Can enzymatic stoichiometry be used to determine growth-limiting nutrients for microorganisms? - A critical assessment in two subtropical soils

The measurement of potential enzymatic activities has been proposed as an efficient method to infer nutrient limitations for microorganisms in environmental samples. To validate this use, confirmation with direct methods of microbial growth responses to resource additions are required. We experimentally manipulated nutrient-poor soils from the afromontane subtropics with relatively low (grassland soils, ca. 4% soil carbon (C)) or high organic matter content (forest soils, ca. 13% soil C) with nutrient additions (plant material added at 8 mg C g−1 soil combined with mineral N and/or P to reach C:N:P mass-ratios of 10:1:1) in a multifactorial design for one month in order to shift the microbial community towards C-, N- or P-limitation. We then measured the responses of the most commonly measured indicator enzymes used to infer growth limiting nutrients, using ß-1,4-glucosidase, ß-1,4-N-acetylglucosaminidase and leucine aminopeptidase, and acid phosphatase as indicators for C-, N- and P-acquiring enzymatic activities, respectively. In the same soil samples, we also determined the responses in bacterial (3H-leucine incorporation) and fungal growth rates (14C-acetate incorporation into ergosterol) to nutrient supplements, and also verified these with biomass responses (microbial PLFA and ergosterol concentrations) to the factorial nutrient loading amendments. Ratios of C-, N-, and P-acquiring enzymes indicated that the grassland soils were primarily P-limited, and secondarily co-limited by C and N, while the forest soils were co-limited by C and P. However, short-term responses in growth rates and respiration to nutrient additions, along with long-term growth rate, respiration and biomass responses to nutrient loading treatments all indicated that bacterial growth, fungal growth and respiration were primarily limited by C in both grassland and forest soils. We conclude that enzymatic ratios do not capture the growth-limiting factors for bacterial growth, fungal growth, or respiration in soil. Furthermore, the addition of C-rich plant material could shift the fungal community into N-limitation, while bacteria were shifted into co-limitation by both C and N, revealing that bacteria and fungi can be limited by different nutrients within the same soil environment