单级自养脱氮系统中功能菌的分子生物学鉴定及氨氧化反应关键酶基因的克隆与表达
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摘要
单级自养脱氮工艺是指在同一反应器内、由自养细菌完成由氨氮至氮气全部转化过程的一类工艺,是近年来出现的一种新型脱氮工艺。该工艺具有简化工艺流程节省供气量和动力消耗、不需外加碳源及减少工艺处理费用等优点,因此有很好的应用前景。但由于该工艺脱氮机理的不明晰,因此影响了其在环保工业上的应用。本研究着眼于该系统的功能菌,从氨氧化菌和厌氧氨氧化菌的种群进化及氨氧化菌功能基因amoA和hao的原核表达入手,以期从生物学角度为该系统的脱氮机理的研究提供理论基础。
本研究对稳定运行、氨氮转化率及总氮去除率分别达到90%及80%左右的单级自养脱氮系统的底部的活性污泥为研究材料,研究结果如下:
(1)采用分子生物学方法提取活性污泥细菌总DNA,利用特异引物Pla46rc/Amx820对单级自养脱氮系统中的厌氧氨氧化菌16S rDNA基因进行PCR扩增,扩增产物经克隆、测序及BLAST分析。研究结果表明,该单级自养脱氮系统中存在的厌氧氨氧化菌与目前鉴定出的厌氧氨氧化菌Candidatus Kuenenia stuttgartiensis和Candidatus Brocadia anammoxidans的16S rDNA序列同源性达99%,进化分析证明与Candidatus Kuenenia stuttgartiensis进化上较为接近。
(2)以活性污泥为材料富集培养氨氧化菌,提取细菌总DNA,分别设计氨氧化菌16S rDNA、氨单加氧酶基因(amoA)及羟胺氧化酶基因(hao)的特异引物,扩增产物经克隆、测序及BLAST分析。研究结果表明该系统中存在的氨氧化菌的16S rDNA、amoA和hao分别与已知具有氨氧化功能的Nitrosomonas europaea ATCC 19178,Uncultured bacterium clone amoA_SBR_JJY clone,Nitrosomonas sp. ENI-11的同源性达到99%,99%,100%。进化分析证明该系统中存在的氨氧化菌和“Nitrosomonas sp. DYS323”、“Nitrosomonas sp. DYS317”、“Uncultured bacterium clone amoA_SBR_JJY 61clone”、“Uncultured bacterium clone amoA_SBR_JJY 70clone”、Nitrosomonas sp.ENI-11hao2进化上较为接近。上述研究结果表明该系统中存在厌氧氨氧化菌和氨氧化菌,其中氨氧化菌属于亚硝化单胞菌属,且在系统中存在几种同属不同种的氨氧化菌。
(3)本研究将成功克隆的氨单加氧酶基因(amoA)和羟胺氧化酶基因(hao)的全长序列,构建pET32a原核表达载体,得到重组质粒后,转化到表达宿主菌BL21(DE3)中进行原核表达。研究结果表明以1.0 mmol/L IPTG诱导6 h的目标融合蛋白的表达量最高,并通过Western Blot鉴定了目的蛋白的正确表达。粗酶液活性测定结果表明,通过原核表达的AMO和HAO均有较强的活性,为后续构建工程菌株打下了基础。
Single-stage autotrophic nitrogen removal process is achieved in one single reactor, where ammonia is oxidized to nitrogen by autotrophic bacteria simultaneously. It is a new nitrogen removal process and has many advantages such as less gas and power supplying, no extra carbon source, fewer costs of processing etc, and it will have a better application prospect in the field of environmental protection. Until now the mechanism of nitrogen removal is not yet clear, which impedes its application. Research on the functional bacterial in the system has been in progress in order to explicate the mechanism of ammonia romoval. Here this study focused on phylogenetic analysis of two important groups in the single-stage autotrophic nitrogen removal system, anaerobic ammonium-oxidizing bacteria (ANAMMOX bacteria) and ammonium-oxidizing bacteria (AOB), and prokaryotic expression of functional gene amoA and hao in order to provide a theoretical foundation for the mechanism of nitrogen removal.
In this study, using the cultivated sludge from the single-stage autotrophic nitrogen removal system with stable operation and 90% of ammonia conversion rate and 80% of total nitrogen removal rate, several aspects were investigated as follows.
(1) With the total DNA extracted from cultivated sludge, partial 16S rDNA sequence of ANAMMOX bacteria was amplified by polymerase chain reaction (PCR) with a pair of specific primers Pla46rc/Amx820. Amplified product was cloned, sequenced and analyzed by BLAST. The result indicated that the sequence has 99% identities with ANAMMOX bacteria Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia anammoxidans. The phylogenetic analysis showed that the anaerobic ammonium-oxidizing bacteria in the single-stage autotrophic nitrogen removal system have closer relationship with Candidatus Kuenenia stuttgartiensis evolutionarily.
(2) With the total DNA extracted from cultivated sludge through four times of enrichments, partial sequence of 16S rDNA, the full length sequences of ammonia monooxygenase (amoA) gene and hydroxylamine oxidoreductase (hao) gene of AOB were amplified by PCR with specific primers which were designed according to sequence published NCBI GenBank. Amplified product was cloned, sequenced and analyzed. The result showed that the sequences of 16S rDNA, amoA and hao have 99%, 99% and 100% identities with Nitrosomonas europaea ATCC 19178, Uncultured bacterium clone amoA_SBR_JJY clone, Nitrosomonas sp.ENI-11, respectively. The phylogenetic analysis revealed that the AOB in the single-stage autotrophic nitrogen removal system have closer relationship with“Nitrosomonas sp. DYS323”、“Nitrosomonas sp. DYS317”、“Uncultured bacterium clone amoA_SBR_JJY 61clone”、“Uncultured bacterium clone amoA_SBR_JJY 70clone”and“Nitrosomonas sp.ENI-11hao2”. The results described above demonstrated that ANAMMOX bacteria and AOB exist in the system, and that the AOB belongs to different species of Nitrosomonas sp.
(3) The full-length sequences of the amoA and hao were successfully amplified with the specific primers based on published amoA and hao sequences in NCBI. The amoA and hao had been cloned into the prokaryotic expression vector pET-32a, generating the recombinant plasmid pET-32a-amoA and pET-32a-hao that were then transformed into E. Coli BL21 (DE3). The best induction of recombinant AMO and HAO was obtained when 1.0 mmol/L IPTG was added into the E. Coli BL21 (DE3) incubated for 6 h at 37℃and 30℃, respectively. Western Blot detection showed that the fusion protein was correctly expressed in E. Coli BL21 (DE3). Detection of crude enzyme activity showed that the activity of AMO and HAO was stronger in vitro. This result established the theoretical basis for building engineering bacteria.
本研究对稳定运行、氨氮转化率及总氮去除率分别达到90%及80%左右的单级自养脱氮系统的底部的活性污泥为研究材料,研究结果如下:
(1)采用分子生物学方法提取活性污泥细菌总DNA,利用特异引物Pla46rc/Amx820对单级自养脱氮系统中的厌氧氨氧化菌16S rDNA基因进行PCR扩增,扩增产物经克隆、测序及BLAST分析。研究结果表明,该单级自养脱氮系统中存在的厌氧氨氧化菌与目前鉴定出的厌氧氨氧化菌Candidatus Kuenenia stuttgartiensis和Candidatus Brocadia anammoxidans的16S rDNA序列同源性达99%,进化分析证明与Candidatus Kuenenia stuttgartiensis进化上较为接近。
(2)以活性污泥为材料富集培养氨氧化菌,提取细菌总DNA,分别设计氨氧化菌16S rDNA、氨单加氧酶基因(amoA)及羟胺氧化酶基因(hao)的特异引物,扩增产物经克隆、测序及BLAST分析。研究结果表明该系统中存在的氨氧化菌的16S rDNA、amoA和hao分别与已知具有氨氧化功能的Nitrosomonas europaea ATCC 19178,Uncultured bacterium clone amoA_SBR_JJY clone,Nitrosomonas sp. ENI-11的同源性达到99%,99%,100%。进化分析证明该系统中存在的氨氧化菌和“Nitrosomonas sp. DYS323”、“Nitrosomonas sp. DYS317”、“Uncultured bacterium clone amoA_SBR_JJY 61clone”、“Uncultured bacterium clone amoA_SBR_JJY 70clone”、Nitrosomonas sp.ENI-11hao2进化上较为接近。上述研究结果表明该系统中存在厌氧氨氧化菌和氨氧化菌,其中氨氧化菌属于亚硝化单胞菌属,且在系统中存在几种同属不同种的氨氧化菌。
(3)本研究将成功克隆的氨单加氧酶基因(amoA)和羟胺氧化酶基因(hao)的全长序列,构建pET32a原核表达载体,得到重组质粒后,转化到表达宿主菌BL21(DE3)中进行原核表达。研究结果表明以1.0 mmol/L IPTG诱导6 h的目标融合蛋白的表达量最高,并通过Western Blot鉴定了目的蛋白的正确表达。粗酶液活性测定结果表明,通过原核表达的AMO和HAO均有较强的活性,为后续构建工程菌株打下了基础。
Single-stage autotrophic nitrogen removal process is achieved in one single reactor, where ammonia is oxidized to nitrogen by autotrophic bacteria simultaneously. It is a new nitrogen removal process and has many advantages such as less gas and power supplying, no extra carbon source, fewer costs of processing etc, and it will have a better application prospect in the field of environmental protection. Until now the mechanism of nitrogen removal is not yet clear, which impedes its application. Research on the functional bacterial in the system has been in progress in order to explicate the mechanism of ammonia romoval. Here this study focused on phylogenetic analysis of two important groups in the single-stage autotrophic nitrogen removal system, anaerobic ammonium-oxidizing bacteria (ANAMMOX bacteria) and ammonium-oxidizing bacteria (AOB), and prokaryotic expression of functional gene amoA and hao in order to provide a theoretical foundation for the mechanism of nitrogen removal.
In this study, using the cultivated sludge from the single-stage autotrophic nitrogen removal system with stable operation and 90% of ammonia conversion rate and 80% of total nitrogen removal rate, several aspects were investigated as follows.
(1) With the total DNA extracted from cultivated sludge, partial 16S rDNA sequence of ANAMMOX bacteria was amplified by polymerase chain reaction (PCR) with a pair of specific primers Pla46rc/Amx820. Amplified product was cloned, sequenced and analyzed by BLAST. The result indicated that the sequence has 99% identities with ANAMMOX bacteria Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia anammoxidans. The phylogenetic analysis showed that the anaerobic ammonium-oxidizing bacteria in the single-stage autotrophic nitrogen removal system have closer relationship with Candidatus Kuenenia stuttgartiensis evolutionarily.
(2) With the total DNA extracted from cultivated sludge through four times of enrichments, partial sequence of 16S rDNA, the full length sequences of ammonia monooxygenase (amoA) gene and hydroxylamine oxidoreductase (hao) gene of AOB were amplified by PCR with specific primers which were designed according to sequence published NCBI GenBank. Amplified product was cloned, sequenced and analyzed. The result showed that the sequences of 16S rDNA, amoA and hao have 99%, 99% and 100% identities with Nitrosomonas europaea ATCC 19178, Uncultured bacterium clone amoA_SBR_JJY clone, Nitrosomonas sp.ENI-11, respectively. The phylogenetic analysis revealed that the AOB in the single-stage autotrophic nitrogen removal system have closer relationship with“Nitrosomonas sp. DYS323”、“Nitrosomonas sp. DYS317”、“Uncultured bacterium clone amoA_SBR_JJY 61clone”、“Uncultured bacterium clone amoA_SBR_JJY 70clone”and“Nitrosomonas sp.ENI-11hao2”. The results described above demonstrated that ANAMMOX bacteria and AOB exist in the system, and that the AOB belongs to different species of Nitrosomonas sp.
(3) The full-length sequences of the amoA and hao were successfully amplified with the specific primers based on published amoA and hao sequences in NCBI. The amoA and hao had been cloned into the prokaryotic expression vector pET-32a, generating the recombinant plasmid pET-32a-amoA and pET-32a-hao that were then transformed into E. Coli BL21 (DE3). The best induction of recombinant AMO and HAO was obtained when 1.0 mmol/L IPTG was added into the E. Coli BL21 (DE3) incubated for 6 h at 37℃and 30℃, respectively. Western Blot detection showed that the fusion protein was correctly expressed in E. Coli BL21 (DE3). Detection of crude enzyme activity showed that the activity of AMO and HAO was stronger in vitro. This result established the theoretical basis for building engineering bacteria.
引文
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