No evidence for increased loss of old carbon in a temperate organic soil after 13 years of simulated climatic warming despite increased CO2 emissions.

Determining the temperature sensitivity of terrestrial carbon (C) stores is an urgent priority for predicting future climate feedbacks. A key aspect to solve this long-standing research gap is to determine whether warmer temperatures will increase autotrophic activities leading to greater C storage or promote heterotrophic activities that will drive these systems to become C sources. We experimentally addressed this critical question by subjecting intact plant-soil systems in a UK upland ecosystem to simulated climate warming under natural field conditions. We report the results of a 13-year field-based climate manipulation experiment combining in situ respiration measurements with radiocarbon (14 C) analyses of respired CO2 , dissolved organic carbon (DOC), soil and the tissue contents of the dominant soil fauna (enchytraeids). We found that warming during the growing season produced the largely expected increases in ecosystem respiration (63%) and leaching of DOC (19%) with no evidence for thermal acclimation or substrate exhaustion over the whole 13-year experimental period. Contrary to expectations, we found no evidence to support an increased release of old soil C after more than a decade of simulated climatic change, and indeed, 14 C analyses indicated that warming caused a significant shift towards mineralisation of more recent plant-derived C inputs. Further support came from the radiocarbon analyses of the enchytraeid tissues, which showed a greater assimilation of the more recent (plant-derived) C sources following warming. Therefore, in contrast to subarctic ecosystems, our results suggest that changes in C storage in this UK upland soil are strongly coupled to plant activities and that increasing temperatures will drive the turnover of organic material fixed only within recent years, without resulting in the loss of existing old carbon stores.

To replicate, or not to replicate - that is the question: how to tackle nonlinear responses in ecological experiments.

A fundamental challenge in experimental ecology is to capture nonlinearities of ecological responses to interacting environmental drivers. Here, we demonstrate that gradient designs outperform replicated designs for detecting and quantifying nonlinear responses. We report the results of (1) multiple computer simulations and (2) two purpose-designed empirical experiments. The findings consistently revealed that unreplicated sampling at a maximum number of sampling locations maximised prediction success (i.e. the R² to the known truth) irrespective of the amount of stochasticity and the underlying response surfaces, including combinations of two linear, unimodal or saturating drivers. For the two empirical experiments, the same pattern was found, with gradient designs outperforming replicated designs in revealing the response surfaces of underlying drivers. Our findings suggest that a move to gradient designs in ecological experiments could be a major step towards unravelling underlying response patterns to continuous and interacting environmental drivers in a feasible and statistically powerful way.

Arbuscular mycorrhizal fungi reduce nitrous oxide emissions from N2 O hotspots.

Nitrous oxide (N2 O) is a potent, globally important, greenhouse gas, predominantly released from agricultural soils during nitrogen (N) cycling. Arbuscular mycorrhizal fungi (AMF) form a mutualistic symbiosis with two-thirds of land plants, providing phosphorus and/or N in exchange for carbon. As AMF acquire N, it was hypothesized that AMF hyphae may reduce N2 O production. AMF hyphae were either allowed (AMF) or prevented (nonAMF) access to a compartment containing an organic matter and soil patch in two independent microcosm experiments. Compartment and patch N2 O production was measured both before and after addition of ammonium and nitrate. In both experiments, N2 O production decreased when AMF hyphae were present before inorganic N addition. In the presence of AMF hyphae, N2 O production remained low following ammonium application, but increased in the nonAMF controls. By contrast, negligible N2 O was produced following nitrate application to either AMF treatment. Thus, the main N2 O source in this system appeared to be via nitrification, and the production of N2 O was reduced in the presence of AMF hyphae. It is hypothesized that AMF hyphae may be outcompeting slow-growing nitrifiers for ammonium. This has significant global implications for our understanding of soil N cycling pathways and N2 O production.

How does elevated ozone reduce methane emissions from peatlands?

The effects of increased tropospheric ozone (O3) pollution levels on methane (CH4) emissions from peatlands, and their underlying mechanisms, remain unclear. In this study, we exposed peatland mesocosms from a temperate wet heath dominated by the sedge Schoenus nigricans and Sphagnum papillosum to four O3 treatments in open-top chambers for 2.5years, to investigate the O3 impacts on CH4 emissions and the processes that underpin these responses. Summer CH4 emissions, were significantly reduced, by 27% over the experiment, due to summer daytime (8hday-1) O3 exposure to non-filtered air (NFA) plus 35ppb O3, but were not significantly affected by year-round, 24hday-1, exposure to NFA plus 10ppb or NFA plus 25ppb O3. There was no evidence that the reduced CH4 emissions in response to elevated summer O3 exposure were caused by reduced plant-derived carbon availability below-ground, because we found no significant effect of high summer O3 exposure on root biomass, pore water dissolved organic carbon concentrations or the contribution of recent photosynthate to CH4 emissions. Our CH4 production potential and CH4 oxidation potential measurements in the different O3 treatments could also not explain the observed CH4 emission responses to O3. However, pore water ammonium concentrations at 20cm depth were consistently reduced during the experiment by elevated summer O3 exposure, and strong positive correlations were observed between CH4 emission and pore water ammonium concentration at three peat depths over the 2.5-year study. Our results therefore imply that elevated regional O3 exposures in summer, but not the small increases in northern hemisphere annual mean background O3 concentrations predicted over this century, may lead to reduced CH4 emissions from temperate peatlands as a consequence of reductions in soil inorganic nitrogen affecting methanogenic and/or methanotrophic activity.

Evaluating the carbon balance estimate from an automated ground-level flux chamber system in artificial grass mesocosms.

Measuring and modeling carbon (C) stock changes in terrestrial ecosystems are pivotal in addressing global C-cycling model uncertainties. Difficulties in detecting small short-term changes in relatively large C stocks require the development of robust sensitive flux measurement techniques. Net ecosystem exchange (NEE) ground-level chambers are increasingly used to assess C dynamics in low vegetation ecosystems but, to date, have lacked formal rigorous field validation against measured C stock changes. We developed and deployed an automated and multiplexed C-flux chamber system in grassland mesocosms in order rigorously to compare ecosystem total C budget obtained using hourly C-flux measurements versus destructive net C balance. The system combines transparent NEE and opaque respiration chambers enabling partitioning of photosynthetic and respiratory fluxes. The C-balance comparison showed good agreement between the two methods, but only after NEE fluxes were corrected for light reductions due to chamber presence. The dark chamber fluxes allowed assessing temperature sensitivity of ecosystem respiration (R eco) components (i.e., heterotrophic vs. autotrophic) at different growth stages. We propose that such automated flux chamber systems can provide an accurate C balance, also enabling pivotal partitioning of the different C-flux components (e.g., photosynthesis and respiration) suitable for model evaluation and developments.

Links between methane flux and transcriptional activities of methanogens and methane oxidizers in a blanket peat bog.

The relationship between biogeochemical process rates and microbial functional activity was investigated by analysis of the transcriptional dynamics of the key functional genes for methanogenesis (methyl coenzyme M reductase; mcrA) and methane oxidation (particulate methane monooxygenase; pmoA) and in situ methane flux at two peat soil field sites with contrasting net methane-emitting and -oxidizing characteristics. qPCR was used to quantify the abundances of mcrA and pmoA genes and transcripts at two soil depths. Total methanogen and methanotroph transcriptional dynamics, calculated from mcrA and pmoA gene : transcript abundance ratios, were similar at both sites and depths. However, a linear relationship was demonstrated between surface mcrA and pmoA transcript dynamics and surface flux rates at the methane-emitting and methane-oxidizing sites, respectively. Results indicate that methanotroph activity was at least partially substrate-limited at the methane-emitting site and by other factors at the methane-oxidizing site. Soil depth also contributed to the control of surface methane fluxes, but to a lesser extent. Small differences in the soil water content may have contributed to differences in methanogen and methanotroph activities. This study therefore provides a first insight into the regulation of in situ, field-level surface CH(4) flux at the molecular level by an accurate reflection of gene : transcript abundance ratios for the key genes in methane generation and consumption.

Short-term dynamics of abiotic and biotic soil 13CO2 effluxes after in situ 13CO2 pulse labelling of a boreal pine forest.

Physical diffusion of isotopic tracers into and out of soil pores causes considerable uncertainty for the timing and magnitude of plant belowground allocation in pulse-labelling experiments. Here, we partitioned soil CO(2) isotopic fluxes into abiotic tracer flux (physical return), heterotrophic flux, and autotrophic flux contributions following (13)CO(2) labelling of a Swedish Pinus sylvestris forest. Soil CO(2) efflux and its isotopic composition from a combination of deep and surface soil collars was monitored using a field-deployed mass spectrometer. Additionally, (13)CO(2) within the soil profile was monitored. Physical (abiotic) efflux of (13)CO(2) from soil pore spaces was found to be significant for up to 48 h after pulse labelling, and equalled the amount of biotic label flux over 6 d. Measured and modelled changes in (13)CO(2) concentration throughout the soil profile corroborated these results. Tracer return via soil CO(2) efflux correlated significantly with the proximity of collars to trees, while daily amplitudes of total flux (including heterotrophic and autotrophic sources) showed surprising time shifts compared with heterotrophic fluxes. The results show for the first time the significance of the confounding influence of physical isotopic CO(2)-tracer return from the soil matrix, calling for the inclusion of meaningful control treatments in future pulse-chase experiments.

A new stable isotope approach identifies the fate of ozone in plant-soil systems.

* We show that the stable isotope (18)O can be used to trace ozone into different components of the plant-soil system at environmentally relevant concentrations. * We exposed plants and soils to (18)O-labelled ozone and used isotopic enrichment in plant dry matter, leaf water and leaf apoplast, as well as in soil dry matter and soil water, to identify sites of ozone-derived (18)O accumulation. * It was shown that isotopic accumulation rates in plants can be used to infer the location of primary ozone-reaction sites, and that those in bare soils are dependent on water content. However, the isotopic accumulation rates measured in leaf tissue were much lower than the modelled stomatal flux of ozone. * Our new approach has considerable potential to elucidate the fate and reactions of ozone within both plants and soils, at scales ranging from plant communities to cellular defence mechanisms.

A new method for using 18O to trace ozone deposition.

Isotopically labelled ozone ((18)O(3)) is an ideal tool to study the deposition of O(3) to plants and soil, but no studies have made use of it due to the technical difficulties in producing isotopically enriched ozone. For (18)O(3) to be used in fumigation experiments, it has to be purified and stored safely prior to fumigations, to ensure that the label is present predominantly in the form of O(3), and to make efficient use of isotopically highly enriched oxygen. We present a simple apparatus that allows for the safe generation, purification, storage, and release of (18)O(3). Following the purification and release of O(3), about half (by volume) of the (18)O is present in the form of O(3). This means that for a given release of (18)O(3) into the fumigation system, a roughly identical volume of (18)O(2) is released. However, the small volume of this concurrent (18)O(2) release (100 nmol mol(-1) in our experiment) results in only a minor shift of the much larger atmospheric oxygen pool, with no detectable consequence for the isotopic enrichment of either soil or plant materials. We demonstrate here the feasibility of using (18)O as an isotopic tracer in O(3) fumigations by exposing dry soil to 100 nmol mol(-1) (18)O(3) for periods ranging from 1 to 11 h. The (18)O tracer accumulation in soil samples is measured using gas chromatography/isotope ratio mass spectrometry (GC/IRMS), and the results show a linear increase in (18)O/(16)O isotope ratio over time, with significant differences detectable after 1 h of exposure. The apparatus is adapted for use with fumigation chambers sustaining flow rates of 1 m(3) min(-1) for up to 12 h, but simple modifications now allow larger quantities of O(3) to be stored and continuously released (e.g. for use with open-top chambers or FACE facilities).

Impact of water table depth on forest soil methane turnover in laboratory soil cores deduced from natural abundance and tracer 13C stable isotope experiments.

We investigated turnover of methane (CH4) in soils from a poorly drained UK forest. In situ, this forest exhibited a negligible soil-atmosphere CH4 flux, whereas adjacent grassland plots were sources of CH4. We hypothesised that the forest plots exhibited reduced anaerobic CH4 production through water-table draw down. Consequently, we exposed soil cores from under oak to high and low water-table conditions in the laboratory. Methane fluxes increased significantly in the high water-table (1925+/-1702 mug CH4 m(-2) h(-1)) compared to the low one (-3.5+/-6.8 microg CH4 m(-2) h(-1)). Natural abundance delta13C values of CH4 showed a strong depletion in high water-table cores (-56.7+/-2.9 per thousand) compared to methane in ambient air (-46.0 per thousand) indicative of methanogenic processes. The delta13C values of CH4 from low water-table cores (delta13C-46.8+/-0.2 per thousand) was similar to ambient air and suggested little alteration of headspace CH4 by the soil microbial community. In order to assess the CH4 oxidizing activity of the two treatments conclusively, a 13CH4 spike was added to the cores and 13CO2 production was measured as the by-product of CH4 oxidation. 13CH4 oxidation rates were 57.5 (+/-12.7) and 0.5 (+/-0.1) microg CH4 m(-2) h(-1) for high and low water-tables, respectively. These data show that the lower water-table hydrology treatment impacted methanogenic processes without stimulating methanotrophy.

Sampling systems for isotope-ratio mass spectrometry of atmospheric ammonia.

Passive and active ammonia (NH(3)) sampling devices have been tested for their nitrogen (N) capture potential and delta(15)N fractionation effects. Several sampling techniques produced significantly different delta(15)NH(3) signals when sampling the same NH(3) source released from field site fumigation campaigns. Conventional passive NH(3)-monitoring systems have shown to provide insufficient N for isotope-ratio mass spectrometry and various modified devices have been developed, based on existing diffusion tube designs, to overcome this problem. The final sampler design was then tested in a wind tunnel to verify that sampling NH(3) in different environmental conditions did not significantly fractionate the delta(15)N signal.

Rising atmospheric CO2 reduces sequestration of root-derived soil carbon.

Forests have a key role as carbon sinks, which could potentially mitigate the continuing increase in atmospheric carbon dioxide concentration and associated climate change. We show that carbon dioxide enrichment, although causing short-term growth stimulation in a range of European tree species, also leads to an increase in soil microbial respiration and a marked decline in sequestration of root-derived carbon in the soil. These findings indicate that, should similar processes operate in forest ecosystems, the size of the annual terrestrial carbon sink may be substantially reduced, resulting in a positive feedback on the rate of increase in atmospheric carbon dioxide concentration.

Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms.

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a 13CO2 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of 13C-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded 13C compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from 12C and 13C RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.

Soil functional responses to excess nitrogen inputs at global scale.

There is little evidence that nitrogen (N) cycling in the highly weathered, low-phosphorus (P), acidic soils found in Southern Hemisphere continents will differ greatly from that in North America and Europe. Evidence from the 'south' shows: the similarity in forms and temporal patterns in losses of N from different land uses; that the C:N ratios of the forest floor/litter layer from different continents are strongly predictive of a range of processes on a global scale; that generalizations based on Northern Hemisphere experience of the impact of N additions to 'P-limited' ecosystems are likely to fail for southern ecosystems where anatomical and physiological adaptation of native plants to low-P soils makes questionable the concept of 'P-limitation'; that the greatest threats in the 'south' are probably changes in land use that may greatly increase N inputs and turnover; that localized increases in N inputs produce similar effects to those seen in the 'north'.