2.3 Estimation of organic and inorganic substrates
Residual sugars quantified using high-performance liquid chromatography (HPLC) which includes the Aminex HPX-87H Ion Exclusion column (300 × 7.8 mm) attached with a refractive index (R.I.) detector (Agilent Techologies, USA)[39]. The supernatant was collected every 48 h and was diluted to 100m× with 4 mM sulphuric acid. Sample of 10 uL injected into the column maintained at 40 ºC using 4 mM H2SO4 as the mobile phase with a flow rate of 0.3 mL min-1 and a run time of 50 min [40]. The peak area of standard glycerol (Sigma Aldrich Pvt. Ltd., USA) used as reference. Nitrate uptake by the cells was measured spectrophotometrically[41]. Briefly, 1 mL of culture was pelleted down at room temperature (RT), and supernatant was diluted 50-fold with deionized water. The residual nitrate content was determined by measuring the absorbance at 220 nm. Urea estimation was done as described in Jung et al[42, 43]. Briefly supernatant was used directly to perform the assay, 50 μL of supernatant was transferred into a clear flat-bottom 96-well plate. Then 200 μL of freshly prepared working reagent was added and mixed quickly by gently rocking the plate. The reaction was incubated for 1 h at room temperature. Optical densities (OD) at 430 nm were measured on the plate reader for measuring the urea content in the medium.