Unraveling community structure, ecophysiology, and regulation of new acid-tolerant denitrifiers driving high N2O emissions from cryoturbated peat soil in acidic tundra
Final Report Abstract
Pristine cryoturbated peat circles of the arctic tundra emit the greenhouse gas nitrous oxide like heavily fertilized agricultural soils in central Europe, while adjacent unturbated tundra does not. Peat circles are characterized by the absence of plant cover, high nitrate (up to 1.5 mM) and a low, acidic pH around 4. Such permafrost affected soils are prone to respond to global warming and nitrous oxide producing key organisms are essentially unknown. Denitrifiers convert nitrate to nitrous oxide when organic carbon in the soils is available as substrate and oxygen is limited or absent. Most known denitrifiers are capable of reducing the nitrous oxide further to molecular nitrogen. However, about 30% of denitrifiers lack the enzyme nitrous oxide reductase responsible for this step. Low pH inhibits the nitrous oxide reduction in model denitrifiers and many pH neutral soils, giving rise to increased nitrous oxide missions. Thus, in depth knowledge on denitrifiers and regulation of their diversity as well as activity are important for understanding emissions of the greenhouse gas nitrous oxide from cryoturbated peat circles. We addressed the hypothesis that the ecophysiology of new and active denitrifier communities in cryoturbated peat circle control N2O emissions by microcosm incubations, denitrifier cell counts, differential RNA and DNA stable isotope probing (DSIP), developing and applying an in vitro subtractive transcriptome analysis (STA) approach, and isolation as well as genome analysis of new denitrifiers. Denitrifiers were more abundant in cryoturbated peat circle than in adjacent unturbated tundra peat soil. Such a finding was reflected in high and instantaneous denitrification potentials of cryoturbated peat circle soils relative to essentially absent denitrification potentials of unturbated tundra soils. Denitrification potentials were higher at pH 6 than at in situ pH of 4. pH optimum for denitrification in peat circle soil was around 5-6, temperature optima around 35 °C. Optima were at higher pH and temperature than usually occurring in situ. At pH 4 and pH 6, nitrate was reduced to molecular nitrogen including nitrous oxide reduction only when easily available organic carbon was supplemented, suggesting that nitrous oxide reduction was not impaired by the low pH and that denitrification was complete in peat circle microcosms. DSIP on RNA and DNA level with 13C-labelled acetate indicated that primary acetate assimilators under denitrifying conditions were Burkholderiaceae, and to a lesser extent Rhodanobacter sp. in cryoturbated peat circle microcosms at pH 4. R. denitificans is a well known acid-tolerant complete denitrifier and was thus used as a model to develop as well as validate the STA. STA was then used to elucidate expression patterns of regulators as well as genes associated with denitrification. STA reduces the abundance of transcripts expressed at similar levels under conditions to be compared, and is thus a promising tool for studying complex environmental samples. For R. denitrificans, several new FNR-type regulators were expressed in response to denitrifying conditions. The R. denitrificans genome encoded three NO reductases, but only one was expressed at higher levels during denitrification relative to aerobic growth. This NO reductase was a hitherto unknown quinoldependent NO reductase. 54 diverse other denitrifiers of the Proteobacteria and Firmicutes were isolated from cryoturbated peat circles and unturbated tundra at low pH. Most of them were capable of complete denitrification to molecular nitrogen. Two strains affiliating with the genus Caballeroni (Burkholderiaceae) represent new species of the genus and were genome sequenced. Thaumarchaeal Nitrosocosmicus sp. were the only Archaea detected and putative nitrifiers in peat circle soil, possibly fueling denitrifiers with nitrate. In conclusion, the present project shows that peat circle denitrifiers are abundant, diverse, acid-tolerant to moderately acidophilic, and contribute to nitrous oxide emissions from peat circles due to limitation in easily available organic carbon rather than a pH inhibition of their nitrous oxide reductase as previously thought. Furthermore, we provide new model denitrifiers from permafrost affected cryoturbated peat soil and a new subtractive transcriptomic approach to study regulation of denitrification.
Publications
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2012. Actinobacterial nitrate reducers and Proteobacterial denitrifiers are abundant in N2O metabolizing palsa peat. Applied and Environmental Microbiology 78: 5584-5596
Palmer, K., and M.A. Horn
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2015. Denitrification activity of a remarkably diverse fen denitrifier community in Finnish Lapland is N-oxide limited. PLOSone 10: e0123123
Palmer, K., and M.A. Horn
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2016. Drying-rewetting and flooding impact denitrifier activity rather than community structure in a moderately acidic fen. Frontiers in Microbiology 7: 727
Palmer, K., Köpp, J., Gebauer, G., and M.A. Horn
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2017. Soil conditions rather than long-term exposure to elevated CO2 affect soil microbial communities associated with N-cycling. Frontiers in Microbiology 8: 1976
Brenzinger, K., Kujala (nee Palmer), K., Horn, M.A., Moser, G., Guillet, C., Kamann, C., Müller, C., and G. Braker
