基于产絮克隆菌的遗传及产絮特性研究
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摘要
生物絮凝剂作为一种高效、无毒、无二次污染、具有生物可降解性和安全性的绿色水处理剂,代表了絮凝剂的重要研发方向之一。本研究对所分离到的高效絮凝菌进行了菌种鉴定,并对其絮凝特性及代谢成分进行了分析。同时以基因定位为基础,进行了絮凝菌的细胞融合,构建了絮凝菌的基因组文库,得到了具有絮凝特性的克隆菌,并将克隆菌应用到两段式发酵工艺中,即以纤维素降解菌的产物为絮凝菌的发酵原料,进行二段式发酵,获得了新型生物絮凝剂。
经生理生化和16SrDNA分析鉴定,絮凝菌F2为芽孢杆菌属(Bacillus sp.)。通过絮凝活性分布测定,絮凝剂的有效成分存在于发酵液中,为絮凝菌的胞外产物。进一步利用红外光谱、紫外扫描、考马斯亮蓝等手段,确定了生物絮凝剂的有效成分为多糖类物质,具有良好的高效产絮遗传稳定性。为研究产絮菌的遗传特性,采用质粒转化技术进行了絮凝基因的定位实验:将絮凝菌的质粒转入无絮凝特性的大肠杆菌感受态细胞中,得到大肠杆菌阳性转化菌株;经检测,转化菌株无絮凝特性,说明絮凝基因存在于染色体DNA,初步确定絮凝菌质粒无絮凝基因控制。从而确定了絮凝菌的代谢途径,为合成新型、经济的代谢产物及分析代谢产物成分提供了理论基础。
以细胞融合技术作为絮凝菌的改良手段,采用促融合剂聚乙二醇(PEG),进行原生质体的融合,得到融合细胞。通过混凝试验验证,融合细胞的絮凝效果与单菌株一致,表明细胞融合技术作为絮凝菌的改良是有效的、可行的。
为进一步分离和研究絮凝基因,构建了絮凝基因组文库。从文库中筛选出了一株表达絮凝活性的大肠杆菌阳性克隆子FC2,其絮凝率为90%,高于原絮凝菌和受体菌,说明克隆菌FC2絮凝性状遗传于原絮凝菌。采用轻敲模式下的原子力显微镜成像技术、光学显微镜、Zeta(ξ)电位对絮凝微观形貌进行了测定。结果显示,加入克隆菌发酵液的高岭土悬浮液形成的絮凝体出现较大而且紧密的球形颗粒结构,且表面积粗糙,凹凸程度大,具有大的比表面积和吸附液体悬浮颗粒的能力。同时,絮凝颗粒由不定形且松散的结构转变为密集分布、水平尺寸均匀的球形结构,表明克隆菌发酵液中的凝集素容易以高岭土悬浮颗粒为中心吸附在其表面,从更直观上进一步证实了克隆菌发酵液的净水效能。Zeta(ξ)电位测定结果表明,离子键作用强度不同,致使絮凝形态存在着差异,为研究生物絮凝剂的絮凝机理提供了有力的依据。
最后,采用纤维素降解菌和絮凝菌组成的复合型生物絮凝剂产生菌群,进行两段式发酵工艺。以HIT-3的纤维素降解产物作为絮凝菌的代谢底物,得到优良的复合型生物絮凝剂。并研究纤维素降解菌HIT-3的酶学特性,对纤维素酶活进行测定,培养6d达到产酶高峰,酶活力可达到0.676U·mL-1,所产生的有机碳完全能被絮凝菌作为发酵的底物利用,产生的复合型生物絮凝剂的絮凝效果高达94.5%,使生物能源再利用成为可能。
As an efficient, untoxic, non-secondary contaminative, biodegradable and environmental-friendly green wastewater disposal agent, bioflocculant is one of the most potential flocculants and would certainly receive more and more concerns. In the present research, the isolated flocculant-producing bacteria were identified and their flocculating characteristics and metabolic components were also analyzed. In addition, based on gene localization, the cell fusion and the gene liberary construction of flocculant-producing bacteria were conducted, by which cloning bacterium with flocculating characteristics was obtained. Then the cloning bacterium was applied in the two-stage fermentation process, i.e., the metabolic products of the cellulose degrading bacteria were utilized by flocculating bacteria as fermentation substance to carry out two-stage fermentation in order to obtain novel bioflocculant.
Through physiological-biochemical and 16SrDNA analysis, the flocculant producing bacteria F2 was identified as Bacillus sp.. Results of flocculating activity distribution tests indicated that the effective component of flocculant existed in fermentation solution and was extracellular product. Moreover, through such as method coomassie blue reaction, ultraviolet spectroscope and infared spectra, the effective component of flocculant was determined as polysaccharide, which has favorable and efficient flocculant producing inherited stability.
The location of flocculating gene was carried out by plasmid transformation to investigate. The plasmid of flocculant the inherited characteristics of flocculating bacteria producing bacteria was extracted and transformed into the competent cells of E. coli without flocculating ability. The results showed That the Tran for mants didn’t have flocculating characteristics and the flocculant gene but not flocculant gene located on chromosomes DNA and not on plasmid. It provide theoretical foundation for the determinationis of metabolic pathway of flocculant-producing bacteria, and the synthes is of novel and economical metabolic product as well as the composition analysis of metabolic products .
Protoplast fusion technology was applied to optimal flocculants producing bacteria to carry out protoplast fusion. Polyethylene glycol (PEG) solution was used as fusion agent to obtain fusion cell.The results showed that the flocculating efficiency of fusion and the single slain were the same, indicating that protoplast fusion technology is feasible and effective for the improvement of flocculant-producing bacteria.
For the further isolation and research of flocculating gene, the flocculent genomic library was constructed. A positive cloning bacterium FC2, which could express flocculating activity, was acquired after selection. Flocculent tests showed that the flocculent efficiency of FC2 was 90%, which was higher than the original flocculent bacterium and the competent cell. It demonstrated that FC2’s flocculent characteristics were inherited from the original flocculant-producing bacterium. By adopting the tapping mode AFM, light microscope technique and Zeta-potential test, the flocculating microtopography was identified. The AFM study revealed that, the kaolin suspending solution, which has cloning bacterium FC2’s fermented liquid had larger flocculent gel and more compact spherical structure, and the surface was rough with high degree concave and convex, and had large specific surface area and strong adsorption ability to the suspending particles in the solution. In addition, the amorphous and incompact flocculent particles transformed into spherical structure and was compact and had even horizontal dimension, which indicated that the agglutinin in the fermentation liquid of cloning bacteria could easily take kaolin suspending particles as its adsorption core and adsorbed on its surface, which gave further evident to the effective pollution removal capability of FC2’s fermented liquid. The results of Zeta-potential test illustrated that the intensity of electrovalent bond was different, resulting in various flocculent morphology, which provided significant evidences for studying flocculent mechanisms of biofloculant.
Finally , two-stage fermentation process was conducted by adopting compound-bioflocculant-producing flora composed of cellulose-degrading bacterium and flocculating bacterium. The cellulose degrading metabolites of HIT-3 was taken by flocculating bacterium F2 as its substrates, by which way excellent compound bio-flocculant was obtained. In addition, the enzymology characteristics of HIT-3 were investigated when cultured in cellulose media which utilized CMC-Na as its sole carbon. The results showed that HIT-3 achieved enzyme producing climax after 6 days’incubation, which reached as high as 0.676U·mL-1, and the organic carbons produced was sufficient as the substrates required by the fermentation of flocculating bacterium F2(flocculating efficiency=94.5%), which make it is feasible to reuse bioenergy.
经生理生化和16SrDNA分析鉴定,絮凝菌F2为芽孢杆菌属(Bacillus sp.)。通过絮凝活性分布测定,絮凝剂的有效成分存在于发酵液中,为絮凝菌的胞外产物。进一步利用红外光谱、紫外扫描、考马斯亮蓝等手段,确定了生物絮凝剂的有效成分为多糖类物质,具有良好的高效产絮遗传稳定性。为研究产絮菌的遗传特性,采用质粒转化技术进行了絮凝基因的定位实验:将絮凝菌的质粒转入无絮凝特性的大肠杆菌感受态细胞中,得到大肠杆菌阳性转化菌株;经检测,转化菌株无絮凝特性,说明絮凝基因存在于染色体DNA,初步确定絮凝菌质粒无絮凝基因控制。从而确定了絮凝菌的代谢途径,为合成新型、经济的代谢产物及分析代谢产物成分提供了理论基础。
以细胞融合技术作为絮凝菌的改良手段,采用促融合剂聚乙二醇(PEG),进行原生质体的融合,得到融合细胞。通过混凝试验验证,融合细胞的絮凝效果与单菌株一致,表明细胞融合技术作为絮凝菌的改良是有效的、可行的。
为进一步分离和研究絮凝基因,构建了絮凝基因组文库。从文库中筛选出了一株表达絮凝活性的大肠杆菌阳性克隆子FC2,其絮凝率为90%,高于原絮凝菌和受体菌,说明克隆菌FC2絮凝性状遗传于原絮凝菌。采用轻敲模式下的原子力显微镜成像技术、光学显微镜、Zeta(ξ)电位对絮凝微观形貌进行了测定。结果显示,加入克隆菌发酵液的高岭土悬浮液形成的絮凝体出现较大而且紧密的球形颗粒结构,且表面积粗糙,凹凸程度大,具有大的比表面积和吸附液体悬浮颗粒的能力。同时,絮凝颗粒由不定形且松散的结构转变为密集分布、水平尺寸均匀的球形结构,表明克隆菌发酵液中的凝集素容易以高岭土悬浮颗粒为中心吸附在其表面,从更直观上进一步证实了克隆菌发酵液的净水效能。Zeta(ξ)电位测定结果表明,离子键作用强度不同,致使絮凝形态存在着差异,为研究生物絮凝剂的絮凝机理提供了有力的依据。
最后,采用纤维素降解菌和絮凝菌组成的复合型生物絮凝剂产生菌群,进行两段式发酵工艺。以HIT-3的纤维素降解产物作为絮凝菌的代谢底物,得到优良的复合型生物絮凝剂。并研究纤维素降解菌HIT-3的酶学特性,对纤维素酶活进行测定,培养6d达到产酶高峰,酶活力可达到0.676U·mL-1,所产生的有机碳完全能被絮凝菌作为发酵的底物利用,产生的复合型生物絮凝剂的絮凝效果高达94.5%,使生物能源再利用成为可能。
As an efficient, untoxic, non-secondary contaminative, biodegradable and environmental-friendly green wastewater disposal agent, bioflocculant is one of the most potential flocculants and would certainly receive more and more concerns. In the present research, the isolated flocculant-producing bacteria were identified and their flocculating characteristics and metabolic components were also analyzed. In addition, based on gene localization, the cell fusion and the gene liberary construction of flocculant-producing bacteria were conducted, by which cloning bacterium with flocculating characteristics was obtained. Then the cloning bacterium was applied in the two-stage fermentation process, i.e., the metabolic products of the cellulose degrading bacteria were utilized by flocculating bacteria as fermentation substance to carry out two-stage fermentation in order to obtain novel bioflocculant.
Through physiological-biochemical and 16SrDNA analysis, the flocculant producing bacteria F2 was identified as Bacillus sp.. Results of flocculating activity distribution tests indicated that the effective component of flocculant existed in fermentation solution and was extracellular product. Moreover, through such as method coomassie blue reaction, ultraviolet spectroscope and infared spectra, the effective component of flocculant was determined as polysaccharide, which has favorable and efficient flocculant producing inherited stability.
The location of flocculating gene was carried out by plasmid transformation to investigate. The plasmid of flocculant the inherited characteristics of flocculating bacteria producing bacteria was extracted and transformed into the competent cells of E. coli without flocculating ability. The results showed That the Tran for mants didn’t have flocculating characteristics and the flocculant gene but not flocculant gene located on chromosomes DNA and not on plasmid. It provide theoretical foundation for the determinationis of metabolic pathway of flocculant-producing bacteria, and the synthes is of novel and economical metabolic product as well as the composition analysis of metabolic products .
Protoplast fusion technology was applied to optimal flocculants producing bacteria to carry out protoplast fusion. Polyethylene glycol (PEG) solution was used as fusion agent to obtain fusion cell.The results showed that the flocculating efficiency of fusion and the single slain were the same, indicating that protoplast fusion technology is feasible and effective for the improvement of flocculant-producing bacteria.
For the further isolation and research of flocculating gene, the flocculent genomic library was constructed. A positive cloning bacterium FC2, which could express flocculating activity, was acquired after selection. Flocculent tests showed that the flocculent efficiency of FC2 was 90%, which was higher than the original flocculent bacterium and the competent cell. It demonstrated that FC2’s flocculent characteristics were inherited from the original flocculant-producing bacterium. By adopting the tapping mode AFM, light microscope technique and Zeta-potential test, the flocculating microtopography was identified. The AFM study revealed that, the kaolin suspending solution, which has cloning bacterium FC2’s fermented liquid had larger flocculent gel and more compact spherical structure, and the surface was rough with high degree concave and convex, and had large specific surface area and strong adsorption ability to the suspending particles in the solution. In addition, the amorphous and incompact flocculent particles transformed into spherical structure and was compact and had even horizontal dimension, which indicated that the agglutinin in the fermentation liquid of cloning bacteria could easily take kaolin suspending particles as its adsorption core and adsorbed on its surface, which gave further evident to the effective pollution removal capability of FC2’s fermented liquid. The results of Zeta-potential test illustrated that the intensity of electrovalent bond was different, resulting in various flocculent morphology, which provided significant evidences for studying flocculent mechanisms of biofloculant.
Finally , two-stage fermentation process was conducted by adopting compound-bioflocculant-producing flora composed of cellulose-degrading bacterium and flocculating bacterium. The cellulose degrading metabolites of HIT-3 was taken by flocculating bacterium F2 as its substrates, by which way excellent compound bio-flocculant was obtained. In addition, the enzymology characteristics of HIT-3 were investigated when cultured in cellulose media which utilized CMC-Na as its sole carbon. The results showed that HIT-3 achieved enzyme producing climax after 6 days’incubation, which reached as high as 0.676U·mL-1, and the organic carbons produced was sufficient as the substrates required by the fermentation of flocculating bacterium F2(flocculating efficiency=94.5%), which make it is feasible to reuse bioenergy.
引文
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