The Optimization Of Cultivation Conditions Of The Deep Sea PAH-degrading Bacterium Cycloclasticus Sp.P1and The Preliminary Study On Its Degradation Pathway Of Pyrene

A highly effective and broad-spectrum PAHs degradation bacterium,Cycloclasticus sp.P1has been isolated in our laboratory. Different from other strainsthat belong to the genus of Cycloclasticus, Cycloclasticus sp.P1can degrade pyrenewithout any other LWM-PAHs as aided carbon source. At the mean time, some of themetabolites of Cycloclasticus sp.P1grew on pyrene were different from that of anyother strains that have been known, which means the pyrene degradation pathway ofCycloclasticus sp.P1maybe different from other strains. So we carried on the study ofthe pyrene degradation pathway of Cycloclasticus sp.P1to know the extact details.The P1strain belongs to "hard-culture" microorganism,could't be cultivatedwith the common media in our laboratory, so the first part of my study was tooptimize the cultivation conditions of the P1strain. As for aquatic cultivation, afterthe analysis of KEGG map of P1and a serial of optimization, the P1strain could beeasily cultivated with these conditions: ML-1medium,32℃of cultivationtemperature,200rpm of shaking speed. And a more thorough optimization with theResponse Surface technique was applied on ML-1medium, resulting in ML-2, whichexhibited much better cultivating performance than MM-2, thus the aqucticcultivation problem of the P1strain was soundly solved.And the petri dish cultivation of the P1strain was a problem for a long time.Firstly, it could only grow on the1/5216E plate with naphthalene on the lib,providing naphthalene vapour as the PAH carbon source. And even the ML-1platecould not surport the growth of P1clone. Altrasoniced the P1cell at the stage ofOD600=0.5, made P0plate with this kind of mixture, P1clone could grow on this plate,then the petri dish cultivation problem was solved. The second part of the study was to figure out the pyrene degradation pathway ofthe P1strain with the polyomics data of genome, transcriptome and proteome.The P1strain harbored five gene clusters that were responsible for the PAHsdegradation, and most of them were responsible for the upstream pathway. From thegene clusters, we could figure out that genes responsible for the upstream pathwaytent to gather together, while the genes responsible for the downstream pathwayscattered in the genome.Combining the analysis of the data of the transcriptome and proteome, togegherwith former research results, a surely conformed pyrene degradation pathway of theP1strain was elucidated. Its upstream pathway was similar to that of the genus ofMycobacterium, pyrene was dioxygenased and cycle-broken two times to form the1-Hydroxy-2-naphthoate. And its dowmstream pathway was similar to Pseudomonassp.PP2, through salicylic acid pathway and TCA cycle,1-Hydroxy-2-naphthoatecould be completely degraded.In sum, we added sodium pyruvate, sodium citrate, yeast extract, sodiumL-glutame to MM medium and after a serial of optimization, the problem of aquaticcultivation of the P1strain with non-PAHs was souldly solved. And ultrasonized theP1cell fermentation broth when its OD600was0.5, added1%agar to the lysate tomake plate, and the P1strain could grow on this plate, so we basically solved the petridish cultivation problem, which provided valuable insigts on the "hard-culture"problem. Meanwhile, analysis the pyrene degradation pathway of the P1strain withpolyomics data provided useful information on pyrene degradation mechanism of thegenus of Cycloclasticus.