Different methods, different growth rates: disentangling in situ microbial growth quantification

Microbial growth drives carbon mineralization, nutrient turnover, and nearly all biogeochemical cycling. Accurately quantifying (in-situ) microbial growth rates is therefore fundamental for linking microbial activity to soil processes and ecosystem functioning, not only in soils but across ecosystems. Numerous methods have been developed for this purpose. Microbial growth varies across soil properties (e.g., soil type, substrate quantity and quality, pH) and microbial life-history strategies (e.g. oligotrophic vs. copiotrophic lifestyles). At the same time, methodological differences, whether or not they depend on soil and microbial characteristics, make it difficult to compare microbial growth rate across studies, with community-level estimates frequently spanning several orders of magnitude.

Here, we aim to benchmark commonly used microbial growth measurements in soil to enable meaningful comparison of in situ microbial growth rates and ultimately improve our understanding of microbial contributions to soil carbon and nutrient dynamics. We conducted a systematic comparison of four widely applied growth methods across contrasting soils and depths. These included two substrate-free stable isotope probing (SIP) approaches: ¹⁸O–DNA-SIP (incorporation of labelled water into DNA) and ²H–FAME-SIP (incorporation of labelled water into phospholipid fatty acids), as well as two radioactive isotope approaches using labeled organic substrates: the ¹⁴C-leucine method (incorporation of labelled leucine into protein) and ³H-thymidine method (incorporation of thymidine into DNA). Soils were collected from four forest sites around Vienna, spanning sandy to clay-rich textures, at two depths. Incubation experiments were initiated under identical conditions with sieved soil in the lab.

Across all four methods, consistent patterns were observed: topsoils exhibited higher microbial growth rates and respiration than subsoils, with higher moisture and organic matter availability. Despite these shared trends, substantial methodological divergence was observed in estimated specific growth rates within the same soils. This divergence is expected, as the four methods target distinct cellular processes and macromolecular pools (DNA, protein, lipids). Comparability among methods implicitly assumes balanced growth, where all cellular components are synthesized at a given rate, that doesn’t change with external conditions. In natural environments, however, microorganisms frequently experience unbalanced growth, where cell division and synthesis of storage compounds or other metabolic processes become decoupled from each other. In addition, radiotracer approaches rely on extraction of microbes from soil and use of carbon-substrates that may not be taken up at the same rate by all microbial taxa, whereas SIP methods are applied directly to intact soils without substrate addition, introducing further variability in growth estimates. Consequently, carbon use efficiencies (CUE), derived by the four methods, were significantly different.

In summary, our study provides the first controlled comparison of four widely used methods to measure in situ soil microbial growth. Our results demonstrate how methodological choices shape apparent microbial growth rate estimates and identify systematic sources of variation among approaches. By deriving empirically based conversion factors between methods, our work facilitates cross-study comparisons and synthesis, ultimately advancing our understanding of microbial growth and its role in soil ecosystem functioning.