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Experiments for studying the effects of climatic change on ecosystems often involve manipulation of one or several quantitative treatment factors of interest. Response surface regression is the method of choice for these types of experiment. Here, we describe the development of a design of a free air CO2 enrichment experiment with two quantitative treatment factors, that is, elevated temperature and CO2 enrichment. The design strategy takes account of budget constraints imposing limitations on the number of plots with elevated temperature and CO2 levels. The approach is based on polynomial regression models and is focussed on an efficient estimation of interaction between the two treatment factors. Extension to more than two factors is straightforward. An analysis of soil moisture data demonstrates the overall suitability of the proposed design to analyse non‐linear interactions of two (or more) global change factors.

Depolymerization of high‐molecular weight organic nitrogen (N) represents the major bottleneck of soil N cycling and yet is poorly understood compared to the subsequent inorganic N processes. Given the importance of organic N cycling and the rise of global change, we investigated the responses of soil protein depolymerization and microbial amino acid consumption to increased temperature, elevated atmospheric CO2, and drought. The study was conducted in a global change facility in a managed montane grassland in Austria, where elevated CO2 (eCO2) and elevated temperature (eT) were stimulated for 4 years, and were combined with a drought event. Gross protein depolymerization and microbial amino acid consumption rates (alongside with gross organic N mineralization and nitrification) were measured using 15N isotope pool dilution techniques. Whereas eCO2 showed no individual effect, eT had distinct effects which were modulated by season, with a negative effect of eT on soil organic N process rates in spring, neutral effects in summer, and positive effects in fall. We attribute this to a combination of changes in substrate availability and seasonal temperature changes. Drought led to a doubling of organic N process rates, which returned to rates found under ambient conditions within 3 months after rewetting. Notably, we observed a shift in the control of soil protein depolymerization, from plant substrate controls under continuous environmental change drivers (eT and eCO2) to controls via microbial turnover and soil organic N availability under the pulse disturbance (drought). To the best of our knowledge, this is the first study which analyzed the individual versus combined effects of multiple global change factors and of seasonality on soil organic N processes and thereby strongly contributes to our understanding of terrestrial N cycling in a future world.

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Abstract. Effects of climate change on the ecosystem productivity and water fluxes have been studied in various types of experiments. However, it is still largely unknown whether and how the experimental approach itself affects the results of such studies. We employed two contrasting experimental approaches, using high-precision weighable monolithic lysimeters, over a period of 4 years to identify and compare the responses of water fluxes and aboveground biomass to climate change in permanent grassland. The first, manipulative, approach is based on controlled increases of atmospheric CO2 concentration and surface temperature. The second, observational, approach uses data from a space-for-time substitution along a gradient of climatic conditions. The Budyko framework was used to identify if the soil ecosystem is energy limited or water limited. Elevated temperature reduced the amount of non-rainfall water, particularly during the growing season in both approaches. In energy-limited grassland ecosystems, elevated temperature increased the actual evapotranspiration and decreased aboveground biomass. As a consequence, elevated temperature led to decreasing seepage rates in energy-limited systems. Under water-limited conditions in dry periods, elevated temperature aggravated water stress and, thus, resulted in reduced actual evapotranspiration. The already small seepage rates of the drier soils remained almost unaffected under these conditions compared to soils under wetter conditions. Elevated atmospheric CO2 reduced both actual evapotranspiration and aboveground biomass in the manipulative experiment and, therefore, led to a clear increase and change in seasonality of seepage. As expected, the aboveground biomass productivity and ecosystem efficiency indicators of the water-limited ecosystems were negatively correlated with an increase in aridity, while the trend was unclear for the energy-limited ecosystems. In both experimental approaches, the responses of soil water fluxes and biomass production mainly depend on the ecosystems' status with respect to energy or water limitation. To thoroughly understand the ecosystem response to climate change and be able to identify tipping points, experiments need to embrace sufficiently extreme boundary conditions and explore responses to individual and multiple drivers, such as temperature, CO2 concentration, and precipitation, including non-rainfall water. In this regard, manipulative and observational climate change experiments complement one another and, thus, should be combined in the investigation of climate change effects on grassland.