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4/2/2019 Effect of CIN Ratio on the Removal of Nitrogen and Microbial Characteristics in the Water Saturated Denitrifying Section of a Two-Stage C... -'EPS in biofilms was extracted as previously described [14,23]. Sand samples(10 g)were mixed with 45 mL extraction buffer(2 mmol/L Na3PO4,4 mmol/L NaH2PO4,9 mmol/L NaCl, 1 mmol/L KCI,pH 7.0), treated by ultrasonication for 5 min and centrifuged(20 min 2000x g). The supernatant was filtered through a milipore filter of 0.45 gm to give the extracted S-EPS fraction. The pellet was resuspended into 45 mL extraction buffer and shaken for 1 h after which centrifugation was performed at 5000x g for 20 min. The filtered supernatant from this second centrifugation step resulted in LB- EPS,while the resuspended pellet was heated to 60°C for 1 h and centrifuged at 10,000x g(20 min). The supernatant of this third centrifugation step was filtered as above to give TB-EPS. The amount of biofilm in the sand samples was estimated as follows. The samples were briefly rinsed in distilled water and then mixed with 1 M NaOH and incubated at 70 °C for 30 min.Following ultrasonic oscillation for 15 min the biofilm containing extracts were filtered(0.45 gm)using pre- weighed membranes. After filtering,the membranes were dried in an oven at 103 °C for 1 h. The amount of biofilm was estimated by the difference in weight of the membrane filters before and after drying(SS). The content of protein and carbohydrates was used to characterize EPS content(mg/g SS)as these are the main components of EPS [14]. The carbohydrate content was measured by means of the phenol- sulfuric method[24] with glucose as the standard. The protein content was measured by Coomassie brilliant blue [18] using bovine serum albumin as the standard. 3. Results and Discussion 3.1. Effect of C/N Ratio on Removal Efficiency of Ammonium and Nitrate In the first stage of the experimental CRI,ammonium is converted to nitrate according to Equation (1): NH4++202=NO3 +H2O+2H+ (1) The concentrations and removal efficiencies of ammonium in the effluent of the second stage over time was determined for T1 (control),T2(glucose added at a ratio of 2:1, C/N), and T3 (C/N increased to 5:1)tThe results are shown in Figure 4a. The relative removal efficiency was calculated as the difference in concentration between the first-stage influent and the second-stage effluent divided by the concentration in the first stage influent. https://www.ncbi.nim.nih.gov/pmc/articles/PMC6069465/ 7/18