Isolation,Identification And Degradation Characteristics Of A Chloramphenicol-degrading Bacteria

Chloramphenicol(CAP)is a kind of amphenicols antibiotics.It has strong antibacterial activity against a variety of mycoplasma,chlamydia and Gram-negative bacteria.CAP is widely used in medical,livestock and poultry breeding industries.CAP can be naturally produced and enriched through the biosynthesis with soil Streptomyces,and transformed and enriched into plants that serve as food supplies for livestock and poultry.Even in countries where its use is prohibited,there are high levels of contamination(?g/kg)in edible animal products.CAP is also commonly detected in different water environments.Researches on the enhanced removal of CAP have mostly focused on physical and chemical methods.So far,there have been relatively few reports on the degradation of CAP by microorganisms.Therefore,it is urgent to discover new resources of high-efficiency CAP-degrading strains.In this paper,the activated sludge from a pharmaceutical factory was used as the source of inoculation.Through continuous enrichment and subculture,a bacterial consortium that can efficiently degrade CAP was obtained.After 35continuous passages,the bacterial consortium still maintains efficient degradation of CAP.CAP(80 mg/L)was completely degraded within 36 hours.The TOC removal efficiency reaches 76.57±1.08%after 7 days of degradation.The microbial community analysis showed that 71.41%of the microorganisms in the bacterial consortium were belonging to Rhodococcus.A strain that can efficiently degrade CAP by utilizing it as the sole carbon and nitrogen source was isolated from the bacterial consortium.Through 16S r RNA gene sequencing,the strain was identified as Rhodococcus and named CAP-2.Under the condition of 30?,p H=7.0,OD_600nm=0.003 and CAP as the sole carbon and nitrogen source,the degradation efficiency of CAP by strain CAP-2 reached 99.61±0.03%within 42 h.The supply of additional carbon source(glucose)significantly increases the degradation efficiency of CAP,and the addition of nitrogen source(ammonium nitrate)has no obvious effect.It is proposed that the CAP degradation pathway by strain CAP-2 is that the side chain of CAP is first oxidized and broken into p-nitrobenzaldehyde,which is then oxidized to p-nitrobenzoic acid and then it is further transformed to protocatechuic acid.Finally,protocatechuic acid enters the tricarboxylic acid cycle by a complete enzyme system to ring-opening and mineralization.Strain CAP-2 has the best degradation performance when the p H is 7.0,and it still maintains efficient degradation of CAP under low temperature and high salinity conditions.Based on whole-genome sequencing analysis,a complete gene cluster responsible for the conversion of CAP degradation intermediate p-nitrobenzoic acid to protocatechuic acid,and further oxidation and ring opening into the tricarboxylic acid cycle was located and screened.Load strain CAP-2 cells with biochar to explore the application potential of engineering.Through the rapid enrichment of CAP by biochar and the immobilization of strain CAP-2 cells,the efficient removal of CAP is realized.It was observed by SEM characterization that the strain CAP-2cells were densely loaded on the biochar.OD_600nm(0.01)and biochar(0.5 g/L)is the best solid-to-liquid ratio for the loaded strain CAP-2.Under this optimal condition,the removal efficiency of 25 mg/L CAP reached 94.46±1.54%within 1 h,and the removal efficiency of low concentration CAP(2 mg/L)reached 100%within 1 h.The biochar-loaded strain CAP-2 cells still maintain high efficiency in removing CAP at low temperature.Strain CAP-2 has great potential for the basic study of the molecular mechanism of chloramphenicol catabolism and the application of in situ bioremediation of CAP-contaminated environment.