Study On The Denitrification Performance And Response Mechanism Of Marine Anaerobic Ammonium-oxidizing Bacteria Under Combined Antibiotic Stres

Anaerobic ammonium oxidation（anammox）has been widely concerned for its energy-saving and high-efficiency.Mariculture wastewater is a kind of nitrogen-laden saline wastewater,which contained multiple antibiotics.The activity of anammox bacteria would be inhibited by high salinity and antibiotics,thus the nitrogen removel performance could be affected.Halophilic marine anammox bacteria（MAB）has natural advantages in treating nitrogen-laden saline wastewater,but the effects of multi-antibiotics on MAB are still unclear.In this work,the nitrogen removal performance and response mechanisms of MAB were investigated under the short-term shock of antibiotics and long-term stress of multi-antibiotics in the treatment of synthetic mariculture wastewater（3.5%salinity）,and the recovery of MAB activity was studied without multi-antibiotic stress.Under the short-term shock of antibiotics,the inhibition percentage of the nitrogen removal performance of MAB-based sludge increased with the increase of antibiotic concentration.The inhibition percentage of three antibiotics were listed:oxytetracycline>sulfamethoxazole>enrofloxacin.At 35 g/L salinity,the inhibition percentages of enrofloxacin,oxytetracycline and sulfamethoxazole at 1000 mg/L were 15.68%,22.13%and 55.44%,respectively.The half inhibitory concentration of OTC on MAB-based sludge was 905.73 mg/L.In addition,the parameter values predicted by the Remodified Logistic model and Modified Gompertz model were close to the results,which are suitable for predicting and analyzing the substrate removal process of MAB under antibiotic shock.Under the long-term stress of antibiotics,MAB-based reactor exhibited good nitrogen removal performance within 4 mg/L multi-antibiotics（enrofloxacin,sulfamethoxazole and oxytetracycline）,during which the mean NH₄⁺-N removal efficiency was higher than 90%.Under 8 mg/L multi-antibiotic stress,the nitrogen removal performance of the reactor was significantly deteriorated,and the total nitrogen removal rate decreased from 1.46 kg/（m³·d）to 1.24 kg/（m³·d）.In the microbial community,the relative abundance of Candidatus Scalindua decreased significantly at 8 mg/L multi-antibiotics,and several antibiotic-resistant bacteria dominated under multi-antibiotic stress（such as Rheinheimera）.MAB-consortia resisted the multi-antibiotic stress by stimulating the secretion of extracellular polymeric substances（EPS）and the up-regulation of antibiotic resistance genes（ARGs）.EPS could prevent antibiotics from entering cells and weaken the induction of ARGs under low-concentration antibiotics.The up-regulation of ARGs alleviated the inhibition of MAB-based sludge by high-concentration antibiotics.The relative abundance of total ARGs increased with the increase of antibiotic concentration,and reached the maximum of2.44×10^-1 gene copies/16s r RNA at 24 mg/L multi-antibiotics.After the relieve of multi-antibiotic stress,the restoration of MAB activity was conducted by the strategy of reducing the influent total nitrogen concentration and prolonging the hydraulic retention time.After 112-day recovery,the nitrogen removal performance of MAB-based sludge was basically restored.NH₄⁺-N could be completely removed,and the total nitrogen removal efficiency of MAB increased from 23.31%to80.44%.Stoichiometric ratios indicated that anammox was the dominant nitrogen removal reaction.The color of MAB granular sludge changed from light red and black to brick red after recovery,and the finely and divided granula aggregated into larger granula.During the recovery,the relative abundance of 7 ARGs decreased significantly,and the relative abundance of total ARGs decreased by 39.8%.In addition,the up-regulation of functional gene played an important role in the recovery of the nitrogen removal capacity of MAB.