厌氧发酵产氢菌筛选及产氢菌突变体库构建
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摘要
扩大产氢微生物种质资源是生物制氢领域重要的研究课题。本论文采用野生型筛选和转座子诱变产氢菌株的手段,研究混合菌群和纯菌株产氢性质,构建产氢菌株的突变体库,旨在获得高效厌氧发酵产氢微生物,为构建可利用海洋有机废水制氢的工程菌株奠定理论基础。
1培养条件对混合菌群产氢的影响
研究预处理、起始pH值和底物种类对潮间带污泥混合菌群的产氢影响,采用PCR-变性梯度凝胶电泳(DGGE)分析混合菌群组成。在酸、热休克、碱和冻融四种预处理方法中,酸处理条件下富集混合菌群的产氢量最高,混合菌群的优势菌为Clostridium sp.和Bacillus sp.;热休克处理富集混合菌群的优势菌为Clostridium sp.和Enterococcus sp.,碱处理和冻融处理下富集混合菌群的优势菌是Clostridium sp.和Lactobacillus sp.。不同起始pH值对混合菌群产氢量有明显影响,pH值为7.0时,混合菌群产氢量达到最高,其它依次为pH6.5> pH7.5 > pH6.0 > pH8.0 > pH5.5 > pH5.0 > pH4.5 > pH4.0。16S rRNA基因的DGGE分析结果表明,在不同起始pH值条件下混合菌群的优势菌基本相同,NCBI-BLAST比对结果与Clostridium sp.存在较高的相似度(98%)。采用葡萄糖、蔗糖、乳糖、淀粉、蛋白胨5种底物进行厌氧发酵产氢,底物为蔗糖时混合菌群的产氢量最高,混合菌群优势菌为Clostridium sp.和Bacillus sp.。
2产氢菌株的分离与产氢条件优化
野生型产氢菌株的分离是扩大产氢微生物种质资源的必要手段。从天津海水浴场潮间带污泥中分离鉴定两株产氢菌。通过形态观察、生理生化特征和16S rRNA序列分析,两株产氢菌分别命名为Bacillus sp.B2和Clostridium sp.T7。在海水培养条件下,起始葡萄糖浓度为20 g/l,pH值为7.0时,菌株Bacillus sp.B2最大产氢量能达到1.65±0.048 mol H2/mol葡萄糖,菌株Clostridium sp.T7最大产氢量能达到1.79±0.062 mol H2/mol葡萄糖。
3成团泛菌突变体库的构建和高效产氢突变菌株的筛选
转座子诱变是获得高效产氢菌的有效途径。利用Tn7转座子体外转座成团泛菌基因组DNA,采用Kan抗性和转座子插入序列鉴定突变菌株。在含卡那霉素浓度为40μg/ml的平板上,随机挑取340个菌落,其中有331株菌通过PCR鉴定证实插入了转座子。产氢菌突变体库的库容量为4.35×108/ml。以产氢量为筛选指标,在已鉴定的331株突变菌中,筛选出一株高效产氢突变菌株TB5,其产氢百分含量和产氢量分别是野生菌株的1.1倍和1.52倍。菌株TB5的产氢最适pH为7.0,而野生菌株为6.0。在成团泛菌Tn7突变体库中,以能利用淀粉产氢为筛选指标,331株突变菌中有17株的产氢能力明显高于野生菌。
Enlarging species resources of hydrogen-producing bacteria is of importance in bio-hydrogen field. We studied the hydrogen-producing characteration of mixed culture and pure bacterium, and constructed mutant library of hydrogen producing bacterium by screening for wild type and transposon mutagenesis of hydrogen-producing bacteria. The aim of this research was to harvest efficient hydrogen-producing microorganisms This research can lay a theoretical foundation for construction of engineering strain that can utilize organic contents in the high-salt organic wastewater to generate hydrogen energy.
1. Effect of culture conditions on hydrogen producing of mixed culture
The effects of pretreatment, initial pH and substrate species on hydrogen producing of mixed culture were determined. Denaturing gradient gel electrophoresis (DGGE) was used for analyzing composition of microbial community. Four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing, acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. Acid pretreatment was favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp. and Bacillus sp. The dominant bacteria of mixed culture pretreated by heat shock were Clostridium sp. and Enterococcus sp. However, besides hydrogen-producing bacteria Clostridium sp., much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Hydrogen production of mixed culture was evidently affected by different initial pH. At the optimal initial pH of 7.0, hydrogen production potential of mixed culture was the highest, gradually decreasing at the following pH sequence of pH 6.5, pH 7.5, pH 6.0, pH 8.0, pH 5.5, pH 5.0, pH 4.5, and pH 4.0. The dominant bands obtained on DGGE of 16S rRNA gene were identical at various initial pH values. These bands were cloned and sequenced. NCBI blast indicated that the segment of 16S rRNA gene was high identical to that of Clostridium sp. (98%). Mixed culture was cultivated by five different substrates (glucose, sucrose, lactose, starch and peptone). The highest hydrogen production potential of mixed culture was observed when using sucrose as substrate. The dominant bacteria were Clostridium sp.and Bacillus sp..
2. Identification of hydrogen-producing bacteria and optimization of hydrogen production
Isolation of wild type hydrogen-producing bacteria is necessary means for enlarging species resource of hydrogen-producing microorganism. Two strain hydrogen-producing bacteria were isolated from sludge collected in the intertidal zone of a bathing beach in Tianjin. Bacteria with high hydrogen-producing ability were isolated and identified using light microscopic examination, physiologic and biochemical characterization and 16S rRNA gene sequence analysis. The isolated bacteria were designated Bacillus sp.B2 and Clostridium sp. T7, respectively. Hydrogen production of two strains was measured under marine condition, companied with an initial glucose concentration of 20g/l and an initial pH value of 7.0. The maximum hydrogen production was 1.65±0.048 mol H2/mol glucose for Bacillus sp.B2, whereas 1.79±0.062 mol H2/mol glucose for Clostridium sp. T7.
3. Construction of mutant library of Pantoea agglomerans and screening of high hydrogen-producing mutants
Transposon mutagenesis is an effective approach for harvesting efficient hydrogen-producing bacteria. A Tn7-based transposon was randomly inserted into genomic DNA of Pantoea agglomerans BH18 in vitro. Mutants were screened by Kanr and amplification of the inserted sequences. 340 colonies were randomly selected on the plate with 40μg/ml kanamycin, and in which 331 transposon insertion mutants were obtained by amplification of inserted sequences. Capacity of mutants was 4.35×108/ml. Using hydrogen production as screening index, a highly effective hydrogen producing strain TB5 was screened among 331 mutants. Its hydrogen content and hydrogen production were 1.1 and 1.52 times as much as that of wild type, respectively. The optimum pH of hydrogen production for strain TB5 was 7.0, whereas 6.0 for wild type. The ability for utilizing starch as hydrogen producing substrate was investigated. 17 strains with higher hydrogen production than wild type were obtained among 331 mutants.
1培养条件对混合菌群产氢的影响
研究预处理、起始pH值和底物种类对潮间带污泥混合菌群的产氢影响,采用PCR-变性梯度凝胶电泳(DGGE)分析混合菌群组成。在酸、热休克、碱和冻融四种预处理方法中,酸处理条件下富集混合菌群的产氢量最高,混合菌群的优势菌为Clostridium sp.和Bacillus sp.;热休克处理富集混合菌群的优势菌为Clostridium sp.和Enterococcus sp.,碱处理和冻融处理下富集混合菌群的优势菌是Clostridium sp.和Lactobacillus sp.。不同起始pH值对混合菌群产氢量有明显影响,pH值为7.0时,混合菌群产氢量达到最高,其它依次为pH6.5> pH7.5 > pH6.0 > pH8.0 > pH5.5 > pH5.0 > pH4.5 > pH4.0。16S rRNA基因的DGGE分析结果表明,在不同起始pH值条件下混合菌群的优势菌基本相同,NCBI-BLAST比对结果与Clostridium sp.存在较高的相似度(98%)。采用葡萄糖、蔗糖、乳糖、淀粉、蛋白胨5种底物进行厌氧发酵产氢,底物为蔗糖时混合菌群的产氢量最高,混合菌群优势菌为Clostridium sp.和Bacillus sp.。
2产氢菌株的分离与产氢条件优化
野生型产氢菌株的分离是扩大产氢微生物种质资源的必要手段。从天津海水浴场潮间带污泥中分离鉴定两株产氢菌。通过形态观察、生理生化特征和16S rRNA序列分析,两株产氢菌分别命名为Bacillus sp.B2和Clostridium sp.T7。在海水培养条件下,起始葡萄糖浓度为20 g/l,pH值为7.0时,菌株Bacillus sp.B2最大产氢量能达到1.65±0.048 mol H2/mol葡萄糖,菌株Clostridium sp.T7最大产氢量能达到1.79±0.062 mol H2/mol葡萄糖。
3成团泛菌突变体库的构建和高效产氢突变菌株的筛选
转座子诱变是获得高效产氢菌的有效途径。利用Tn7转座子体外转座成团泛菌基因组DNA,采用Kan抗性和转座子插入序列鉴定突变菌株。在含卡那霉素浓度为40μg/ml的平板上,随机挑取340个菌落,其中有331株菌通过PCR鉴定证实插入了转座子。产氢菌突变体库的库容量为4.35×108/ml。以产氢量为筛选指标,在已鉴定的331株突变菌中,筛选出一株高效产氢突变菌株TB5,其产氢百分含量和产氢量分别是野生菌株的1.1倍和1.52倍。菌株TB5的产氢最适pH为7.0,而野生菌株为6.0。在成团泛菌Tn7突变体库中,以能利用淀粉产氢为筛选指标,331株突变菌中有17株的产氢能力明显高于野生菌。
Enlarging species resources of hydrogen-producing bacteria is of importance in bio-hydrogen field. We studied the hydrogen-producing characteration of mixed culture and pure bacterium, and constructed mutant library of hydrogen producing bacterium by screening for wild type and transposon mutagenesis of hydrogen-producing bacteria. The aim of this research was to harvest efficient hydrogen-producing microorganisms This research can lay a theoretical foundation for construction of engineering strain that can utilize organic contents in the high-salt organic wastewater to generate hydrogen energy.
1. Effect of culture conditions on hydrogen producing of mixed culture
The effects of pretreatment, initial pH and substrate species on hydrogen producing of mixed culture were determined. Denaturing gradient gel electrophoresis (DGGE) was used for analyzing composition of microbial community. Four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing, acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. Acid pretreatment was favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp. and Bacillus sp. The dominant bacteria of mixed culture pretreated by heat shock were Clostridium sp. and Enterococcus sp. However, besides hydrogen-producing bacteria Clostridium sp., much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Hydrogen production of mixed culture was evidently affected by different initial pH. At the optimal initial pH of 7.0, hydrogen production potential of mixed culture was the highest, gradually decreasing at the following pH sequence of pH 6.5, pH 7.5, pH 6.0, pH 8.0, pH 5.5, pH 5.0, pH 4.5, and pH 4.0. The dominant bands obtained on DGGE of 16S rRNA gene were identical at various initial pH values. These bands were cloned and sequenced. NCBI blast indicated that the segment of 16S rRNA gene was high identical to that of Clostridium sp. (98%). Mixed culture was cultivated by five different substrates (glucose, sucrose, lactose, starch and peptone). The highest hydrogen production potential of mixed culture was observed when using sucrose as substrate. The dominant bacteria were Clostridium sp.and Bacillus sp..
2. Identification of hydrogen-producing bacteria and optimization of hydrogen production
Isolation of wild type hydrogen-producing bacteria is necessary means for enlarging species resource of hydrogen-producing microorganism. Two strain hydrogen-producing bacteria were isolated from sludge collected in the intertidal zone of a bathing beach in Tianjin. Bacteria with high hydrogen-producing ability were isolated and identified using light microscopic examination, physiologic and biochemical characterization and 16S rRNA gene sequence analysis. The isolated bacteria were designated Bacillus sp.B2 and Clostridium sp. T7, respectively. Hydrogen production of two strains was measured under marine condition, companied with an initial glucose concentration of 20g/l and an initial pH value of 7.0. The maximum hydrogen production was 1.65±0.048 mol H2/mol glucose for Bacillus sp.B2, whereas 1.79±0.062 mol H2/mol glucose for Clostridium sp. T7.
3. Construction of mutant library of Pantoea agglomerans and screening of high hydrogen-producing mutants
Transposon mutagenesis is an effective approach for harvesting efficient hydrogen-producing bacteria. A Tn7-based transposon was randomly inserted into genomic DNA of Pantoea agglomerans BH18 in vitro. Mutants were screened by Kanr and amplification of the inserted sequences. 340 colonies were randomly selected on the plate with 40μg/ml kanamycin, and in which 331 transposon insertion mutants were obtained by amplification of inserted sequences. Capacity of mutants was 4.35×108/ml. Using hydrogen production as screening index, a highly effective hydrogen producing strain TB5 was screened among 331 mutants. Its hydrogen content and hydrogen production were 1.1 and 1.52 times as much as that of wild type, respectively. The optimum pH of hydrogen production for strain TB5 was 7.0, whereas 6.0 for wild type. The ability for utilizing starch as hydrogen producing substrate was investigated. 17 strains with higher hydrogen production than wild type were obtained among 331 mutants.
引文
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