The Impact Mechanism Of Typical Plastic Particles On Microbial Nitrogen Removal In Constructed Wetlands

Constructed wetland（CW）is a water treatment system simulating the natural purification process and is the key barrier to prevent contaminants from entering the open water.Micro-and nano-sized plastics existing in a variety of environmental media are constantly transferred to the surface runoff and wastewater system through rainwater scouring and sewage discharge.Eventually,they may enter the wetland system and interfere with the wetland performance.At present,the research on the impact mechanism of micro-and nano-sized plastics on nitrogen removal in CW system is still in the initial stage.Based on this,polystyrene（PS）plastic particles with different particle sizes（50nm,100μm,1000μm）and different concentrations（0,10μg/L,100μg/L,1000μg/L）were used in this study to carry out a 370-day experiment in the CW devices.The impact of micro-and nano-sized plastics on nitrogen removal in CW system were investigated from the aspects of macroscopical substrate characteristic to microscopical microorganism community and metabolism by using fluorescent staining,electrochemistry,isotope labeling and tracer,enzyme spectroscopy,16S amplicon and metagenomics,and so on.Conclusions and finds were listed below:（1）By investigating the nitrogen removal efficiency and the release of N₂O in CW system,it was found that the accumulation of macro-and micro-sized plastics for a long time（>80 days）would have an irreversible negative effect on the nitrification process.As for the denitrification process,the different responses were found in different sized plastic particles,such as no significant difference among all macro-sized groups was found,and the micro-sized plastics showed a promoting effect on the denitrification process,while the accumulation of nano-sized plastics inhibited the denitrification process.Meanwhile,all sized plastics can inhibit the release of N₂O in the process of ammonia oxidation,nitrification-coupled denitrification,and denitrification,but can promote the generation of N₂O in the process of nitrifier denitrification.In general,the accumulation of all sized plastics can slow down the generation and release of N₂O in CW system,and the slowing down degree is proportional to the concentration and inversely proportional to the particle size.（2）By observing the changes in the substrate characteristics inside of CW system,it was found that the extracellular total organic carbon（TOC）content on the surface of macro-sized plastics was sufficient,but the total protein content was low;In contrast,the surface of gravel treated by nano-sized plastics showed that the extracellular TOC and total protein secretions in biofilm were significantly inhibited.In addition,the long-term accumulation of macro-and micro-sized plastics increased the electrical resistivity of CW system,aggravated the wetland clogging,enhanced the oxygen mass transfer coefficient,and accelerated the oxygen consumption rate（OUR）.Acceleration of OUR in CW system affected the nitrification process of CW system.（3）Further analyzing the microflora characteristics of CW system showed that the accumulation of micro-and nano-sized plastics,as a deterministic disturbance factor,accelerated the process of microbial community succession in CW system,and made it developed towards the direction of decreased diversity and abundance and dominant microorganisms.The results of co-occurrence and network interaction analysis of microbial population showed that the accumulation of micro-and nano-sized plastics had a significant impact on the microbial niche of CW system,which made it more"small-world"and modular.Additionally,nitrogen removal microorganisms as the dominant bacteria showed a strong interaction with other microorganisms.The accumulation of micro-and nano-sized plastics also affected the growth and succession of microorganisms in the wetland system.The nano-sized plastics inhibited most of the nitrifying bacteria（e.g.,Nitrospira and Nitrosomonas）,but had no obvious negative effects on most of the denitrifying bacteria（e.g.,Dechloromonas,Thauera,and Zoogloea）and ammonia-oxidizing archaea（e.g.,Nitrososphaeria）.Most of the nitrifying bacteria in the macro-and micro-sized plastics treatment groups showed a decreasing trend in the relative abundance of community genera at the later stage of the experiment（e.g.,Ellin6067 sp.:declined by 64.6%-67.9%）,while a significant increasing trend in the abundance of denitrifying bacteria（e.g.,Dechloromonas,Thauera and Zoogloea increased by 5-15times,4-6 times and 3-7 times,respectively）.（4）By exploring the interaction between micro-and nano-sized plastics and microorganisms and deeply analyzing the response of microbial metabolism process,it is found that different action modes led to different influence degree on microorganisms.Firstly,for nitrifying microorganisms,the abundance of amo A/B/C and hao functional genes and the activity of the corresponding protease（AMO）were increased in the macro-and micro-sized plastic treatment groups,while the activity of HAO protease was significantly decreased in the three plastic treatment groups.For denitrifying microorganisms,under the accumulation of micro-and nano-sized plastics,the abundance of nar G/H/I functional genes decreased significantly,while the abundance of nir K/S,nor B/C and nos Z increased in each size-level group.On the contrary,corresponding to the functional genes,the activity of NAR protease was increased in each size level group,and the activities of NIR,NOR and N₂OR decreased.By analyzing the metabolic response of energy substances（ATP and NADH）,it was found that the long-term accumulation of micro-and nano-sized plastics had a significant effect on the glycolysis process.Under the impact of micro-and nano-sized plastics accumulation,the abundance of hk,pfk,pk and gap functional genes and the activities of the corresponding proteases HK,PFK,PK and GAPDH showed a downward trend.The quantitative analysis of ATP and NADH showed that their contents increased in the macro-and micro-sized groups,but decreased significantly only in the nano-sized plastic group.Through ETSA analysis,it was found that the accumulation of micro-and nano-sized plastics significantly promoted the electrochemical activity of microorganisms,while the accumulation of nano-sized plastics still showed significant inhibition.（5）Statistical methods and structural equation modeling were used to identify the key impact factors of micro-and nano-sized plastics on nitrogen removal in CW system.The results showed that compared with the differences of various factors caused by the“concentration variable”,the"particle size"of micro-and nano-sized plastics had a more significant effect under prolonged exposure time.Meanwhile,with the accumulated intervention of high concentration micro-and nano-sized plastics,the increase of OUR,the change of nitrifying bacteria population and abundance,and the decrease of enzyme activities in nitrogen（AMO,HAO,NAR,NIR,NOR,and N₂OR）/glycolysis（HK,PFK,PK,and GAPDH）metabolisms were the four key factors that caused the decline in nitrogen removal efficiency and the release of N₂O in CW system.Overall,this study took the CW system as the research carrier to explore the impact mechanism of different sized plastic particles on nitrogen removal process.The results can expand people’s cognition to the correlation between emerging pollutants and nitrogen removal process and add a new understanding basis for the research of different sized plastic particles in wetland and freshwater ecosystem.