Hydrogen stable isotope probing of lipids demonstrates slow rates of microbial growth in soil

Abstract

The rate at which microorganisms grow and reproduce is fundamental to our understanding of microbial physiology and ecology. While soil microbiologists routinely quantify soil microbial biomass levels and the growth rates of individual taxa in culture, there is a limited understanding of how quickly microbes actually grow in soil. For this work, we posed the simple question: what are the growth rates of soil microorganisms? In this study, we measure these rates in three distinct soil environments using hydrogen-stable isotope probing of lipids with 2 H-enriched water. This technique provides a taxa-agnostic quantification of in situ microbial growth from the degree of 2 H enrichment of intact polar lipid compounds ascribed to bacteria and fungi. We find that growth rates in soil are quite slow and correspond to average generation times of 14 to 45 d but are also highly variable at the compound-specific level (4 to 402 d), suggesting differential growth rates among community subsets. We observe that low-biomass microbial communities exhibit more rapid growth rates than high-biomass communities, highlighting that biomass quantity alone does not predict microbial productivity in soil. Furthermore, within a given soil, the rates at which specific lipids are being synthesized do not relate to their quantity, suggesting a general decoupling of microbial abundance and growth in soil microbiomes. More generally, we demonstrate the utility of lipid-stable isotope probing for measuring microbial growth rates in soil and highlight the importance of measuring growth rates to complement more standard analyses of soil microbial communities.

Soil fungi remain active and invest in storage compounds during drought independent of future climate conditions

“…In a recent survey it was demonstrated that aw can range from values close to 0.1 up to values close to 1 (76). In complex communities, such as soil, the assimilation constant is difficult to estimate, but assuming all microbes are heterotrophs, this value is on average 0.71 (35), which we also applied in this study. However, it is important to note that using a different aw factor could lead to strong changes in the final quantification.…”

“…FA representing the natural 2 H abundance of the same fatty acid (sample taken from the same plot and incubated with natural abundance water), at%soil water indicating the 2 H atom% in the soil water estimated by the equilibration curves described in Supplementary methods. The factor aw represents the assimilation efficiency of deuterium into fatty acids and it has been estimated at a value of 0.71 for heterotrophs (35,74). 'FA C' is the C concentration of the respective fatty acid (average of labelled and natural abundance sample) calculated relative to the known amount of C of the internal standard 19:0.…”

“…Deuterium ( 2 H) incorporation into fatty acids can serve as an alternative SIP approach to measure microbial growth, as all microorganisms synthesize lipids and fatty acids to build and maintain cell membranes regardless of their metabolic activity and cell cycle stage. 2 H-labelling has been used in single cell studies (33), pure cultures (34) and soil microbial communities (35). Lipids likely require less investment into repair mechanisms compared to nucleic acids or proteins, which can be resynthesized during repair and cellular maintenance potentially affecting growth rates calculation (35,36), and provide a precise measure of microbial membrane production rates and cell growth.…”

“…2 H-labelling has been used in single cell studies (33), pure cultures (34) and soil microbial communities (35). Lipids likely require less investment into repair mechanisms compared to nucleic acids or proteins, which can be resynthesized during repair and cellular maintenance potentially affecting growth rates calculation (35,36), and provide a precise measure of microbial membrane production rates and cell growth. The major advantages of tracing 2 H into fatty acids ( 2 H-PLFA-SIP) are that it allows the simultaneous and sensitive quantification of bacterial and fungal replication rates (37) and the concurrent measurement of storage compound production (38).…”

Single-cell measurement of microbial growth rate with Raman microspectroscopy

“…Not only does the rate of microbial growth represent a geochemical transformation, but it is also a parameter that microorganisms modulate in response to changing conditions: microorganisms may more readily replicate when conditions are favorable to them and adopt alternative physiologic states under adverse conditions. Methods for measuring microbial growth often rely upon the addition of an isotopically labeled substrate to a natural sample and measuring its incorporation into microbial biomass, a method generally termed stable isotope probing (SIP) (1)(2)(3). SIP has been applied with the stable isotopes of various elements including H, C, N, O, S, etc., through measurement of single-cells (4)(5)(6)(7)(8), membrane lipids (3,9,10), and nucleic acids (2,(11)(12)(13).…”