Screening Of Dominant Strains For High-salinity Landfill Leachate Degradation And Construction Of Subtractive Library Related To COD Degradation

The leachate generated from landfill site in the coastal area not only has higher COD concentration and is difficult to be treated by biodegradation as the general leachate but also contains higher salt, which can lead to biological treatment system running unstable. Several dominant strains were isolated from leachate and dominant bacteria consortium was built in this paper. The biochemical treatment efficiency of leachate was improved by bioaugmentation technology. The dominant strains were identified using molecular biology techniques. The subtractive library was constructed with suppression subtractive hybridization (SSH), which laid a good foundation for building engineered bacteria of landfill leachate.According to the composition and characteristics of high-salinity landfill leachate, ten strains were isolated using selective medium and were screened with the method of gradient increasing percentage of landfill leachate in this study. In laboratory degradation experiments, six dominant strains (TJ02, TJ03, TJ05, TJ06, TJ07, TJ09) were screened and effects of temperature, inoculum size, pH on growth of the 6 strains were studied.The COD degradation tests were carried out to build dominant bacteria consortium. The result showed the COD removal efficiency of the bacteria consortium composed of TJ06 and TJ09 was the highest. The COD removal efficiency of the dominant bacteria consortium increased with the COD concentration increasing when COD concentration was less than 7375mg /L, while the COD removal efficiency decreased with the COD concentration increasing when COD concentration was more than 7375mg /L.Strains TJ06 and TJ09 had good salt-tolerant features. When Cl- concentration was less than 30000mg/L in the medium, the relative growth rates of strain TJ06 and TJ09 were more than 75% and 60% respectively. With the Cl- concentration increased, the relative growth rates of the two strains decreased. The relative growth rate of strain TJ06 was only 1.11% after cultured 24h when the Cl- concentration was 70000mg/L, and the relative growth rates of the strain TJ06 and TJ09 after cultured 48h were about 40%. When the Cl- concentration was 90000mg /L, the relative growth rates of the two strains were less than 1%, which suggested the dominant strains almost stopped growing.The growth of strains TJ06 and TJ09 was inhibited by Zn2+ and Cd2+. As the Zn2+ and Cd2+ concentration increased, the inhibition on strains became greater. The relative growth rate of strain TJ06 was about 40% when the Zn2+ concentration was 100mg /L, while the strain TJ06 almost stopped growing when Zn2+ concentration was more than 100mg /L. Strain TJ09 was sensitive to Zn2+, the relative growth rates of strain TJ09 were less than 10% when the Zn2+ concentrations were more than 50mg /L. The growth of TJ06 and TJ09 significantly inhibited when the concentration of Cd2+ was 50mg /L, their relative growth rates were 40.59% and 34.89% respectively. When the Cd2+ concentrations were higher than 150mg / L, the relative growth rates of TJ06 and TJ09 were less than 10%.According to the sequence analysis of 16S rDNA, compared the gene sequence of TJ06 and TJ09with the published Genbank sequence strain, the result showed that TJ06 shared homology of 99% with Bacillus cereus (EU931563)and Bacillus thuringiensis(AY138288), 98% with Bacillus mycoides(EU162011). TJ09 shared homology of 99% with Bacillus marisflavi (FJ554665), Bacillus aquimaris(EU835730) and Bacillus baekryungensis (EU835731), 97% with Bacillus ferrariarum(EF451043). The result suggested that the TJ06 and TJ09 belonged to the genus Bacillus.To identify unique DNA fragments associated with strain TJ06, suppression subtractive hybridization (SSH) was used. The genome of Bacillus cereus AS 1.196 was subtracted from the genome of strain TJ06. Subtractive hybridization library containing 230 positive clones was built, and 205 different fragments were amplified by bacterial suspension PCR. Amplified DNA fragments were analyzed using the software of GelComparâ…¡. Sequence homology analysis of 12 specific fragments was done through BLAST at the NCBI web. The result showed that two fragments had no similar sequences, which may be new gene fragments, and their functions should be study further; seven fragments had high similarity with the nucleotide sequence which functions were unknown; other three fragments were similar to the encoding genes of peptide methionine sulfoxide reductase, shikimate transport protein and ubiquinol-cytochrome C reductase iron-sulfur subunit or cytochrome c oxidase subunitâ… , and the similarities were 90%, 93% and 98% respectively. The study laid a good foundation for researching the functions of specific genes and for building the related engineering bacteria.