Study On Constructed Wetlands Augmentation Technology Based On Root Exudates

Constructed wetlands,as a common aquaculture wastewater treatment method,have some problems,such as too long start-up time,reduced effect after long-term use and occupying a lagre area.Increasing COD/N in influent can increase the treatment efficiency.However,long-term and massive replenishment of carbon source causes the cost to rise significantly.Consequently,there is concern about whether there are alternatives with higher cost performance.Previous studies have shown that root exudates released by plants play a small and efficient role in promoting nitrogen utilization,especially denitrification.However,there are few studies on root exudates of plants in constructed wetlands treating aquaculture wastewater,and few cases have been reported on how to efficiently use root exudates to enhance constructed wetlands treating performance.This study systematically analyzed the types and contents of root exudates in constructed wetlands treating aquaculture wastewater,and further selected citric acid in root exudates as a supplement to enhance the performance of constructed wetland,and studied its addition method.The details are as follows:1.Composition of root exudates of different plants in constructed wetlands and their effects on the treatment of aquaculture wastewaterThe root exudates of Iris tectorum Maxim and Cyperus involucratus Rottboll in constructed wetlands treating aquaculture wastewater were comprehensively detected by liquid chromatography-tandem mass spectrometry,and the correlation between root exudates and treatment effect was analyzed.The results showed that planting significantly increased the removal rate of pollutants in constructed wetlands.A total of 84 compounds with CAS Registry Number were detected in the samples,among which 82 were from Iris and 24 were from cyperus.The relative concentration of citric acid detected in iris group was 62.3±9.65μg/m L,which was the highest relative concentration of root exudates detected,and it was significantly positively correlated with the removal rate of NO₃^--N and TN.The removal effect of NO₂^--N in planting plant group was more stable and significantly higher than that in NP（no plant）group.The NO₃^--N removal rate of planting plant group was significantly higher than that of NP group,and that of YW group was significantly higher than that of FCC group.L-phenylalanine and citric acid were positively correlated with NO₃^--N removal rate.The removal rates of NH₄⁺-N and TN in the planting group were significantly higher than those in the NP group,and the removal rates of NH₄⁺-N in the FCC group were higher than those in the YW group.The removal rates of NH₄⁺-N were positively correlated with L-valine and choline.In conclusion,planting plants can significantly improve the treatment effect of constructed wetlands on N and P.The types and amounts of root exudates released by different plants are different and have some correlation with the removal of pollutants.Both the type and quantity of root exudates of iris had certain advantages,and the removal rate of TN and TP of iris was higher than that of Cyperus,indicating that root exudates had a positive effect on pollutant removal.2.Effect and mechanism of the addition of small molecular organic acids on the treating effect of the constructed wetland.Three small molecular organic acids were used to replace 20%of COD in the influent（A:tartaric acid,B:succinic acid,C:citric acid）to simulate adding carbon source to the low COD/N wastewater.In Phase A,the average removal rates of NO₃^--N,NO₂^--N,TN and COD increased by 1.04%,2.44%,1.18%and 2.86%respectively for the iris group,and by 6.53%,4.70%,5.24%and 3.00%respectively for the NO₃^--N,NO₂^--N,TN and COD of the cyperus group.In Phase B,the average removal rates of NO₃^--N,NO₂^--N,TN and COD increased by 3.01%,4.15%,2.82%and 3.57%respectively for the iris group,and by 8.69%,3.60%,6.67%and 3.58%respectively for the cyperus group.In stage C,the average removal rates of NO₃^--N,NO₂^--N,TN and COD of iris were increased by 11.56%,1.95%,8.82%and 9.29%,respectively,and by 15.53%,3.18%,11.97 and7.43%respectively for the cyperus group.The three carbon sources had little effect on the average removal rate of NH₄⁺-N and TP.The effect of citric acid on the activity of denitrifying enzymes and denitrifying genes was further analyzed.Replacing 20%of the carbon source with citric acid significantly increased the activity of Nar and Nir in the upper layers of the cyperus and iris,but had little effect on the denitrifying enzyme activity in the lower layers.The effect of citric acid on Nar activity in the upper layer of cyperus（11.32%）was higher than that of Iris（3.9%）,and the effect of citric acid on Nir activity in the upper layer of iris（20.3%）was higher than that of cyperus（14.5%）.For the nir S gene,substituting 20%of the carbon source with citric acid did not significantly affect the number of copies of the upper layer gene in each group.For nir K gene,replacement of 20%carbon source with citric acid significantly increased the number of upper-layer rhizosphere copies in the Cyperus group（55%）and Iris group（18.8%）.In this study,the community composition of nitrite-reducing bacteria was determined based on the nir S and nir K genes,as their role is a key rate-limiting step in denitrification.In summary,different small molecular organic acids promote the removal of NO₃^--N,NO₂^--N,TN,and COD,but do not significantly affect the removal of NH₄⁺-N and TP.Citric acid worked best.The mechanism is that citric acid promotes the activity of the denitrification enzymes Nar and Nir,increasing the number of nir K gene copies and enriching Acidithiobacillale,Burkholderiales,Rhizobiaceae and other microorganisms.It facilitates the removal of TN and COD in the constructed wetland system.3.Effect of the addition of citric acid to the plant rhizosphere on the treatment effect of constructed wetlands treating aquaculture wastewater.By adding carbon sources to the rhizosphere of constructed wetlands using a device that mimics the release of carbon from plant roots,the treatment effect of this method was compared to that of a normal way.It was shown that the direct addition of citric acid to the rhizosphere facilitated the removal of various forms of N and COD in the wetlands.The direct addition of carbon sources to rhizosphere significantly increased Nar（22.56%in the cyperus group and 21.38%in the iris group）and Nir（46.42%in the cyperus group and 14.36%in the iris group）activities in the upper layer.For the lower bulk regions,rhizospheric addition of citric acid did not have a significant effect on Nar,but significantly increased Nir activity,by 22.29%in the iris group and 27.97%in the cyperus group.High-throughput sequencing and bioinformatics analyses were performed on different taxa of rhizosphere microorganisms.The results showed that shannon index in rhizosphere treatment increased significantly,from 5.38±0.15 to 5.78±0.22 in iris group and from 5.31±0.15 to 5.86±0.04 in cyperus group.The sobs index of iris rhizosphere addition group increased significantly from 1861±93.7 to 2052±31.6 compared with the normal addition group.The simpson index of the cyperus rhizosphere treatment group was significantly decreased from 0.03±0.01 to 0.01±0.00 compared with the normal addition group.The microorganisms of each group share certain similarities at the level of phylum,class and genus.The Partial least squares Discriminant Analysis showed that the structure of the microbial community in the iris is more similar to that of the Cyperus group under the normal addition circumstances.These results suggest that the direct addition of citric acid to the rhizosphere drives the changes in the microbial community.The Lef Se analysis was further used to find different species in different groups.The normal carbon source addition group of iris enriched p＿Firmicutes、o＿Myxococcales、c＿Myxococcia、f＿Myxococcaceae、f＿B1-7BS and that of Cyperus enriched o＿Chloroplast、f＿JG30-KF-CM45.Another treatment of iris enriched g＿Aeromonas、o＿Chloroflexales and that of Cyperus enriched f＿Rhodobacteraceae、o＿Saccharimonadales、g＿Gemmobacter、o＿Pe M15.In addition,KEGG and FAPROTAX function prediction were carried out.Adding carbon sources to the rhizosphere promoted the increase of various substances metabolism functions of rhizosphere microorganisms.Among the functions related to the nitrogen cycle,the addition of citric acid to the rhizosphere of both plants promoted an increase in nitrogen respiration,nitrate respiration,nitrite oxidation,nitrous oxide denitrification,nitrite denitrification,nitrate denitrification and other functions.Conclusions:Adding citric acid to the plant rhizosphere in constructed wetlands promotes denitrification enzyme activity in the rhizosphere and improves the treatment effect on aquaculture wastewater.The addition of carbon sources to the rhizosphere alters the microbial community structure primarily by increasing the abundance of microbes that metabolize macromolecules or complex carbon sources,such as Sporacetigenium,Clostridium＿sensu＿stricto＿13,norank＿f＿A4b.In addition,it promotes the abundance of related denitrification bacteria,such as Bacillus,Aeromonas,Saccharimonadales,which together improve denitrification ability in two ways,and subsequently the removal rates of NO₃^--N,TN,and COD.