高盐渗滤液COD降解优势菌的筛选及相关基因消减文库的构建
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摘要
滨海地区垃圾填埋场产生的渗滤液,不仅具有一般渗滤液的COD浓度高且生物难降解的特性,而且还含有较高的盐量,常常使得生物处理工艺很难稳定运行。本文从渗滤液中分离出多株优势菌,构建优势菌群,通过生物强化技术,提高渗滤液生化处理效能。利用分子生物技术,鉴别优势菌株。利用抑制消减杂交技术,构建优势菌株的消减文库,为构建垃圾渗滤液工程菌奠定基础。
本研究根据高盐垃圾渗滤液的组成及特点,通过梯度增加培养基中垃圾渗滤液比例的方法,分离、筛选出垃圾渗滤液中的10株耐盐菌株。再经实验室摇瓶降解试验,从中筛选出6株降解COD的优势菌株,并研究了温度、接种量、pH对6株优势菌生长的影响。
将不同优势菌株混合配比进行COD降解试验,发现了以TJ06和TJ09菌株为混合配比的优势菌群具有较高的有机物去除效率。当COD浓度小于7375mg/L时,该优势菌群对渗滤液的COD去除效率随着COD初始浓度的增加而增加。当COD浓度大于7375mg/L时,优势菌群对渗滤液的COD去除效率随着COD初始浓度的增加而减少。
菌株TJ06和TJ09具有良好的耐盐特性。当培养基中Cl-浓度小于30000mg/L时,菌株TJ06和TJ09的相对生长率分别大于75%和60%。随着培养基中Cl-浓度的增加,两株菌的相对生长率均呈下降趋势。当Cl-浓度为70000mg/L时,菌株TJ06培养24h,其相对生长率只有1.11%,48h时菌株TJ06和TJ09的相对生长率为40%左右;当Cl-浓度为90000mg/L时,两株菌的相对生长率均小于1%,几乎不再生长。
重金属离子Zn2+、Cd2+对菌株TJ06和TJ09的生长具有明显的抑制作用,这种抑制作用大小与培养基中Zn2+、Cd2+浓度呈正比。当培养基中Zn2+浓度为100mg/L时,菌株TJ06的相对生长率为40%左右;当Zn2+浓度大于100mg/L时,TJ06几乎不再生长。菌株TJ09对Zn2+更敏感,当Zn2+浓度大于50mg/L时,TJ09的相对生长率低于10%。培养基中Cd2+浓度为50mg/L时,菌株TJ06和TJ09的生长就明显受到抑制,其相对生长率分别为40.59%和34.89%;当Cd2+浓度大于150mg/L时,TJ06和TJ09的相对生长率均小于10%。
利用16S rDNA分析技术,对优势菌株进行序列分析。发现菌株TJ06与Bacillus cereus (EU931563)和Bacillus thuringiensis(AY138288)的同源性最高,达到99%。菌株TJ09与Bacillus marisflavi (FJ554665)、Bacillus aquimaris(EU835730)和Bacillus baekryungensis (EU835731)的同源性达99%。并根据序列同源性分析建立相关菌种的系统进化树,确认TJ06和TJ09均属于Bacillus属。
为筛选优势菌株中COD降解相关基因,利用抑制消减杂交技术(SSH)对菌株TJ06和Bacillus cereus SA1.196基因组进行比较,构建一个库容量为230个阳性克隆的消减杂交文库。通过菌液PCR进行鉴定,扩增出差异片段205条。对12个差异片段进行核苷酸序列相似性比对和功能分析。结果显示其中2个差异片段未检索到同源序列,可能为新基因片段,其功能有待进一步研究;7个差异表达片段,虽然找到了与其相似性很高的序列,但功能未知;其他3个差异表达片段分别与肽甲硫氨酸亚砜还原酶的编码基因同源性达90%、与编码莽草酸转运蛋白的基因同源性达93%、与辅酶Q细胞色素C还原酶Fe-S亚单位编码基因的同源性达98%。
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 GelComparⅡ. 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 subunitⅠ, 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.
本研究根据高盐垃圾渗滤液的组成及特点,通过梯度增加培养基中垃圾渗滤液比例的方法,分离、筛选出垃圾渗滤液中的10株耐盐菌株。再经实验室摇瓶降解试验,从中筛选出6株降解COD的优势菌株,并研究了温度、接种量、pH对6株优势菌生长的影响。
将不同优势菌株混合配比进行COD降解试验,发现了以TJ06和TJ09菌株为混合配比的优势菌群具有较高的有机物去除效率。当COD浓度小于7375mg/L时,该优势菌群对渗滤液的COD去除效率随着COD初始浓度的增加而增加。当COD浓度大于7375mg/L时,优势菌群对渗滤液的COD去除效率随着COD初始浓度的增加而减少。
菌株TJ06和TJ09具有良好的耐盐特性。当培养基中Cl-浓度小于30000mg/L时,菌株TJ06和TJ09的相对生长率分别大于75%和60%。随着培养基中Cl-浓度的增加,两株菌的相对生长率均呈下降趋势。当Cl-浓度为70000mg/L时,菌株TJ06培养24h,其相对生长率只有1.11%,48h时菌株TJ06和TJ09的相对生长率为40%左右;当Cl-浓度为90000mg/L时,两株菌的相对生长率均小于1%,几乎不再生长。
重金属离子Zn2+、Cd2+对菌株TJ06和TJ09的生长具有明显的抑制作用,这种抑制作用大小与培养基中Zn2+、Cd2+浓度呈正比。当培养基中Zn2+浓度为100mg/L时,菌株TJ06的相对生长率为40%左右;当Zn2+浓度大于100mg/L时,TJ06几乎不再生长。菌株TJ09对Zn2+更敏感,当Zn2+浓度大于50mg/L时,TJ09的相对生长率低于10%。培养基中Cd2+浓度为50mg/L时,菌株TJ06和TJ09的生长就明显受到抑制,其相对生长率分别为40.59%和34.89%;当Cd2+浓度大于150mg/L时,TJ06和TJ09的相对生长率均小于10%。
利用16S rDNA分析技术,对优势菌株进行序列分析。发现菌株TJ06与Bacillus cereus (EU931563)和Bacillus thuringiensis(AY138288)的同源性最高,达到99%。菌株TJ09与Bacillus marisflavi (FJ554665)、Bacillus aquimaris(EU835730)和Bacillus baekryungensis (EU835731)的同源性达99%。并根据序列同源性分析建立相关菌种的系统进化树,确认TJ06和TJ09均属于Bacillus属。
为筛选优势菌株中COD降解相关基因,利用抑制消减杂交技术(SSH)对菌株TJ06和Bacillus cereus SA1.196基因组进行比较,构建一个库容量为230个阳性克隆的消减杂交文库。通过菌液PCR进行鉴定,扩增出差异片段205条。对12个差异片段进行核苷酸序列相似性比对和功能分析。结果显示其中2个差异片段未检索到同源序列,可能为新基因片段,其功能有待进一步研究;7个差异表达片段,虽然找到了与其相似性很高的序列,但功能未知;其他3个差异表达片段分别与肽甲硫氨酸亚砜还原酶的编码基因同源性达90%、与编码莽草酸转运蛋白的基因同源性达93%、与辅酶Q细胞色素C还原酶Fe-S亚单位编码基因的同源性达98%。
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 GelComparⅡ. 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 subunitⅠ, 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.
引文
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