Hydrilla verticillata–Sulfur-Based Heterotrophic and Autotrophic Denitrification Process for Nitrate-Rich Agricultural Runoff Treatment

Hydrilla verticillata–sulfur-based heterotrophic and autotrophic denitrification (HSHAD) process was developed in free water surface constructed wetland mesocosms for the treatment of nitrate-rich agricultural runoff with low chemical oxygen demand/total nitrogen (C/N) ratio, whose feasibility and mechanism were extensively studied and compared with those of H. verticillata heterotrophic denitrification (HHD) mesocosms through a 273-day operation. The results showed that the heterotrophic and autotrophic denitrification can be combined successfully in HSHAD mesocosms, and achieve satisfactory nitrate removal performance. The average NO(3)(−)-N removal efficiency and denitrification rate of HSHAD were 94.4% and 1.3 g NO(3)(−)-N m(−3)·d(−1) in steady phase II (7–118 d). Most nitrate was reduced by heterotrophic denitrification with sufficient organic carbon in phase I (0–6 d) and II, i.e., the C/N ratio exceeded 4.0, and no significant difference of nitrate removal capacity was observed between HSHAD and HHD mesocosms. During phase III (119–273 d), sulfur autotrophic denitrification gradually dominated the HSHAD process with the C/N ratio less than 4.0, and HSHAD mesocosms obtained higher NO(3)(−)-N removal efficiency and denitrification rate (79.1% and 1.1 g NO(3)(−)-N m(−3)·d(−1)) than HHD mesocosms (65.3% and 1.0 g NO(3)(−)-N m(−3)·d(−1)). As a whole, HSHAD mesocosms removed 58.8 mg NO(3)(−)-N more than HHD mesocosms. pH fluctuated between 6.9–9.0 without any pH buffer. In general, HSHAD mesocosms were more stable and efficient than HHD mesocosms for NO(3)(−)-N removal from agricultural runoff during long-term operation. The denitrificans containing narG (1.67 × 10(8) ± 1.28 × 10(7) copies g(−1) mixture-soil(−1)), nirS (8.25 × 10(7) ± 8.95 × 10(6) copies g(−1) mixture-soil(−1)), and nosZ (1.56 × 10(6) ± 1.60 × 10(5) copies g(−1) mixture-soil(−1)) of litter bags and bottoms in HSHAD were higher than those in HHD, which indicated that the combined heterotrophic and autotrophic denitrification can increase the abundance of denitrificans containing narG, nirS, and nosZ, thus leading to better denitrification performance.