Mechanism Of Phanerochaete Chrysosporium Reducing Tetracycline Resistance Genes In Cultured Feces

China is a large producer and consumer of tetracycline antibiotics.Tetracycline plays an important role in animal husbandry.With the increasing need in livestock and poultry breeding,the abuse of antibiotics causes tetracycline residues in feces,and induces the generation of antibiotic resistance genes(ARGs).ARGs pose a great threat to the ecological environment and human health through migration and transformation via various environmental media.Composting technology is widely used in harmless treatment of farm feces,and Phanerochaete chrysosporium has an application potential in many fields due to containing a variety of peroxidase systems.Therefore,in this experiment,the degradation ability of P.chrysosporium‘s in tetracycline was explored and we also discussed the bioaugmentation mechanism of tetracycline resistance gene(TRG)reduction in pig mannre.The content is divided into the following parts.Firstly,P.chrysosporium is labeled with GFP through Agrobacterium mediated fungal transformation method to express green fluorescence,which is convenient for the fungal track and observation.The experimental device was designed and white-rot fungus P.chrysosporium was added to the experimental reactor,and 30 TRGs were screened,and 21 TRGs were detected in the initial fecal samples from farms.The relative abundance(RA)of five TRGs including tet W,tet M,tet(32),tet PA,and tet O was higher.Secondly,five TRGs with higher RAs were detected,and the effect of P.chrysosporium on tetracycline removal was studied.The results showed that,compared with the control,the addition of P.chrysosporium increased the reduction efficiency of the five TRGs by 7.9-100 times,proving that it is a potential method to reduce TRGs in pig manure.On the 35 th day,the removal rate of tetracycline in the treatment group was 83.55%,which was 11.93% higher than that of the control group,indicating that bioaugmentation enhanced the degradation ability of tetracycline.Thirdly,the bioaugmentation mechanism of P.chrysosporium to reduce resistance genes was discussed.The conclusions are as follows: Fungal bioaugmentation further enhanced the ability of reactor in degrading tetracycline,which may be one of the reasons for the reduction of TRGs in the treatment group.The decrease of both bacteria and tetracycline resistant bacteria can directly explain the decrease in TRGs.From the perspective of nutrition,reduction of of ammonium nitrogen loss and improvement of the quality of compost indirectly lead to TRGs reduction in the treatment group.Results of high-throughput sequencing showed that the phenomenon of ecological succession of bacterial communities was not the reason why TRGs in treatment group was less than that in control group.On the 35 th day,the RA of Phanerochaete in the fungal community of the treatment group was 62.7%,and the number of P.chrysosporium detected by GFP by plate counting method reached 4.3×105 cfu/g.In addition,P.chrysosporium existed in the form of chlamydospores in compost,and the chlamydospores of P.chrysosporium can capture bacteria,which is also an important reason for the reduction of TRGs.Fourthly,the transfer of bacteria into P.chrysosporium chlamydospores was investigated after fluorescent labeling of bacteria.The transfer of Escherichia coli MG1655 and Bacillus subtilis 92082 was observed at 72 h and 96 h,respectively.With the increase of co-culture time,it was found that the number of bacteria with green and red fluorescence in chlamydospores decreased.After 216 h co-culture,the fluorescence was basically invisible under microscope,and the number of bacteria in the culture broth also decreased significantly.Finally,the pathway of bacteria entering chlamydospores was preliminarily studied.The results showed that E.coli MG1655 entered the fungal cell from the wound and crack of cell wall.B.subtilis can enter the chlamydospore via the swollen part of hypha middle and the ruptured site of the chlamydospores located the top of the hypha.Extensive tests have confirmed that transfer into fungal cells is a common phenomenon for bacteria including gram-negative and gram-positive bacteria.