Study On The Properties Of A Cr(?) Reducing Bacteria And Its ARTP Mutagenesis To Optimize Cr(?) Reducing Ability

Chromium and its compounds are widely used in metallurgy,chemical industry,leather,printing and dyeing,electroplating and other fields,bringing great economic benefits to our country,but at the same time it also brings serious pollution to the environment and threatens human health.In recent years,microbiological methods have opened up a new field with potential for Cr(?)pollution remediation.Microorganisms can continue to grow and reproduce,have a strong ability to adapt to the environment,and the use of microorganisms to treat chromium pollution has the characteristics of low investment cost,low energy consumption,and no secondary pollution.At present,more and more Cr(?)reducing strains are reported by researchers.In this study,a Cr(?)reducing strain Ylb10 was isolated from a group of bacteria YEM001 that effectively reduces Cr(?)through the method of gradient dilution plate coating,and its biological characteristics and reduction of Cr(?)was studied.In order to improve the ability of Ylb10 bacteria to reduce Cr(?),the Atmospheric and Room Temperature Plasma(ARTP)was used for mutagenesis screening.A mutant strain L1-3with higher ability to reduce Cr(?)was obtained,and the Cr(?)reduction performance of the original strain Ylb10 and the mutant strain L1-3 were compared.The results are as follows:(1)The Ylb10 strain isolated from the YEM001 flora,through physiological and biochemical methods and 16 S r DNA sequencing,the similarity rate between Ylb10 strain and Alicycliphilus denitrificans(Alicycliphilus denitrificans)was 98.07%,and Gram staining showed negative bacteria.Through the analysis of the substrate utilization of Ylb10 strain through the GEN ? identification board,it was known that the main substrates used by Ylb10are:methyl pyruvate,?-hydroxy-butyric acid,L-lactic acid,?-keto-butyric acid,and propionic acid,acetic acid,?-hydroxy-D,L butyric acid,L-alanine,L-aspartic acid,L-glutamic acid,L-serine,etc,and for ?-D-glucose,D-galactose,?-D-lactose,D-maltose,sucrose,stachyose,L-rhamnose,melibiose,dextrin and D-mannose and other sugars have not shown metabolic ability.(2)Using the diphenylcarbazide spectrophotometric method to determine Cr(?),the Ylb10 strain's Cr(?)reduction properties were studied.The results showed that the shaking table culture seed liquid will make the Ylb10 strain lose Cr(?)reduction ability.The static culture condition of Ylb10 was more conducive to the reduction of Cr(?).The biomass of Ylb10 bacteria grows rapidly under shaking(aerobic)conditions,but it was difficult to reduce Cr(?),and only 18.24% of Cr(?)is reduced at 100 mg/L in 72 hours.Under static culture conditions,100 mg/L of Cr(?)can be reduced by 98.70% in 36 hours.Ylb10 had a high reduction ability to Cr(?).When the concentration of Cr(?)was 50,100,200 mg/L and 300mg/L,after 18,24,60 and 84 hours,the reduction rate of Cr(?)was 96.45%,93.83%,99.06% and 2.15% respectively.The optimum pH for growth of Ylb10 bacteria was 7,and the optimum pH for reducing Cr(?)was 8-9.(3)Using ARTP mutagenesis technology,the Ylb10 strain was mutagenized.After preliminary screening of LB liquid medium containing 450 mg/L Cr(?),ten mutagenic groups were obtained,namely: A5,B5,B7,B9,L1,L2,J1,J2,A6,J5,to screen for mutagenic groups that can tolerate higher concentrations.Through the Cr(?)reduction ability experiment,the results showed that L1 and L2 showed higher tolerance to Cr(?)among more mutagenized strains.On the 6th day,the biomass of L1 reached 1.43 OD,the reduction rate of Cr(?)was 21.02%,the biomass of L2 reached 1.52 OD,and the reduction rate of Cr(?)was 24.89%.(4)The mutagenized bacterial groups L1 and L2 were spread on LB solid medium containing 300 mg/L Cr(?)by gradient dilution,and 6 strains of single bacteria with better growth ability were isolated,namely: L1-3,L1-5,L1-15,L2-7,L2-9,L2-15.Through the Cr(?)reduction ability experiment,the results showed that the mutagenized strain L1-3 had higher Cr(?)tolerance and Cr(?)reduction ability.By comparing the Cr(?)reduction ability of the original strain Ylb10 and the mutant strain L1-3,the results showed that at 400mg/L Cr(?),the original strain Ylb10 only reduced Cr(?)by 0.16% at 72 hours.The mutant strain L1-3 can reduce 17.72%.The biological growth of the original strain Ylb10 is-0.08 OD,and the biological growth of the mutant strain L1-3 is 0.788 OD.