Orion ea 940

Carbon oxidation and ammonium stabilization were assessed in batch cultures by cultivating strain R31 in a basal medium (all units g/L): K₂HPO₄ 14; KH₂PO₄ 6; MgSO₄·7H₂O 0.2; trace mineral solution 2 mL; pH (at 25°C) = 7 ± 0.2. The trace mineral solution consisted of (all units g/L), EDTA 0.01; ZnSO₄·7H₂O 0.0001; CaCl₂·2H₂O 0.1; MnCl₂·2H₂O 0.008; FeCl₃·6H₂O 0.71; (NH₄)₆Mo₇O₂₄ 0.00011; CuSO₄·5H₂O 0.0001; CoCl₂·6H₂O 0.2. The carbon (glucose) concentration was 1.251 g/L (carbon substrate 500 mg/L) while nitrogen (ammonium chloride) was 0.191 g/L (nitrogen substrate 50 mg/L) forming C/N ratio (w/w) of 10. The sterilized media were inoculated with 1% (v/v) of the isolate and incubated at 37°C with shaking (100 rpm) for 48 h. Aliquots were removed from each Erlenmeyer flask at four hour intervals and growth was recorded by measuring OD₆₀₀. Next, the liquid was centrifuged at 10,000 rpm for 10 minutes and the supernatant was analyzed for COD (by closed reflux method using dichromate), ammonia, nitrite, and nitrate nitrogen by using an expandable ion electrode analyzer (Orion EA 940, Thermo Fisher Scientific, USA). Medium pH was measured using Cyberscan 510 pH meter (Eutech Instruments, Singapore). Hydroxylamine was measured spectrophotometrically according to Frear and Burrell [28 ]. Experiments were done thrice in triplicate sets.

Fluoride concentrations were measured using an ion-specific fluoride electrode (Orion 9609 BNWP, Thermo Fisher Scientific Inc., Waltham, MA, USA) coupled to an ion analyzer (Orion EA-940 Thermo Fisher Scientific Inc., Waltham, MA, USA). Before each reading, samples were shaken with a vibrator (Classic Vortex Mixer, Velp Scientifica, Italy) to homogenize the sample. The electrode was calibrated beforehand with standard solutions from 0.125 to 2.0 ppm F, mixing 1 mL of each standard solution with 1 mL of TISAB II (1.0 M pH acetate buffer 5.0; 1.0 M NaCl and 4% CDTA). Once calibrated, the samples were read, for which we mixed 1 mL of each saliva sample with 1 mL of TISAB II (Hanna Instruments, Woonsocket, RI, USA). The results in mV were converted into fluoride concentrations (ppm) using the standard calibration curves measured immediately before the analysis.