We investigated the effects of substrate (cellulose or starch) and different clay contents on the production of microbial extracellular polymeric substances (EPS) and concomitant development of stable soil aggregates. Soils were incubated with different amounts of montmorillonite (+ 0.1%, + 1%, + 10%) both with and without two substrates of contrasting quality (starch and cellulose). Microbial respiration (CO₂), biomass carbon (C), EPS-protein, and EPS-polysaccharide were determined over the experimental period. The diversity and compositional shifts of microbial communities (bacteria/archaea) were analysed by sequencing 16S rRNA gene fragments amplified from soil DNA. Soil aggregate size distribution was determined and geometric mean diameter calculated for aggregate formation. Aggregate stabilities were compared among 1–2-mm size fraction. Starch amendment supported a faster increase than cellulose in both respiration and microbial biomass. Microbial community structure and composition differed depending on the C substrate added. However, clay addition had a more pronounced effect on alpha diversity compared to the addition of starch or cellulose. Substrate addition resulted in an increased EPS concentration only if combined with clay addition. At high clay addition, starch resulted in higher EPS concentrations than cellulose. Where additional substrate was not provided, EPS-protein was only weakly correlated with aggregate formation and stability. The relationship became stronger with addition of substrate. Labile organic C thus clearly plays a role in aggregate formation, but increasing clay content was found to enhance aggregate stability and additionally resulted in the development of distinct microbial communities and increased EPS production.

Adsorption of extracellular enzymes to soil minerals is assumed to protect them against degradation, while modifying their activities at the same time. However, the persistence of the activity of adsorbed enzymes remains poorly understood. Therefore, we studied the persistence of cellulase and α-amylase activities after adsorption to soil amended with various amounts (+1, +5, and +10 wt.%) of three typical soil minerals, montmorillonite, kaolinite, and goethite. Soil without mineral addition (pure soil), pure minerals, and pure dissolved enzymes were used as references. Soil mineral–enzyme complexes were prepared and then incubated for 100 days; temporal changes in enzyme activities were analyzed after 0, 0.1, 1, 10, and 100 days. The specific enzyme activities (activities normalized to protein content) and their persistence (activities relative to activities at day 0) were compared to enzyme activities in solution and after sorption to the control soil. Amylase adsorption to pure minerals increased in the following order: montmorillonite > kaolinite > goethite. That of cellulase increased in the following order: goethite > montmorillonite > kaolinite. Adsorption of enzymes to soils did not increase in the same order of magnitude as the addition of reactive binding sites. Based on inverse relationships between the amount of enzyme adsorbed and the specific enzyme activity and their persistency, we showed that a limited availability of sorption sites is important for high specific activity and persistence of the enzymes. This is probably the consequence of less and weaker bonds, as compared to a high availability of sorption sites, resulting in a smaller impact on the active sites of the enzyme. Hence, we suppose that the soil mineral phase supports microorganisms in less-sorptive environments by saving energy on enzyme production, since small enzyme release could already result in sufficient activities to degrade respective target carbon substrates.

Knowledge of how interactions of clay minerals and extracellular enzymes (EEs) influence organic matter turnover in soils are still under discussion. We studied the effect of different montmorillonite contents on EE activities, using two experiments—(1) an adsorption experiment with a commercially available enzyme (α-glucosidase) and (2) an incubation experiment (10 days) where microorganisms were stimulated to produce enzymes through organic carbon (OC) addition (starch and cellulose). Soil mixtures with different montmorillonite contents were created in four levels to a sandy soil: +0% (control), +0.1%, +1%, and +10%. The potential enzyme activity (pEA) of four enzymes, α-glucosidase, β-glucosidase, cellobiohydrolase, and aminopeptidase, involved in the soil carbon and nitrogen cycle were analysed. The adsorption experiment revealed a reduction in the catalytic activity of α-glucosidase by up to 76% with increasing montmorillonite contents. However, the incubation experiment showed an inhibitory effect on pEA only directly after the stimulation of in-situ EE production by OC addition. At later incubation stages, higher pEA was found in soils with higher montmorillonite contents. This mismatch between both experiments, with a transient reduction in catalytic activity for the incubation experiments, points to the continuous production of enzymes by soil microorganisms. It is conceivable that microbial adaptation is characterized by higher investment in EEs production induced by increasing clay contents and a stabilisation of the EEs by clay minerals. Our results point to the need to better understand EE-clay mineral-OC interactions regarding potential microbial adaptations and EE stabilisation with potentially prolonged activities.