| Antibiotics and antibiotic resistance genes（ARGs）have become common emerging pollutants in the environment.Fluoroquinolones（FQs）antibiotic is one of the focuses which are paid wide attention due to the large use,poor biodegradability,and high detection frequency.The conventional activated sludge treatment process has low removal efficiency of antibiotic pollutants and may induce the production of resistant bacteria（ARB）.Bioaugmentation can improve the treatment efficiency of refractory organic pollutants,but few efficient strains can be used for enhanced treatment,and the research on the enhanced degradation mechanism is not deep enough.In this study,ciprofloxacin（CIP）,a widely used FQs antibiotic,was selected as the target pollutant and a strain of bacteria screened from soil showed a high degradation ability to ciprofloxacin.The biodegradation characteristics,degradation mechanism,and genomics of the strain were explored to provide theoretical basis and technical support for the biological enhanced treatment of antibiotic wastewater.The main research contents and conclusions are as follows:（1）A CIP degrading strain was isolated by plate screening method,the physiological and biochemical characteristics and biosafety were analyzed,and the effects of different conditions on its growth were investigated.Results showed that the degrading bacteria,which was identified and named as Pseudomonas sp.CIP-10 by 16S r DNA,was a gram-negative（G-）bacterium with a rod shape and a length of 1-2μm.The optimum growth conditions of the strain were:sodium acetate as a carbon source,culture temperature of30°C,initial p H=7.0,and salinity=1%.The drug sensitivity test showed that strain CIP-10 had no amino-acid-decarboxylase activity,was sensitive to most antibiotics except FQs antibiotics,penicillin and tetracycline antibiotics,and was easy to be killed by ultraviolet light(>18 m J?cm-2),meaning that the strain had high biosafety.（2）The effects of reaction conditions on CIP degradation were investigated by shake flask batch experiment and response surface method,and the degradation pathway and degradation mechanism were further analyzed.The results showed that the optimal degradation conditions were:sodium acetate of 1100 mg?L-1,shaking speed of 120 rpm,and culture temperature of 35°C,and under these conditions,the removal efficiency of CIP(10 mg?L-1)was 99.8%.The degradation process of CIP could be well explained by the first-order kinetic equation.After inoculation,the half-life of CIP was shortened from277.2 d to 7.57 h,with a biodegradation rate of 0.0958 h–1.It was speculated that the degradation pathways mainly included piperazine epoxidation and cracking,decarboxylation,hydroxyl substitution and cyclopropyl cleavage based on 10 kinds of degradation intermediates determined by LC/MS.After biodegradation,the toxicity of CIP to E.coli and Chlorella was reduced by 96.2%and 83.2%,respectively.The calculation of toxicity value based on the quantitative structure-activity relationship（QSAR）showed that the acute（or slow）toxicity threshold of CIP degradation intermediates to aquatic organisms was greatly improved,indicating that the risk of aquatic organisms being poisoned was greatly reduced.（3）The whole genome of strain CIP-10 was analyzed based on the third-generation sequencing technology,and the important functional genes and proteins were annotated.The results showed that the total length of the whole genome was 6.01 Mb.There were5998 genes,of which the number of resistance genes to antibiotics and toxic compounds was 71,including heavy metal resistance,FQs resistance,β-Lactamase,and other functional genes,and no functional genes leading to human diseases were found.The main cause for the resistance of the strain to FQs antibiotics is the mutation of the gyr A gene,and the strain also has multiple efflux pump functions since there are many ABC family transporters and MFS multidrug resistance transporter genes in its genome.The KEGG metabolic pathway annotation showed that the enzymes related to CIP biodegradation mainly included acetoacetate isomerase,amide transferase,benzoyl-coenzyme A dehalogenase,ethanol dehydrogenase,amine oxidase,and P450 enzyme.（4）Strain CIP-10 was applied to enhance activated sludge,and the bioaugmentation effects on the pollutant removal efficiency were explored.The sequencing batch biofilm reactor（SBBR）system was constructed with enhanced activated sludge to treat the CIP pharmaceutical wastewater.Results showed that the degradation rate constant of CIP by activated sludge increased from 0.005 h-1 to 0.055 h-1 and the CIP removal efficiency increased from about 10%to about 68%after bioaugmentation.The bioaugmentation by strain CIP-10 improved the species abundance and diversity in activated sludge,increased the abundance of nitrogen and phosphorus removal bacteria such as Nitrospira,Nitrosomonas,and Accumulibacter,and there was a positive interaction between bioaugmentation bacteria and autochthonous bacteria of activated sludge.Biofilm prevented the enhanced bacteria from being preyed on by protozoa but reduced the mass transfer efficiency of CIP.This problem could be solved by adding biochar to the biofilm.The BC-BSBBR,which had been improved by biochar based on SBBR,could operate stably for a long time with an average CIP removal efficiency of more than 65%.（5）The distribution of antibiotic-resistant bacteria（ARB）and ARGs in different reactors were studied by heterotrophic plate culture and fluorescence quantitative PCR（q PCR）.The results showed that Proteobacteria played a major role in ARGs transmission.BC-BSBBR could inhibit ARB escape,reduce the absolute abundance of int I1 in activated sludge and wastewater,and help to reduce the ARGs horizontal transfer.Compared with traditional SBR,the abundance level of FQs-ARGs in BC-BSBBR effluent was reduced by 0.8-1.9 orders of magnitude. |
PDF Full Text Request