Validity of eco-enzymatic stoichiometry to reveal microbial C and nutrients limitation: Evidence from six long-term field experiments

The soil eco-enzymatic stoichiometry approach has been widely used in terrestrial ecosystems for decades to assess microbial carbon (C), nitrogen (N), and phosphorus (P) limitation based on the ratios of five arbitrary-selected enzyme activities. As numerous enzymes are involved in soil C, N, and P cycling, it remains uncertain whether the stoichiometric approach will be valid if it is based on different set of eco-enzymes.

To address this issue, soils were collected from six long-term field experiments (12–123 years in duration) at Bad Lauchstädt, central Germany. These experiments encompass a wide range of soil organic matter contents (1.4–6.9%) and include contrasting field treatments such as fertilization regimes, land-use intensity, and fallow periods. In addition to the five basic enzymes (β-glucosidase, cellobiohydrolase, N-acetyl-glucoseaminidase, leucine aminopeptidase, and acid phosphatase), lipase activity was measured and incorporated into the stoichiometric analysis.

The additional C-cycling enzyme (lipase) increased vector length by 12–90% across all experiments and treatments, in numerous cases increasing a threshold value 0.6 and indicating microbial C limitation, which was not evident by basic set of enzymes. Vector angles showed variable responses to lipase addition. For example, vector angles increased by 13–41% under natural succession and excessive manure application, suggesting reduced N limitation, whereas no effect of lipase addition was observed on vector angles under poor soil conditions (no fertilization and 36 years fallow). However, soil microbial biomass C:N ratios ranged from 20 to 45 under poor soil conditions, indicating strong microbial N limitation, which contradicts the stoichiometry results.

Overall, our findings highlight the considerable uncertainty and potential biases of the enzyme stoichiometry approach and emphasize the need to identify more reliable ecological indicators of microbial nutrient limitation.