The transfers from plate to flask were repeated every 3–4 weeks. Selleckchem Nec-1s anaerobic nitrate turnover The capability of An-4 to reduce nitrate anaerobically was investigated in two experiments: (1) An-4 was cultivated in Erlenmeyer flasks under oxic vs. anoxic

conditions in the presence of both NO3 – and NH4 +, and (2) An-4 was pre-cultivated in Erlenmeyer flasks under oxic conditions in the presence of 15NO3 – and then exposed to anoxic conditions in gas-tight incubation vials. In Experiment 1, the fate of NO3 – and NH4 + added to the liquid media was followed during aerobic and anaerobic cultivation of An-4. Six replicate selleck liquid cultures were prepared P005091 ic50 as described above, but with the YMG broth adjusted to nominal concentrations of 50 μmol L-1 NO3 – and 50 μmol L-1 NH4 + using aseptic NaNO3 and NH4Cl stock solutions, respectively. Three cultures

were incubated aerobically, whereas the other three cultures were incubated anaerobically by flushing the Erlenmeyer flasks with dinitrogen for 30 min and then closing them with butyl rubber stoppers. Subsamples of the liquid media (1.5 mL) were taken after defined time intervals using aseptic techniques. Anaerobic cultures were sampled in an argon-flushed glove box to avoid intrusion of O2 into the Erlenmeyer flasks. Samples were immediately frozen at −20°C for later analysis of NO3 – and NH4 + concentrations. In Experiment 2, the precursors, intermediates, and end products of dissimilatory nitrate reduction by An-4 were investigated in a 15N-labeling experiment, involving an oxic-anoxic shift imposed on axenic mycelia. For the aerobic pre-cultivation,

a liquid culture was prepared as described above, but with the YMG broth Amylase adjusted to 120 μmol L-1 15NO3 – (98 atom% 15N; Sigma-Aldrich). For anaerobic incubation, fungal aggregates were transferred to gas-tight glass vials (5.9-mL exetainers; Labco, Wycombe, UK) filled with anoxic NaCl solution (2%) amended with nitrate as electron acceptor and glucose as electron donor. Using aseptic techniques, equally-sized subsamples of fungal aggregates were transferred from the aerobic pre-cultures into 30 replicate exetainers. The wet weight of the aggregates was determined. Then the exetainers were filled with anoxic NaCl solution adjusted to 120 μmol L-1 15NO3 – and 25 μmol L-1 glucose. Care was taken not to entrap any gas bubbles when the exetainers were closed with the septum cap. The exetainers were fixed in a rack that was continuously rotated to keep the aggregates in suspension and were incubated at 26°C in the dark for 24 days. The anaerobic incubation was terminated in batches of three exetainers after defined time intervals.