Soil metabolome response to whole-ecosystem warming at the Spruce and Peatland Responses under Changing Environments experiment

Wilson, Rachel M.; Tfaily, Malak M.; Kolton, Max; Johnston, Eric R.; Petro, Caitlin; Zalman, Cassandra A.; Hanson, Paul J.; Heyman, Heino M.; Kyle, Jennifer E.; Hoyt, David W.; Eder, Elizabeth; Purvine, Samuel O.; Kolka, Randy; Sebestyen, Steven; Griffiths, Natalie; Schadt, Christopher W.; Keller, J K.; Bridgham, Scott D.; Chanton, Jeffrey; Kostka, Joel E.

doi:10.1073/pnas.2004192118

Title: Soil metabolome response to whole-ecosystem warming at the Spruce and Peatland Responses under Changing Environments experiment

Journal Article · Tue Jun 22 00:00:00 EDT 2021 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.2004192118· OSTI ID:1814492

Wilson, Rachel M. ^[1]; Tfaily, Malak M. ^[2]; Kolton, Max ^[3]; Johnston, Eric R. ^[4]; Petro, Caitlin ^[3]; Zalman, Cassandra A. ^[5]; Hanson, Paul J. ^[4]; Heyman, Heino M. ^[6]; Kyle, Jennifer E. ^[7];

^[7]; Eder, Elizabeth ^[7];

^[7]; Kolka, Randy ^[8]; Sebestyen, Steven ^[8]; Griffiths, Natalie ^[4]; Schadt, Christopher W. ^[4]; Keller, J K. ^[5]; Bridgham, Scott D. ^[9]; Chanton, Jeffrey ^[10]; Kostka, Joel E. ^[10]

FLORIDA STATE UNIVERSITY
University of Arizona
Georgia Institute of Technology
Oak Ridge National Laboratory
Chapman University
Bruker Daltonics Inc
BATTELLE (PACIFIC NW LAB)
US Forest Service
University of Oregon
Florida State University

In this study, a suite of complementary environmental geochemical analyses, including NMR and gas chromatography-mass spectrometry (GC-MS) analyses of central metabolites, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of secondary metabolites, and lipidomics, was used to investigate the influence of organic matter (OM) quality on the heterotrophic microbial mechanisms controlling peatland CO2, CH4, and CO2:CH4 porewater production ratios in response to climate warming. Our investigations leverage the Spruce and Peatland Responses under Changing Environments (SPRUCE) experiment, where air and peat warming were combined in a whole-ecosystem warming treatment. We hypothesized that warming would enhance the production of plant-derived metabolites, resulting in increased labile OM inputs to the surface peat, thereby enhancing microbial activity and greenhouse gas production. Because shallow peat is most susceptible to enhanced warming, increases in labile OM inputs to the surface, in particular, are likely to result in significant changes to CO2 and CH4 dynamics and methanogenic pathways. In support of this hypothesis, significant correlations were observed between metabolites and temperature consistent with increased availability of labile substrates, which may stimulate more rapid turnover of microbial proteins. An increase in the abundance of methanogenic genes in response to the increase in the abundance of labile substrates was accompanied by a shift toward acetoclastic and methylotrophic methanogenesis. Our results suggest that as peatland vegetation trends toward increasing vascular plant cover with warming, we can expect a concomitant shift toward increasingly methanogenic conditions and amplified climate–peatland feedbacks.

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Research Organization:: Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE

DOE Contract Number:: AC05-76RL01830

OSTI ID:: 1814492

Report Number(s):: PNNL-SA-152358

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 118, Issue 25

Country of Publication:: United States

Language:: English

References (69)

Carbon Dioxide and Methane Exchange of a Subarctic Heath in Response to Climate Change Related Environmental Manipulations Christensen, T. R.; Michelsen, A.; Jonasson, S. Oikos, Vol. 79, Issue 1 https://doi.org/10.2307/3546087	journal	May 1997
Estimating carbon accumulation rates of undrained mires in Finland–application to boreal and subarctic regions Turunen, Jukka; Tomppo, Erkki; Tolonen, Kimmo The Holocene, Vol. 12, Issue 1 https://doi.org/10.1191/0959683602hl522rp	journal	January 2002
Rapid expansion of northern peatlands and doubled estimate of carbon storage Nichols, Jonathan E.; Peteet, Dorothy M. Nature Geoscience, Vol. 12, Issue 11 https://doi.org/10.1038/s41561-019-0454-z	journal	October 2019
Greenhouse gas balance over thaw-freeze cycles in discontinuous zone permafrost: C Balance of Permafrost Peat Wilson, R. M.; Fitzhugh, L.; Whiting, G. J. Journal of Geophysical Research: Biogeosciences, Vol. 122, Issue 2 https://doi.org/10.1002/2016JG003600	journal	February 2017
Moving Beyond Global Warming Potentials to Quantify the Climatic Role of Ecosystems Neubauer, Scott C.; Megonigal, J. Patrick Ecosystems, Vol. 18, Issue 6 https://doi.org/10.1007/s10021-015-9879-4	journal	May 2015
Contribution of hydrogen to methane production and control of hydrogen concentrations in methanogenic soils and sediments Conrad, R. FEMS Microbiology Ecology, Vol. 28, Issue 3 https://doi.org/10.1111/j.1574-6941.1999.tb00575.x	journal	March 1999
Hydrogenation of organic matter as a terminal electron sink sustains high CO2:CH4 production ratios during anaerobic decomposition Wilson, Rachel M.; Tfaily, Malak M.; Rich, Virginia I. Organic Geochemistry, Vol. 112 https://doi.org/10.1016/j.orggeochem.2017.06.011	journal	October 2017
Variation in methane production pathways associated with permafrost decomposition in collapse scar bogs of Alberta, Canada: VARIATION IN METHANE PRODUCTION Prater, James L.; Chanton, Jeffrey P.; Whiting, Gary J. Global Biogeochemical Cycles, Vol. 21, Issue 4 https://doi.org/10.1029/2006GB002866	journal	October 2007
Carbon loss from northern circumpolar permafrost soils amplified by rhizosphere priming Keuper, Frida; Wild, Birgit; Kummu, Matti Nature Geoscience, Vol. 13, Issue 8 https://doi.org/10.1038/s41561-020-0607-0	journal	July 2020
Methane Concentration and Stable Isotope Distribution as Evidence of Rhizospheric Processes: Comparison of a Fen and Bog in the Glacial Lake Agassiz Peatland Complex Chasar, L. Annals of Botany, Vol. 86, Issue 3 https://doi.org/10.1006/anbo.2000.1172	journal	September 2000
Radiocarbon and stable carbon isotopic evidence for transport and transformation of dissolved organic carbon, dissolved inorganic carbon, and CH ₄ in a northern Minnesota peatland Chasar, L. S.; Chanton, J. P.; Glaser, P. H. Global Biogeochemical Cycles, Vol. 14, Issue 4 https://doi.org/10.1029/1999GB001221	journal	December 2000
Radiocarbon evidence for the importance of surface vegetation on fermentation and methanogenesis in contrasting types of boreal peatlands: IMPORTANCE OF SURFACE VEGETATION Chanton, J. P.; Glaser, P. H.; Chasar, L. S. Global Biogeochemical Cycles, Vol. 22, Issue 4 https://doi.org/10.1029/2008GB003274	journal	December 2008
Organic matter transformation in the peat column at Marcell Experimental Forest: Humification and vertical stratification: Organic matter dynamics Tfaily, Malak M.; Cooper, William T.; Kostka, Joel E. Journal of Geophysical Research: Biogeosciences, Vol. 119, Issue 4 https://doi.org/10.1002/2013JG002492	journal	April 2014
Does dissolved organic matter or solid peat fuel anaerobic respiration in peatlands? Hopple, Anya M.; Pfeifer-Meister, Laurel; Zalman, Cassandra A. Geoderma, Vol. 349 https://doi.org/10.1016/j.geoderma.2019.04.040	journal	September 2019
Changes in peat chemistry associated with permafrost thaw increase greenhouse gas production Hodgkins, S. B.; Tfaily, M. M.; McCalley, C. K. Proceedings of the National Academy of Sciences, Vol. 111, Issue 16 https://doi.org/10.1073/pnas.1314641111	journal	April 2014
Characterization of dissolved organic matter in northern peatland soil porewaters by ultra high resolution mass spectrometry D’Andrilli, Juliana; Chanton, Jeffrey P.; Glaser, Paul H. Organic Geochemistry, Vol. 41, Issue 8 https://doi.org/10.1016/j.orggeochem.2010.05.009	journal	August 2010
Methane fluxes show consistent temperature dependence across microbial to ecosystem scales Yvon-Durocher, Gabriel; Allen, Andrew P.; Bastviken, David Nature, Vol. 507, Issue 7493 https://doi.org/10.1038/nature13164	journal	March 2014
Stoichiometry and temperature sensitivity of methanogenesis and CO ₂ production from saturated polygonal tundra in Barrow, Alaska Roy Chowdhury, Taniya; Herndon, Elizabeth M.; Phelps, Tommy J. Global Change Biology, Vol. 21, Issue 2 https://doi.org/10.1111/gcb.12762	journal	November 2014
Impact of Warming on Greenhouse Gas Production and Microbial Diversity in Anoxic Peat From a Sphagnum-Dominated Bog (Grand Rapids, Minnesota, United States) Kolton, Max; Marks, Ansley; Wilson, Rachel M. Frontiers in Microbiology, Vol. 10 https://doi.org/10.3389/fmicb.2019.00870	journal	April 2019
Stability of peatland carbon to rising temperatures Wilson, R. M.; Hopple, A. M.; Tfaily, M. M. Nature Communications, Vol. 7, Article No. 13723 https://doi.org/10.1038/ncomms13723	journal	December 2016
Attaining whole-ecosystem warming using air and deep-soil heating methods with an elevated CO ₂ atmosphere Hanson, Paul J.; Riggs, Jeffery S.; Nettles, W. Robert Biogeosciences, Vol. 14, Issue 4 https://doi.org/10.5194/bg-14-861-2017	journal	January 2017
Rapid loss of an ecosystem engineer: Sphagnum decline in an experimentally warmed bog Norby, Richard J.; Childs, Joanne; Hanson, Paul J. Ecology and Evolution, Vol. 9, Issue 22 https://doi.org/10.1002/ece3.5722	journal	October 2019
Ecosystem warming extends vegetation activity but heightens vulnerability to cold temperatures Richardson, Andrew D.; Hufkens, Koen; Milliman, Thomas Nature, Vol. 560, Issue 7718, p. 368-371 https://doi.org/10.1038/s41586-018-0399-1	journal	August 2018
Massive peatland carbon banks vulnerable to rising temperatures Hopple, A. M.; Wilson, R. M.; Kolton, M. Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-16311-8	journal	May 2020
Automated Analysis of Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectra of Natural Organic Matter Kujawinski, Elizabeth B.; Behn, Mark D. Analytical Chemistry, Vol. 78, Issue 13 https://doi.org/10.1021/ac0600306	journal	July 2006
Advanced Molecular Techniques Provide New Rigorous Tools for Characterizing Organic Matter Quality in Complex Systems Wilson, Rachel M.; Tfaily, Malak M. Journal of Geophysical Research: Biogeosciences, Vol. 123, Issue 6 https://doi.org/10.1029/2018JG004525	journal	June 2018
Carbon and hydrogen isotope systematics of bacterial formation and oxidation of methane Whiticar, Michael J. Chemical Geology, Vol. 161, Issue 1-3, p. 291-314 https://doi.org/10.1016/S0009-2541(99)00092-3	journal	September 1999
Characterization of dissolved organic matter in Lake Superior and its watershed using ultrahigh resolution mass spectrometry Minor, Elizabeth C.; Steinbring, Carla J.; Longnecker, Krista Organic Geochemistry, Vol. 43 https://doi.org/10.1016/j.orggeochem.2011.11.007	journal	February 2012
Degradation of natural organic matter: A thermodynamic analysis LaRowe, Douglas E.; Van Cappellen, Philippe Geochimica et Cosmochimica Acta, Vol. 75, Issue 8 https://doi.org/10.1016/j.gca.2011.01.020	journal	April 2011
Are oxygen limitations under recognized regulators of organic carbon turnover in upland soils? Keiluweit, Marco; Nico, Peter S.; Kleber, Markus Biogeochemistry, Vol. 127, Issue 2-3 https://doi.org/10.1007/s10533-015-0180-6	journal	January 2016
Targeted Profiling: Quantitative Analysis of ¹ H NMR Metabolomics Data Weljie, Aalim M.; Newton, Jack; Mercier, Pascal Analytical Chemistry, Vol. 78, Issue 13 https://doi.org/10.1021/ac060209g	journal	July 2006
Comparative metabolic profiling of pigmented rice (Oryza sativa L.) cultivars reveals primary metabolites are correlated with secondary metabolites Kim, Jae Kwang; Park, Soo-Yun; Lim, Sun-Hyung Journal of Cereal Science, Vol. 57, Issue 1 https://doi.org/10.1016/j.jcs.2012.09.012	journal	January 2013
MetaboliteDetector: Comprehensive Analysis Tool for Targeted and Nontargeted GC/MS Based Metabolome Analysis Hiller, Karsten; Hangebrauk, Jasper; Jäger, Christian Analytical Chemistry, Vol. 81, Issue 9 https://doi.org/10.1021/ac802689c	journal	May 2009
FiehnLib: Mass Spectral and Retention Index Libraries for Metabolomics Based on Quadrupole and Time-of-Flight Gas Chromatography/Mass Spectrometry Kind, Tobias; Wohlgemuth, Gert; Lee, Do Yup Analytical Chemistry, Vol. 81, Issue 24 https://doi.org/10.1021/ac9019522	journal	December 2009
LIQUID: an-open source software for identifying lipids in LC-MS/MS-based lipidomics data Kyle, Jennifer E.; Crowell, Kevin L.; Casey, Cameron P. Bioinformatics, Vol. 33, Issue 11 https://doi.org/10.1093/bioinformatics/btx046	journal	January 2017
The PRIDE database and related tools and resources in 2019: improving support for quantification data Perez-Riverol, Yasset; Csordas, Attila; Bai, Jingwen Nucleic Acids Research, Vol. 47, Issue D1 https://doi.org/10.1093/nar/gky1106	journal	November 2018
KEGG: Kyoto Encyclopedia of Genes and Genomes Kanehisa, Minoru; Goto, Susumu Nucleic Acids Research, Vol. 28, Issue 1, p. 27-30 https://doi.org/10.1093/nar/28.1.27	journal	January 2000
New approach for understanding genome variations in KEGG Kanehisa, Minoru; Sato, Yoko; Furumichi, Miho Nucleic Acids Research, Vol. 47, Issue D1 https://doi.org/10.1093/nar/gky962	journal	October 2018
Toward understanding the origin and evolution of cellular organisms Kanehisa, Minoru Protein Science, Vol. 28, Issue 11 https://doi.org/10.1002/pro.3715	journal	September 2019
Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample Caporaso, J. G.; Lauber, C. L.; Walters, W. A. Proceedings of the National Academy of Sciences, Vol. 108, Issue Supplement_1 https://doi.org/10.1073/pnas.1000080107	journal	June 2010
Cutadapt removes adapter sequences from high-throughput sequencing reads Martin, Marcel EMBnet.journal, Vol. 17, Issue 1 https://doi.org/10.14806/ej.17.1.200	journal	May 2011
DADA2: High-resolution sample inference from Illumina amplicon data Callahan, Benjamin J.; McMurdie, Paul J.; Rosen, Michael J. Nature Methods, Vol. 13, Issue 7 https://doi.org/10.1038/nmeth.3869	journal	May 2016
The SILVA ribosomal RNA gene database project: improved data processing and web-based tools Quast, Christian; Pruesse, Elmar; Yilmaz, Pelin Nucleic Acids Research, Vol. 41, Issue D1 https://doi.org/10.1093/nar/gks1219	journal	November 2012
Differential abundance analysis for microbial marker-gene surveys Paulson, Joseph N.; Stine, O. Colin; Bravo, Héctor Corrada Nature Methods, Vol. 10, Issue 12 https://doi.org/10.1038/nmeth.2658	journal	September 2013
PEAR: a fast and accurate Illumina Paired-End reAd mergeR Zhang, J.; Kobert, K.; Flouri, T. Bioinformatics, Vol. 30, Issue 5 https://doi.org/10.1093/bioinformatics/btt593	journal	October 2013
SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data Cox, Murray P.; Peterson, Daniel A.; Biggs, Patrick J. BMC Bioinformatics, Vol. 11, Issue 1 https://doi.org/10.1186/1471-2105-11-485	journal	September 2010
Responses of tundra soil microbial communities to half a decade of experimental warming at two critical depths Johnston, Eric R.; Hatt, Janet K.; He, Zhili Proceedings of the National Academy of Sciences, Vol. 116, Issue 30 https://doi.org/10.1073/pnas.1901307116	journal	July 2019
The Sequence Alignment/Map format and SAMtools Li, H.; Handsaker, B.; Wysoker, A. Bioinformatics, Vol. 25, Issue 16 https://doi.org/10.1093/bioinformatics/btp352	journal	June 2009
Partitioning pathways of CO2 production in peatlands with stable carbon isotopes Corbett, J. Elizabeth; Tfaily, Malak M.; Burdige, David J. Biogeochemistry, Vol. 114, Issue 1-3 https://doi.org/10.1007/s10533-012-9813-1	journal	December 2012
Vertical Stratification of Peat Pore Water Dissolved Organic Matter Composition in a Peat Bog in Northern Minnesota: Pore Water DOM composition in a peat bog Tfaily, Malak M.; Wilson, Rachel M.; Cooper, William T. Journal of Geophysical Research: Biogeosciences, Vol. 123, Issue 2 https://doi.org/10.1002/2017JG004007	journal	February 2018
Microbial Metabolic Potential for Carbon Degradation and Nutrient (Nitrogen and Phosphorus) Acquisition in an Ombrotrophic Peatland Lin, Xueju; Tfaily, Malak M.; Green, Stefan J. Applied and Environmental Microbiology, Vol. 80, Issue 11 https://doi.org/10.1128/AEM.00206-14	journal	March 2014
Microbial Community Stratification Linked to Utilization of Carbohydrates and Phosphorus Limitation in a Boreal Peatland at Marcell Experimental Forest, Minnesota, USA Lin, Xueju; Tfaily, Malak M.; Steinweg, J. Megan Applied and Environmental Microbiology, Vol. 80, Issue 11 https://doi.org/10.1128/AEM.00205-14	journal	March 2014
Betaine lipids and zwitterionic phospholipids in plants and fungi Künzler, Katharina; Eichenberger, Waldemar Phytochemistry, Vol. 46, Issue 5 https://doi.org/10.1016/S0031-9422(97)81274-5	journal	November 1997
Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined? Michaeli, Simon; Fromm, Hillel Frontiers in Plant Science, Vol. 6 https://doi.org/10.3389/fpls.2015.00419	journal	June 2015
Methylotrophic methanogenesis in Sphagnum-dominated peatland soils Zalman, C. A.; Meade, N.; Chanton, J. Soil Biology and Biochemistry, Vol. 118 https://doi.org/10.1016/j.soilbio.2017.11.025	journal	March 2018
Discovery of a novel methanogen prevalent in thawing permafrost Mondav, Rhiannon; Woodcroft, Ben J.; Kim, Eun-Hae Nature Communications, Vol. 5, Issue 1 https://doi.org/10.1038/ncomms4212	journal	February 2014
An evolving view of methane metabolism in the Archaea Evans, Paul N.; Boyd, Joel A.; Leu, Andy O. Nature Reviews Microbiology, Vol. 17, Issue 4 https://doi.org/10.1038/s41579-018-0136-7	journal	January 2019
Molecular transformation and degradation of refractory dissolved organic matter in the Atlantic and Southern Ocean Lechtenfeld, Oliver J.; Kattner, Gerhard; Flerus, Ruth Geochimica et Cosmochimica Acta, Vol. 126 https://doi.org/10.1016/j.gca.2013.11.009	journal	February 2014
Unifying Concepts Linking Dissolved Organic Matter Composition to Persistence in Aquatic Ecosystems Kellerman, Anne M.; Guillemette, François; Podgorski, David C. Environmental Science & Technology, Vol. 52, Issue 5 https://doi.org/10.1021/acs.est.7b05513	journal	February 2018
Untargeted metabolomic profiling of Sphagnum fallax reveals novel antimicrobial metabolites Fudyma, Jane D.; Lyon, Jamee; AminiTabrizi, Roya Plant Direct, Vol. 3, Issue 11 https://doi.org/10.1002/pld3.179	journal	November 2019
Aerobic and Anaerobic Catabolism of Vanillic Acid and Some Other Methoxy-Aromatic Compounds by Pseudomonas sp. Strain PN-1 Taylor, Barrie F. Applied and Environmental Microbiology, Vol. 46, Issue 6 https://doi.org/10.1128/AEM.46.6.1286-1292.1983	journal	December 1983
Effects of temperature on biological degradation of phenols, benzoates and phthalates under methanogenic conditions Levén, Lotta; Schnürer, Anna International Biodeterioration & Biodegradation, Vol. 55, Issue 2 https://doi.org/10.1016/j.ibiod.2004.09.004	journal	March 2005
Sugars in soil and sweets for microorganisms: Review of origin, content, composition and fate Gunina, Anna; Kuzyakov, Yakov Soil Biology and Biochemistry, Vol. 90 https://doi.org/10.1016/j.soilbio.2015.07.021	journal	November 2015
Glutamine signalling in bacteria Forchhammer, Karl Frontiers in Bioscience, Vol. 12, Issue 1 https://doi.org/10.2741/2069	journal	January 2007
Role of glutamate metabolism in bacterial responses towards acid and other stresses Feehily, C.; Karatzas, K. A. G. Journal of Applied Microbiology, Vol. 114, Issue 1 https://doi.org/10.1111/j.1365-2672.2012.05434.x	journal	September 2012
Methane emissions from wetlands: biogeochemical, microbial, and modeling perspectives from local to global scales Bridgham, Scott D.; Cadillo-Quiroz, Hinsby; Keller, Jason K. Global Change Biology, Vol. 19, Issue 5 https://doi.org/10.1111/gcb.12131	journal	February 2013
Small differences in ombrotrophy control regional-scale variation in methane cycling among Sphagnum-dominated peatlands Zalman, C.; Keller, J. K.; Tfaily, M. Biogeochemistry, Vol. 139, Issue 2 https://doi.org/10.1007/s10533-018-0460-z	journal	June 2018
The potential importance of methylated substrates in methane production within three northern Minnesota peatlands Hanna, Emily; Keller, Jason K.; Chang, Daniel Soil Biology and Biochemistry, Vol. 150 https://doi.org/10.1016/j.soilbio.2020.107957	journal	November 2020
Disproportionate increase in freshwater methane emissions induced by experimental warming Zhu, Yizhu; Purdy, Kevin J.; Eyice, Özge Nature Climate Change, Vol. 10, Issue 7 https://doi.org/10.1038/s41558-020-0824-y	journal	June 2020

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