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To identify the microbial factors that cause the differences in the purification performance of constructed wetlands with different substrate structures, the relationship between the substrate structure and the microbial community composition in horizontal subsurface flow constructed wetlands (HSSFCWs) was studied by high throughput sequencing. The results revealed that the purification performance of a six-layer constructed wetland (CW6), of which the permeability coefficient gradually increased from the surface layer to the bottom layer, was the highest among the three constructed wetland systems. The average concentrations of COD, TN, NO 3 - -N, and NH 4 + -N in the effluent were 39, 11, 0.35, and 4 mg·L -1 , respectively. The monolayer structure constructed wetland (CW1) had the worst purifying efficiency, with average effluent concentrations of 95, 21, 0.60 and 12 mg·L -1 for COD, TN, NO 3 - -N, and NH 4 + -N, respectively. The results of the high-throughput sequencing showed that the number of microbial OTUs in multilayer structure wetlands was slightly lower than that in the monolayer structure wetland, but the relative abundance of the dominant phylum Proteobacteria and the nitrifying and denitrifying bacteria in the genus was significantly higher than the monolayer structure wetland. The results of PCA and heatmap indicated that there were significant differences in the spatial distribution of microbes in the genus of Proteobacteria in CW3 and CW6, which facilitated the degradation of pollutants. No significant differences were found in the community structure of CW1.

The correlation of substrate structure and hydraulic characteristics was studied by numerical simulation combined with experimental method. The numerical simulation results showed that the permeability coefficient of matrix had a great influence on hydraulic efficiency in subsurface flow constructed wetlands. The filler with a high permeability coefficient had a worse flow field distribution in the constructed wetland with single layer structure. The layered substrate structure with the filler permeability coefficient increased from surface to bottom could avoid the short-circuited flow and dead-zones, and thus, increased the hydraulic efficiency. Two parallel pilot-scale constructed wetlands were built according to the numerical simulation results, and tracer experiments were conducted to validate the simulation results. The tracer experiment result showed that hydraulic characteristics in the layered constructed wetland were obviously better than that in the single layer system, and the substrate effective utilization rates were 0.87 and 0.49, respectively. It was appeared that numerical simulation would be favorable for substrate structure optimization in subsurface flow constructed wetlands.