| In this study, a simple pre-column derivatization-high performance liquid chromatographic (HPLC) with p-tosyl chloride as derivation agent method was established for the determination of anhydrous piperazine in water. Using orthogonal experiment, the optimum conditions of derivatization reaction were:reaction temperature55℃, reaction time10min, pH of buffer10and derivation agent overdose. The method established is easy operating, good reproducible and results reliable, and can be widely used in laboratory analysis.A highly effective piperazine-degrading bacterium was isolated from the acclimated activated sludge of a pharmaceutical plant by enrichment and separation technology, which could utilize piperazine as the sole source of carbon, nitrogen and energy for growth and the strain was named as TOH. It was preliminary identified as Paracoccus aminovorans according to its physiological and biochemical properties and combined with16S rRNA gene sequence analysis of the strain and its homology comparison.The strain TOH grew well in LB liquid medium, after8h bacteria into the logarithmic growth and34-54h was the stable growth period. The optimum temperature and pH for the growth of strain TOH were30℃and7.0～8.0, respectively. Little liquid volume was better for the growth of the strain TOH. Ventilation had effect on the growth of the strain; larger dissolved oxygen in the water was more benefit for the growth. The strain could highly utilize organic carbon source such as fructose, xylose, sucrose, maltose, glucose and so on; for nitrogen source using experiments, TOH could highly utilize peptone and followed are nitrate-N, nitrite-N and ammonium nitrogen. The strain TOH with salt tolerance could grow well in the range of5-30g/1NaCl concentration, so it has the outstanding advantages in wastewater treatment applications.The strain TOH could completely degrade100mg L-1piperazine in24h in MSM culture; when cultured in the medium, the OD600increased with the degradation of piperazine, and along with the release of ammonia, which indicated that the strain could use piperazine as the sole carbon and nitrogen source for growth. The optimum temperature and pH for piperazine degradation by strain TOH were30℃and7.0～8.0, respectively, the same as its growth characteristics. The strain was more sensitive to acidic environment than alkaline environment. The lower initial concentration, the easier utilization of piperazine by the strain, generally, the strain had a higher resistance to piperazine. A certain concentration of Mg2+can promote the degradation of piperazine while Co2+、Cd2+and Ni2+had the greatest effect on the bacteria growth and inhibited the piperazine degradation significantly. Addition of glucose as the co-substrates could promote the growth of the bacterial biomass and increase the rate of degradation of the piperazine while ammonium chloride has little effect.The metabolites were detected by LC-MS, and the pathway of piperazine biodegradation by strain TOH was proposed. Through the mass spectrometry analysis, speculated biodegradation pathway was:The first step of the biodegradation pathway was cleavage of a C-N bond by the release of NH3, leading to N, N-diacetaldehyde amine. The next step was cleavage of another C-N bond to form2-aminoacetaldehyde. Next,2-aminoacetaldehyde was degraded to ammonia and glyoxal. Then, glyoxal was converted to oxalic acid and then into the metabolic cycle. Finally, piperazine was mineralized to carbon dioxide and water. The inner and extra-cell crude enzyme from bacteria TOH were also be determined. Activities were measured by the degradation that extracellular enzyme activity was higher in the degradation of piperazine while intracellular enzyme activity was lower. Results showed that the piperazine degrading enzyme was extracellular enzyme. |
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