Title: Untargeted soil metabolomics methods for analysis of extractable organic matter

Abstract

The cycling of soil organic matter (SOM) by microorganisms is a critical component of the global carbon cycle but remains poorly understood. There is an emerging view that much of SOM, and especially the dissolved fraction (DOM), is composed of small molecules of plant and microbial origin resulting from lysed cells and released metabolites. Unfortunately, little is known about the small molecule composition of soils and how these molecules are cycled (by microbes or plants or by adsorption to mineral surfaces). The water-extractable organic matter (WEOM) fraction is of particular interest given that this is presumably the most biologically-accessible component of SOM. Here we describe the development of a simple soil metabolomics workflow and a novel spike recovery approach using ¹³C bacterial lysates to assess the types of metabolites remaining in the WEOM fraction. Soil samples were extracted with multiple mass spectrometry-compatible extraction buffers (water, 10 mM K₂SO₄ or NH₄HCO₃, 10–100% methanol or isopropanol/methanol/water [3:3:2 v/v/v]) with and without prior chloroform vapor fumigation. Profiling of derivatized extracts was performed using gas chromatography/mass spectrometry (GC/MS) with 55 metabolites identified by comparing fragmentation patterns and retention times with authentic standards. As expected, fumigation, which is thought to lyse microbial cells, significantly increasedmore » the range and abundance of metabolites relative to unfumigated samples. To assess the types of microbial metabolites from lysed bacterial cells that remain in the WEOM fraction, an extract was prepared from the soil bacterium Pseudomonas stutzerii RCH2 grown on ¹³C acetate. This approach produced highly labeled metabolites that were easily discriminated from the endogenous soil metabolites. Comparing the composition of the fresh bacterial extract with what was recovered following a 15 min incubation with soil revealed that only 27% of the metabolites showed >50% recovery in the WEOM. Many, especially cations (polyamines) and anions, showed <10% recovery. These represent metabolites that may be inaccessible to microbes in this environment and would be most likely to accumulate as SOM presumably due to binding with minerals and negatively-charged clay particles. Here this study presents a simple untargeted metabolomics workflow for extractable organic matter and an approach to estimate microbial metabolite availability in soils. These methods can be used to further our understanding of SOM and DOM composition and examine the link between metabolic pathways and microbial communities to terrestrial carbon cycling.« less

Authors:

Swenson, Tami L. ^[1]; Jenkins, Stefan ^[1]; Bowen, Benjamin P. ^[1]; Northen, Trent R. ^[1]

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+ Show Author Affiliations

1. Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Publication Date:

Wed Oct 22 00:00:00 EDT 2014

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1511423

Alternate Identifier(s):

OSTI ID: 1254534

Grant/Contract Number:  

AC02-05CH11231; SC0010566

Resource Type:

Accepted Manuscript

Journal Name:

Soil Biology and Biochemistry

Additional Journal Information:

Journal Volume: 80; Journal Issue: C; Journal ID: ISSN 0038-0717

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; soil organic matter; metabolomics; gas chromatography/mass spectrometry; fumigation