Study On The Efficient Degradation Of Tetracycline By Two Intestinal Functional Bacteria In Synergistic Black Soldier Fly And Their Functional Genes

The residue of antibiotics in livestock and poultry manure has always been an important issue in the harmless treatment process of manure in China.Among the current numerous treatment technologies,the transformation technology of Hermetia Illucens L.(BSFL)has demonstrated efficient degradation of antibiotic residues in manure,and the gut microbiota of BSFL play an important role.In the early stage of our laboratory,two strains of bacteria,Serratia marcescensis BSFL-6 and Trichosporon asahii BSFL-2,were isolated from the intestine of BSFL and have the ability to efficiently degrade tetracycline in a single bacterial environment.However,the genetic mechanism of their synergistic degradation is still unclear.Therefore,this study mainly focuses on the tetracycline degradation of the two functional bacteria in cooperation with the BSFL.Based on the sequencing analysis of the genome and Transcriptome,we screened the related functional genes of the two functional bacteria and verified them using the Escherichia coli heterologous expression system,in order to lay the foundation for analyzing the mechanism of tetracycline degradation of the functional bacteria in cooperation with the BSFL,It will also provide theoretical guidance for the technology of functional microorganisms collaborating with water flies to promote antibiotic degradation.The main research findings are as follows:(1)Toxicity evaluation of tetracycline degradation products by functional bacteriaTo evaluate the safety of tetracycline degradation products from functional bacteria,this study used Escherichia coli as a sensitive bacterium and established an in vitro degradation system to evaluate the toxicity of tetracycline degradation products from Serratia marcescensis BSFL-6 and Trichosporon asahii BSFL-2.The results showed that the metabolites of tetracycline degraded by these two strains of intestinal functional bacteria did not show significant biological toxicity,and can be safely applied.(2)The characteristics of the efficient degradation of tetracycline by functional bacteria in BSFL.Based on the normal intestinal transformation system of BSFL,this study analyzed the characteristics of functional bacteria promoting the efficient degradation of tetracycline in the transformation system by directly inoculating functional bacterial agents.The results showed that adding functional bacteria could significantly increase the average weight of the BSFL body under tetracycline stress,with little impact on material p H and moisture content.It could also significantly promote the degradation of tetracycline in BSFL transformation system for 5 to 10 days,with a final degradation rate of 97.05%.In addition,this study found that the addition of functional bacteria can significantly reduce the relative abundance of typical tetracycline resistance genes tet A,tet B,and tet M in the material transformation residue,with a decrease more than 75%.This indicates that the addition of functional bacteria can be applied to the BSFL transformation system and significantly enhance the tetracycline degradation effect,reducing the abundance of typical tetracycline resistance genes.(3)Screening and verification of tetracycline-promoting degradation gene in Serratia marcescensis BSFL-6Based on the genome data of Serratia marcescens BSFL-6,six potential degradation promoting genes were screened in this study,and Functional verification was carried out by constructing an E.coli heterologous expression system.The results showed that the Glutathione peroxidase genes GSHPX1,GSHPX2,efflux pump genes oprm,mexa,tet41,and enzyme modified gene tet34 in Serratia marcescens BSFL-6 increased the MIC of tetracycline.Minimum inhibitory concentration of heterologous expression host E.coli BL21 by 1.5-2 times,and the expression of tet34 enables E.coli BL21 to degrade nearly80% of 10 μg/kg tetracycline within 72 hours,while the expression of other genes can promote the degradation of tetracycline by about 35.14%-55.67%.This indicates that the Serratia marcescens BSFL-6 carries multiple tetracycline degrading genes,and the expression of these genes can achieve accelerated tetracycline degradation.(4)Discovery of tetracycline degrading genes in Trichosporon asahii BSFL-2In order to analyze the potential tetracycline degrading genes in Trichosporon asahii BSFL-2,this study established degrading system of Trichosporon asahii BSFL-2,which uses tetracycline or glucose as the only carbon source.The potential tetracycline degrading genes were analyzed using the whole genome and Transcriptome sequencing analysis techniques.The results showed that the manganese peroxidase,Glutathione S-transferase,coenzyme A transferase and salicylate dehydrogenase of Trichosporon asahii BSFL-2might be potential degradation genes induced by tetracycline.Further validation of gene expression through q RT-PCR revealed that the relative expression levels of the four genes were upregulated 2.6,3.8,2.4,and 115 times compared to the control group,indicating that they may all be functional genes that degrade tetracycline.Among them,the expression level of salicylate dehydrogenase was upregulated most significantly,indicating that it is highly likely to play a key role in the utilization of tetracycline as a single carbon source in Trichosporon asahii BSFL-2.In addition,after supplementing 0.2% glucose in the original system,the expression levels of the first three genes were significantly increased,indicating that low carbon source supplementation may be more conducive to the degradation and utilization of tetracycline by Trichosporon asahii BSFL-2.In conclusion,the research results of this study will provide a theoretical basis for the application of functional bacteria in the tetracycline degradation transformation system of BSFL in actual production,and provide relevant functional gene information for further research on the synergistic degradation mechanism of tetracycline by intestinal bacteria and BSFL.