Does long-term soil warming affect microbial element limitation? A test by short-term assays of microbial growth responses to labile C, N and P additions

Increasing global temperatures have been reported to accelerate soil carbon (C) cycling, but also to promote nitrogen (N) and phosphorus (P) dynamics in terrestrial ecosystems. However, warming can differentially affect ecosystem C, N and P dynamics, potentially intensifying elemental imbalances between soil resources, plants and soil microorganisms. Here, we investigated the effect of long-term soil warming on microbial resource limitation, based on measurements of microbial growth (18O incorporation into DNA) and respiration after C, N and P amendments. Soil samples were taken from two soil depths (0–10, 10–20 cm) in control and warmed (>14 years warming, +4°C) plots in the Achenkirch soil warming experiment. Soils were amended with combinations of glucose-C, inorganic/organic N and inorganic/organic P in a full factorial design, followed by incubation at their respective mean field temperatures for 24 h. Soil microbes were generally C-limited, exhibiting 1.8-fold to 8.8-fold increases in microbial growth upon C addition. Warming consistently caused soil microorganisms to shift from being predominately C limited to become C-P co-limited. This P limitation possibly was due to increased abiotic P immobilization in warmed soils. Microbes further showed stronger growth stimulation under combined glucose and inorganic nutrient amendments compared to organic nutrient additions. This may be related to a prolonged lag phase in organic N (glucosamine) mineralization and utilization compared to glucose. Soil respiration strongly positively responded to all kinds of glucose-C amendments, while responses of microbial growth were less pronounced in many of these treatments. This highlights that respiration–though easy and cheap to measure—is not a good substitute of growth when assessing microbial element limitation. Overall, we demonstrate a significant shift in microbial element limitation in warmed soils, from C to C-P co-limitation, with strong repercussions on the linkage between soil C, N and P cycles under long-term warming

Long-term soil warming decreases microbial phosphorus utilization by increasing abiotic phosphorus sorption and phosphorus losses

Phosphorus (P) is an essential and often limiting element that could play a crucial role in terrestrial ecosystem responses to climate warming. However, it has yet remained unclear how different P cycling processes are affected by warming. Here we investigate the response of soil P pools and P cycling processes in a mountain forest after 14 years of soil warming (+4 °C). Long-term warming decreased soil total P pools, likely due to higher outputs of P from soils by increasing net plant P uptake and downward transportation of colloidal and particulate P. Warming increased the sorption strength to more recalcitrant soil P fractions (absorbed to iron oxyhydroxides and clays), thereby further reducing bioavailable P in soil solution. As a response, soil microbes enhanced the production of acid phosphatase, though this was not sufficient to avoid decreases of soil bioavailable P and microbial biomass P (and biotic phosphate immobilization). This study therefore highlights how long-term soil warming triggers changes in biotic and abiotic soil P pools and processes, which can potentially aggravate the P constraints of the trees and soil microbes and thereby negatively affect the C sequestration potential of these forests

Spatial variability shapes microbial communities of permafrost soils and their reaction to warming

Climate change threatens the Earth’s biggest terrestrial organic carbon reservoir: permafrost soils. With climate warming, frozen soil organic matter may thaw and become available for microbial decomposition and subsequent greenhouse gas emissions. Permafrost soils are extremely heterogenous within the soil profile and between landforms. This heterogeneity in environmental conditions, carbon content and soil organic matter composition, potentially leads to different microbial communities with different responses to warming. The aim of the present study is to (1) elucidate these differences in microbial community compositions and (2) investigate how these communities react to warming. We performed short-term warming experiments with permafrost soil organic matter from northwestern Canada. We compared two sites characterized by different glacial histories (Laurentide Ice Sheet cover during LGM and without glaciation), three landscape types (low-center, flat-center, high-center polygons) and four different soil horizons (organic topsoil layer, mineral topsoil layer, cryoturbated soil layer, and the upper permanently frozen soil layer). We incubated aliquots of all soil samples at 4 °C and at 14 °C for 8 weeks and analyzed microbial community compositions (amplicon sequencing of 16S rRNA gene and ITS1 region) before and after the incubation, comparing them to microbial growth, microbial respiration, microbial biomass and soil organic matter composition. We found distinct bacterial, archaeal and fungal communities for soils of different glaciation history, polygon types and for different soil layers. Communities of low-center polygons differ from high-center and flat-center polygons in bacterial, archaeal and fungal community compositions, while communities of organic soil layers are significantly different from all other horizons. Interestingly, permanently frozen soil layers differ from all other horizons in bacterial and archaeal, but not fungal community composition. The 8-week incubations led to minor shifts in bacterial and archaeal community composition between initial soils and those subjected to 14 °C warming. We also found a strong warming effect on the community compositions in some of the extreme habitats: microbial community compositions of (i) the upper permanently frozen layer and of (ii) low-center polygons differ significantly for incubations at 4 °C and 14 °C. Yet, the lack of a community change in horizons of the active layer suggests that microbes are adapted to fluctuating temperatures due to seasonal thaw events. Our results suggest that warming responses of permafrost soil organic matter, if not frozen or water-saturated, may be predictable by current models. Process changes induced by short-term warming can be rather attributed to changes in microbial physiology than community composition. This work is part of the EU H2020 project “Nunataryuk”

Long-term responses of soil microbial activities to soil warming in a temperate forest demonstrate strong changes in element cycling and microbial element limitation

Trotz der verstärkten Bemühungen, die Auswirkungen des Klimawandels auf die Kohlenstoff (C) Dynamik des Waldbodens zu verstehen, haben sich nur wenige Studien mit den langfristigen Auswirkungen der Erwärmung auf durch Mikroben vermittelte Boden-C- und Nährstoffprozesse befasst. Aufgrund der thermischen Akklimatisierung der mikrobiellen Gemeinschaft oder der Erschöpfung des labilen Boden-C als Hauptsubstrat für heterotrophe Bodenmikroben, lässt die anfängliche Stimulation des C-Zyklus des Bodens mit der Zeit in Langzeitversuchen zur Bodenerwärmung nach. Die thermische Akklimatisation kann wegen einer längeren Erwärmung auftreten und ist definiert als die direkte Reaktion des Organismus auf erhöhten Temperaturen über jährliche bis dekadische Zeitskalen, die sich als physiologische Veränderungen der mikrobiellen Gemeinschaft im Boden manifestieren. Dieser Mechanismus unterscheidet sich deutlich von der offensichtlichen thermischen Akklimatisation, bei der die abgeschwächte Reaktion von mikrobiellen Prozessen im Boden auf die Erwärmung auf die Erschöpfung der labilen C-Pools im Boden zurückzuführen ist. Das Achenkirch-Experiment in den nördlichen Kalksteinalpen in Österreich (47° 34' 50'' N; 11° 38' 21'' O; 910 m ü.M.) ist ein langfristiges Experiment zur Bodenerwärmung (> 15 Jahre, +4 °C Erwärmung über der Umgebung), die wichtige Einblicke in die Auswirkungen der globalen Erwärmung auf den C-Zyklus des Waldbodens liefert. Am Standort Achenkirch haben wir nach neun Jahren (2013) eine anhaltend positive Reaktion der heterotrophen Bodenatmung und des CO2-Ausflusses auf die Erwärmung beobachtet, die zu einer geeigneten Umgebung wurde für die Prüfung von Hypothesen über anhaltende oder abnehmende Erwärmungseffekte auf dekadischen Skalen. Wir haben im Oktober 2019 Erdeproben von sechs beheizten und sechs Kontrollflächen mit Bodentiefe von 0-10 cm und 10-20 cm gesammelt und bei drei verschiedenen Temperaturen inkubiert: Außentemperatur, +4 ° C und +10 ° C. Wir haben die potenzielle Bodenenzymaktivitäten mit fluorometrischen Assays, die Bruttoraten der Protein-depolymerisation, Stickstoff (N)-Mineralisierung und Nitrifikation anhand den 15N-Isotopenpool-Verdünnungsansätzen sowie die mikrobiellen Wachstum- und CO2 Produktion- und die Effizienz der C- und N-Nutzungen basierend auf dem 18O-Einbau in DNA gemessen. Unsere Ergebnisse zeigten, dass die potenziellen Enzymaktivitäten, einschließlich Leucinaminopeptidase, N-Acetylglucosaminidase, -Glucosidase und saurer Phosphatase, durch dekadische Bodenerwärmung signifikant 43 hochreguliert wurden (1,3- bis 3,3-fach in R10). Im Gegensatz dazu nahm ihre Temperaturempfindlichkeit (Q10) mit der Erwärmung des Bodens ab, insbesondere für C- und N-bezogene Enzymaktivitäten, was auf eine thermische Akklimatisierung der Mikroben und die Produktion neuer Isoenzyme hinweist. Bei mikrobiellen C-Prozessen fanden wir keine Herunterregulierung (R10) mit chronischer Erwärmung, während die Bodenerwärmung die Q10 von mikrobiellen anabolen Prozessen merklich verringerte (Wachstum), blieb diese Verringerung bei Q10 von katabolen Prozessen (Atmung) aus. Dies führte effektiv zu einer Abnahme der thermischen Empfindlichkeit von mikrobiellem CUE in erwärmten Böden. Gleichzeitig hatten die Q10-Werte der mikrobiellen CUE weniger als eins, was zeigt, dass die mikrobielle CUE mit kurzfristigen Temperaturerhöhungen abnimmt. Dies löste in der Zukunft unter erwärmten Bodenbedingungen eine Abnahme des mikrobiellen CUE aus, während die Umschlagsdauer für mikrobielle Biomasse abnahm, was auf einen schnelleren mikrobiellen Umsatz und eine schnellere Bildung von Nekromassen hinweist. Boden-N-Prozesse wurden ebenfalls nicht herunterreguliert (R10), obwohl wir eine thermische Akklimatisierung der Protein-Depolymerisation (Abnahme von Q10) fanden. Unter Feldbedingungen (Rfield) zeigten wärmere Böden keine Unterschiede in den Boden-N-Prozessen außer der Protein-depolymerisation (Abnahme). Insgesamt deuteten unsere Ergebnisse auf komplexe Reaktionen von mikrobiellen C- und N-Prozessen hin. Mikrobielle C- und N-Prozesse wurden nicht herunterreguliert (R10), aber die Temperaturempfindlichkeit (Q10) der meisten mikrobiellen C- und N-Prozessen nahm ab, was zu einer leichten Abschwächung aber dennoch zu einer Stimulierung dieser Prozessen in erwärmten Böden (Rfield) führte. Die Stimulation von Bodenenzymen führte daher nicht zu höheren C- und N-Prozessraten, was auf eine erhöhte Substratbegrenzung in wärmeren Böden hinweist.Despite the intensified efforts to understand the impacts of climate change on forest soil C dynamics, few studies have addressed the long-term effects of warming on microbial mediated soil C and nutrient processes. In long-term soil warming experiments the initial stimulation of soil C cycling diminished with time, due to thermal acclimation of the microbial community or due to depletion of labile soil C as the major substrate for heterotrophic soil microbes. Thermal acclimation can arise because of prolonged warming and is defined as the direct organism response to elevated temperature across annual to decadal timescales which manifest as a physiological change of the soil microbial community. This mechanism is clearly different from apparent thermal acclimation, where the attenuated response of soil microbial processes to warming is due to the exhaustion of the labile soil C pool. The Achenkirch experiment, situated in the Northern Limestone Alps, Austria (47°34’ 50’’ N; 11°38’ 21’’ E; 910 m a.s.l.) is a long term soil warming experiment (>15 yrs, +4 °C warming above ambient) that has provided key insights into the effects of global warming on the forest soil C cycle. At the Achenkirch site, we have observed a sustained positive response of heterotrophic soil respiration and of soil CO2 efflux to warming after nine years (2013), making it an appropriate setting for testing hypotheses about continued or decreasing warming effects at decadal scales. We collected soil from six heated and six control plots in October 2019, from 0-10 cm and 10-20 cm soil depth, and incubated them at three different temperatures: ambient, +4 °C, and +10 °C. We measured potential soil enzyme activities with fluorometric assays, gross rates of protein depolymerization, N mineralization, and nitrification with 15N isotope pool dilution approaches, and microbial growth, respiration, and C and N use efficiencies based on the 18O incorporation in DNA. Our results show that the potential enzyme activities including leucine aminopeptidase, N-acetylglucosaminidase, B-glucosidase, and acid phosphatase were significantly up-regulated by decadal soil warming (1.3- to 3.3-fold in R10). In contrast, their temperature sensitivity (Q10) decreased with soil warming, particularly for C- and N-related enzyme activities, indicating thermal acclimation of the microbes and the production of new isoenzymes. For microbial C processes, we found no down-regulation (R10) due to chronic warming while soil warming notably decreased the Q10 of microbial anabolic processes (growth) but not of catabolic processes (respiration). This effectively caused a decrease in the thermal sensitivity of microbial CUE in warmed soils. At the same time, microbial CUE had Q10 values lower than one, demonstrating that in the short-term microbial CUE decreases with short-term temperature increases. This triggered a decrease in microbial CUE under warmed soil conditions in a future world, while microbial biomass turnover time decreased, pointing to faster microbial turnover and necromass formation. Soil N cycle processes were also not down-regulated (R10) though we found thermal acclimation on protein depolymerization (decrease in Q10). Under field conditions (Rfield), warmer soils did not show differences in soil N processes other than protein depolymerization (decrease). Overall, our results indicate a complex response of microbial C and N cycle processes. Microbial C and N processes were not down-regulated (R10), but the temperature sensitivity (Q10) of most of them decreased, leading to a slight attenuation but still a stimulation of these processes in warmed soils (Rfield). The stimulation of soil enzymes therefore did not translate into higher C and N process rates, indicating increased substrate limitation in warmer soils

Seasonal dynamics of soil microbial growth, respiration, biomass, and carbon use efficiency in temperate soils

Soil microbial growth, respiration, and carbon (C) use efficiency (CUE) are essential parameters to understand, describe and model the soil carbon cycle. While seasonal dynamics of microbial respiration are well studied, little is known about how microbial growth and CUE change over the course of a year, especially outside the plant growing season. In this study, we measured soil microbial respiration, gross growth via 18O incorporation into DNA, and biomass in an agricultural field and a deciduous forest 16 times over the course of two years. We sampled soils to a depth of 5 cm from plots at which harvest residues or leaf litter remained on the plot or was removed. We observed strong seasonal variations of microbial respiration, growth, and biomass. All these microbial parameters were significantly higher at the forest site, which contained 4.3 % organic C compared to the agricultural site with 0.9 % organic C. CUE also varied strongly (0.1 to 0.7) but was overall significantly higher at the agricultural site compared to the forest site. We found that microbial respiration and to a lesser extent microbial growth followed the seasonal dynamics of soil temperature. Microbial growth was further affected by the presence of plants in the agricultural system or foliage in the forest. At low temperatures in winter, both microbial respiration and gross growth showed the lowest rates, whereas CUE (calculated from both respiration and growth) showed amongst the highest values determined during the two years, due to the higher temperature sensitivity of microbial respiration. Microbial biomass C strongly increased in winter. Surprisingly, this winter peak was not connected to high microbial growth or an increase in DNA content. This suggests that microorganisms accumulated C and N, potentially in the form of osmo- or cryoprotectants or increased in cell size but did not divide. This microbial winter bloom and following decline, where C is released from microbial biomass and freely available, might constitute a highly dynamic time in the annual C cycle in temperate soil systems. Highly variable CUE, which was observed in our study, and the fact that CUE is calculated from independently controlled microbial respiration and microbial growth, ask for great caution when CUE is used to describe soil microbial physiology, soil C dynamics or C sequestration. Instead, microbial respiration, microbial growth, and microbial biomass C should be investigated individually in combination to better understand the soil C cycle

How do microorganisms from permafrost soils respond to short-term warming?

Arctic ecosystems outpace the global rate of temperature increases and are exceptionally susceptible to global warming. Concerns are raising that CO2 and CH4 released from thawing permafrost upon warming may induce a positive feedback to climate change. This is based on the assumption, that microbial activity increases with warming and does not acclimate over time. However, we lack a mechanistic understanding of carbon and nutrient fluxes including their spatial control in the very heterogeneous Arctic landscape. The objective of this study therefore was to elucidate the microbial controls over soil organic matter decomposition in different horizons of the active layer and upper permafrost. We investigated different landscape units (high-centre polygons, low-centre polygons and flat polygon tundra) in two small catchments that differ in glacial history, at the Yukon coast, Northwestern Canada. In total, 81 soil samples were subjected to short-term (eight weeks) incubation experiments at controlled temperature (4 °C and 14 °C) and moisture conditions. Heterotrophic respiration was assessed weekly, whereas physiological parameters of soil microbes and their temperature response (Q10) were determined at the end of the incubation period. Microbial growth was estimated by measuring the incorporation of 18O from labelled water into DNA and used to calculate microbial carbon use efficiencies (CUE). Microbial biomass was determined via chloroform fumigation extraction. Potential activities of extracellular enzymes involved in C, N, P and S cycling were measured using microplate fluorimetric assays. Cumulative heterotrophic respiration of investigated soil layers followed the pattern organic layers > upper frozen permafrost > cryoturbated material > mineral layers in both catchments. Microbial respiration responded strongly in all soils to warming in all soils, but the observed response was highest for organic layers and cryoturbated material at the beginning and end of the experiment. Average Q10 values at the beginning of the experiment varied between 1.7 to 4.3 with differences between horizons but converged towards Q10 values between 2.0min to 2.9max after eight weeks of incubation. Even though microbial biomass C did not change with warming, microbial mass specific growth was enhanced in organic, cryoturbated and permafrost soils. Overall, warming resulted in a 65% reduced CUE in organic horizons. Our results show no indication for physiological acclimatization of permafrost soil microbes when subjected to 8-weeks of experimental warming. Given that the duration of the season in which most horizons are unfrozen is rarely longer than 2 months, our results do not support an acclimation of microbial activity under natural conditions. Instead, our data supports the current view of a high potential for prolonged carbon losses from tundra soils with warming by enhanced microbial activity. This work is part of the EU H2020 project “Nunataryuk”

Subjecting permafrost microorganisms to short-term warming

Arctic environments are a prime example for ecosystems facing manifold vast and rapid changes in the wake of climate change, outpacing the global rate of temperature increases. The risk of thawing permafrost soils raises concerns about a positive feedback process being mediated by increased microbial activity that does not acclimate over time freeing greenhouse gases. However, the mechanistic understanding of the controls on microbial carbon cycling upon warming is still vague. In the following study we investigate microbial growth and soil organic matter decomposition in different soil horizons of the active layer and upper permafrost, covering different polygonal landscape units in two small catchments at the Canadian Yukon Coast. 81 soil samples were subjected to a short-term warming experiment under controlled temperature (4 °C and 14 °C) and moisture conditions. Microbial respiration was measured weekly whereas microbial biomass and physiological parameters were determined at the end of the incubation period and used to assess temperature responses. Microbial growth was estimated by measuring the incorporation of 18O from labelled water into DNA and used to calculate CUE. Microbial biomass was determined via chloroform fumigation. Potential activities of extracellular enzymes were measured using microplate fluorometric assays. Microbial biomass carbon was not affected by warming except for permafrost layers where it either increased or decreased depending on the examined catchment. Microbial respiration strongly responded to warming following the pattern organic layers > upper frozen permafrost > cryoturbated material > mineral layers. Mass specific growth and extracellular enzymatic activities were also enhanced with short-term warming in all soil horizons. This led to rather variable CUE being unaffected in mineral and cryoturbated layers whereas we could observe a minor reduction in organic and permafrost layers where the response of respiration outpaced the one of microbial growth. Our results are not indicative for any physiological acclimatization of permafrost microbes when subjected to 8 weeks of experimental warming and hence support the current concern for potential prolonged carbon losses from warming tundra soils. This work is part of the EU H2020 project “Nunataryuk”