Cometabolic Mechanism And Enhancement Strategy Of 17?-ethynylestradiol Removal By Ammonia Oxidizing Bacteria

The synthetic estrogen 17?-ethynylestradiol(EE2)is a micropollutant(MP)with strong endocrine disrupting effects.Its increasing concentration in water environments has raised global concerns.Ammonia oxidizing bacteria(AOB),the functional group responsible for nitrogen removal at wastewater treatment plants are considered as the main contributor to EE2 removal,which can biodegrade the non-growth substrate EE2 via cometabolism in the presence of the growth substrate ammonia.However,EE2 cometabolic kinetics and molecular mechanism remain unclear.By utilizing the model AOB,Nitrosomonas europaea,this thesis investigated the cometabolic kinetics of EE2,its transformation products and key regulatory genes involved in the cometabolic process.The potential enhancement of EE2 cometabolism by adding alternative energy source during ammonia oxidation was also evaluated.The main findings are as follows:1)During EE2-ammonia cometabolism by N.europaea,the non-linear correlations between EE2 degradation and ammonia oxidation can be divided into three phases according to reducing power availability,namely "activation","coupling" and "saturation".When the initial ammonia concentration was relatively low(<38 mg N/L in this study),the reducing power was insufficient to initiate EE2 cometabolism("activation").With initial ammonia concentration of 38?180 mg N/L,EE2 removal was linearly correlated with ammonia oxidation("coupling").When initial ammonia concentration was relatively high(>180 mg N/L),EE2 degradation activity did not increase with higher ammonia("saturation").Key kinetic parameters of EE2 cometabolism were obtained by applying the reductant model,including maximum specific EE2 transformation rate,EE2 half-saturation coefficient and transformation capacity.Nine biotic and abiotic transformation products of EE2 were identified,five of which were newly discovered.2)EE2 cometabolism resulted in elevated NO accumulation and upregulation of the nitrite reductase encoding gene,nir K.In chemostat degradation tests,headspace NO concentration significantly increased by 12 ± 2% during EE2 cometabolism.Thirty-four differentially expressed genes were identified via RNA-seq,among which nir K cluster genes were up-regulated by 2.04-to 3.97-fold during EE2 cometabolism,whereas amo A gene was down-regulated by 1.72-fold,indicating that long-term EE2 exposure may have an inhibitory effect on ammonia oxidation.3)Organics and heavy metal would greatly impact EE2 cometabolism by N.europaea.Pyruvate at 10 m M made the growth mode of bacteria shift from autotrophic to both autotrophic and heterotrophic,leading to an increased EE2 removal by 13 ± 4%.Two pyruvate utilization-related genes ppc and ace E were up-regulated by 1.63-and 1.95-fold,respectively.The upregulation of ncg A(by1.19-fold)suggested its potential role in EE2 degradation enhancement.In contrast,copper ions above 50 ?g/L would inhibit EE2 cometabolism.This study expended our knowledge of MP cometabolism by typical functional microorganisms in wastewater treatment,and will be useful for predicting the transport and transformation of EE2,assessing its ecological toxicity,as well as enhancing the simultaneous removal of traditional pollutants and MPs during wastewater treatment.