The Characteristics Of Antibiotic Biodegradation Through Co-metabolism By Antibiotic-resistant Denitrifying Bacteria And Esearch Of Microorganism Community Structure

Antibiotics are widely and widely used in the production of human medical,animal husbandry and aquaculture industries.Studies have found that only a part of antibiotic drugs are absorbed and metabolized,and about 40%-90%are excreted in the form of original drugs or primary metabolites.Although the half-life of antibiotics is not long,due to continuous entry into the environment,antibiotics exhibit pseudo-persistent characteristics in the natural environment.Antibiotics that persist in the natural environment have the potential risk of entering the body through drinking water or the food chain.Even at low doses,long-term exposure of antibiotics may induce microbes in the human body and the natural environment to develop resistance.The resistance genes will be transferred horizontally,resulting in increased resistance of pathogenic bacteria,which will bring human health and safety.Great risk.The removal rate of some antibiotics in urban sewage by conventional processes is low,leading to ecological risks of increased microbial resistance in natural water environments.It is of great significance to reduce the amount of antibiotics discharged into the natural environment and control ecological risks.This project proposes a risk control technology that uses the co-metabolism of drug-resistant denitrifying bacteria to degrade trace antibiotics.Based on the selection of the most suitable co-matrix and the construction of denitrifying bacteria,this project selected Ofloxacin?OFX?,representing fluoroquinolone antibiotics as the research object,to study the effect of different antibiotic concentrations on the denitrification process.OFX adopted the degradation effects of solid phase and liquid phase reverse nitrification co-metabolism and the effect of drug-resistant denitrifying bacteria on the degradation process of antibiotics,using molecular biology methods to study the evolution process and community structure of denitrifying bacteria in different antibiotic concentrations in the growth environment of denitrifying bacteria Influence.The research results are as follows:?1?The COD release effect of the three solid carbon sources is from good to poor in the order of PCL6500,PCL6800,and PHA,with little difference between them.PHA,PCL6500and PCL6800 solid carbon sources adsorb OFX from strong to weak in the order of PHA?PCL6800?PCL6500.Low concentration antibiotics?1?g/L?did not significantly inhibit the denitrification process;high antibiotics?1mg/L?significantly inhibited the denitrification process.High antibiotic concentration environment will lead to the accumulation of nitrous nitrogen.?2?Using sodium acetate as the electron donor and maintaining the concentration of ofloxacin?OFX?at 1?g/L to gradually increase the predominant growth of antibioti c-degrading bacteria,and cultured separately to obtain the reaction in the presence of sodium acetate and nitrogen only.Nitrifying bacteria?DnB₁?,trace antibiotics,denitrify ing bacteria?DnB₂?in the presence of sodium acetate and nitrogen.The results show that DnB₂ has a significant degradation effect on OFX compared to DnB₁,and the de gradation amounts of OFX by DnB₁ and DnB₂ are 0.31?g/g and 16.14?g/g,respecti vely.Increasing OFX concentration will inhibit DnB₁ denitrification activity in the sho rt term.The denitrification process of DnB₂ is less affected by OFX.At the same tim e,using the Illumina MiSeq platform for high-throughput sequencing,based on the O TUs information formed by the clustering of the sequencing results,comparative analy sis of the diversity of each sample,the results show that the relative abundance of D nB₁ microbial community and the diversity of microbial community are higher than D nB₂.?3?Twelve groups of denitrifying bacteria were cultivated and domesticated.The solid carbon source was PCL6500 and PCL6800.The antibiotic concentration was 0,1?g/L,1 mg/L and 10mg/L respectively.The average denitrification rate of the four groups of denitrifying bacteria with the antibiotic concentration of 0 and 1?g/L is significantly higher than that of the four groups of antibiotics with the antibiotic concentration of 1 mg/L and 10mg/L.The high concentration of antibiotics has a significant inhibitory effect on the denitrification process.The removal rate of NFX by PCL6800 group of denitrifiers was 2.05times that of OFX,indicating higher biodegradability of NFX.?4?Study the difference of the degradation rate of ofloxacin under the respective growth conditions of each reaction bottle.The removal rate of ofloxacin in the reaction bottle with a concentration of 10 mg/L ofloxacin is obviously higher.Comparing the degradation effects of 8 kinds of denitrifying bacteria on the same concentration of ofloxacin,the removal rate of ofloxacin in the 10mg/L reaction flask was the fastest.Investigating the removal effect of different denitrifying bacteria on the same concentration of norfloxacin under their respective growth conditions,it was found that denitrifying bacteria cultured in an environment containing a certain concentration of norfloxacin also had a certain degree of removal of norfloxacin with similar molecular structure effect.?5?Ofloxacin did not undergo a decarboxylation reaction at the beginning of the solid-phase nitration process,but a substitution reaction of a hydroxyl group occurred at the terminal C of the piperazinyl group,and a bond break of the quinolone group and piperazinyl group occurred..In the last step of the reaction,the oxazinyl group was broken and a decarboxylation reaction occurred.?6?Through 16S rDNA amplicon sequencing,study the community structure of 8groups of denitrifying colonies.The three types with the largest proportion at the door level in each sample are Proteobacteria,Actinomycetes,and Bacteroides.At the class level,the types of Proteobacteria are mainly?-Proteobacteria,?-Proteobacteria,and?-Proteobacteria.Among them,?-Proteobacteria and?-Proteobacteria are dominant.At the genus level,the microbial genus accounted for the largest proportion in the sample.In the four groups of microorganism samples with high antibiotic concentration,there were 7.0%-10.4%of Xanthomonas.The richness and diversity of the original sludge samples are the highest.The microbial community of 8-A1 group had poor uniformity,while the denitrifying bacteria of8-A4 and 8-A3 groups after domestication were better.In terms of similarity,the microbial samples with the highest similarity and relatively independent of other sludge samples were two groups of denitrifying colonies cultured at 10 mg/L antibiotic concentration,followed by the denitrifying bacteria community cultivated in 1 mg/L antibiotic environment.The relative abundance of microbial communities and diversity of microbial communities in DnB₁ are higher than those in Dn B₂.From the analysis of relative abundance of species at the gate level,it can be seen that the categories with more content in DnB₂ and DnB₁ are:Proteobacteria,Proteobacteria,Bacteroidetes,Green Chloroflexi,Acidobacteria.