Laboratory analysis
Plant and faecal samples were air-dried on the spot, and later
oven-dried at 40oC for 24 hours. Plant biomass samples
were then milled to a fine powder in liquid nitrogen using a pestle and
mortar. From each burrow, we randomly selected 3 sets of 3 faecal
pellets. We milled each faecal pellet set together in liquid nitrogen to
form 3 mixed samples from each burrow. From each sample (plant or
faeces) we weighted approx. 5 mg into a tin capsule and measured total N
(expressed as % of dry mass) using a Flash 2000 Elemental Analyser. To
measure P content approx. 100 mg of each sample (plant or faeces) was
microwave digested in nitric acid. The P content was measured using the
molybdate method on a continuous flow San++ Skalar analyser. Between 1
and 3 mg of each plant or faeces sample (depending on N content) was
weighed into a tin capsule and measured for nitrogen stable isotope
composition using a Delta V Plus Isotope Ratio Mass Spectrometer,
coupled with a Flash 2000 Elemental Analyser via continuous flow. Stable
isotope ratios were expressed as δ, i.e. as the deviation in per-mille
(‰) from the international standard, atmospheric nitrogen, in line with
the equation: δsample =
(Rsample/Rstandard - 1) x 1000, where R
is the isotopic ratio, 15N/14N.
International standards were measured for calibration and measurement
precision, which was <0.1‰. All chemical laboratory analyses
were done in the Laboratory of Biogeochemistry and Environmental
Protection, Biological and Chemical Research Centre, University of
Warsaw.