Nessler s reagent

Ethanol (99%), acetone (≥99%), sulfuric acid (96%), oxygen peroxide (50%), and Nessler’s reagent were obtained from VWR chemicals (Leuven, Belgium). Celite, sodium hydroxide (1N solution), and hydrochloric acid (≥32%) were acquired from Sigma-Aldrich (Steinheim, Germany). MES hydrate (>99%) and ammonium sulfate were provided by Alfa Aesar (Kandel, Germany). TRIS (>99.8%) was obtained from Acros Organics (Geel, Belgium). Petroleum ether (60‒80 °C) was supplied by LAB-SCAN analytical sciences (Gliwice, Poland). Amylase thermostable (3000 U/mL), protease (9 tyrosine equivalent units/mg), and amyloglucosidase (3260 U/mL), suitable for AOAC International total dietary fiber and starch analytical procedures, were obtained from Megazyme (Te Huissen, The Netherlands). Ultrapure water was prepared in a Simplicity UV water purification system (Millipore, Molsheim, France).

Ammonia production was analyzed by colorimetric assays using Nessler’s reagent (VWR Chemicals). The cells were grown in 50ml rMB for 24 or 48h, as described in section Growth Conditions. Cells were removed by centrifugation for 5min at 3200g, 4°C, after which 10ml of supernatant was collected. Subsequently, 1ml of Nessler’s reagent was added to the supernatant and the formation of a dark blue color indicating the presence of ammonia.

The ammonia production activity of bacteria was performed as previously described [39 ]. A bacterial suspension (100 μL (OD_600nm = 0.8)) was inoculated into tubes containing 10 mL of sterile peptone water (peptone 10 g; sodium chloride 5 g and distilled water 1 L and pH was adjusted to 6.75 ± 0.25 and incubated at 30 °C with shaking at 150 rpm for 96 h. Uninoculated medium and QF11 isolate served as negative control (C−) and positive controls, respectively. Afterward, aliquots (1 mL) were taken and centrifuged at 10,000 rpm for 10 min. Then, 0.5 mL of Nessler’s reagent (VWR Chemicals, Rosny-sous-Bois cedex, France) was added to each aliquot. The ammonia production was considered positive following the development of a brownish coloration and absorbance was measured at 450 nm using a spectrophotometer. Ammonia produced was estimated using a standard curve of ammonium sulfate for concentrations in the 0–0.3 µmol/mL range [40 ].