A novel approach for calorespirometry: Integrating a CO2 sensor into an isothermal microcalorimeter for simultaneous measurement of microbial heat evolution and mineralization

Soil, as the largest terrestrial carbon sink, plays a crucial role in carbon sequestration. Within soil systems, microorganisms decompose soil organic matter to generate energy and obtain carbon for growth, concomitantly release heat and CO 2 as metabolic byproducts. The calorespirometric (CR) ratio – defined as the ratio of heat production to CO 2 evolution, is a key indicator of carbon use efficiency and soil anaerobicity. However, conventional methodologies typically measure heat and CO 2 separately, with CO 2 often quantified by intermittent sampling. This discontinuous approach, compounded by the inherent heterogeneity of soil, introduces uncertainties in calorespirometric analysis. To address this limitation, an infrared CO 2 sensor was mounted onto a stainless-steel calorimetric ampoule, containing soil-glucose mixtures, enabling simultaneous real-time measurements within an isothermal microcalorimeter. The novel configuration permits continuous monitoring of both parameters, validated through comparative analysis with traditional methods. The derived CR ratios aligned with theoretical predictions for carbohydrates metabolism. Furthermore, parallel oxygen measurements enabled quantification of CR ratio based on O 2 (heat-to-O 2 ), and the respiratory quotient (CO 2 -to-O 2 ), offering deeper insight into microbial carbon-energy coupling and turnover in soil systems. This methodological advancement enhances the capacity to interrogate soil biogeochemical processes under dynamic environmental conditions. • Set-up for simultaneous heat and CO 2 measurement developed. • Validation confirms sensor integration does not affect heat production accuracy. • Gas diffusion errors minimized. • Innovation enables real-time calculation of calorespirometric ratios.