Microbial Ecological Mechanism For Degradation Of Norfloxacin During Chicken Manure Composting

Antibiotics were not only used for disease therapy of human beings, but also applied to livestock and poultry industry as the feed additive. However, part of the used drug was excreted through feces and urine because of its incomplete absorption by animals. And the organic fertilizer containing antibiotics went into agriculture fields, which would pose great threat to agriculture ecological environments and human health. In the present thesis, a high performance liquid chromatography（HPLC） method was developed for meanwhile determination of 11 antibiotics residues in excrements of livestock as the foundation of solving antibiotics residue problem. In addition, norfloxacin was chosen as a model compound of fluoroquinolones, and the foam insulation boxes severed for composting reactors. The effects of the C/N and exogenous microbial agent on norfloxacin degradation and compost parameters were mainly investigated during chicken manure composting using the method of aerobic-composting. And microbial ecological mechanism for degradation of norfloxacin were also researched from the molecular level using modern sequencing technology. The specific results of the survey as follows:（1）A simple and highly efficient economic method- solid phase extraction（SPE） and high performance liquid chromatography（HPLC）- for the analysis of eleven antibacterial drugs in livestock manure was established through the optimization of extractant, SPE column, leacheate and eluent. Recoveries calculated from spiked samples of animal manures ranged from 62.7% to 99.2% for eleven antibiotics. Limits of detection ranged from 0.1 μg?kg^-1 to 1.9 μg?kg^-1, and limits of quantification ranged from 0.3 μg?kg^-1 to 5.9 μg?kg^-1. The results show that the established SPE-HPLC is an inexpensive and practical method for rapid detection of multiple antibiotics in animal manure. Using the above method, antibiotic residues were determined in cow manure, pig manure and chicken manure from large-scale farms of Beijing suburb. The results found that tetracyclines, fluoroquinolones and sulfonamides were given priority to antibiotic residues, accounting for more than 99% of total residues in the animal manure. Chicken manure are the most antibiotic-contaminated feces, and three kinds of antibiotics could be detected in all of samples.（2）The pile initial C/N and exogenous microbial agents had effects on norfloxacin(100 mg?kg^-1) degradation in compost of chicken manure. The removal efficiency of norfloxacin with C/N of 25 was superior to that of other treatments. At the end of composting, the removal rate of norfloxacin with C/N of 25 was 71.37%, increasing by 13.18%（C/N of 10）, 1.76%（C/N of 15）, 0.25%（C/N of 20）, 15.69%（C/N of 35）, respectively. And the germination index with C/N of 25 was 82.14%, compared with other treatments, increasing by 3.90%-25.42%. To a certain degree exogenous microbial agents improved the degradation of norfloxacin. Thereinto, bacteria had an insignificant advantage than fungus. And exogenous bacteria reduced the toxicity of compost and promoted the decomposition. At the end, the germination indexes with exogenous bacteria were 82.28%（high concentration） and 80.55%（low concentration）, while others were below 80%. The high concentration of exogenous fungi decreased the loss of nitrogen in the composting, and the reduction rate of total nitrogen was 11.31%, which was lower 1.84%-7.93% than others.（3）Learned from the degradation regularity of norfloxacin in compost of chicken manure, the existence of norfloxacin shortened the time of thermophilic, reduced the pile pH, and increased the total nitrogen content. The degradation of norfloxacin during the composting satisfied the first-order kinetic equation of the correlation coefficient between 0.7357-0.9396. But the degradation of different concentration existed differences, the removal efficiency with the added concentration of 50 mg·kg^-1 was the best and the removal rate was 80.78%. High concentration of norfloxacin would suppress the microorganism activeness and low content could promote the absorption of antibiotic on compost substrates, which had adverse effects on drug removal. The treatments with norfloxacin more than 50 mg?kg^-1 lowered the maturity significantly（GI<80%）. The dynamic of microbial diversity in different treatments showed that norfloxacin could improve the abundance of microbial community in the thermophilic and the fungal diversity in the whole process of compost, and had no effect on bacterial diversity. The main bacteria including bacillus, sporosarcina and bacteroides and the main fungi covering issatchenkia, aspergillus, wallemia and an unidentified genus may exist intimate connection with norfloxacin degradation.