Preparation, Purification And Application Of Bioflocculant Excreted From Agrobacterium Radiobacter

Bioflocculants are newly developed natural macromolecular organic substances produced by microorganisms, which have the ability to flocculate the suspensions and separate solid from the mixed. Compared with traditional inorganic and organic flocculants, they have the characteristic of biodegradability, high performance, harmlessness to environment, and heavy metal biosorption. So bioflocculants have drawn more and more attentions and become front line of flocculants investigation.Twenty-eight strains that could produce bioflocculants were screened from ten samples. A novel bioflocculant-procucing bacterium M-503 was isolated from activated sludge taken from sewage plants, and the bioflocculant it produced had the excellent flocculating ability (910U), while M-503 could not utilize polysaccharide (eg. starch) as the sole carbon source. Through 16S rDNA sequencing method and physiological experiments, M-503 was identified to be Agrobacterium radiobacter. There was no report about bioflocculant produced by Agrobacterium radiobacter before.Bioflocculant ZL5-2 produced by Agrobacterium radiobacter M-503 was a kind of extracellular bioflocculant, and it consisted of 93.5% neutral sugar, 3.5% uronic acid, and 3.0% protein. Result of infared spectra analysis: the absorbance peak around 3417cm reflected -OH stretching vibration, and the absorbance peak around 2928cm reflected C-H stretching vibration, and the absorbance peak around 1619cm'1 reflected amid I group stretching vibration, and the absorbance peak around 1400cm1 reflected C-H bending vibration, and the absorbance peak around 1100 cm"1 reflected C—O and C—OH stretching vibration. Through composition analysis and infared spectra analysis, ZL 5-2 was proved to be a kind of polysaccharide substance containing little protein.Culturing in bioflocculant-producing medium, Agrobacterium radiobacter M-503 came into exponential phase at 12 hour, and came into stationary phase at 24 hour. ZL 5-2 began to produce in exponential phase, men paralleled with culturing time extending, production was accumulated largely in stationary phase, and ZL 5-2 reached its maximal yield at 48hour. When prolonged culturing time, Agrobacterium radiobacter M-503 could not utilize ZL 5-2 because Agrobacterium radiobacter could not utilize polysaccharide as the sole carbon source, which was propitious to accumulation of ZL 5-2.The optimal carbon source was sugar and the optimal nitrogen source was yeast extract. The highest yield was acquired after culturing 48 hours with the shake speed of 120r/min in 30°C, and the quantity was 14.9g/L while the flocculating ability was 991U. The yield of ZL 5-2 was very low with the medium initial pH<7, while the yield was high and stable with the medium initial pH 7~12. The yield of ZL 5-2 was affected little by the changing of aeration, which was propitious to save the production cost.ZL 5-2 was applied effectively within pH 6~12. Both Excessive and deficient bioflocculant were adverse to the flocculation because deficient flocculent could not flocculate the suspensions effectively, while excessive flocculent would make the kaolin stabilize again. Flocculation mechanism: a flocculent molecular adhering to the kaolin molecular extended to the solution, and the extending part of the flocculent combined other kaolin molecular, and other kaolin molecular combined other flocculent molecular, then flocculation happened. Combining positions on kaolinsurface could not be held abundantly in deficient flocculent concentration, while excessive flocculent would make the kaolin stabilize again because the saturation of the combining position in the kaolin surface prevented flocculation.Tervalent and bivalent positive ions could improve the flocculation, because they had several binding positions to combine kaolin, while monovalent positive ions did not have the characteristic. Ca2+ was the best positive improving ion and Ca2+ was not harmful to the environment. With the heat-treatment among 4-121 "C in 30mins, the flocculating ability of ZL 5-2 retained more than 98%. Pepsin, trypsin, trichloroacetic acid, a-amylase and cellulase had no significant effect on the flocculating ability of ZL 5-2.Cr(VI) existed in the form of Cr2O72" .Biosorption of Cr(VI) by ZL5-2 was studied, and infrared spectra of native, absorbing and desorbing ZL 5-2 were compared, and it was the first report about biosorption by bioflocculant ZL 5-2 with the method of Fourier infrared spectrometry. The initial pH was important for biosorption. The optimal effect on Cr(VI) biosorption was acquired within initial pH 0.5-1.5,then with the pH increasing, the effect on Cr(VI) biosorption was reduced. ZL 5-2 consisted of polysaccharide and protein with functional group (such as -OH and -NH) in its surface. With pH falling, ZL 5-2's surface would accumulate more and more positive charge, then combining position with Cr2C>72" increased. So the falling of pH would be propitious to the biosorption of Cr(VI). In the biosorption progress, H* exchange happened firstly, then $t combined with functional group (such as -OH and -NH), finally they chelated Cr2O72". The biosorption for Cr(VI) was a quick process ,only within lOmin 65.4% Cr(VI) is biosorbed, and the biosorption was in equilibrium after 60 min, then all the Cr(VI) was biosorbed after 80 min. After ZL 5-2 biosorbed Cr(VI), there was no greenish - gray deposition in the supernatant, which proved that biosorption of Cr(VI) was not the result of oxidoreduction. The absorbed Cr(A/I) could be recovered by desorption and the desorption rate reached 13.6—67.9%. The absorbance peak around 3400cm"1, which reflected -OH stretching vibration,moved about 8 cm"1, and its absorbance decreased; the absorbance peak around 2900 cm'1 decreased, which reflected C-H stretching vibration; and the absorbance peak around 1600 cm'1, which reflected amid I group stretching vibration moved about 13 cm"1, and its absorbance decreased. After desorption, the absorbance peaks which reflected OH, C—H were increased to the initial degree, and the absorbance peak which reflected NH and amid I group did not increase. The biosorption are fitted with Langmuir and Freundlich isotherm adsorption equations. Therefore, we presumed mat the progress of biosorption was reversible adsorption and irreversible adsorption simultaneously, however the reversible adsorption was more important in the biosorption.ZL-P was purified from supernatant by ethanol precipitation, ion-exchange chromatography and gel chromatography. The flocculating ability of purified ZL-P was 1.449 times than that of ZL 5-2, and the total flocculating ability of ZL-P was retained 53.07%. Its molecular weight was determined by using GPC Column-Ultrahydrogel Linear in HPLC system and the refractive index detector was used as the detector, while the Dextran series were used as the molecular weight standard, at last the molecular weight was determined about 81, OOODa. Through element analysis, the content of N, C and H in ZL-P were 0.007%, 38.410% and 6.662% separately. The Fourier transform infrared spectra and UV spectra showed the presence of hydroxyl, carboxyl and methoxyl groups which were the typical characteristics of polysaccharides in its molecule, while no protein and nucleic acid was found in ZL-P. Chemical and chromatographic analysis indicated that it mainly consisted of glucose by using the method of GC-MS. We presumed that glucose residues were connected with P (1 —6 ) glycosidic bond through lH NMR and 13C NMR analysis. The molecular of ZL-P was lower compared with the reported bioflocculant, and the configuration of ZL-P was not reported before.Bioflocculation of the wastewater let off from printworks and refinery and microbe from fennentation by the bioflocculant ZL5-2 was studied and comparedwith the flocculating ability that poly aluminum flocculent flocculated the waste water and the microbe. The flocculating ability of the wastewater from printworks and refinery was 74.7% and 65.8% separately, and COD reduced 32.4% and 49.5% separately, and BOD reduced 55.4% and 62.3% separately. The flocculating ability of the penicillium ferment and the actinomycetes ferment was 68.4% and 69.0% separately. The flocculating ability of ZL 5-2 was better than the flocculating ability of equal volume of poly aluminum flocculent. It was the first report about bioflocculation of the wastewater let off from refinery.