Enhanced Rhamnolipid Production By Genetically Engineered Strain And Research On Bioremediation Of Petroleum Contamianted Soil By Pseudomonas Aerguginosa DN1

Petroleum pollution has drawn the worldwide concern and bioremediation is considered to be a promising method.The low water solubility of petroleum contaminants have greatly restricted the bioavailability,thereby the addition of surfactants is a good option to achieve much more efficient biodegradation.Rhamnolipids,as one of biosurfactants,have a great prospect with advantages of non-toxicity,easy degradation,and solubilization of hydrophobic carbon.However,the high-cost and low yield of rhamnolipids have been an obstacle in the application widely.In this research,the construction of engineered strains and optimization of nutritional parameters for enhancing the yields of rhamnolipids in Pseudomonas aeruginosa DN1 was investigated.Then experiments were designed to explore how to stimulate indigenous bacteria for the degradation of crude oil pollutants by microbial consortium supplemented with rhamnolipids.A molecular biology strategy was used to construct the engineered strains DNAB and DNC through introducing rhlAB and rhlC genes respectively to Pseudomonas aeruginosa wild-type strain DN1,and optimization of nutritional parameters such as carbon and nitrogen sources were assessed simultaneously.The optimum carbon and nitrogen source were olive oil and sodium nitrate respectively,when six carbon sources?palm oil,rapeseed oil,soybean oil,glycerol,corn oil,and olive oil?and six nitrogen sources?ammonium chloride,sodium nitrate,ammonium nitrate,potassium nitrate,ammonium sulfate,and urea?were chose as the sole carbon and nitrogen source.In addition,the ideal C/N ratio was found to be 20 that increased specific rhamnolipid productivity to 19.3 g/L and 22.5 g/L of the engineered strains,a certain amount to 1.35-fold and 1.58-fold of DN1 strain respectively.Thus,rhamnolipids productivity of strain DN1 could be enhanced by means of increasing the copy number of rhlAB and rhlC genes respectively,as well as optimization of nutritional parameters.Meanwhile,Rha-Rha-C₁₀0 and Rha-Rha-C₁₀-C₁₀were the dominant structure of rhamnolipids for the engineered strains DNAB and DNC through ESI-MS analysis.Furthermore,bioremediation of petroleum-contaminated soil was conducted to explore how to stimulate indigenous bacteria for the crude oil degradation by microbial consortium supplemented with rhamnolipids.The results showed that the degradation efficiency of total petroleum hydrocarbon?TPH?in natural amendment?NA?,bioremediation under the addition of rhamnolipid?BR?,bioremediation through microbial consortium?BM?and bioremediation by microbial consortium supplemented with rhamnolipids?BMR?were 43%,39%,63%and 81%after 35 days to cultivation,respectively.Bioremediation by microbial consortium supplemented with rhamnolipids?BMR?significantly enhance TPH biodegradation compared to the other treatments.Bacterial diversity analysis showed that BM,BR and BMR increased the richness of indigenous microorganism communities,it also demonstrated the richness and diversity of BMR which was the highest in the all treatment.For the NA sample,Gordonia?25%?and Nocardioides?15%?were the most abundant.In the BR and BM samples,Gordonia was the most abundant,ranging from 40%to 42.3%.Pseudomonas came second?7.3-8.1%?,and Nocardioides was the third?1.7-2%?.Other predominant genera were well represented,based on the average abundance analysis.The generic profile in the BMR sample was unique except Gordonia?23%?was the most abundant,while Pseudomonas?7.8%?and Pseudoxanthomonas?7.3%?were the second most dominant genus.In addition,some genera which occupied a relatively small proportion of the community composition were also found in the BMR sample,including Mycobacterium?5.6%?,Cavicella?5.6%?,Rhizobiaceae?4.6%?and Acinetobacter?3.9%?,which have been associated with biodegradation of petroleum hydrocarbons.Moreover,BMR treatment increased the enzyme numbers associated with hydrocarbon degradation and transporters enhance the transport of degrading bacteria to the bioremediated position.Our findings suggested that petroleum hydrocarbons in contaminant soil can be removed efficiently under the influence of microbial consortium supplemented with rhamnolipids by changing soil microbial community structure enrich the degradative bacteria,which is in favor of bioremediation of pollutants.