Effect Of Macrolide Antibiotic On Anammox Nitrogen Removal Performance And Its Mechanism

Anaerobic ammonia oxidation?Anammox?,as a type of low energy consumption,sustainable and efficient biological denitrification technology,has broad prospects in the field of ammonia nitrogen wastewater treatment.However,the environmental sensitivity of anaerobic ammonia oxidizing bacteria?AAOB?and typical inhibitors such as antibiotics widely present in the actual ammonia nitrogen wastewater limit the engineering application of the Anammox process.Therefore,based on the existing Anammox sludge mixture of the research group,this article first obtained a more active AAOB by further enrichment culture;on this basis,the macrolide antibiotic telmicosin?TIL?And spiramycin?SPM?in the short-term and long-term system under the Anammox process denitrification performance.And through the analysis of EPS composition analysis of Anammox sludge,detection of metabolic key enzyme activity,microbial flora conversion,functional genes and antibiotic resistance genes?ARGs?response,the mechanism of inhibition of Anammox by TIL and SPM was analyzed at various levels,the main conclusions as follows:?1?With the method of gradually increase fill nitrogen load,through the 128 days of training,won the highly active AAOB.Under a 2.61kg N/m³/d volumetric nitrogen load?NLR?medium temperature system,the removal rates of ammonia nitrogen nitrite reached98.7%and 99.2%,respectively,?NO₂^--N:?NO₃^--N:?NH₄⁺-N stoichiometric ratio was close to 1.32:0.26:1,and the enriched Anammox sludge was dominated by unique brick red particles.?2?With the increase of the concentration of the two antibiotics,the inhibitory effect of Anammox's nitrogen removal performance is more significant.Through optimization of denitrification conditions,the initial sludge concentration of the batch experiment was2g VSS/L,and the initial substrate concentration was 80mg/L.The short-term effect of antibiotic concentration?100mg/L has no obvious effect on Anammox;while when the concentration of TIL and SPM is 200-1000mg/L,the ammonia nitrogen removal rate drops 11.58-53.72%and 37.43-64.20%,respectively.Under maximum inhibition,Anammox The nitrogen removal capacity of sludge is reduced by at least half.Under the long-term influence of antibiotics?5mg/L,the inhibitory effect on the nitrogen removal performance of the Anammox reactor can be restored within a few days;while the long-term inhibition of TIL and SPM of 20mg/L,the reactor appears unstable and the total nitrogen in the effluent?TN?concentration is as high as 147.81 and 203.09 mg/L,and the nitrogen removal load?NRR?drops to 0.79 and 0.69 kg N/m³/d.The overall inhibitory effect of SPM on Anammox denitrification is stronger than that of TIL.?3?AAOB can resist the inhibition of macrolide antibiotics to a certain extent by increasing EPS secretion.Under the short-term effects of antibiotics and the long-term effects of?1mg/L,the EPS content increased to 50.81 and 77.66mg/g VSS,and EPS?PN?was secreted on the cell surface to relieve the biological toxicity of antibiotics.When the long-term action concentration of TIL or SPM is 20mg/L,the final content of EPS drops to 74.17 and 58.70%of the initial level.TIL or SPM will enter the cell and irreversibly bind to specific target sites on the ribosome,resulting in the inhibition of specific protein synthesis.A 3D-EEM scan of EPS found that with the increase of antibiotic concentration,the fluorescence intensity of the corresponding peak of microbial metabolism and decay products increased,and there was cumulative toxicity inhibition;under the long-term influence of SPM,the fluorescence peak of fulvic acids gradually disappeared,which can Can be insufficient and inhibit bacterial activity.?4?The short and long term inhibition of the key enzyme activity and the coenzyme Heme c in Anammox metabolism was different between the two antibiotics.1000 mg/L or TIL SPM short-term will of nitrite reductase?Ni R?,hydrazine synthetase?HZS?,diamine oxidase?HZO?and four kinds of enzyme activity of restraining the coenzyme Heme c,but the SPM main effect on nitrite reductase?Ni R?is the largest,the active damping by42.38%,while TIL mainly affects diamine oxidase?HZO?,its activity was reduced by32.88%;When 20mg/L TIL or SPM has a long-term effect,SPM still mainly affects nitrite reductase?Ni R?,but TIL mainly inhibits hydrazine synthase?HZS?and hydrazine oxidase?HZO?.?5?The long-term effects of TIL or SPM have a significant effect on the microbial community structure.When the TIL or SPM concentration was 20 mg/L,the Simpson index increased by 44.0 and 88.9%,respectively,and species diversity decreased;while the abundance of the Planctomycetes in the phylum Aspergillus fell to 13.11%and 10.52%,respectively,and dominant anaerobic ammonia-oxidizing bacteria Candidatus?Brocadia Abundance was significantly reduced;Redundancy analysis?RDA?showed that with the increase of antibiotic concentration,the structure difference of microbial flora in the reactor became larger.Among them,Limnobacter abundance was positively correlated with antibiotic concentration,which is likely to be a potential resistant bacteria,while Candidatus?Jettenia Only have good tolerance to SPM.?6?Under the long-term effects of the two antibiotics,the response of Anammox functional genes and antibiotic resistance genes?ARGs?is related to their concentration.When the antibiotic concentration is less than or equal to 1 mg/L,the inhibition will be reduced by increasing the abundance of resistance genes nir S,hzs A,hzo and erm F;and when the antibiotic concentration is 20 mg/L,it will continue to poison AAOB,resulting in a decrease in the abundance of functional genes And erm F expression is restricted.Among them,TIL will cause the abundance of hzs A and hzo genes to decrease by 23.96%and 31.02%compared with the initial level,which limits the synthesis and oxidation of hydrazine,while SPM mainly affects the abundance of nir S and hzo genes,which are reduced by 36.65%and 46.16%,respectively The reduction of nitrous nitrogen to NO and hydrazine oxidation process,and its erm F abundance is also reduced by 46.16%;SPM at the gene level of the comprehensive inhibition effect is more significant.