Screening Of Tylosin Degrading Bacteria And Preparation Of Microbial Agents

In recent years,with the continuous improvement of medical level,antibiotics have been widely used in the treatment of human diseases,livestock,fodder and other fields.In the process of application,some antibiotic residues will be discarded or some cannot be fully utilized by livestock and poultry.As a result,they will be brought into the environment through excretion and other ways.These wastes may be used as organic fertilizers for crops and eaten by some aquatic animals like fish,and eventually harm the human body.In order to avoid more hazards,it is necessary to remove antibiotic residues in the environment as soon as possible to ensure the landscaping of the environment and human health.Therefore,the study of antibiotic removal methods has important basic theoretical guiding significance for environmental remediation.In this paper,tylosin(Tylosin,TYL)was used as the experimental object.Firstly,the strain was isolated and purified from dregs,identified and optimized its degrading conditions,and applied to the sewage treatment to test the degrading effect.Secondly,the degrading bacteria were combined with the substrates by adsorption method to study the adsorption mechanism and influence factors.Finally,the best substrate was selected to prepare the microbial agent,and the results were obtained as follows:1.Eight strains of microorganisms were screened from pharmaceutical residues,and four kinds of bacteria had the ability to degrade TYL,among which TYL2 had the most obvious effect,and the degradation rate was 46.15%.This strain was identified as Brevibacillus borstelensis by its morphology,physiological and biochemical reactions and sequencing the PCR-amplified ITS fragments of its rRNA-coding genes.2.The degradation of TYL by TYL2 strain under different environmental conditions was observed,and the optimum conditions for TYL2 were screened.The results showed that:The degradation rate of TYL by this strain could reach to 75%with an initial concentration of 25 mg/L within 7 days under the inoculum size of1.4×10~9 CFU,at pH 7 and at 35°C.3.The bacteria was detected using a kind of protein mensuration,and the results showed that it is feasible through comparison of the linear relationship with the determination of bacterial count.4.The adsorption kinetics of TYL2 on millet shell,sawdust,bran,vermiculite and medical stone were studied.The results showed that:during the adsorption process,with the increase of time,the adsorbtion of five substrates and bacteria also increased with time,and vermiculite and medical stone reached the maximum adsorption value at 20 minutes,and millet shell,sawdust,and bran reached the maximum adsorption value at 30 minutes.Combined with the kinetic model,the adsorption speeds of the five substrates at the saturation time were as follows:millet shell>sawdust>bran>vermiculite>medical stone.The isothermal adsorption of TYL2 on the substrate was also discussed.The adsorption amount of TYL2 on five substrates increased with the increase of TYL2 concentration,and eventually reached equilibrium.This phenomenon was similar to the Langmuir equation.Based on this result,the adsorption capacity of the five substrates for TYL2 were judged as:millet shell>sawdust>bran>vermiculite>medical stone.5.In order to further study the mechanism,the zeta potential on the degradation bacteria,the substrate and the substrate with the degradation bacteria was measured.It was found that the Zeta potential of TYL2 changes from 20 mV to-24.8 mV at pH3-8,and when the pH is in the range of 5-6,it is the transition point of Zeta potential,so it is speculated that the isoelectric point of TYL2 is between pH5 and pH6.Within this range,the zeta potentials of the millet shell and the medical stone are negative from beginning to end and gradually become larger,and the zeta potential of the degrading bacteria attached to the substrate changes from a positive number to a negative number.By comparing the changes of Zeta potential before and after the degradation bacteria on millet shell and medical stone,it can be judged that electrostatic effect was generated between them.6.The effect of the influencing factors(such as substrate particle size,time,temperature and desorption)on the adsorption of degradation bacteria on the substrate was studied.The results showed that:(1)the optimal adsorption particle size of millet shell,vermiculite,bran and medical stone was 60 mesh,and the protein contents were30.5,23.24,22.32,21.77μg/m L,respectively.The maximum protein content of sawdust at 40 meshes was 27.08μg/mL.(2)When the adsorption time was 30minutes,the protein content on the millet shell and sawdust showed the maximum saturation,and the bran,vermiculite and medical stone showed adsorption saturation at 20 minutes.(3)The maximum adsorption temperature of sawdust,millet shell and vermiculite was 35°C;the maximum adsorption temperature of bran and medical stone were 30°C and 25°C,respectively.(4)The ability of degrading bacteria TYL2to adhere on the substrates was observed by desorption reaction.With the prolongation of time,the protein content of the five substrates decreased and the desorption rate increased slowly.At 60 minutes,the order of desorption capacity of five substrates was medical stone>vermiculite>bran>sawdust>millet shell.7.The preparation of microecological agents:millet shell was chosen as substrate,the mass ratio of bacteria suspension and millet shell was 2:1,the drying temperature was 65°C,the culture pH of degrading bacteria was 7,the final number of living bacteria was 2.4×10~8 CFU/g.