Using 13C to Unravel the Molecular Mechanisms of Microbiome Response to Rewetting in Soil

Abstract

<p>Hydration levels influence carbon and nutrient cycles in soils. The rapid carbon release and nutrient utilization upon hydration in desiccated soils has been observed for decades, but little is known about the controls and timing of the underlying molecular events. This research is aimed at developing and using novel isotope based techniques to map the interdependence of carbon and nutrient cycling in soils using <sup>13</sup>C labeled substrates as a tracer of nutrient fluxes in desiccated soil microbiomes after rewetting in combination with our novel Real Time Mass Spectrometry (RTMS) system<sup> 1</sup>, isotope ratio mass spectrometry (IRMS) and omics measurements.</p><p>Our experiments involve mapping the initial microbial response to wetting, by using a combination of an atmospheric monitoring RTMS instrument that measures the levels of CO<sub>2</sub>, O<sub>2</sub>, N<sub>2</sub>, H<sub>2</sub>O and their isotopologues, simultaneously in real time and IRMS which will be used to determine the biological fate of the carbon from each substrate. In each experiment desiccated soil that has been kept at drought conditions for 2 weeks was placed in the incubation chamber. Deionized water (control), <sup>13</sup>C glucose and <sup>13</sup>C alanine dissolved in water was added to the soil through a syringe to mimic a rewetting event. An identical initial hydration response measured by tracing the production of <sup>12</sup>CO<sub>2</sub> every 5 sec for the first 10 minutes after the addition of water was seen for all the samples potentially indicating that the carbon respired in this initial burst had formed an association with the cell (either intracellular or EPS bound) during dry down. The metabolism and respiration of glucose and alanine, measured by the production of <sup>13</sup>CO<sub>2</sub> occurred at a much slower time frame (60 to 90 minutes) where the rate of <sup>13</sup>CO<sub>2</sub> production of the glucose was about 10x that of alanine.</p><p>IRMS measurements were used to determine the preferential metabolic pathway of each substrate. The soil was removed from the chamber, treated with a mixture of methanol/chloroform/water, beadbeat, sonicated and centrifuged in a modified Folch extraction.  The resulting supernatant was allowed to separate into 3 fractions of polar metabolites (methanol layer), proteins (middle layer) and lipids (chloroform layer).  Each fraction was analyzed by IRMS to quantify the extent of <sup>13</sup>C label incorporation into the soil phase to help guide interpretation of the <sup>13</sup>CO<sub>2</sub> production rates (measured via RTMS), the preferential metabolic pathway of each substrate will be determined. While it was seen that glucose was taken up into all of the biomass partitions as well as respired into CO<sub>2</sub>, alanine was metabolized to a lesser extent and was predominantly used in protein synthesis.</p><p>These studies represent approaches that showcase advanced stable isotope analyses to determine molecular provenance in ecosystems by developing an understanding of the molecular mechanisms involved in carbon and nutrient cycles in terrestrial ecosystems.</p>