 3/7, 5/5, 7/3, 9/1 at 20˚C. Each combination of concentrations and ratios was replicated six to seven times.

To investigate the influence of temperature on the calibration of the constant A, the experiment with 0.5 M KOH and 0.25 M K₂CO₃ was conducted again at six different temperatures (5˚C, 10˚C, 12˚C, 15˚C, 20˚C and 25˚C) with three experimental replicates for each temperature. According to Equation 2, constant A was calculated from the linear relationship between the equivalent amount of CO₂(Eq(CO₂)), and the corresponding measured conductance:

where C_KOH was the conductivity of the original KOH solution.

To detect a possible temperature dependency of the constant A, measurements at 6 temperatures from 5˚C to 25˚C were fitted to the following linear function:

where T₂₀ = 20˚C, T is the actual temperature, A₂₀ is the value of the constant A (mg CO₂) at 20˚C and B is the regression coefficient indicating temperature dependency.

Linear regressions and corresponding coefficients of determination were calculated. An analysis of variance was performed to identify significant differences between calibrations with different molarity of the KOH solution.

The conductance of the KOH solution is positively correlated with temperature. Equation (1) assumes that the temperature is constant during incubation. Thus, the conductance must be recalculated if the temperature changes. During incubation at a constant temperature (ideal condition), the conductance in vessels without soil does not change and any change in conductance measured in these control vessels is only related to changes in temperature during incubation. Fluctuations of conductance in vessels without soil can, thus, be used to calculate a correction factor (F_corr(t₀,t₁)) for changes in temperature between times t₀ and t₁:

where C_empty(t₀) and C_empty(t₁) are the conductances measured in the vessel without soil at time t₀ and t₁, respectively. By assuming equal temperatures in vessels with and without soil, the temperature corrected conductance C_corr(t₁) in the vessels with soil can be calculated for t₁ using the following equation:

To verify this method, we performed the following experiment. Soils were placed into plastic vessels with hermetic lids and were pre-incubated at 20˚C for 24 hours before starting the experiment. The temperature scheme of the incubation experiment in the Respicond VI water bath was ten days at 20˚C, eight days at 25˚C and another four days at 20˚C. Two vessels without soil were used to calculate the correction factor F_corr(t₀,t₁). Then, this correction factor was used to correct the conductance in vessels with soil. Five soil samples (0 - 60 cm) from two experimental sites in the state of Baden Württemberg (Southwest Germany) were used: one sample from a Calcaric Cambisol (silty loam, 0.7% organic C, pH(CaCl₂) 6.4) and four samples from a Rendzic Leptosol (clay loam, 1.9% organic C, pH(CaCl₂) 7.9). Soil samples were sieved ( 2 mm ) and adjusted to water content of about 50% maximum water holding capacity. Three replicates were performed for each soil sample resulting in a total of 15 vessels.