Soil zymography: A decade of rapid development in microbial hotspot imaging

This review summarizes the most important methodological developments and applications of soil zymography – the technique used to visualize enzyme activities in soil to identify and localize hotspots. After the first application of fluorogenic substrates for enzyme activity imaging in soil 10 years ago, zymography has been used in many studies. This reflects its universality, simplicity and visualization of the hidden microbial and root life in soil, which is a very heterogeneous and “dark” environment. After a short description of zymography background, we present the 10-year advances in the spectrum of enzyme activity imaging, zymography resolution, standardization and hotspot identification. A special focus is placed on combining the zymography with visualizing the localization of soil acidification, root exudation, pore distribution, nutrient and water fluxes. To date, most applications have been related to enzyme activities in the rhizosphere to visualize plant-soil-microbial interactions. Here, we go a step further and show many other important directions (i.e., biopores, detritusphere, microplastisphere) to identify the heterogeneity and localize hotspots of microbial activities and processes in soil. The past progress, current possibilities and challenges, as well as future perspectives of soil zymography are discussed. Soil zymography is widely applicable in natural and agricultural ecosystems in the field and in laboratory studies, scaling down from the whole root system (dm) to microbial communities (μm). In the decade ahead, enzyme research and specifically zymography imaging will continue to expand microbial studies and hotspot localization. This will involve intersecting with (bio)chemical, physico-chemical, microbial cell imaging and isotope applications. In summary, over the past 10 years, zymography based on fluorogenic substrates has illuminated the hidden microbial transformations of nutrients in soil hotspots. This has opened new ways to combine the localization of microbial processes with physico-chemical soil heterogeneity, and to identify the biochemical prerequisites for hotspot formation in soil.