Study On Flocculent Characteristics And Heredity Of Flocculent Producing Cloning Bacteria

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.