Death – The elusive side of microbial turnover in soil

To counteract climate change and to naturally mitigate rising levels of carbon dioxide (CO2 ) in the atmosphere, efforts to increase stabile soil carbon have been propagated. Carbon that has been fixed by plants is mainly stabilized in soils after it has passed through the microorganisms living in soils. Microorganisms like Bacteria and fungi act as a carbon pump in soils. They take up carbon from dead plant material or root exudates, to produce energy and to grow. When soil microorganisms die, carbon is released back into the soil, where it can attach to clay minerals, and thus become stabilized for decades to centuries before it is released back to the atmosphere as CO2. While the carbon cycle concept from atmosphere to plants, to soil microorganism, to soil minerals, and back to the atmosphere, is well established, it is difficult to quantify and to assess the role of soil microorganisms in stabilizing carbon in soils. The major reason for this is that we still lack a method to measure microbial death rates. In this project we aim to measure death rates of soil microorganisms. To achieve this, we will make use of the fact that growing microorganisms synthesize new Deoxyribonucleic acid (DNA); while DNA is released from the cell when microorganisms die. Growth, and therefore the production of DNA can already be measured by the incorporation of isotopically labelled oxygen into DNA during synthesis. To measure microbial death, we will reverse this approach and trace once isotopically labelled living DNA into so called environmental DNA which remains outside of living cells after cell death. If the change in labelled DNA in the living DNA pool and the environmental DNA pool is measured over time, microbial death rates can be calculated. In a second approach, we will add labelled environmental DNA and monitor the dilution of the environmental DNA pool over time. Newly released DNA will decrease, dilute, the concentration of label in the environmental DNA pool over time. If we can determine the isotopic signal of the environmental DNA pool at two time points, death rates can again be calculated. Both of these approaches will be first tested in pure cultures of soil bacteria and fungi before we will apply them to field fresh soil to measure microbial death rates. To our knowledge this is the first project to measure microbial death rates in soils. The combination of pure culture studies and laboratory experiments with field fresh soil will clarify the role of microbial death in soil microbial biomass dynamics and the formation of stabile soil carbon. Quantifying microbial death rates will close the knowledge gap in soil carbon concepts and models and will help efforts to transfer atmospheric carbon into soils which could contribute to mitigate climate change.

Soil microorganisms are a driving force in the soil carbon cycle. They take up C previously fixed by plants to build biomass and produce energy. Upon death, C is then again released from the microorganisms. Microbial C can then bind to soil minerals and form up to 50% of soil organic matter. While the microbial pathway of soil organic carbon formation and stabilization is essential to the terrestrial soil C cycle, we lack methods to quantify microbial death rates. In this project we developed a method to determine microbial death rates. The method combines sequential DNA extraction to separately extract extracellular DNA (eDNA) and DNA inside of living cells (iDNA), and microbial growth measurements using 18O incorporation into DNA. In brief: eDNA and iDNA is first extracted form soil samples to determine the initial pool sizes. Then soil samples are incubated with 18O-labelled water. After the incubation of 24 h, eDNA and iDNA is extracted from the incubated samples. After quantification of DNA and determination of the 18O content of the iDNA, microbial growth rates are determined for iDNA. Death rates can be calculated as the net change in iDNA minus the iDNA gross growth rates. We tested this method in two soils, one from an agricultural field, the other from a beech forest in Austria with similar climate but large differences in soil properties. For this test, microbial growth and death, along with respiration was determined at 20 , 30 and 45 . This range in temperatures covers temperatures that soil microorganisms of these soils regularly encounter, to peak optimum temperatures, and temperatures that have been shown to be over the microbial temperature optimum and would likely cause either inhibition of microbial growth or even increased microbial death. Indeed, we found, that while respiration but also 18O-incorporation into iDNA increased with temperature up to 45, the iDNA pool size drastically decreased from 30 to 45 in both soils. Calculated death rates thus showed a steep increase above 30 . With our new method, developed in this project we could thus demonstrate temperature induced microbial death.