RFLP Analysis Of Bacterial Communities Of Remedied Chromium Contaminated Soil By Different Methods

Microbial community can reflect the situation of their survival environment and bio-diversity. Soil bacterial community is an important criterion of soil quality evaluation. The study about soil bacterial diversity during the process of biological remediation plays a significant role in the discussion of biological theory for environment remediation.The simulation Cr(VI) contaminated soil was used as test material, which had been treated by waterlogging for 10 days (S2), adding ferrihydrite and waterlogging 10 days (S3) and 20days (S4). As control, the nature soil by waterlogging 10 days (S1) was used. Total genomic DNAs of bacteria were extracted directly from soil treatments by three method. The products were purified by Agarose gel electrophoresis. The 16S rDNAs were amplified by PCR with the bacterial universal primers 63F/1387R. Connecting the partial sequence of 16S rDNA into pMD19-T vector, transforming into E. coli JM109, screening positive clone by white bacterial colony. By bacteria colony PCR, about 200 positive clone had been got randomLy, then amplified the inserted partial sequence of 16S rDNA using universal primer M13 of pMD19-T vector, purified the PCR production and digested them separately by restrict endonuclease Rsa I. After running 8% PAGE and stained by AgNO3, RFLP patterns were obtained by statistic analysis of gel. Usingα&?-measurement analyzed the bacterial diversity indexes of different samples. of Shannon-Wiener(H'), Gini(D), species richness index dMa, evenness index Jgi were calculated according to the RFLP patterns.The 16S rDNAs of predominant bacteria in S3&S4 were digested by HhaI. The sequencing 16S rDNAs is domiant bacteria. The species messages were obtained from NCBI by 16S rDNAs sequence BLAST. Phylogenetic analysis was done based on partial 16S rDNA sequencing of predominant bacteria of treatment S3 and S4. Some results were obtained as following:1. Total OTUs of four treatments are 123(S1), 120(S2), 97(S3)and 69(S4), respectively. The OTUs of bacterium indicates the different bacterial distribution in four soil treatments. The number of predominant clone was 7, 5, 11 and 11, especially in S4 treatment, which has 2 OTUs with percentage of more than 10%.2. From S1 to S4, Shannon-Wiener index (H') was 4.616, 4.588, 4.091 and 3.589; Gini index (D) was 0.9873, 0.9868, 0.9708 and 0.9492; dMa was 23.18, 22.61, 12.988and.13; Jgi were 0.9953, 0.9951, 0.9809 and 0.9632. They represented an identical order S1>S2>S3>S4. Based on the bacterial diversity index clustering analysis, treatments of S1and S2 were classified into the same group, while S3 and S4 was showing similarity on some level.3. Phylogenetic analysis of partial 16S rDNA revealed that the dominant bacteria in S3 and S4 were highly similar, belonging to the same bacterial phylum or family in evolution. The dominant bacteria in S3 and S4 manly distributed in clustering of : Acinetobacter, Pseudomonas fluorescens, gamma, Endophyte bacterium and Enterobacter sp. All these bacterium are proteobacterium, which, as kind of anaerobic bacteria , abroadly consist in soil and have the ability of reducing Cr(VI) and Fe(III).