Effect of Aeration and External Carbon Source on Nitrogen Removal and Distribution Patterns of Related-Microorganisms in Horizontal Subsurface Flow Constructed Wetlands

More than 200 genera were identified in each sample. Among these, the genera related to nitrogen removal (ammonia-oxidizing, nitrite-oxidizing, denitrification, and anaerobic ammonium-oxidizing) were selected (Figure 3). The relative abundance of nitrifiers was 2.76–5.72% and 4.03–5.31% at the fore and rear ends of HSSFCWs, respectively. The highest relative abundance of nitrifiers was observed in F_CW_A and R_CW_CA at the fore and rear ends of HSSFCWs, respectively. The lowest relative abundance of nitrifiers in both the fore and rear ends of HSSFCWs was observed in the CW_C treatment. Four types of ammonia-oxidizing bacteria (Sphingomonas, Arthrobacter, Nitrosospira, and Prosthecobacter) and three types of nitrite-oxidizing bacteria (Nitrospira, Candidatus_Nitrotoga, and Nitrobacter) were identified in all samples (Table S1). Sphingomonas was the predominant ammonia-oxidizing bacterium, and Nitrospira was the predominant nitrite-oxidizing bacterium. Aeration was found to be beneficial in promoting the growth and reproduction of nitrifiers in the CWs [16]. Compared to CW_CK, the CW_A and CW_CA treatments led to a rise in the relative abundance of Sphingomonas and Arthrobacter at the fore ends of HSSFCWs, indicating that aeration could provide suitable conditions for these two bacteria. Simultaneously, the relative abundance of Nitrospira in the CW_A and CW_CA treatments was also boosted at the rear ends of HSSFCWs. The increase in the related nitrifier revealed that the CW_A and CW_CA treatments enhanced the ammonia-oxidizing and nitrite-oxidizing processes at the fore and rear ends of HSSFCWs, respectively.

The relative abundance of denitrifying bacteria accounted for 7.35% to 35.56% and 7.26% to 25.79% at the fore and rear ends of HSSFCWs, respectively. The highest relative abundance of denitrifying bacteria at the fore and rear ends of HSSFCWs was found in F_CW_C and R_CW_CA, respectively. The addition of carbon sources significantly increased the relative abundance of denitrifying bacteria at the fore ends of HSSFCWs, while it had no evident impact on the denitrifying bacteria at the rear end of CW_C. The high relative abundance of nitrifiers and denitrifying bacteria in the substrate sample of CW_CA led to the concurrent conversion of ammonium and nitrate. This could explain the high TN removal rate observed in the CW_CA treatment. These findings were consistent with previous studies. Xia et al. [44] reported that the removal of total inorganic nitrogen primarily occurred in the zone with a high relative abundance of nitrifiers and denitrifying bacteria in the vertical subsurface flow constructed wetlands. Thirty-eight and forty-one genera related to denitrification were detected at the fore and rear ends of the four treatments, respectively. The leading genera responsible for denitrification were Acinetobacter (0.48–30.07%), Gaiella (0.71–1.08%), Pseudomonas (0.47–3.09%), and Rhizobium (0.66–1.02%) (Table S1). Most of the denitrifying bacteria identified in this study were heterotrophic and enriched in the CW_C and CW_CA treatments. For instance, CW_C and CW_CA exhibited the highest relative abundance of Acinetobacter at the fore and rear ends of HSSFCWs, reaching 30.07% and 14.21%, respectively. These heterotrophic denitrifying bacteria played a significant role in the removal of both COD and TN in HSSFCWs [44]. Moreover, autotrophic denitrifying genera, such as Thiobacillus and Lysobacter, were also observed in various treatments, which could complete the denitrification process using sulfide as electron donors [16].

The contribution of nitrifiers and denitrifying bacteria to nitrogen removal was indicated by the abundance of functional genes related to nitrogen [45,46]. The absolute abundance of amoA, nxrA, nirS, nirK and nosZ at both the fore and rear ends of HSSFCWs is depicted in Figure 4. The functional genes amoA and nxrA are associated with the nitrification process. The process of aeration (CW_A and CW_CA) significantly increased the absolute abundance of amoA (p amoA in CW_A and CW_CA amounted to 4.56 × 10⁴ and 3.72 × 10⁴ copies·g⁻¹ substrate, respectively. The absolute abundance of amoA in all treatments decreased in the rear ends of HSSFCWs, but the abundance of amoA in the CW_A and CW_CA treatments remained higher than that in other treatments at the rear ends of HSSFCWs. The absolute abundance of amoA in CW_A was higher than that in CW_CA at the fore ends of HSSFCWs, while the opposite results appeared at the rear ends of HSSFCWs. These results indicated that the addition of carbon sources partially inhibited nitrification even under the aeration condition. nxrA is another important gene related to nitrification. Aeration significantly increased the abundance of nxrA at the fore ends of HSSFCWs. The absolute abundance of amoA in CW_A and CW_CA treatments was higher than that in other treatments. The highest absolute abundance of nxrA in the fore and rear ends of HSSFCWs was observed in the CW_A and CW_CA treatments, reaching 8.22 × 10³ and 6.07 × 10³ copies·g⁻¹ substrate, respectively. The aeration process provided sufficient DO to facilitate the proliferation, breeding, and biochemical activities of aerobic ammonia-oxidizing bacteria, thereby enhancing their activity [36]. The copy number of the amoA gene was 2.60 to 6.12 times higher than that of nxrA, aligning with the findings of Pelissari et al. [47]. Ammonia-oxidizing bacteria oxidize NH₄⁺-N to NO₂^–-N, thereby providing substrates for nitrite-oxidizing bacteria to oxidize NO₂^–-N to NO₃⁻-N [48]. At the rear ends of HSSFCWs, a steady decrease was observed in the disparity between the absolute abundance of amoA and nxrA, indicating that the conversion rate of NH₄⁺-N to NO₂⁻-N by ammonia-oxidizing bacteria was reduced in the rear ends of HSSFCWs.

Denitrification is a stepwise process facilitated by reductase. The absolute abundances of nirS, nirK, and nosZ serve as strong indicators of denitrification activity [49]. nirS and nirK were the key genes related to the transformation of NO₂⁻ to NO, while the nosZ gene played a crucial role in the conversion from N₂O to N₂. The addition of carbon sources led to a rise in the abundance of these genes. The highest abundance of nirS and nirK at the fore end of HSSFCWs was observed in the CW_C treatment, reaching 2.86 × 10⁶ copies·g⁻¹ substrate and 2.74 × 10⁶ copies·g⁻¹ substrate, respectively. Comparable findings were also observed in the research by Hou et al. [50]; there was a notable increase in the absolute abundance of nirS and nirK when the COD/TN ratio ranged from 1 to 4 in the constructed wetlands. In addition to CW_C, CW_CA treatment also enhanced the gene abundance of nirS and nirK. Previous research also found that the absolute abundance of nirS and nirK significantly increased in the vertical subsurface flow constructed wetlands with limited aeration (1.00 L·min⁻¹) when the COD/TN reached 9.4 [35]. The impact of an external carbon source on the abundance of nirS and nirK in aerated environments was complex, and it varied depending on the state of CWs. Compared to the absolute abundance of nirS and nirK at the fore ends of HSSFCWs, the absolute abundance of nirS and nirK in the CW_C treatment decreased at the rear ends of HSSFCWs, whereas it increased in the CW_CA treatment at the rear ends of HSSFCWs. These results were consistent with the variation in the abundance of denitrifying bacteria (Figure 3). At the rear ends of HSSFCWs, a higher absolute abundance of nirS and nirK was observed in CW_CA treatment compared to CW_C, suggesting that the CW_CA treatment was more conducive to the process of microbial denitrification at the rear ends of HSSFCWs. The high absolute abundance of the nosZ gene served as an indicator for complete heterotrophic denitrification [51]. The abundance of nosZ ranged from 4.03 × 10⁴ to 1.70 × 10⁵ copies·g⁻¹ substrate. The highest absolute abundance of nosZ at both the fore and rear ends of HSSFCWs was observed in the CW_C treatment, suggesting that CW_C created favorable conditions for complete denitrification in HSSFCWs. Above all, treatments with carbon and aeration provided suitable conditions for the survival of microbes. CW_C and CW_A enhanced the removal rate of TN by amplifying bacteria and genes involved in nitrogen transformation at the fore ends of HSSFCWs. Nonetheless, CW_CA improved the removal rate of TN by increasing bacteria and genes related to nitrogen transformation at both the fore and rear ends of HSSFCWs.