Reax 2

Birch pollen (Betula Pendula) was purchased from Allergon (Allergon AB, Ängelholm, Sweden). Proteins were extracted from birch pollen with Tris-Borate buffer (8mM Tris with 10mM (NH₄)₂B₁₀O₁₆; pH8.5; in 1/10 w/v ratio) for 6h at 4°C at 500 rpm (Reax 2, Heidolph Instruments Schwabach, Germany), followed by centrifugation at 16 000 g, and filtration using a 0.22μm nitro cellulose membrane (Sartorius AG, Goettingen, Germany). The protein concentration was determined by the Bradford assay using bovine serum albumin as standard. The extract was stored at -80°C.

1 mg lyophilized S-layer protein was dissolved in 1 mL 5 M GHCl 50 mM Tris/HCl buffer (pH 7.2). The solutions were dialysed against distilled water at least 24 h at 20 °C. For recrystallization of the S-layer protein on emulsomes, the S-layer protein solution was mixed with the emulsome suspension and diluted with Milli-Q water to achieve final protein and DPPC concentrations of 300 μg mL⁻¹ and 150 μg mL⁻¹, respectively. Recrystallization of the S-layer protein was carried out for 3 h at room temperature in a test tube rotator (REAX2, Heidolph, Germany) with a rotation speed of 32–36 rpm. Excess non-assembled S-layer protein was removed by centrifugation at 14,100 × g for less than 1 min. The pellet containing the S-layer coated emulsomes was resuspended in Milli-Q water and stored at 4 °C until further analysis.

For the determination of the nitrate concentrations in the two soil habitats, one gram of soil was diluted with two milliliters of deionized water, mixed in an overhead shaker (Reax 2, Heidolph Instruments, Schwalbach, Germany) and centrifuged at 16.249 × g (Mikro 20, Hettich GmbH, Tuttlingen, Germany). The supernatant was filtered through a 0.2 µm regenerated cellulose membrane (Rotilabo, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and analyzed by ion chromatography [42 (link)]. For the analyses of the ammonium concentrations in the two soil habitats, two grams of soil were extracted with 8 mL 1 M KCl, and the concentration was determined by a colorimetric assay [43 (link)].
pH values of soil solutions (extraction with 0.01M CaCl₂) were measured with a pH meter (Ino Lab pH 720, WTW 82362 Weilheim, Germany).
Statistical comparisons were done with ANOVA and Tukey HSD Test, using SPSS version 20. The model included the factors “treatment”, “soil habitat”, their interaction, and “block” as a random factor. The main effect of factors “mineral-N” and “Manure” was also tested.

The polystyrene PS168N pellets were grinded in a ZM200 freezer mill (Retsch, Haan, Germany) using a mesh size of 1000 μm at a rotation speed of 18.000 rpm. The powdered PS was mixed with 1.0 wt.% (batch foaming) or 5.0 wt.% (extrusion foaming) of the finely powdered corresponding kinked bisamide. A Reax 2 overhead shaker (Heidolph, Schwabach, Germany) was used for 24 h at 50 rpm to achieve a homogeneous powder–powder mixture of the PS and kinked bisamides.