Isolation,Identification Of Acinetobacter Pittii L1 And The Analysis On Indole Degradation Mechanism

Nitrogen Heterocyclic Compounds?NHCs?are the class of heterocyclic compounds containing N atoms.Nitrogen heterocyclic compounds are widely found in environment,including medical,pesticide,coking,oil smelting,dyes waste water and other industrial wastewater.NHCs are covering a wide scope of pollution-prone and refractory organic matter with a wide range of environmental effects,which poses a great threat to the environment and human beings.Indole,pyridine,quinoline,isoquinoline,pyrrole,imidazole and its derivatives are common nitrogen-containing heterocyclic compounds.Researching for a suitable method to remove such compounds have become an urgent problem.In this paper,indole was the main research object.We screen out a strain that could degrade indole efficiently under aerobic condition.The degradation of indole,the change of biotoxicity,the pathways of metabolites using this strain and the purification of indole degrading enzymes were investigated by microbiology and molecular biology.The main results are as follow:1.A bacillus L1 that could degrade indole effectively under aerobic conditions was isolated from the sludge water samples collected from the coking plant wastewater treatment system.Based on the physiological and biochemical experiments and 16 S rRNA gene homology analysis,the strain was confirmed to be Acinetobacter pittii,and then named Acinetobacter pittii L1.2.The degradation of indole by strain L1 was studied.The results showed that the strain had the best degradation effect at 37 °C,pH 7.0 and 150 r/min.While low concentrations?100 mg/L?of indole were completely degraded at 18 h,400 mg/L indole required 2 d to be completely degraded.And it also showed a better degradation effect on low concentration of quinoline and indole,but it was not ideal for the degradation of phenol and pyridine.3.The removal of TOC and the change of toxicity were investigated during the reaction of the system.The results show that when the indole was completely degraded TOC and TC were not completely removed.Most of the organic carbon is converted to CO₂ to the atmosphere,and a small fraction is converted to CO32-and then to metal salt with ions in the solution.There may be indole decomposition into small molecules,transferring to the atmosphere after the volatile products.The results of the biotoxicity test show that most of the indole is slowly degraded during the process,partially converted to CO₂ and H2 O,and diffused into the atmosphere,which indicates that the indole has not been completely metabolized as intermediates.In addition,the process of indole biodegradation produced some metabolites more toxic than indole itself.4.The products of indole biodegradation were estimated to be indigo,isatin and 4-?3-hydroxy-pyrrole?-2-oxobutenic acid by the analysis of secondary mass spectra of HPLC-MS/MS.On one hand,Indigo was produced by the dimerization of oxindole,and isatin was the result of the indole oxidation to indolone then farther.4-?3-hydroxy-pyrrole?-2-oxobuturic acid,on the other hand,was the products of dihydroxyindolewhich was formed by the C4 position of the benzene ring in indole being attacked and opening the benzene ring.According to these biodegradation metabolites,the three metabolic routes of indole were proposed.5.The experiment also studied the characterization of the purified indole degrading enzymes.The results showed that it was mainly composed of intracellular protein.The specific activity of the enzyme protein purified by DE-52 method was about 9 times of the crude enzyme solution,and the purification effect was ideal.The optimum temperature for the enzymatic reaction was 35 °C,the range of pH was 6.0-7.0.Different metal ions were tested to have a great impact on the enzyme,so that it was suspected to be a metal enzyme.While 1.0 mM Ca²⁺ and Mg²⁺ promoted the enzymatic reaction,the same concentration of Zn²⁺ showed a strong inhibitory effect on the enzymatic reaction.