Effect And Mechanism Of Polystyrene Nanoplastics Exposure On Anammox Granular Sludge

With the increasing use of plastics,a large number of microplastics enter the wastewater treatment plant with wastewater,and were broken into nanoplastics with due to the action of mechanical stirring,air and water shear force.Nanoplastics are easy to be adsorbed by sludge and have strong biological toxicity.Anammox is an economical and efficient biological nitrogen removal process,which has been widely used in the supernatant from sludge digestion.Anammox granular sludge had high biomass and compact spatial structure,which can process higher nitrogen loading and reduce the diffusion of toxic substances.Therefore,Anammox granular sludge was increasingly used in anammox process.Anammox granular sludge process has low biological yield and long sludge retention time.The long-term exposure of granular sludge with nanoplastics is likely to be negatively affected.At present,little is known about the exposure mechanism of nanoplastic and its effects on the nitrogen removal performance,structural stability and microbial community structure of Anammox granular sludge.This study took anammox granular sludge as the research object and polystyrene nanoparticles(PS-NPs)as the model nanoplastic,studied the effects of PS-NPs short-term exposure on the nitrogen removal performance and structural stability of Anammox granular sludge,compared the differences of short-term exposure tolerance of PS-NPs between Anammox granular sludge and activated sludge;analyzed the effects of PS-NPs long-term exposure on the performance and community structure of Anammox granular sludge,revealed the exposure mechanism of PS-NPs by proteomics;and investigated the effect and mechanism of continuous exposure of low concentration PS-NPs on the performance of Anammox reactor.The research results could provide theoretical basis and technical support for evaluating the potential risk of nanoplastics on the actual operation of Anammox process.Conclusions and finds were listed below:(1)PS-NPs can be rapidly adsorbed by Anammox granular sludge and enriched on the sludge surface.The effect of PS-NPs short-term exposure on the nitrogen removal performance of Anammox granular sludge showed concentration dose effect.Compared with activated sludge,Anammox granular sludge had compact structure and stronger adaptability to short-term exposure of 100 mg/L PS-NPs.However,after 500 mg/L and1000 mg/L PS-NPs exposure,the specific anammox activity(SAA)of Anammox granular sludge decreased significantly.The increase in the proportion of dead bacteria and the obstruction of mass transfer were the reasons for the decline of sludge nitrogen removal performance under short-term exposure of PS-NPs.(2)The influence of PS-NPs short-term exposure on the structural stability of Anammox granular sludge also showed concentration dose effect.After short-term exposure of 100 mg L PS-NPs,the structural stability of Anammox granular sludg did not change significantly,while after short-term exposure of 500 mg/L and 1000 mg/L PS-NPs,the instability coefficient of granular sludge increased by 42.9%and 85.7%,respectively.The decrease of structural stability of granular sludge was mainly due to the increase of electrostatic repulsion between microbial cells caused by the adsorption of negatively charged PS-NPs,the changes of sludge hydrophobicity based extracellular polymer substances(EPS)content,components,functional groups and secondary structure,increased the number of dead bacteria in sludge.Compared with activated sludge,the effect of PS NPs short-term exposure on the structure of Anammox granular sludge was less than activated sludge flocs because the structure of Anammox granular sludge was more compact.(3)The effect of PS-NPs long-term exposure on the nitrogen removal performance of Anammox granular sludge raised with the increase of PS-NPs concentration.Anammox granular sludge had a certain adaptability to the long-term exposure of 200mg/L PS-NPs,and the nitrogen removal performance of sludge decreased continuously under 500 mg/L PS-NPs.PS-NPs long-term exposure changed the community structure of Anammox granular sludge.The abundance of Anammox bacteria decreased with the increase of PS-NPs concentration.In addition,PS NPs long-term exposure also affected the abundance of nir S、hzs A and kdh.Compared with short-term exposure,long-term exposure had a greater impact on the nitrogen removal performance because the decline of Anammox bacteria abundance and functional gene abundance.(4)Proteomics technology was used to compare the differences in protein expression of Anammox granular sludge after 30 days of exposure of 0 mg/L and 500mg/L PS-NPs.Through the GO function annotation of the differential protein,it was found that the protein expression related to the metabolic process was significantly down-regulated,indicating that PS-NPs exposure affected the metabolism of microorganisms.Based on the analysis of KEGG metabolic pathway,the expression of NADH dehydrogenase subunits Nuo M and Nuo L in the dominant Anammox bacteria Candidatus Kuenenia were significantly up-regulated after long-term exposure of PS-NPs,resulting in the increase of reactive oxygen species(ROS)which caused dead bacteria.In Candidatus Kuenenia,the down-regulated expression of NAD~+oxidoreductase containing FAD and 5,10-methylenetetrahydrofolate dehydrogenase(Fol D)respectively influenced the electron transport and anabolism.Besides,the down-regulated expression of UDP Glc NAc protein and Gsp G preprotein translocase affected the synthesis of extracellular polysaccharide and extracellular protein,resulting in the decrease of Candidatus Kuenenia abundance,which further affect the nitrogen removal performance of Anammox granular sludge.In addition,the hindered extracellular polysaccharide and extracellular protein synthesis also reduced the structural stability of granular sludge.Compared with Candidatus Kuenenia,although Candidatus Jettenia was affected by PS-NPs long-term exposure in electron transport and anabolism,the enhancement of its tendency and group sensing effect might help to resist the toxicity of PS-NPs.Therefore,Candidatus Jettenia had better adaptability to PS-NPs exposure.(5)Continuous exposure of low concentration PS-NPs have a negative impact on the nitrogen removal performance and stability of Anammox reactor,and the degree of impact depends on the cumulative exposure of PS-NPs.When the cumulative exposure of PS-NPs exceeds 100 mg PS-NPs/g VSS,the nitrogen removal performance of sludge decreased,and resulted in disintegration and flotation.Furthermore,the amount of PS-NPs exposure that sludge can withstand under low concentration continuous exposure was higher than that under high concentration long-term exposure.With the increase of cumulative exposure of PS-NPs,the protective effect of EPS on cells was weakened,resulting in the increase of ROS content and LDH release of sludge cells,which affected the abundance of Anammox bacteria.Compared with the non-disintegrated granular sludge,the disintegrated sludge suffered from stronger toxicity,but had higher SAA,which proved that PS-NPs hindered the mass transfer of granular sludge.Moreover,the content and surface characteristics of EPS also changed with the increase of cumulative exposure of PS-NPs,which affected the structural strength of granular sludge.Also,PS-NPs blocked the holes on the surface of granular sludge,resulted in disintegration and flotation due to the formation of air bags and excessive air pressure.The reduction of the abundance of Anammox bacteria,the disintegration and flotation of granular sludge lead to the worse of the nitrogen removal performance of the reactor.