Temperature and livestock grazing trigger transcriptome responses in bumblebees along an elevational gradient.

Climate and land-use changes cause increasing stress to pollinators but the molecular pathways underlying stress responses are poorly understood. Here, we analyzed the transcriptomic response of Bombus lucorum workers to temperature and livestock grazing. Bumblebees sampled along an elevational gradient, and from differently managed grassland sites (livestock grazing vs unmanaged) in the German Alps did not differ in the expression of genes known for thermal stress responses. Instead, metabolic energy production pathways were upregulated in bumblebees sampled in mid- or high elevations or during cool temperatures. Extensive grazing pressure led to an upregulation of genetic pathways involved in immunoregulation and DNA-repair. We conclude that widespread bumblebees are tolerant toward temperature fluctuations in temperate mountain environments. Moderate temperature increases may even release bumblebees from metabolic stress. However, transcriptome responses to even moderate management regimes highlight the completely underestimated complexity of human influence on natural pollinators.

Can flooding-induced greenhouse gas emissions be mitigated by trait-based plant species choice?

Intensively managed grasslands are large sources of the potent greenhouse gas nitrous oxide (N2O) and important regulators of methane (CH4) consumption and production. The predicted increase in flooding frequency and severity due to climate change could increase N2O emissions and shift grasslands from a net CH4 sink to a source. Therefore, effective management strategies are critical for mitigating greenhouse gas emissions from flood-prone grasslands. We tested how repeated flooding affected the N2O and CH4 emissions from 11 different plant communities (Festuca arundinacea, Lolium perenne, Poa trivialis, and Trifolium repens in monoculture, 2- and 4-species mixtures), using intact soil cores from an 18-month old grassland field experiment in a 4-month greenhouse experiment. To elucidate potential underlying mechanisms, we related plant functional traits to cumulative N2O and CH4 emissions. We hypothesized that traits related with fast nitrogen uptake and growth would lower N2O and CH4 emissions in ambient (non-flooded) conditions, and that traits related to tissue toughness would lower N2O and CH4 emissions in flooded conditions. We found that flooding increased cumulative N2O emissions by 97 fold and cumulative CH4 emissions by 1.6 fold on average. Plant community composition mediated the flood-induced increase in N2O emissions. In flooded conditions, increasing abundance of the grass F. arundinacea was related with lower N2O emissions; whereas increases in abundance of the legume T. repens resulted in higher N2O emissions. In non-flooded conditions, N2O emissions were not clearly mediated by plant traits related with nitrogen uptake or biomass production. In flooded conditions, plant communities with high root carbon to nitrogen ratio were related with lower cumulative N2O emissions, and a lower global warming potential (CO2 equivalent of N2O and CH4). We conclude that plant functional traits related to slower decomposition and nitrogen mineralization could play a significant role in mitigating N2O emissions in flooded grasslands.

Organic Residue Amendments to Modulate Greenhouse Gas Emissions From Agricultural Soils.

Organic fertilizers have been shown to stimulate CH4 uptake from agricultural soils. Managing fertilizer application to maximize this effect and to minimize emission of other greenhouse gasses offers possibilities to increase sustainability of agriculture. To tackle this challenge, we incubated an agricultural soil with different organic amendments (compost, sewage sludge, digestate, cover crop residues mixture), either as single application or in a mixture and subjected it to different soil moisture concentrations using different amounts of organic amendments. GHG fluxes and in vitro CH4 oxidation rates were measured repeatedly, while changes in organic matter and abundance of GHG relevant microbial groups (nitrifiers, denitrifiers, methanotrophs, methanogens) were measured at the end of the incubation. Overall the dynamics of the analyzed GHGs differed significantly. While CO2 and N2O differed considerably between the treatments, CH4 fluxes remained stable. In contrast, in vitro CH4 oxidation showed a clear increase for all amendments over time. CO2 fluxes were mostly dependent on the amount of organic residue that was used, while N2O fluxes were affected more by soil moisture. Several combinations of amendments led to reductions of CO2, CH4, and/or N2O emissions compared to un-amended soil. Most optimal GHG balance was obtained by compost amendments, which resulted in a similar overall GHG balance as compared to the un-amended soil. However, compost is not very nutrient rich potentially leading to lower crop yield when applied as single fertilizer. Hence, the combination of compost with one of the more nutrient rich organic amendments (sewage sludge, digestate) provides a trade-off between maintaining crop yield and minimizing GHG emissions. Additionally, we could observe a strong increase in microbial communities involved in GHG consumption in all amendments, with the strongest increase associated with cover crop residue mixtures. Future research should focus on the interrelation of plants, soil, and microbes and their impact on the global warming potential in relation to applied organic amendments.

Explaining the doubling of N2 O emissions under elevated CO2 in the Giessen FACE via in-field 15 N tracing.

Rising atmospheric CO2 concentrations are expected to increase nitrous oxide (N2 O) emissions from soils via changes in microbial nitrogen (N) transformations. Several studies have shown that N2 O emission increases under elevated atmospheric CO2 (eCO2 ), but the underlying processes are not yet fully understood. Here, we present results showing changes in soil N transformation dynamics from the Giessen Free Air CO2 Enrichment (GiFACE): a permanent grassland that has been exposed to eCO2 , +20% relative to ambient concentrations (aCO2 ), for 15 years. We applied in the field an ammonium-nitrate fertilizer solution, in which either ammonium ( NH4+ ) or nitrate ( NO3- ) was labelled with 15 N. The simultaneous gross N transformation rates were analysed with a 15 N tracing model and a solver method. The results confirmed that after 15 years of eCO2 the N2 O emissions under eCO2 were still more than twofold higher than under aCO2 . The tracing model results indicated that plant uptake of NH4+ did not differ between treatments, but uptake of NO3- was significantly reduced under eCO2 . However, the NH4+ and NO3- availability increased slightly under eCO2 . The N2 O isotopic signature indicated that under eCO2 the sources of the additional emissions, 8,407 µg N2 O-N/m2 during the first 58 days after labelling, were associated with NO3- reduction (+2.0%), NH4+ oxidation (+11.1%) and organic N oxidation (+86.9%). We presume that increased plant growth and root exudation under eCO2 provided an additional source of bioavailable supply of energy that triggered as a priming effect the stimulation of microbial soil organic matter (SOM) mineralization and fostered the activity of the bacterial nitrite reductase. The resulting increase in incomplete denitrification and therefore an increased N2 O:N2 emission ratio, explains the doubling of N2 O emissions. If this occurs over a wide area of grasslands in the future, this positive feedback reaction may significantly accelerate climate change.

Soil Conditions Rather Than Long-Term Exposure to Elevated CO2 Affect Soil Microbial Communities Associated with N-Cycling.

Continuously rising atmospheric CO2 concentrations may lead to an increased transfer of organic C from plants to the soil through rhizodeposition and may affect the interaction between the C- and N-cycle. For instance, fumigation of soils with elevated CO2 (eCO2) concentrations (20% higher compared to current atmospheric concentrations) at the Giessen Free-Air Carbon Dioxide Enrichment (GiFACE) sites resulted in a more than 2-fold increase of long-term N2O emissions and an increase in dissimilatory reduction of nitrate compared to ambient CO2 (aCO2). We hypothesized that the observed differences in soil functioning were based on differences in the abundance and composition of microbial communities in general and especially of those which are responsible for N-transformations in soil. We also expected eCO2 effects on soil parameters, such as on nitrate as previously reported. To explore the impact of long-term eCO2 on soil microbial communities, we applied a molecular approach (qPCR, T-RFLP, and 454 pyrosequencing). Microbial groups were analyzed in soil of three sets of two FACE plots (three replicate samples from each plot), which were fumigated with eCO2 and aCO2, respectively. N-fixers, denitrifiers, archaeal and bacterial ammonia oxidizers, and dissimilatory nitrate reducers producing ammonia were targeted by analysis of functional marker genes, and the overall archaeal community by 16S rRNA genes. Remarkably, soil parameters as well as the abundance and composition of microbial communities in the top soil under eCO2 differed only slightly from soil under aCO2. Wherever differences in microbial community abundance and composition were detected, they were not linked to CO2 level but rather determined by differences in soil parameters (e.g., soil moisture content) due to the localization of the GiFACE sets in the experimental field. We concluded that +20% eCO2 had little to no effect on the overall microbial community involved in N-cycling in the soil but that spatial heterogeneity over extended periods had shaped microbial communities at particular sites in the field. Hence, microbial community composition and abundance alone cannot explain the functional differences leading to higher N2O emissions under eCO2 and future studies should aim at exploring the active members of the soil microbial community.

Potential N2O Emissions from the Tanks of Bromeliads Suggest an Additional Source of N2O in the Neotropics.

We studied the propensity of the tank bromeliad Werauhia gladioliflora to emit the greenhouse gas nitrous oxide (N2O) at current and at increased N deposition levels in the range of predicted future scenarios. Potential production rates and net accumulation of N2O from tank substrate corresponded to N availability. N2O was produced in excess at all N levels due to a low level of N2O reductase activity which agreed well with a low abundance of N2O reducers compared to nitrite reducers. Transcriptional activation, however, indicated that expression of denitrification genes may be enhanced with increasing N supply eventually leading to more efficient N2O turnover with potential for adaptation of denitrifier communities to higher N levels. Our findings indicate that tank bromeliads may constitute a novel source of N2O in Neotropical forest canopies but further studies are required to understand the size and significance of in situ N2O fluxes from tank bromeliads to the environment.

pH-driven shifts in overall and transcriptionally active denitrifiers control gaseous product stoichiometry in growth experiments with extracted bacteria from soil.

Soil pH is a strong regulator for activity as well as for size and composition of denitrifier communities. Low pH not only lowers overall denitrification rates but also influences denitrification kinetics and gaseous product stoichiometry. N2O reductase is particularly sensitive to low pH which seems to impair its activity post-transcriptionally, leading to higher net N2O production. Little is known about how complex soil denitrifier communities respond to pH change and whether their ability to maintain denitrification over a wider pH range relies on phenotypic redundancy. In the present study, we followed the abundance and composition of an overall and transcriptionally active denitrifier community extracted from a farmed organic soil in Sweden (pH H2O = 7.1) when exposed to pH 5.4 and drifting back to pH 6.6. The soil was previously shown to retain much of its functioning (low N2O/N2 ratios) over a wide pH range, suggesting a high functional versatility of the underlying community. We found that denitrifier community composition, abundance and transcription changed throughout incubation concomitant with pH change in the medium, allowing for complete reduction of nitrate to N2 with little accumulation of intermediates. When exposed to pH 5.4, the denitrifier community was able to grow but reduced N2O to N2 only when near-neutral pH was reestablished by the alkalizing metabolic activity of an acid-tolerant part of the community. The genotypes proliferating under these conditions differed from those dominant in the control experiment run at neutral pH. Denitrifiers of the nirS-type appeared to be severely suppressed by low pH and nirK-type and nosZ-containing denitrifiers showed strongly reduced transcriptional activity and growth, even after restoration of neutral pH. Our study suggests that low pH episodes alter transcriptionally active populations which shape denitrifier communities and determine their gas kinetics.

Impact of Land Use Management and Soil Properties on Denitrifier Communities of Namibian Savannas.

We studied potential denitrification activity and the underlying denitrifier communities in soils from a semiarid savanna ecosystem of the Kavango region in NE Namibia to help in predicting future changes in N(2)O emissions due to continuing changes of land use in this region. Soil type and land use (pristine, fallow, and cultivated soils) influenced physicochemical characteristics of the soils that are relevant to denitrification activity and N(2)O fluxes from soils and affected potential denitrification activity. Potential denitrification activity was assessed by using the denitrifier enzyme activity (DEA) assay as a proxy for denitrification activity in the soil. Soil type and land use influenced C and N contents of the soils. Pristine soils that had never been cultivated had a particularly high C content. Cultivation reduced soil C content and the abundance of denitrifiers and changed the composition of the denitrifier communities. DEA was strongly and positively correlated with soil C content and was higher in pristine than in fallow or recently cultivated soils. Soil type and the composition of both the nirK- and nirS-type denitrifier communities also influenced DEA. In contrast, other soil characteristics like N content, C:N ratio, and pH did not predict DEA. These findings suggest that due to greater availability of soil organic matter, and hence a more effective N cycling, the natural semiarid grasslands emit more N(2)O than managed lands in Namibia.

Impact of short-term storage temperature on determination of microbial community composition and abundance in aerated forest soil and anoxic pond sediment samples.

Sampling strategy is important for unbiased analysis of the characteristics of microbial communities in the environment. During field work it is not always possible to analyze fresh samples immediately or store them frozen. Therefore, the effect of short-term storage temperature was investigated on the abundance and composition of bacterial, archaeal and denitrifying communities in environmental samples from two different sampling sites. Oxic forest soil and anoxic pond sediment were investigated by measuring microbial abundance (DNA) and transcriptional activity (RNA). Prior to investigating the effect of storage temperature, samples were immediately analyzed, in order to represent the original situation in the habitat. The effect of storage temperature was then determined after 11 days at different low temperatures (room temperature, 4 °C, −22 °C and −80 °C). Community profiling using terminal restriction fragment length polymorphism (T-RFLP) showed no significant differences between the immediately analyzed reference sample and the samples stored at different incubation temperatures, both for DNA and RNA extracts. The abundance of microbial communities was determined using quantitative PCR and it also revealed a stable community size at all temperatures tested. By contrast, incubation at an elevated temperature (37 °C) resulted in changed bacterial community composition. In conclusion, short-term storage, even at room temperature, did not affect microbial community composition, abundance and transcriptional activity in aerated forest soil and anoxic pond sediment.