Domestication Of CIP-degrading Bacterial Consortium And Preliminary Study On Their Degradation Characteristics

The widely use of antibiotics for large-scale animal farming,sewage irrigation and the application of bio-organic fertilizer leads to serious threats to soil antibiotic comtanination.The antibiotics accumulated in soils will be uptake by crops,seriously affecting quality and safty of agricultural products.The consumption of agricultural products containing antibiotics in diet,leading to long-term low-dose exposes and posing risks to human health.Therefore,the effective and advanced remediation technologies for removal of these contaminants are critically needed.Currently,the main remediation technologies to remove antibiotics are physical and chemical methods.By contrast,biodegradation has advantages with low consumption,high efficiency and non-secondary pollution,and it can effectively remove the organic pollutants in environment.At present,there are few reports about CIP-degradable bacteria.Studies show that Alphaproteobacteria and T.versicolor can degrade CIP.Therefore,in this study,quinolones antibiotics CIP as the main subject,manure samples were collected as inoculum for isolation of efficient degrading bacterial consortium HD-1.High-throughput molecular technology was used to determine structure diversity of bacterial consortium HD-1.The Biolog-ECO plates were applied to study the utilization of different carbon sources by flora HD-1 and screened out optimal carbon sources for CIP-cometabolism.The effects of different co-metabolised carbon sources on degradation flora growth and CIP degradation was also studied.At last,the effects of plant-microbial symbiosis system on the accumulation of CIP in Chinese flowering cabbages with higher and lower accumulation of CIP were further studied.The following results were obtained in this study:(1)The methods of domesticating microorganism degrading CIP was estabolished.CIP-degrading community HD-1 was obtained from livestock and poultry manure through enrichment and domestication method with different kinds and concentration of carbon sources.PCR-DGGE technique was used to monitor the community composition change.The result showed that microorganisms from genera Rhizobium anf Ochrobactrum showed dominance during domestication process.High-throughput sequencing technique was applied to depict the structure of acclimated microbiota HD-1,and the results showed that HD-1 was dominant by phyla Proteobacteria,Firmicute and Bacteriodetes.And at class level,Betaproteobateria,Bacilli and Alphaproteobacteria dominated in the HD-1.At genus level,microorganisms from genera Achromobater,Bacillus,Lactococcus,Ochrobactrum and Enterococcus showed dominance in community HD-1.(2)A strain obtained from CIP-degrading community HD-1 can degrade CIP named JOB.Based on the morphology,physio-biochemical characteristics,and 16 Sr DNA gene sequence analysis,the degrading strain were identified as Ochrobactrum.sp.The degradation ability of CIP by community HD-1 and single strain JOB was investigated,and the result showed that the addition of carbon sources could significantly enhance the degradation ability.In addition,the degradation rate of community HD-1 was obviously higher than that of single bacterium.(3)The effect of CIP concentration on utilization of different carbon sources by consortium HD-1 was also investigated by Biolog-ECO plates.The promoted utilization of different carbon sources by consortium HD-1 at a lower concentration(?5 mg·L-1)of CIP was observed.The higher concentration(>5 mg·L-1)of CIP inhibited the utilization of different carbon sources by consortium HD-1.Principal component analysis of the utilization of 31 carbon sources showed that the co-metabolic carbon sources of ammonia acyl-L-glutamic acid,itaconic acid and D-cellobiose enhanced the biodegradation of CIP by 62%,56%,and 51%,respectively.In addition,a considerable biomass increase was occurred in the microcosms.(4)CIP-degrading community HD-1 could reduce the toxic effect on high-and low-CIP accumulation cultivars of Chinese flowering cabbages and reduce the content of CIP in shoot,in which the CIP content of the high cumulative cabbage was much more decreased.