高氯酸盐降解菌的鉴定及特性研究
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摘要
近年来,高氯酸盐在大量水、土壤和食物中均被检测到,已经引起全世界范围内人们的高度重视。由于高氯酸盐摄入人体后会导致甲状腺激素的分泌不足,进而抑制人体正常的新陈代谢和生长发育,因此,寻找消除此类污染物的有效方法成为全世界关注的重大环境问题之一。微生物降解作用是环境中高氯酸盐污染去除的最主要途径,利用厌氧微生物在缺氧条件下将高氯酸盐最终降解为无毒无害的氯化物,而且微生物降解是最为经济简便、无二次污染的有效方法,倍受关注。本论文通过分离纯化得到两种菌株,并借助分子生物学方法和生物信息学技术鉴定其菌属,并通过试验确定其最佳生长条件。
主要研究内容及结果如下：
(1)以从江滨泵站污水中分离所得的菌株JD15和从大港污水处理厂污水中分离得到的菌株JD22为研究对象,纯化后在PCA培养基中富集培养,提取培养基中总细菌的DNA,利用PCR扩增技术克隆水体细菌的16S rDNA基因片段,测序结果在NCBI数据库进行序列检索,通过EBI比对序列和构建系统发育树,并与数据库中已收录的细菌16SrDNA序列进行相似性比较分析。分析结果显示：JD15和Dechloromonas sp SIUL序列相似度高达100%,JD22和Dechlorospirillum序列相似度达97%,两者分别具有同源性。
(2)以JD15和JD22为研究对象,利用分子生物学技术和生物学信息学技术对两种菌株进行了进一步的分析,分析结果显示：JD15与Dechloromonas agitate菌的ClO_4还原酶a亚基基因片段相似率达99%,JD22和Dechlorospirillum sp菌的CLO_4还原酶a亚基基因部分片段相似率达99%。
(3)改变影响Dechloromonas agitate和Dechlorospirillum的生长条件,通过OD值测定和离子色谱仪分析,分别绘制生长曲线和降解曲线。结果显示：Dechloromonas agitate和Dechlorospirillum在24℃和pH=7时生长较快且生长状态较好。在此条件下培养14d,高氯酸盐的降解率可达86%±6.5%。随着菌株生长进入衰退期,降解能力减弱。利用同种培养基,分别在不同温度下进行培养,结果发现菌株Dechloromonas agitate和Dechlorospirillum降解底物的最适温度为24℃-30℃,温度高于37℃或低于15℃时降解率明显降低。不同pH值对菌株降解底物的速率也有影响,实验表明在pH=7时,降解速率最高,而在酸性或碱性条件下,降解速率均显著下降。
Recently, the discovery of perchlorate, ClO4, in a large number of water, ground and foods, has been gaining increasing attention all over the world due to its interference with the function of the thyroid gland as well as its potential health impact at low-dose exposure. Therefore, to search for the effective method for removing these pollutants, became the main environmental problem world widely. Research showed that micro-biological degradation play an important role in the transport and transformation of these pollutants, finally disappearing from the environment. It also was the primary handiest and economy way of removing contaminants. Under the anaerobic surroundings, bacteria reduced the perchlorate to innocuous chloride iron with the degradation via the sequence:ClO4→ClO3→ClO2→Cl+O2. Transformation of perchlorate to chlorate, ClO3, was the rate-limiting step with complete conversion to chloride. Perchlorate reducing microorganisms (PRMs) were ubiquitous in the natural environment, and applied to perchlorate remedation through enrichment. After a series of separation and purification procedures, two strains were identified by molecular biological methods and biotechnology informatics, two important elements of the growth conditions, temperature and pH, were determined after a train of tests.
The main contents and results of the research as following:
Firstly, total DNA was extracted from the effluent of JD15 and JD22, which were from Jiangbin and Dagang wastewater treatment plant, respectively, and 16S rDNA fragments were amplified, cloned and sequenced, the sequencing results were blasted in the Genebank of NCBI, the corresponding bacterial species were compared with the micro-organisms in EBI and constructing the phylogenetic tree. The analysis results showed that the 16S rDNA of JD15 showed 100% homology to the Dechloromonas sp SIUL, and JD22 showed 97% homology to the Dechlorospirillum.
Secondly, the research analyzed JD15 and JD22 further by molecular biological methods and biotechnology informatics, the results showed that JD15 was found up to 99% similarity to Dechloromonas agitate in a subunit of perchloric reductase, and JD22 was found up to 99% similarity to Dechlorospirillum sp in a subunit of perchloric reductase.
Thirdly, changing their growth factors, bacterial growth curves were drew by measuring the OD value of Dechloromonas agitate and Dechlorospirillum every other day, while bacterial degradation curves were drew by using the ion chromatography. The results showed that Dechloromonas agitate and Dechlorospirillum grow faster and better under the conditions of 24℃and pH7. Biomass reached the maximum within 7-10 days, then turned down and began to decline. Bacteria consumed a large number of nutrients, the metabolites accumulated rapidly. Eventually, they died and autolyzed. The removal rate of perchlorate reached 86%±6.5% in 14 days, which was gradually weakened with the decline of the strains. Through the experiment discovered the most suitable temperature is 24～30℃, clearly, when the temperature was above 37℃or below 15℃, the efficiencies decreased. Degradation rate can be influenced by other factors such as pH, it will achieve maximum under the condition of pH=7.
主要研究内容及结果如下：
(1)以从江滨泵站污水中分离所得的菌株JD15和从大港污水处理厂污水中分离得到的菌株JD22为研究对象,纯化后在PCA培养基中富集培养,提取培养基中总细菌的DNA,利用PCR扩增技术克隆水体细菌的16S rDNA基因片段,测序结果在NCBI数据库进行序列检索,通过EBI比对序列和构建系统发育树,并与数据库中已收录的细菌16SrDNA序列进行相似性比较分析。分析结果显示：JD15和Dechloromonas sp SIUL序列相似度高达100%,JD22和Dechlorospirillum序列相似度达97%,两者分别具有同源性。
(2)以JD15和JD22为研究对象,利用分子生物学技术和生物学信息学技术对两种菌株进行了进一步的分析,分析结果显示：JD15与Dechloromonas agitate菌的ClO_4还原酶a亚基基因片段相似率达99%,JD22和Dechlorospirillum sp菌的CLO_4还原酶a亚基基因部分片段相似率达99%。
(3)改变影响Dechloromonas agitate和Dechlorospirillum的生长条件,通过OD值测定和离子色谱仪分析,分别绘制生长曲线和降解曲线。结果显示：Dechloromonas agitate和Dechlorospirillum在24℃和pH=7时生长较快且生长状态较好。在此条件下培养14d,高氯酸盐的降解率可达86%±6.5%。随着菌株生长进入衰退期,降解能力减弱。利用同种培养基,分别在不同温度下进行培养,结果发现菌株Dechloromonas agitate和Dechlorospirillum降解底物的最适温度为24℃-30℃,温度高于37℃或低于15℃时降解率明显降低。不同pH值对菌株降解底物的速率也有影响,实验表明在pH=7时,降解速率最高,而在酸性或碱性条件下,降解速率均显著下降。
Recently, the discovery of perchlorate, ClO4, in a large number of water, ground and foods, has been gaining increasing attention all over the world due to its interference with the function of the thyroid gland as well as its potential health impact at low-dose exposure. Therefore, to search for the effective method for removing these pollutants, became the main environmental problem world widely. Research showed that micro-biological degradation play an important role in the transport and transformation of these pollutants, finally disappearing from the environment. It also was the primary handiest and economy way of removing contaminants. Under the anaerobic surroundings, bacteria reduced the perchlorate to innocuous chloride iron with the degradation via the sequence:ClO4→ClO3→ClO2→Cl+O2. Transformation of perchlorate to chlorate, ClO3, was the rate-limiting step with complete conversion to chloride. Perchlorate reducing microorganisms (PRMs) were ubiquitous in the natural environment, and applied to perchlorate remedation through enrichment. After a series of separation and purification procedures, two strains were identified by molecular biological methods and biotechnology informatics, two important elements of the growth conditions, temperature and pH, were determined after a train of tests.
The main contents and results of the research as following:
Firstly, total DNA was extracted from the effluent of JD15 and JD22, which were from Jiangbin and Dagang wastewater treatment plant, respectively, and 16S rDNA fragments were amplified, cloned and sequenced, the sequencing results were blasted in the Genebank of NCBI, the corresponding bacterial species were compared with the micro-organisms in EBI and constructing the phylogenetic tree. The analysis results showed that the 16S rDNA of JD15 showed 100% homology to the Dechloromonas sp SIUL, and JD22 showed 97% homology to the Dechlorospirillum.
Secondly, the research analyzed JD15 and JD22 further by molecular biological methods and biotechnology informatics, the results showed that JD15 was found up to 99% similarity to Dechloromonas agitate in a subunit of perchloric reductase, and JD22 was found up to 99% similarity to Dechlorospirillum sp in a subunit of perchloric reductase.
Thirdly, changing their growth factors, bacterial growth curves were drew by measuring the OD value of Dechloromonas agitate and Dechlorospirillum every other day, while bacterial degradation curves were drew by using the ion chromatography. The results showed that Dechloromonas agitate and Dechlorospirillum grow faster and better under the conditions of 24℃and pH7. Biomass reached the maximum within 7-10 days, then turned down and began to decline. Bacteria consumed a large number of nutrients, the metabolites accumulated rapidly. Eventually, they died and autolyzed. The removal rate of perchlorate reached 86%±6.5% in 14 days, which was gradually weakened with the decline of the strains. Through the experiment discovered the most suitable temperature is 24～30℃, clearly, when the temperature was above 37℃or below 15℃, the efficiencies decreased. Degradation rate can be influenced by other factors such as pH, it will achieve maximum under the condition of pH=7.
引文
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[2]Sorial G A. The perchlorate dilemma in drinking water. Journal of Environmental Engineering, 2004, (1):122.
[3]Motzer W E. Perchlorate:problems, detection, and solutions. Environmental Forensics,2001, (2):3012 311.
[4]Okamoto H S, Rishi D K, SteeberW R, et al. Using ion chromat ography t o detect perchl orate. Journal AWWA,1999, (10):73284.
[5]Krynitsky A J, Niemann R A, Nortrup D A. Determination of perchlorate anion in foods by ion chromatography tandem mass spectrometry[J].Anal Chem,2004,76 (18):5518—5522.
[6]Kirk A B, Martinelango P K, Tian K, et al. Perchlorate and iodide in dairy and breast milk[J].Environ Sci.Technol,2005,39 (7):2011—2017.
[7]Yali Shi, Ping Zhang, Yawei Wang, et.al. Perchlorate in sewage sludge, rice, bottled water and milk collected from different areas in China[J]. Environment International,2007,33:955-962.
[8]Logan, B.E., Lapoint, D. Treatment of perchlorate-and nitrate-contaminated groundwater in an autotrophic, gas phase, packed-bed bioreactor. Water Res.2002;36;3647—3653
[9]Rikken G. B, Kroon A G. M, Ginkel C G. Transformation of (per) chlorate into chloride by a newly isolated bacterium:reduction and dismutation. Appl. Microbiol.1996;45 (3):420—426
[10]Gullick R w, Lechevallier M W, Barhorst T S. Occurrence of perchlorate in drinking water sources. JAW WA,2001;93 (1):66—77
[11]Urbansky E T. Perchlorate in the environment. Dordrecht, New York:Kluwer Academic/ Plenum Publishers;2000
[12]Bruce Logan, Huse Zhang, Peter Mulvaney, et al. Kinetics of perchlorate and chlorate-respiring bacteria[J].Applied and Environmental Microbiology,2001,67 (6):2499-2506.
[13]Jiasheng Cao, Daniel Elliott, Wei-xian Zhang. Perchlorate reduction by nanoscale iron particles[J].Journal of Nanoparticle Research,2005,7:499—506.
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