Incorporation of mineral nitrogen into the soil food web as affected by plant community composition

We set up a field experiment in experimental grasslands (Jena Experiment) with different levels of plant species richness (2, 4, 8, and 16 plant species), and plant functional group richness (1, 2, 3, and 4 plant functional groups; legumes, grasses, small herbs, tall herbs). The experimental subplots were labeled with 15N at the beginning of the growing season in 2011 (18- 19th April). The 15N tracer solution (0.01 mol 15NH415NO3/L deionized water; 98 atom %; Cambridge Isotope Laboratories, Tewksbury, MA, USA) was injected into pre-drilled holes of a depth of 7 cm in the soil arranged along gridlines (distance within grid lines 8.7 cm, distance between grid lines 10 cm, resulting in 49 holes per subplot). The tracer solution was injected using a 3 mm thick four-side port needle (2 mL per injection point) connected with a silicon tube to a bottle top dispenser (Socorex Isba SA, Switzerland) on a 1 L glass bottle. We measured incorporation of mineral-derived 15N into soil microorganisms and mesofauna over three months. For measuring the time-integrated incorporation of 15N into soil microorganisms, five sampling campaigns were carried out: 2, 15, 30, 60, and 120 days after labeling, respectively. At each sampling campaign, three soil cores were taken per subplot for microbial biomass (Ø 5 cm, 0-5 cm depth). Microbial biomass N was extracted from soil by chloroform fumigation-extraction (CFE). Two subsamples (10 g soil fresh weight each) were taken from each pre-extracted soil sample. One subsample was fumigated with chloroform vapor for 24 h, the other remained unfumigated. Both subsamples were extracted with 60 ml 0.05 M K2SO4 , the extracts were filtered and frozen at -18°C until further analysis. Before analyzing stable isotope ratios of the subsamples, a fraction of the samples (15 mL) was freeze-dried (VaCo2, Zirbus Technology, Bad Grund, Germany) at -30°C for 3 d and stored in plastic vessels in a desiccator. For referring results of 15N measurements to one gram dry soil, gravimetric soil water content was measured by drying 10 g of fresh soil subsamples of each sample at 105°C for 48 h. For analyses of 15N/14N ratios in microbial biomass N, appropriate amounts of the freeze-dried microbial N extract (60-65 μg) were transferred into tin capsules. Stable isotope ratios were measured with a coupled system of an elemental analyzer (NA 1500, Carlo Erba, Milan, Italy) and a mass spectrometer (MAT 251, Finnigan, Bremen, Germany). Microbial biomass N was calculated as Nmic = EN/kEN, with EN being the difference between total N extracted from fumigated soil and total N extracted from unfumigated soil, and kEN the extractable fraction of microbial biomass N after fumigation. Soil microbial biomass 15N (μg 15N/ g dry soil) was calculated as 15Nmic (μg/ g dry soil) = 15N (μg / g dry soil) of fumigated subsample – 15N (μg /g dry soil) of unfumigated subsample. Atom percent excess (APE, isotopic enrichment) of 15N in microbial biomass N was calculated as the difference in atom% between labelled and natural abundance level of 15N in soil microbial biomass. For measuring the time-integrated incorporation of 15N into soil microorganisms and mesofauna, five sampling campaigns were carried out: 5, 15, 30, 60, and 120 days after labeling, respectively. At each sampling campaign, one soil core per subplot was taken for mesofauna (Ø 20 cm, 0-10 cm depth). The following mesofauna species were used for stable isotope analyses: Tectocepheus velatus sarekensis (Oribatida, primary decomposer), Lepidocyrtus cyaneus, Isotoma viridis, Parisotoma notabilis, Ceratophysella sp. and Stenaphorura denisi (all Collembola, secondary decomposers), as well as Lasioseius berlesei (Gamasina, predator). Tectocepheus velatus sarekensis (Oribatida, primary decomposer), Lepidocyrtus cyaneus, Isotoma viridis, Parisotoma notabilis, Ceratophysella sp. and Stenaphorura denisi (all Collembola, secondary decomposers), as well as Lasioseius berlesei (Gamasina, predator). For analyses of 15N/14N ratios in soil mesofauna, appropriate numbers of animals (10-120 individuals weighing 10-200 μg and containing 1-20 μg N) were transferred into tin capsules. In few cases individuals from the same sampling campaign but different plots with similar plant community composition were pooled. Stable isotope ratios were measured with a coupled system of a micro- elemental analyzer system (Euro-EA 300, Eurovector, Milano, Italy) allowing the analysis of small amounts of animal tissue, and a mass spectrometer (MAT 251, Finnigan, Bremen, Germany). Isotope signatures are expressed using the δ notation with δ15N (‰) = (Rsample/Rstandard -1) x 1000, where R is the molar ratio of heavy to the light isotope (15N/14N). Acetanilide (C8H9NO, Merck, Darmstadt, Germany) was used for internal calibration. As standard for δ15N, atmospheric nitrogen was used. Shifts in 15N/14N ratios in mesofauna species due to labeling with 15NH415NO3 were inspected by calculating the difference between δ15N values of specimens inside and outside the subplots, i.e. Δ values.