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4/2/2019 Effect of ON Ratio on the Removal of Nitrogen and Microbial Characteristics in the Water Saturated Denitrifying Section of a Two-Stage C... second stage influent was favorable to enhance the secretion of protein and carbohydrates in total EPS. This would lead to an improved ability of biofilm formation,which would protect the microbial cells and allow more efficient pollutants removal. It has been reported that the protein fraction of EPS could promote biofilm and granular sludge formation,and maintain the stability of microbial aggregates [33]. The carbohydrate fraction serves as the skeleton of these microbial aggregates, and when a mutation was introduced in a gene that abolishes carbohydrate biosynthesis,the bacteria could no longer form mature biofilms [34]. However, the contents of protein and carbohydrate in biofilm LB-EPS both showed a opposite trend to that of biofilm S-EPS, which may be because the carbon source was primarily used to generate carbohydrate and protein, which was then gradually degraded and utilized by the microorganisms when the carbon source became restricted over time. 4. Conclusions This work shows that the removal efficiency of TN by a two-stage CRI could reach 75.4 f 2.8%, provided the C/N ratio in the influent of the second stage was elevated to 5 by the addition of glucose. We have shown that elevating the C/N ratio can promote the removal of nitrate and TN in the second- stage effluent,without impairing the COD removal. The quantity of denitrifying bacteria in the sand of the permanently submerged denitrifying section increased as a result of the higher C/N ratio, and the concentrations of NO3--N and TN in the subsequent effluent both decreased as the quantity of denitrifying bacteria increased. Moreover,analysis of the bacterial biofilms that had formed in the sand of the denitrifying section showed that the elevated C/N ratio had enhanced secretion of the total biofilm EPS,which leads to improved biofilm formation and enhances the pollutant's removal in the two-stage CRI. Author Contributions W.X. conceived and designed the experiments and wrote the paper; Q.F. and Z.P. performed the experiments and contributed reagents and analysis tools; G.X. analyzed the data. Funding This research is funded by Chinese National Natural Science Foundations(41502333), Sichuan science and technology support project(2017JY0141,2018GZ0416), China Postdoctoral Science Foundation (2017M610598,2018T110963), and the State Key Laboratory of Geohazard Prevention and Geoenvironment Protection Foundation (SKLGP2016Z019). We received above funds for covering the costs to publish in open access. Conflicts of Interest The authors declare no conflict of interest. References 1. He J.T.,Zhong Z.S.,Tang M.G., Chen H.H. Experimental research of constructed rapid infiltration wastewater treating system. China Environ. Sci. 2002;22:239-243. [Google Scholar] 2. He J.T.,Zhong Z.S.,Tang M.G.New method of solving contradiction of rapid infiltration system land using. Geoscience. 2001;15:339-345. [Google Scholar] 3. Xu W.L.,Zhang W.,Jian Y. Analysis of nitrogen removal performance of constructed rapid infiltration system(CRIS) Appl. Ecol. Environ. Res. 2017;15:199-206. doi: 10.15666/aeer/1501_199206. [CrossRefJ [Google Scholar] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069465/ 15/18