Separating cellular metabolism from exoenzyme activity in soil organic matter decomposition

Soil organic matter (SOM) decomposes both inside and outside of cells. Cellular metabolism and extracellular depolymerization normally operate simultaneously in soil but are difficult to separate in practice. To learn more about the extracellular component of SOM decomposition, we sterilized a semiarid annual grassland soil to inhibit cellular metabolism, and then assayed cell viability, exoenzyme activities, and pathways of carbon dioxide (CO₂) emission. Chloroform (CHCl₃) fumigation was intended to disrupt cellular activities while leaving biochemical processes intact. Gamma (γ) irradiation and autoclaving were intended to disrupt both cellular and extracellular biochemical processes while leaving abiotic processes intact. We measured the potential activities of eight enzymes (six hydrolytic, two oxidative) and CO₂ emission induced by seven substrates (glucose, three amino acids, three tricarboxylic acid [TCA] cycle intermediates). We found that all three sterilization techniques clearly disrupted cellular metabolism. Chloroform and irradiation decreased cultivable cell counts by 2-3 orders of magnitude, inhibited CO₂ emission pathways associated with glucose and amino acids, and decreased the hydrolytic activities of α-glucosidase and xylosidase by 72-82%. The other hydrolytic enzymes (β-glucosidase, cellobiohydrolase, NAGase, phosphatase) were less sensitive to both CHCl₃ and irradiation. All hydrolytic activities that we assayed were inhibited by autoclaving, indicating that biochemical reactions and other extracellular processes drive hydrolytic SOM decomposition. Oxidative activities, on the other hand, did not stop after autoclaving or even combusting at 500°C. This supports other studies which have found that mineral catalysts partly drive oxidative SOM decomposition. Unexpectedly, CO₂ emission from TCA intermediates decreased by only 26-47% after sterilization suggesting that the required dehydrogenase enzymes for decarboxylation are still active when cells are dead but relatively intact. Because CHCl₃ had slightly smaller effects on exoenzyme activities compared to irradiation, and because it may be continuously applied, limiting the potential for recolonization and regrowth (unlike irradiation), we suggest it is an adequate and more accessible method for separating the activity of exoenzymes from cellular metabolism under realistic soil conditions.