Study On Toxic Effects Of Biosurfactant Produced By Bacillus Sp. D32 And Its Effect On Bacterial Community

Compared with chemical oil spill dispersants,biosurfactant-based oil spill dispersants are less toxic and easily degraded.It shows more superiority to deal with oil pollution in ecologically sensitive areas.However,there are limited research on it until now,and the current related researches mainly focus on the theoretical research of promoting the dissolution,emulsification and degradation of petroleum hydrocarbons by biosurfactant,while few on its toxic effects and influence on bacterial community.In this paper,the petroleum degrading bacterium Bacillus sp.D32 was used as the biosurfactant producter.Biosurfactant production,ecotoxicity and effects on bacterial community were studied,and the mechanism of biosurfactants strengthening bioremediation of oil spill was analyzed on the gene level.The main research results of this paper are as follows:(1)The strain Bacillus sp.D32 could use diesel as carbon source to grow and produce BS-D32.After the addition of 0.5%,1% and 10% biosurfactant,the effect of degradation increased from 46.1 to 52.6%,65.3% and 62.3%.(2)Bacillus sp.D32 can use a variety of carbon and nitrogen sources,and diesel fuel,n-cetane and glycerin were the suitable carbon sources to produce surfactant.Peptone,yeast extract and ammonium nitrate are suitable nitrogen sources.The optimal temperature range for the production of BS-D32 was 15-20℃,the optimal salinity range was 15-35 g/L,the optimal p H range was 6-8,and the optimal bacterial solution addition range was 5%-10%.The results of response surface analysis by Design-Expert software showed that the optimal fermentation conditions were temperature 16.2℃,p H 6.8,Na Cl concentration 21 g/L,adding7.9 % of bacterial solution,and the surface tension of fermentation solution could be reduced to 28.4 m N/m.(3)The toxic effect of BS-D32 produced by Bacillus sp.D32 was studied using Chlorella as the test organism.The results showed that after adding 1% BS-D32 for 120 h,the density of Chlorella increased by 13.8% ~ 40% and the soluble protein content increased by4.1%~5.8%.When the amount of biosurfactant exceeded 1%,the growth of Chlorella was inhibited.(4)High-throughput sequencing was used to study the effect of adding BS-D32 on bacterial community structure.The results showed that the abundance of bacterial community increased significantly after 7 days of adding biosurfactant in seawater contaminated with crude oil.When low concentrations of biosurfactants were added,microbial diversity increased.The bacteria with significant changes in dominance included Alcanivorax,Rhodococcus,Marinobacter,Sphingobium,Pontivivens,tc.(5)The function of the bacterial community also changed significantly after the addition of biosurfactants to the crude oil contaminated seawater.The analysis of functional genes showed that the content of functional genes related to petroleum hydrocarbon degradation increased significantly.For example,when 0.5%-10% biosurfactants were added,the naphthalene degrading gene,cytochrome P450 and toluene degrading gene increased by1.4~2.3 times,0.4~0.6 times and 0.9~1.9 times respectively.Therefore,the bacterial community in oily seawater can significantly improve the degradation function of petroleum hydrocarbons after the addition of surfactants.