Positive relationship between substrate-induced respiration rate and translationally active bacteria count in soil

Soil microorganisms carry out many processes that are fundamental to soil functions. Among the millions of microbial cells present in a gram of soil, however, less than 2% are commonly estimated to be active at any point in time. Because the respiratory response of a bulk soil to carbon substrate addition would be expected to reflect the number of active cells, we hypothesized a positive correlation between active cells and soil respiration rates during substrate-induced respiration (SIR) assays. To test this, we monitored respiration and active cell counts during 24-h incubations of agricultural soil subsamples after treating with two carbon substrates or a water-only control. We enumerated active cells with the Bioorthogonal Non-canonical Amino Acid Tagging (BONCAT) method. BONCAT provides a labeled amino acid for active cells to incorporate into newly synthesized proteins, which can then be tagged with a fluorescent dye to enable enumeration by flow cytometry. Both respiration rates and active cell counts increased over time and were positively correlated with each other after 6 h of incubation. After 24 h, increases in active cells were proportionally greater than increases in respiration. Additionally, carbon-amended soils had higher respiration rates than water-only soils with similar active cell counts, suggesting differences in carbon use efficiency. Our study documents for the first time the respiratory response from in-situ microbial activation induced by substrate amendment of soil within 6 h, a short enough timescale to exclude most cell replication. This study also demonstrates that the correlation between active cell numbers and respiration is substrate-dependent. IMPORTANCEWhile many critical ecosystem services provided by soil are known to rely on microbial activity, the soil microbial community largely remains a black box. While respiration is a common indicator of bulk soil microbial activity, this study demonstrates that the relationship between respiration and the number of active cells differs based on available carbon substrates. Advancing knowledge in this area will both enable better interpretation of biological soil tests by land managers and inform researchers modeling contributions of soil microbial respiration to global carbon dynamics.