| Residual sulfonamides antibiotics(mainly sulfamethoxazole),and their induced antibiotic resistance genes(ARGs)have been highly detected in a variety of environmental media.Especially in animal husbandry wastewater and landfill leachate,contains high concentration of sulfonamides antibiotics,ammonia-N,heavy metals and other pollutants.Antibiotic compound pollution would cause the ecological environment risk,when it discharge into the aquatic environment.Thus,screen of function strain with capacities of SMX degradation,nitrogen conversion,and heavy metal tolerance from industrial sludge sediments is a key biochemical technology strategy to solve antibiotic compound pollution.A novel sulfamethoxazole degrading and aerobic denitrification strain(Achromobacter sp.JL9),was isolated from aerobic activated sludge after long-term domestication,screenin and purification.And explore the physiological and biochemical characteristics of strain JL9,pollution degradation and kinetics characteristics,antibiotics degradation pathway and mechanism,nitrogen transformation mechanism,antibiotics compound pollution degradation characteristics,and the efficiencies of bioaugmented MBBR to degradation different kinds of antibiotics in simulated aquaculture wastewater.The main research results are as follows:1.Using the aerobic activated sludge of pharmaceutical wastewater treatment plant and industrial wastewater treatment plant as seed sludge,a novel strain(Achromobacter sp.JL9)with capacities of SMX degradation and nitrogen conversion was isolated through enrichment,acclimation,screening and purification techniques.Through alignment of gene sequences,confirm that strains JL9 belongs to Achromobacter.sp,and can use 33 kinds of carbon sources.The growth curve of strain JL9 directly into the logarithmic phase,without adaptive phase.The aerobic denitrification genes(napA)have been amplified from strain JL9(gene fragment of 857 bp).2.Strain JL9 have high nitrate-N,ammonia-N and total nitrogen(TN)removal efficiencies,and removal ratios were 94.61%,87.07%and 83.44%.Meanwhile,the total organic carbon and sulfamethoxazole degradation ratios were 75.43%and 91.60%.The conclusions of environmental factors on the sulfamethoxazole degradation and nitrogen removal were as follows:when C/N ratio 3.0-5.0,sulfaethoxazole concentration 20-40 mg·L-1,dissolved oxygen 2-6 mg·L-1,initial pH 6.0-8.0,temperature 25-35 oC,strain JL9 exhibit high sulfamethoxazole degradation and total nitrogen-N removal efficiencies.And the SMX degradation and nitrogen-N removal characters of strain JL9 was consistent with the first-order kinetic model.Response surface methodology(RSM)analysis showed that the degradation efficiency of SMX would reached to 96.11%when the C/N ratio was 6,shaking speed of 148 rpm·min-1,and SMX concentration of 25.67 mg·L-1.Carbon and nitrogen source dependence tests showed that carbon resource is more important for the growth and metabolism of strain JL9 than the nitrogen source.The degradation of SMX was mainly through the extracellular substances and intracellular substances and cell membrane,and cell membrane take the mainly contribution for SMX degradation.The metabolites of SMX degradation were identified by Qex Active LC-MS/MS,and the mainly reaction for SMX degradation included:S-N bond breaks,isoxazole ring-opening,benzene ring-opening and hydroxylation;The ECOSAR program showed that the toxicity of some metabolites was higher than the SMX,but the toxicity of the final metabolites was lower.With the degradation process,the biotoxicity of the products is gradually reduced.3.Strain JL9 has abilities to degrade different sulfonamides antibiotics,and the degradation efficiency of SMX was the highest.The degradation efficiency of trimethoprim(TMP)by strain JL9 was very low,while almost not ciprofloxacin(CIP)was been degradation.The differences metabolic processes of strain JL9 with exposed to different antibiotics were found by the Non-target metabolomic sequencing(UHPLC-QTOF-MS),results indicated that sulfamethoxazole,trimethoprim and ciprofloxacin stress showed no significant impact on the synthesis and conversion of dihydrofolate and tetrahydrofolate in strain JL9,while the stress of trimethoprim and ciprofloxacin could cause the changes of aminoyl-t RNA synthesis and purine metabolism pathway.Meanwhile,the ABC transporter,amino acid biosynthesis,phenylalanine,tyrosine,tryptophan biosynthesis,and Fc RI signaling pathways were enriched very significant.These enrichment was the main reasons for the difference of antibiotic degradation performance and antibiotic resistance of strain JL9.4.Addition of heavy metals would significantly inhibit the growth of the strain JL9 and the degradation activity of SMX.However,addition of Mn(?)can improve the growth and metabolic activity of strain JL9 and the SMX degradation efficiency.Appropriate concentration of Mn(?)could improve the stability and temperature tolerance of sulfamethoxazole degradation by strain JL9.The excellent pollutant removal efficiency was obtained with the Mn(?)concentration of 2 mg·L-1,the SMX degradation,nitrogen-N removal and Mn(?)oxidation ratios were 97.43%,85.61%and 85.78%,respectively.The contributions of microbial degradation and biogenic manganese oxides(Bio Mn Ox)catalytic oxidation for SMX degradation were approximately 80.17%and 15.54%,respectively.However,the contributions of microbial degradation and Bio Mn Ox catalytic oxidation for TN removal were approximately 85.45%and 3.36%,respectively.The degradation of SMX mainly occurred at sites of the oxazole ring,S-N bond,and amino group under the combined contamination of antibiotics and Mn(?),and the evolution of environmental toxicity is mainly through microbial co-metabolic.With the oxidation of heavy metals and the degradation of antibiotic,the toxicity of the solution was significantly reduced.5.A Moving Bed Biofilm Reactor(MBBR)system bioaugmented with Achromobacter strain JL9 was used to treat the simulated wastewater,results showed that the optimal removal efficiency of SMX,nitrate-N and ammonia-N were 80.49,94.70 and 95.38%,respectively.The bioaugmented strain(strain JL9)can improve the removal efficiency of SMX,nitrate-N and ammonia-N.Carbon resource concentration would influence the replication and propagation probability of antibiotics resistance genes(ARGs),but almost no influence on the biological toxicity of effluent.Under the conditions of C/N ratio 3.0-6.0,initial p H 7.0,HRT10 h,the relative abundance of Acromobacter(1%)in the experimental group was one of the most abundant genus,while the abundance very low in the control group,The bioaugmented strain(strain JL9)plays an important role for simultaneously degradation of SMX and aerobic denitrification in the MBBR.6.Explore the removal efficiency of bioaugmented MBBR for total nitrogen(TN)and antibiotics under different antibiotic stress.The maximum removal efficiency of TN was91.83%,and the removal ratios of various antibiotics were 85.16%(SMX),65.34%(TMP),75.53%(TEC),and 27.11%(CIP),respectively.Under the stress of 5 mg·L-1sulfamethoxazole,trimethoprim,tetracycline and ciprofloxacin,the removal ratios of total nitrogen by bioenhanced MBBR were 93.28 79.78 84.42 and 77.48%,respectively.Trimethoprim and ciprofloxacin would lead to the accumulation of nitrite-N,while tetracycline and ciprofloxacin could lead to the accumulation of nitrate-N.Biofilm damage caused by antibiotic stress is a reversible damage,and different antibiotics follows the same degradation pathways:ring-opening,S-N bond cleavage,amination,hydroxylation,and methylation.The relative abundance of antibiotic-resistant bacteria and denitrifying function bacteria on biofilm gradually increased under the stress of different antibiotic,including:Paenarthrobacter,Plasticicumulans,Pseudomonas,Hydrogenophaga,Thauera and Rivibacter.Meanwhile,some functional and bioenhanced bacteria was obvious decreased after treatment by antibiotics.Such as Achromobacter and Nitrospira.These above research results provide theoretical and technical basis and reference cases for further understanding the microbial degradation of SMX,and provide theoretical basis and technical support for biological treatment of animal husbandry wastewater and landfill leachate. |
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