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4/2/2019 Effect of C/N Ratio on the Removal of Nitrogen and Microbial Characteristics in the Water Saturated Denitrifying Section of a Two-Stage C... Table 1 Water quality parameters of influent of the first stage. Water Quality Parameters Mean Concentration(mg/L) Chemical Oxygen Demand(COD) 268.3 t 20.0 NH4 _N 50.4 f 3.5 NO3 -N 2.6 t 0.3 NO2 -N 0.039 t 0.06 Total nitrogen(TN) 60.8 f 2.0 pH 8.2 2.3. Batch Experiments To study the effect of different C/N ratios of the second stage influent,three experimental setups were constructed as described in Section 2.2 and these were started under the same conditions,to give Test 1,Test 2 and Test 3 (T1,T2, T3). After operation for 30 days,the removal percentages of COD and NH4 _N from effluent were all stabilized,reaching levels up to 90%, indicating that biofilms had formed successfully in the filling medium [8]. From this time point onwards, Tl served as control treatment not receiving additives; the C/N ratio in the second stage influent of T2 was increased by adding a carbon source at twice the amount of COD per nitrate-nitrogen(C/N 2:1) and that of T3 was increased to a C/N ratio of 5:1. Glucose was used for this,as it is cheap,easy to obtain,non-toxic, and easily biodegraded [20]. Influent and effluent of all three experiments was sampled every two days and used for analysis.At the end of the experiment, after the removal efficiency of TN in the second stage effluent of all experimental tests had stabilized, the filling medium(sand)was sampled from the second stage column for analysis. 2.4. Analytical Methods 2.4.1. Water Quality Analytical Methods The concentration of COD in the water was determined using the potassium dichromate method,the TN concentration was measured by UV spectrometry and the concentration of NH4+-N was determined by the Nessler's reagent colorimetric method. Lastly,NO3--N was measured by UV spectrometry, using standard procedures [21]. 2.4.2. Microbiological Analysis and EPS Quantization Bacterial counts of nitrifying and denitrifying bacteria were determined by the most probable number method(MPN) [22].For this, 10 g of sand was collected from the position 10 cm above the supporting layer of the water-saturated denitrifying column and this was added to 100 mL sterile water in conical flasks that were shaken for 30 min. The suspension was then serially diluted by 10-fold steps and this dilution series was inoculated into culture medium and cultured for 14 days at 28 °C. Finally, the culture medium was titrated by a chromogenic agent, and the number of bacteria was obtained by comparing the number of tubes in the chromogenic medium with the MPN value table. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069465/ 6/18