Study On The Isolation, Identification And Biodegradation Characteristics Of Lincomycin-Degrading Bacteria

Lincomycin, a member of broad-spectrum antibiotics, is produced by Streptomyces lincolnensis, and after the solid-liquid separation of the fermentation broth, the resulting solid phase is called waste mycelium containing rich crude proteins, inorganic salt, essential amino acid, vitamin and microelement. But the waste mycelium poses as a potential health risk because of its stable physiochemical properties, long-term residues, biological accumulation and drug resistance to residual lincomycin and can not be used as animal feeds or fertilizer. According to the international and Chinese legislations and regulations, the waste mycelium is required to be disposed and decontaminated by incineration as biological hazardous wastes. How to change the waste into valuables and reduce its pollution to the surrounding environment becomes an urgent problem need to be solved for the antibiotic production enterprises.Microorganisms are well known to play an important role in the control of environmental pollutions, and it is one of the most important pathways of the antibiotic degradation. However, the microbial degradation of lincomycin has not been reported. In this paper, some lincomycin-degrading bacteria were isolated and identified to lay a foundation for the study of functional gene and the construction of genetic engineering bacteria. And then, the degrading conditions of these strains were optimized to provide reference for the microbial degradation of lincomycin in the waste mycelium. The main research contents and conclusion were as follows:(1) Many methods, including traditional enriched domestication method, concentration gradient plate method, culture in situ method and double agar layer capping method, were employed to isolate 52 strains of lincomycin-degrading bacteria from the samples which were collected from the waste mycelium storage place and activated sludge in the Nanyang Pukang Group Chemical Pharmaceutical Factory.(2) Ultraviolet spectrophotometry, visible spectrophotometry and high performance liquid chromatography were adopted to determine the lincomycin in the basal culture. Herein we have validated the rationality and feasibility of the three methods by determining their linear correlation, precision, reproducibility, stability and recovery of lincomycin in the basal culture. At last, we found that visible spectrophotometry was convenient and rapid to determine the lincomycin at the initial stage of the isolation for many samples, the ultraviolet spectrophotometry was a relatively accurate method for the biodegradation characteristics research, and high performance liquid chromatography was the best method to validate the optimized conditions.(3) We established two new methods to screen lincomycin-degrading bacteria by agar plate and silica gel plate and optimize their conditions. Finally, three strains labeled as LD9#, LD19# and LD25# were obtained with lincomycin degrading ability exceeding 50% by these two methods and the traditional screening methods.(4) The results of morphological observation, physiological and biochemical identification and 16S rDNA sequence analysis indicated that LD9#, LD19# and LD25# belonged to Ochrobactrum sp., Sphingobacterium sp. and Stenotrophomonas sp., respectively.(5) Single-factor, two-factors and orthogonal tests were adopted to inspect the effects of pH, inoculation amount, culture time and rotation speed on the degradation rate of the three strains and to optimize the degradation conditions. And the results showed that:For the LD 9# strain, the optimal degradation conditions of external carbon source, the initial pH of the culture, culture time, rotation speed, the inoculation and lincomycin concentration were 0.1 g/L starch,9.0,37℃,80 rpm/min,10% and 1.5 g/L, respectively.For the LD 19# strain, the optimal degradation conditions of external carbon source, the initial pH of the culture, culture time, rotation speed, the inoculation and lincomycin concentration were 0.1 g/L starch,7.0,37℃,80 rpm/min,10% and 1.5 g/L, respectively.For the LD 25# strain, the optimal degradation conditions of external carbon source, the initial pH of the culture, culture time, rotation speed, the inoculation and lincomycin concentration for the were 0.25 g/L starch,7.0,37℃,80 rpm/min,10% and 1.0 g/L, respectively. Under the optimal conditions, the results of the verification test demonstrated that the degradation rates of the three strains exceeded 90%.