| Flocculation process is a most important one in the water and wastewater treatment, downstream processing of bio-industry, and processes of foodstuff production and fermentation, and the trend of research is transferred from optimization of process in the past to the development of agent at present. As a new type product, bioflocculant (BF) is a safe and non-toxic substance, with high efficiency, non-secondary pollution and shows a great importance to human health and environmental protection. However, most of researches are in bench-scale stage at present. Because of high cost of cultural medium, the production and application of BF are restricted. Therefore, this papaer studies on fermentation kinetics of flocculant-producing bacteria F2, separation and purification, composition and structure, synthetic metabolism of bioflocculant, so as to provide reference for theoretical research and technical guarantee for industrialization of bioflocculant.Method of plate streak was used in rejuvenating flocculant-producing bacteria F2 whose ability of flocculantion become lower and lower. After rejuvenation, flocculation effect of bioflocculant produced by F2 strain returened to 95% of original level, F2 strain grow well and is used as original strain. In this study, the growth curve of F2 strain was measured by three method, the experimental result showed that exponential growth phase was 0-15h, stationary phase was 15-24h, decline phase was 24h later, and generation time was 44.1min. This paper made tracing monitoring on dissolved oxygen, pH, dry cell weight, active component content, flocculating rate, protein content, glucose content, total nitrogen, and total phosphorus of F2 strain fermentation procession. Fermentation medium were optimized, the experimental result showed that K2HPO4 was essential, its content was no fewer than 5.0 g/L, and could be added or not. For depicting the cell growth, bioflocculant-producing and depletion of glucose, the Logistic Equations, Luedeking-Priet are used in quantifying the proeessing. The biofloeeulant-producing dynamics of F2 strain were demonstrated. The models about the strain growth, production of bioflocculant, and consumption of glucose were deseribed. Through the comparison of experimental data and the corresponding calculated values from the models,we found that the data joint well.The flocculation activated substances of BF distributed mainly in upper phase, so flocculationg efficiency of BF does not caused by bacteria cell. BF possed good thermal staility, but its activity change acutly with pH varying. The components of BF are complex, mainly composed of polysaccharide and protein. Chemical properties of BF were also analyzed. All above results demonstrated that BF contained polysaccharide and protein, the content ratio was 97.4: 2.2 and polysaccharide was bearer of flocculating function. BF was extracted by organic solvent, the experimental result showed that the optimum extracting conditions were that contrating multiple is 1/2, ethanol consistency is 100%, ethanol quantity is 1:2, precipitating time is 2h, and pH is 6. There was some free protein in BF, so was used to eliminate free protein. The optimum eliminating conditions were that Vsample: Vsevage is 2:3, Vchloroform: Vbutanol is 5:2, eliminating time is 30 min. The experimental result showed that the active component of BF was polysaccharide, and the protein was lower after process and the flocculation activity maintained high. According to the properties of BF, three week anion exchange DEAE Sephadex A-25,DEAE Sepharose Fast Flow and DEAE-650M were slected for purifying BF, and the best one was DEAE-650M. Chromatographic conditions were determined: week anion exchange DEAE-650M, 2.6?20 cm column, pH8.0 Tris-HCl as initial buffer, 0-1.5 mol/LNaCl as eluent, 6mL as sample content, 54mL/h as elution speed, 3mL/tube as collection amount.The flocculating active substances through purification were amprphism solid, milk white. Detecting with agarose gel electrophoresis, there was only a blue band. The UV spectra showed the presence of groups which were the typical characteristics of polysaccharide and protein, while no nucleic acid was found in BF. Its molecular weight was determined by using GPC Column-Ultrahydrogel Linear in HPLC system, while the Dextran series were used as the molecular weight standard, at last the molecular weight was determined about 1156, 000Da. The thin layer chromatography (TLC), GC and GC-MS all showed BF was consisted by glucose, galactose and mannose. It could be seen from the scanning of infared spectrograph that the purified BF not only had amino group (or amide group) which were the typical characteristics of protein, but also had carboxyl and hydroxyl groups which were the typical characteristics of polysaccharide in its molecule, there were ?-pyranose. 1H NMR spectrum exhibitepolysaccd the sample was polysaccharide composed of three kind of single sugar, there wereα-galactopyranose,?-mannopyranose andα-glucopyranose, the mass ratio was 1:3:5, therefore there existed two kinds of glycosidic bonds withαand ? configuration respectively, and the chemical formula was [C54H108O54]n.Based on composition and structure of BF, F2 strain utilization of different carbon source and intermediate, a hypothetic metabolic pathway of BF was proposed. |
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