For NO3 -, NO2 -, and NH4 + total analysis, 1.5 mL of the liquid media was immediately frozen at −20°C. For N2O analysis, 1 mL of the liquid media was immediately transferred into an N2-purged 3-mL exetainer and fixed with 100 μL ZnCl2 (50%). For 15NH4 + analysis, 0.5 mL of the liquid media was transferred into a 3-mL exetainer and frozen at −20°C. The liquid media remaining in the incubation exetainers were fixed
with 100 μL ZnCl2 (50%) for later 15N-N2O and 15N-N2 analysis. For technical reasons, 15N-N2O could not be quantified for this specific experiment, but only for a slightly modified twin experiment the results of which are presented in the Supporting Information. Additional exetainers with fungal aggregates were prepared and treated in the same way as the other exetainers for verifying that An-4 remained axenic throughout the anaerobic incubation. At the end of the experiment, these exetainers were opened using check details aseptic techniques and Selleckchem SN-38 Subsamples of both fungal aggregates (at least two) and liquid medium (100 μL) were plated
on YMG agar. After incubation at 26°C for 15 days, the fungal colonies were carefully checked by microscopy for the presence of bacteria and xenic fungi. All microscopic checks were negative. Additionally, Selleck Akt inhibitor DNA was extracted from fungal aggregates and liquid medium with the UltraClean™ Soil DNA Isolation Kit (Mo Bio, Carlsbad, CA) and used as template for PCR targeting the 16S rRNA gene with the universal bacterial primers GM3F/GM4R . All molecular checks were negative, since agarose gel electrophoresis did not reveal any specific amplification product except for in the positive control, a laboratory strain of Agrobacterium sp. Intracellular nitrate storage The capability of An-4 to store nitrate intracellularly Etomidate was investigated during both aerobic and anaerobic cultivation (Experiment 3). Liquid cultures were prepared as described above, but with the YMG broth adjusted to 50 μmol L-1 NO3 -. After defined time intervals, YMG broth and fungal aggregates were subsampled for analysis of NO3 – freely dissolved in the broth (i.e., extracellular
nitrate = ECNO3) and NO3 – contained within the fungal hyphae (i.e., intracellular nitrate = ICNO3). Subsamples for ECNO3 analysis (1.5 mL) were cleared from suspended hyphae by mild centrifugation at 1000× g for 10 min and the supernatants (S0) were stored at −20°C for later analysis. Fungal aggregates for ICNO3 analysis were collected in a 2-mL centrifugation tube and the adhering YMG broth was siphoned off using a hypodermic needle. The aggregates were washed with 1 mL nitrate-free NaCl solution (2%) and blotted dry on nitrate-free filter paper. The aggregates were then equally distributed among two 15-mL centrifugation tubes, one for ICNO3 analysis and one for protein analysis. Aggregates intended for ICNO3 analysis were weighed and thoroughly mixed with 2.5 mL nitrate-free NaCl solution (2%) and centrifuged at 1000× g for 5 min.