Isolation, Identification Of Petroleum-degrading Bacteria And Analysis Of Intestinal Microbial Diversity In The Digestive Tract Of Perinereis Aibuhitensis (Polychaete)

Polychaetes are important model organisms of marine invertebrates in estuarine sediments witha wide geographical distribution. Considering their short-distance migration characteristic,polychaetes are known to accumulate significant amounts of organic matter from theenvironment and steady-state body burdens are a function of biotransformation and eliminationprocesses. In the present study, methods of microbial cultivation were used to isolate thepetroleum-degrading bacteria in the digestive tract of Perinereis aibuhitensis (Polychaete),furthermore, DGGE was also played to analyse the distribution of isolated bacteria and theintestinal microbial diversity.In the present study, the Perinereis aibuhitensis were collected from the shoreline of Panjin(Liaoning Province, China) and transformed to the laboratory. After being starved for24hours,the gut samples of5healthy worms were removed and the diluted homogenate was daubed onsolid2216E agar plate media. Then incubated at25℃for24-48h,7strains were eventuallyselected and purified according to different colony morphology and3of them can grow on themineral medium supplemented with diesel oil as a sole source of carbon, number SC11-1、SC11-2、SC11-3. and SC11-3strain was selected as the test strain for its relatively good growth.After determining degradation efficiency quantitatively by UV spectrophotometry, strain SC11-3was selected as the test strain for its relatively good growth and degradation ability. Detailedanalysis of morphological, biochemical and16S rDNA sequence were used to identify the isolateand its growth characteristics were determined, meanwhile the oil-degrading efficiency was alsostudied associated with different concentrations of glucose. Moreover, in order to further revealthe intestinal microflora structure-distribution related biological characteristics of Pernereisaibuhitensis presented as polychaetes, stating the bioavailability and tolerability mechanisms ofpersistent organic pollutants of marine sedimentary inverabrates, the total genomic DNA wasisolated from gut samples of5healthy Perinereis aibuhitensis, respectively. Then the V3regionof the16S rRNA genes (approximately230bp) was amplified with specific primers set ofGC-341f/534r. The DGGE band profiles of immobilized bacteria in the digestive tract ofPerinereis aibuhitensis were finally generated. The results indicate: strain SC11-3wasGram-negative, rod shaped and16S rRNA sequencing showed that strain SC11-3was closelyrelated to Peudoalteromonas haloplanktis, and the homology was100%. The experimentalresults of growing characteristics showed the optimum growth temperature and pH of strainSC11-3was25℃and8, respectively; the adaptive NaCl concentration was in the range of1-3%.After being oscillation incubated (150rpm/min) at25℃for15days, about40%diesel oil wasinitially consumed by SC11-3strain. The degrading efficiency of strain SC11-3was significantlyenhanced when adding glucose to a specific concentration of0.4g/L, and the degradation of diesel achieved more than50mg. But as more glucose was added, the degradation efficiencydropped. The semi-quantitative analysis of DGGE map showed that22,21,18,18,19bandswere generated in5gut samples, respectively and the common predominant band was found, butthe relative content of each predominant band was different. The sequencing and blast results ofthe DGGE common bands are listed as follows: Pseudoalteromonas sp., UnculturedPseudoalteromonas sp., Elizabethkingia sp., Uncultured Propionigenium sp., Unculturedbacterium, Bacterium, Uncultured Pseudoalteromonas sp., Alteromonadaceae bacterium,Uncultured γ-proteobacterium, Pseudoalteromonas sp. and the results indicate thatPropionigenium sp. is the predominant intestinal microflora in the5samples, respectively andPseudoalteromonas sp. is widely distributed in the digestive tract of Perinereis aibuhitensis.