Study On Degradation Efficiency And Mechanism Of Tetracycline By "Lolium Multiflorum L.-Microbial Inoculantion"

In order to prevent and treat infectious diseases and promote the growth of livestock,veterinary tetracycline is widely used in large-scale farms,usually 40%-90%of veterinary tetracycline enters the soil in the form of livestock excrement（urine or feces）,and the residual tetracycline in soil poses a potential threat to soil quality,biodiversity and human health through enrichment.Compared with single-use microorganisms,“plant-microbe”degrading tetracycline has the advantages of high efficiency,strong adaptability,wide application range and no secondary pollution.Therefore,screening complex strains is a key strategy for“plant-microbe”degradation of tetracycline.In this study,cow dung from large-scale farms was used as the material to carry out“enrichment and domestication”of tetracycline-degrading microflora,the concentration of tetracycline(50 mg L^-1-200 mg L^-1)in the process of microflora acclimation was gradually increased,and a stable microflora capable of degrading tetracycline efficiently was obtained by“subculture”;A single strain was further screened in the tetracycline-degrading microflora to construct a composite strain（MI）,and explore the degradation characteristics,simultaneous nitrogen transformation and degradation pathways of tetracycline by MI;Finally,a coupling system of“Lolium multiflorum L-compound strain”was constructed to repair the tetracycline pollution,and provide a“plant-microbe”restoration scheme for veterinary tetracycline pollution,the main research results are as follows.（1）The microflora degradation test showed that the effect of inoculation amount at1.0%–3.0%on the tetracycline degradation rate was not significant（P<0.05）,the degradation rate of the microflora under the alkaline condition was greater than that under the acidic condition,and the addition of carbon sources（glucose,maltose,lactose and sucrose）would weaken the ability of the microflora to degrade tetracycline.The degradation ability of tetracycline to the same concentration of microflora showed the order of generation 0<5<10<20<30≈40,and tetracycline could significantly reduce polyphenol oxidase and dehydrogenase activities of stable bacteria（P<0.05）.Response surface optimization showed that the optimal degradation conditions of tetracycline were as follows,the incubation time was 7 d,the temperature was 29.9°C,the p H was 7.34,the inoculum amount was 2.39%,and the degradation efficiency of tetracycline was 73.01%.（2）The microflora domestication test showed that the dominant microbial communities at the phylum level were mainly Proteobacteria,Actinobacteria,Bacteroidetes,Firmicutes and Ascomycota;at genus level are Alcaligenes,Pseudomonas and Bacillus;and the relative abundances of functional genes tet33 and tet Z ranged from 1.41 to 10.74 and 0.1to 6.48,respectively.The relative abundance of Metabolism in metabolic pathways was16.7%-18.7%,and Environmental Information Processing and Metabolism also showed a similar trend,ranging from 6.01%-7.45%.The structural equation modeling found that the abundances of functional microbial communities Bacillus（r=-0.0886）and Pseudomonas（r=-0.1417）significantly affected the tetracycline degradation rate（r=0.75,P<0.001）,Amino acid（r=0.3094）and Metabolic pathway of Carbohydrate（r=0.5729）;The functional genes positively correlated with the microbial community were mainly tet33（r=0.0988）,sul3（r=0.0874）,emr B（r=0.4501）and mef B（r=0.877）.During the domestication of tetracycline-degrading microflora,we found the Function dominates Resistant,and the Sensitive threatens Function.The succession model of functional microorganisms under tetracycline stress was explained for the first time,which was visualized as“Paper-rock-scissors-good”.（3）The composite strains construction test showed that the degradation rates of bacteria（60 strains）and yeast（14 strains）screened from the microflora were 50.23%-62.18%and50.44%-66.35%,respectively;A composite strain（Bacillus albus:Yarrowia lipolytica=1:2,V/V）was constructed by antagonistic experiments,and the degradation rate of tetracycline reached 74.02%when the bacterial inoculation amount was 3%;Under the influence of temperature（20°C,30°C and 40°C）,p H（6,7,8 and 9）and concentration(50mg·L^-1,100 mg·L^-1 and 150 mg·L^-1),The degradation rate of compound strains was in accordance with the first-order biodegradation kinetic model（ln C=0.2047 t-0.0407;Km=0.2047,R²=0.9914）.The alkaline environment is helpful for the degradation of tetracycline,and the effect of p H on the degradation of tetracycline is alkaline>neutral>acidic.（4）The study of tetracycline degradation and nitrogen transformation showed that tetracycline and NO₃-N were simultaneously reduced by the MI with the addition of nitrate nitrogen（Na NO₃）.Under the condition of adding ammonium nitrogen（NH₄⁺-N）,the first 24 h of tetracycline degradation was dominated by NH₄⁺-N assimilation and tetracycline degradation,with the assimilation of the strain as the dominant.In the logarithmic phase of tetracycline degradation,tetracycline degradation was concurrent with the nitrification-denitrification reaction,and NH₄⁺-N decreased from 12.94 mg L^-1-3.33 mg L^-1,and NO₃^--N concentration decreased from 4.25 mg L^-1-2.72 mg L^-1.Through PCA,sample clustering and inter-group differential screening,we found that the differential changes of tetracycline degradation mainly concentrated in the 1-5 days,and the composition,number and type of the degradation products decreased after the 7th day.The degradation of tetracycline by the strain was mainly through demethylation（-CH₃）,decarbonylation（-C=O-）,deamination（-NH₂）,dehydroxylation（-OH）,and oxidation.（5）The degradation test of tetracycline by the coupling system of“Lolium multiflorum L.-compound strain”showed that 100 mg L^-1 tetracycline can make ryegrass root tip edge,root cap area and interior ulceration,can not form a complete cap covering the root tip,but adding MI can improve the number and shape of root tip cells,root length and Bud length increased by 23.92%-21.83%,respectively;When the concentration of tetracycline was 50 mg·L^-1,100 mg·L^-1 and 150 mg·L^-1,the relative conductivity of Lolium multiflorum L.was significantly reduced by adding MI by 34.34%,13.67%and 11.94%（P<0.05）,the aboveground fresh weight increased by 11.22%,21.60%and 1.78%;When the tetracycline concentration was 150 mg L^-1,the addition of MI could significantly increase the malondialdehyde（MDA）content and peroxidase（POD）activity by 25.29%and 12.68%（P<0.01）,and significantly reduce the superoxide dismutase（SOD）by 6.36%（P<0.05）;In addition,when the concentration of tetracycline was 50mg·L^-1-150 mg·L^-1,the coupled degradation system of“Lolium multiflorum L.-compound strain”can not only increase the degradation rate of tetracycline by 40.63%-45.74%,but also significantly increase the tetracycline residues in the underground and aboveground parts of Lolium multiflorum L.by 24.48%-42.43%and 29.92%-42.40%.In conclusion,tetracycline-degrading microflora were obtained with high efficiency,and revealed the succession mechanism of microflora under tetracycline stress;A complex strain（Bacillus albus:Yarrowia lipolytica=1:2,V/V）was constructed,the relationship between the complex strain on tetracycline degradation and nitrification-denitrification was clarified,and the tetracycline degradation pathway was speculated;It was found that the degradation rate of tetracycline by the coupling system of“Lolium multiflorum L.-compound strain”was higher than that of the strains and ryegrass alone,which provided a coupled restoration scheme of“Lolium multiflorum L.-compound strain”for veterinary tetracycline pollution.