絮凝基因的克隆及其絮凝形态表征

分离出一株具有强絮凝特性的芽孢杆菌Bacillus sp. F2, 其絮凝率达到84%, 并构建了絮凝基因组文库, 从中筛选并获得表达絮凝活性的大肠杆菌阳性克隆子FC2. 絮凝试验测定FC2的絮凝率为90%, 稍高于原絮凝菌F2, 高于受体菌JM109(6.9%), 说明FC2絮凝性状遗传于原絮凝菌F2. 采用轻敲模式下的原子力显微镜成像技术、光学显微镜、ζ电位等对加入FC2与F2及不加入絮凝剂的絮凝微观形貌等进行了测定. 原子力显微成像显示, 加入克隆菌FC2发酵液的高岭土悬浮液形成的絮凝胶体出现大而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. 向高岭土悬浮液中加入克隆菌FC2发酵液后, 絮凝颗粒由松散的不定形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FC2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, 而且絮凝率达到90%, 进一步证实了克隆菌FC2发酵液的除污染效能. ζ电位测定结果表明, 离子键作用强度不同, 致使絮凝形态存在差异, 为研究生物絮凝剂的絮凝机理提供了有力的依据.

Cloning of Flocculent Gene and Morphology Characterization of Flocculent

One strain, named as Bacillus sp. F2, with the flocculent ability as high as 84%, was separated. Flocculent genomic library was constructed and a Bacillus coli. Positive clone FC2, which could express flocculent activity, was acquired after selection. The flocculent tests showed that the flocculent efficiency of FC2 was 90%, which was slightly higher than the original flocculent bacterium F2 and greatly higher than the competent cell JM109(6.9%), which demonstrated that FC2's flocculent characteristics inherited from the original flocculent bacterium F2. By adopting the tapping mode AFM, light microscope technique and ζ potential test, the flocculent microtopography of FC2, F2 and pure Kaolin suspending solution was identified. The AFM study revealed that, compared to the Kaolin suspending solution with and without F2's fermented liquid, the kaolin suspending solution to which fermented liquid with cloning bacterium FC2 was added had larger flocculent gel and more compact spherical structure, and the surface was rough with high degree concave and convex, had large specific surface area and strong adsorption ability to the suspending particles in the solution. After delivering the fermented solution of cloning bacterium FC2 into kaolin suspending solution, the amorphous and incompact flocculent particles transformed into spherical structure which was compact and had even horizontal dimension, this indicates that the agglutinin in FC2's fermented liquid could easily take Kaolin suspending particles as its adsorption core and adsorbed on its surface and the flocculent efficiency was about 90%, which gave further confirmation to the favorable pollution removal capability of FC2's fermented liquid. The results of ζ-potential test illustrates that the intensity of electrovalent bond was different, resulting in various flocculent morphology, which provided significant evidences for studying flocculent mechanisms of biofloculant.