Coping with artifacts induced by CaCO 3–CO 2–H 2O equilibria in substrate utilization profiling of calcareous soils

Abstract

This study was undertaken to cope with artifacts derived from CaCO 3–CO 2–H 2O equilibria that occur in substrate-induced respiration (SIR) measurements in calcareous soils. Considering that partitioning of respired CO 2 between headspace and soil solution becomes increasingly important above a pH value of 6 and when headspace-to-solution ratios are low, an underestimation in respiration measurements are to be expected under such conditions. In order to evaluate the relative magnitude of this bias in respiration measurements under a wide range of soil conditions, we investigated soils from four sites along a climatic gradient in Israel. Even though all soils were formed over calcareous bedrock, their CaCO 3 contents increased with increasing climate aridity. We calculated the theoretical distribution of CO 2 between the gaseous and solution phases for each soil, based on the equilibrium reactions between CO 2, water, and the various carbonate species. Theoretically calculated factors required for the correction of biased CO 2 evolution data were compared to those obtained by actually measuring CO 2 retention in the soils, and were found to be smaller, although reasonably close to them. CO 2 retention levels determined by experiment were substantial and followed the order: Mediterranean (74% of initial headspace CO 2) > semi-arid (70%) > humid-Mediterranean (64%) > arid soil (58%). An additional potential artifact in substrate utilization profiling was addressed when soil respiration was found to be overestimated when induced by considerably acidic substrates such as carboxylic acids. This overestimation results from soil pH lowering which reduces the CO 2 retention potential. Hence, if CO 2 retention correction factors are applied to carboxylic acid-induced respiration measurements, they must be reduced according to any pH decline. Moreover, CaCO 3 dissolution may follow pH decline, and be accompanied by abiotic CO 2 evolution that may be mistakenly regarded as respired CO 2. We suggest taking the following sequence of measures when generating substrate utilization profiles for calcareous soils: (1) experimental determination of CO 2 retention levels and retention correction factors; (2) comparison with theoretical calculations; (3) assessment of pH changes induced by substrates and modification of retention factors necessitated by these changes; (4) measurement of abiotic substrate-induced CO 2 evolution rates and their subtraction from measured SIR data; and (5) multiplication of biotic SIR data by CO 2 retention correction factors. The comparative analysis of utilization profiles obtained from corrected SIR data with those obtained from the originally measured CO 2 evolution rates emphasized the immense underestimation of non-acidic SIRs, concurrent with an overestimation of SIRs obtained for appreciably acidic substrates such as carboxylic acids, in ordinary SIR assays.