Soil zymography as a powerful tool for exploring hotspots and substrate limitation in undisturbed subsoil

It is widely accepted that soil microorganisms are not evenly distributed but are often concentrated in spatially segregated hotspots that are characterized by higher substrate availability compared to the surrounding bulk soil. However, microorganisms outside of hotspots may be in a dormant or inactive state, since they have depleted all available substrates within their vicinity. So far, the knowledge about the spatial distribution and dynamics of microbial activity in subsoil is very scarce, since most available data has been acquired from either homogenized soil samples or as bulk signals from undisturbed soil cores. In this study, we introduced a new incubation approach combining soil zymography and substrate addition on undisturbed soil core surfaces. We mapped three extracellular enzymes (β-glucosidase, chitinase and acid phosphatase) on a subsoil sample from 60 cm depth and analyzed their activity-patterns using different geostatistical and spatial analyses. After initial enzyme mapping, the soil was homogenously sprayed with 14C glucose as model substrate and incubated for 14 days. Soil zymography was suitable for detecting hotspots in undisturbed soil, making up a proportion of 2.4% on average of the total area. Consequently, microbial-driven biogeochemical processes can be expected to be limited to small areas in this subsoil, while the major part of the soil volume is not contributing. Glucose additions considerably increased enzyme activities up to 900% in initial non-hotspots, while the effect was far lower in initial hotspots. These results show that microorganisms in the subsoil outside of hotspots can be activated and release enzymes when substrate is supplied. Thus, dormant or inactive microorganisms outside of hotspots are able to contribute to SOC mineralization when substrate limitation is overcome, thus most likely inducing positive priming effects. Our results clearly demonstrate the benefits of combining enzyme mapping with substrate additions on undisturbed soil to gain new insights about microbial hotspots and C-cycling in subsoils using spatial analyses. In contrast to traditional incubation experiments, this method gives high spatial information about microbial activity, allowing a more differentiated interpretation of incubation results.