Study On The Screening,Identification And Mercury Conversion Mechanism Of Mercury Tolerant Strains

As one of the most toxic heavy metals,mercury pollution has the characteristics of non-biodegradability,concealment,long-term accumulation and biological amplification in the food chain.It will have an irreversible impact on the environment and cause serious harm to organisms.Microorganisms can degrade and convert organic and inorganic mercury into volatile zero-valent mercury.Although mercury pollution can not be completely eliminated,they can effectively reduce its toxicity and adverse effects on the environment.Microbial remediation is widely used in environmental pollution remediation because of its low cost,convenient operation and no secondary pollution.It is one of the most effective ways to solve environmental pollution problems.Therefore,the screening of indigenous microorganisms with degradation and transformation of mercury pollution in the environment and their application in the remediation of mercury pollution have important theoretical value and application prospects.In this study,mercury-contaminated water samples from a chemical plant in Lanzhou were collected,and bacterial strains with resistance to mercury were screened and isolated.Their tolerance to mercury,transformation ability,optimal culture conditions,mercury tolerance mechanism and tolerance to other heavy metals were studied,which provided valuable germ resources,theoretical basis and data support for bioremediation of mercury-contaminated water.The main results were as follows:1.Three strains with strong resistance to mercury were isolated.All of them could grow on solid medium containing 120 mg/L HgCl2.The strain numbers were H1,H8 and H10.16S rRNA gene sequence analysis showed that the similarity between H1 strain and Pseudomonas putida N-408 strain was 99.65%,and H1 strain was identified as Pseudomonas putida.H8strain was identified as a strain of Pseudomonas sp because its sequence was 99.93%similarity with the Pseudomonas JCM 5482 strain.The similarity between strain H10 and Brevundimonas H20069 strain was 99.67%.Therefore,H10 was identified as a strain of Brevundimonas sp.The 24 h Hg²⁺conversion rate of strain H1 was 93.53%,and that of strain H8 was 92.75%,their transformation ability of Hg²⁺was higher than that of mercury-resistant strains reported in the literature.2.In liquid medium,the maximum Hg²⁺tolerance concentration of H1 strain was 90mg/L,and the maximum tolerance concentration of H8 and H10 were 60 mg/L.The optimum growth conditions of the three strains were at pH 8,temperature 30?.However,H1and H8 were better adapted to alkaline environment.3.Quantitative polymerase chain reaction?Q-PCR?confirmed that merA?mercury ion reductase gene?gene was contained in the genome of the three strains.The merA gene copies of H1 strain was the highest,attained to 5.995×10⁶/?L DNA.The merA gene copies of H8and H10 strains were much lower than that in H1 strain,only 102.57 and 10.61/?L DNA.The results of real-time fluorescence Q-PCR showed,under the induced by 10 mg/L Hg²⁺,the expression of merA,merC,merR and merT genes,which are mainly involved in the transformation of Hg²⁺,was up-regulated compared with CK,suggesting that the mer operon played an important role in the transformation of Hg²⁺.The mer operon of H1 strain was started earliest?before 4 h?,and the transformation process was completed at 8 h.The mer operon of H8 strain was started latest?at 8 h?,the transformation process lasted 12 hours,or even longer.The mer operon of H10 strain was started at 4 h,the secondary expression of merC and merA gene appeared at 12 h,so the transformation process of the strain continued until 12 h or later.4.Under the presence of 10 mg/L HgCl2,the tolerance of H1 strain to Pb²⁺could reach40 mg/L,to As⁵⁺and Cr⁶⁺were 20 mg/L,and to Cd²⁺was only 10 mg/L.The degradation rate of Pb²⁺,Cr⁶⁺,As⁵⁺and Cd²⁺at 48 h were 49.6%,45.4%,50.4%and 59.8%.H8 strain could tolerate 20 mg/L Cr⁶⁺,10 mg/L Cd²⁺,As⁵⁺and Pb²⁺.The degradation rates of Pb²⁺,Cr⁶⁺,As⁵⁺and Cd²⁺at 48 h were 70.9%,75.4%,48.3%and 46.1%.The tolerance of H10 strain to As⁵⁺,Cr⁶⁺,Cd²⁺and Pb²⁺were coincidence,the maximum tolerable concentration was 10 mg/L,and the degradation rate of Pb²⁺,Cr⁶⁺,As⁵⁺,and Cd²⁺at 48 h were 71.9%,79.9%,50.7%,and72.2%.The results showed that the three strains could be applied to the waste water containing Hg²⁺,Cr⁶⁺,Cd²⁺,As⁵⁺and Pb²⁺.Among them,H1 had the best ability to tolerate the other heavy metals and H10 had the highest degradation rate to the other heavy metals.