Salt-tolerant Mechanism Of A High-efficiency Diesel-degrading Strain HX-2 And Properties Of Exopolysaccharide

Oil spills caused by oil extraction and transportation process are becoming more serious to the environment.Compared with traditional physical or chemical remediation,biological remediation does not cause secondary pollution,and is energy-saving and environmentally friendly.However,the actual oil-contaminated environment is often accompanied by very high salinity,and these salts will seriously inhibit the growth of microorganisms,causing the restoration effect to be greatly reduced.In view of this situation,we screened an efficient diesel-degrading strain to investigate its salt tolerance mechanism and the properties and structure of extracellular polymers.(1)The strain HX-2 isolated from the Karamay oilfield of Xinjiang,it was identified as Gram-positive bacteria by Gram staining and scanning electron microscopy.It was not moving,short rod-shaped,no spores,and had typical splayed arrangement.The colony morphology on the LB solid plate was colony size 1-2 mm,yellow,intact,round,opaque,and shiny.The physiological and biochemical characteristics of the comprehensive strain HX-2 and the results of 16S r RNA sequence analysis confirmed that the strain HX-2 was Rhodococcus sp.,and named it Rhodococcus sp.HX-2.HX-2 can tolerate 10%salinity(Na Cl)and 8000 mg/L diesel concentration,respectively;the optimum p H,temperature for growth and degradation are 7.0 and 25°C,respectively;the salinity is within 5%,p H=7.0,temperature 25°C and 2%inoculum size,the percentage degradation of diesel with initial concentration of 4000 mg/L after 4d is more than 50%and the percentage degradation is still 10.3%at 10%Na Cl.(2)Studies on the tolerance mechanism of HX-2 showed that there was no significant difference in intracellular K~+/Na~+when the salt concentration changed,and the compatible solute(betaine)accumulated with the increase of Na Cl concentration.It indicates that the accumulation of betaine is the main mechanism for HX-2 to cope with high salt environment.(3)The response of four betaine transporters H0,H1,H3,H5 and betaine-producing gene bet B to Na Cl stress was observed in HX-2.Transcriptional analysis indicated that Na Cl stress induced the production of H0,H1,H3,H5 and bet B;SEM data showed that the addition of exogenous betaine promoted salt tolerance by stimulating the production of exopolysaccharides(EPS).In high-salt solutions or soils,the addition of exogenous betaine promotes cell growth and diesel degradation.(4)HX-2 EPS produced was optimized by RSM to achieve a yield of 8.957 g/L in MSM medium.HPS was separated and purified by ethanol precipitation,DEAE-cellulose column and Sepharose column,the yield of HPS(pure product)reached 3.736 g/L.The molecular weight of HPS was determined by gel chromatography to be 1.04×10~6 Da.The total carbohydrate,protein and lipid content in HPS was 79.24%,5.204%and 8.45%(w/w).HPS consists of fucose,glucuronic acid,galactose,mannose and glucose with mass percentages of 4.79%,15.84%,24.83%,26.66%and 27.29%,respectively.(5)FT-IR spectroscopy indicates the presence of a large amount of hydroxyl groups in the HPS.NMR spectroscopy indicated that the sugars have both?and?-configuration in HPS.The degradation temperature of HPS was determined by thermogravimetric analysis(TGA)to be 255.4°C.The HPS has a network structure by SEM,and AFM analysis reveals that it exhibits a linear shape.At the same time,the WSI and WHC of HPS are 92.15±3.05%and 189.45±5.65%,respectively.The solution viscosity was 18 m Pa·s at HPS concentrations of 1 mg/ml and 30 rpm.Finally,in vitro anticancer activity was assessed on L929 normal cells,A549,SMMC-7721 and He La cancer cells.HPS inhibits the growth of cancer cells at a certain concentration without damaging normal cells.This paper reports for the first time that the salt tolerance mechanism of Rhodococcus sp.HX-2.It is the accumulation of compatible solute(betaine),and the exogenous addition of betaine can promote the production of EPS and help it better resist the high salt environment.The expression levels of betaine transporter gene and betaine production gene under high salt conditions in Rhodococcus sp.were also studied for the first time.Analysis of EPS indicates its potential application value in the food,industrial and pharmaceutical sectors.In summary,Rhodococcus sp.HX-2can provide valuable reference value for strains in salt tolerance and industrial production of EPS.