Effect of soil minerals on the production of extracellular polymeric substance by Bacilli subtilis

Soil microbes produce extracellular enzymes that play a crucial role in organic matter degradation. However, the adsorption of these enzymes to clay mineral surfaces could impede the functioning of extracellular enzymes. Microbes are thought to counteract this adsorption of extracellular enzymes by producing more extracellular polymeric substances (EPS) under adverse conditions. This led to the hypothesis that enzyme adsorption to clay minerals could stimulate EPS production, potentially mitigating the negative effects of adsorption on enzyme activities.

To test this, we conducted a mini-incubation experiment with artificial sterile soil and Bacillus subtilis to investigate i) whether different clay minerals increase EPS production by B. subtilis and ii) whether increasing EPS contents preserve the activity of extracellular enzymes in the presence of clay minerals. Artificial soils were prepared (%/w) with sand (75%), silt (15%), and either an expandable clay mineral, montmorillonite (MT) or non- expandable, kaolinite (KL) clay (10%) and then sterilized using gamma radiation. We included a control treatment containing an additional 10% sand in place of clay minerals. The soil samples (20 g) were supplemented with LB-Lenox media (20 g/l) as a substrate, inoculated with B. subtilis, and incubated in three replicates for 3 days at 25 °C. CO₂ production was monitored with GC measurements at 24-hour intervals. On day 3, soils were destructively sampled and analyzed for amylase enzyme activity and colony-forming units (CFU). EPS was extracted and quantified for protein and polysaccharide content. All procedures were carried out under sterile conditions, and data collected were normalized to the number of bacterial cells added to each treatment.

The results showed a significant (p < 0.05) higher level of EPS production (EPS-protein and EPS-polysaccharide) in the soil amended with MT compared to the control and KL-amended soil. There was no significant difference between the EPS production in control soil and KL-amended soil. Clay minerals did not significantly influence amylase activities (p < 0.05), contradicting previous reports of reduced enzyme activities due to mineral adsorption. However, there was a significant reduction in the CFU in soil with clay minerals compared to the control. This might be an indication of struggle for nutrient availability by B.subtillis and hence the need for EPS production. The observation of high EPS production in the presence of high MT content with no adsorption effects on enzyme activities may be due to a near-steady enzyme-mediated degradation of organic matter in the soil, which is crucial for C cycling. Future experiments should clarify the effect of clay minerals and bacterial EPS production on extracellular enzymes activities in other biogeochemical cycles like the N and P cycle.