Functional Optimization Of Seawater Diesel Oil-degrading Bacteria And Its Biological Carrier Establishment

ObjectiveTo find marine diesel oil-degrading bacteria, construct higher effective flora fordegrading oil pollution and establish immobilization of bacterial consortium, whichused for supplying an evidence in bioremediation of marine diesel pollution.Methods1. Seawater obtained from diesel fuel pollution at harbor was served as a source ofseparation, which was inoculated into the man made marine culture (MMC) containingdiesel fuel for5periods (each period last5days) of enrichment and culturing, and thensample was separated and purified to acquire single bacterial colony named F9.Above-mentioned bacteria was conducted with morphological observation,physiological and biochemical property analysis and16s rDNA sequencing foridentification.2. Property of degrading diesel fuel with F9was investigated by measuring varioustemperature, pH value, initial inoculum concentration and oil concentration withultraviolet spectrophotometry and degrading characteristics of each component from thediesel fuel was also examined with gas chromatography-mass spectrometry (GC-MS).3. Four bacterial strains showing high degradation obtained from diesel pollutedseawater via bacterial enrichment and screening were combined and optimized toexplore its degrading ability. Oil degrading rate by flora and diesel components changeswere respectively observed with ultraviolet spectrophotometry and GC-MS.4. Bacterial consortium were immobilized by sodium alga acid-chitosan-millet, Oildegrading rate by flora and diesel components changes were respectively observed withultraviolet spectrophotometry and GC-MS. Results1. Identification showed that the strain F9was Acinetobacter, whose sequence was99.9%identical with Acinetobacter venetianus. F9was strictly aerobic,non-spore-forming, Gram-negative rods, catalase-positive and oxidase-negative, whichcould not utilize D-glucose, D-xylose, L-arabinose, and D-ribose, but growed in theculture medium containing sodium citrate as carbon source and used potassium nitrateor ammonium sulfate as nitrogen source. It did not require growth factor and survived at37℃but not44℃.2. The results from present study showed that the optimum condition of F9growthwas in50mL MMC, pH7.5,30℃,0.5%of the initial inoculum concentration and0.5%of diesel concentration, which made the diesel oil degradation rate highest at90.24%after7day culture, and it was capabel of degrading n-alkanes in diesel fuelfrom C11~C18with GC-MS.3. Combination and optimization of4strains was carried out under lab condition tofind that flora was named S1、F9、Y9、W3together and their proportion was1:1:2:1correspondingly, which displayed the best degrading rate with condition of50mLMMC, pH7.5,30℃,1%of initial inoculum concentration and0.5%of dieselconcentration. Degrading rate of mixed flora reached71.25%after2day culture. Thebacterial consortium was capable of degrading n-alkanes in diesel from C11~C19withGC-MS.4. Mixed flora were immobilized by complex of sodium alga acid-chitosan-millet,which showed the diesel oil degradation highest at94.98%after3day culture and at95.15%after7day culture that not only absorbed and disaggregated diesel oil inseawater but also used up effects of combined flora on biodegradation for diesel oil inseawater. Compared with dissociated flora, immobilization of mixed flora displayedhigher degradation rate benefiting the environment protective repair from seawaterpollution with diesel oil at harbor.ConclusionAcinetobacter venetianus named F9is an efficient bacterium for degrading dieselfuel in polluted seawater. An immobilization of mixed flora express the strongerdiesel-degrading ability in seawater and their substrate range is also widespread and it is a big hope to utilize mixed flora repairing polluted seawater with diesel oil biologically.