Isolation And Characterization Of A Bioflocculant Produced By Arthrobacter Sp. LF-Tou2

1. Facing to startling water pollution, the study on industrial microbial technologies and microbial flocculant leads a new direction for new century water purification. Microbial flocculants are natural biological macromolecules and biopolymers with flocculating function, high efficiency, safe, biodegradation, without environmental pollution. Because such kinds of modern biochemical products are new inexhaustible natural resources they are displaying stronger momentum in todays economic society of resource and ecological environmental crisis.2. Isolate is capable of quickly growing and producing high activity microbial flocculant by the selective medium with the organic contaminant as the sole carbon source. This strain was identified as Arthrobacter sp. and further named as LF-Tou2 through analyses of morphological, physiological and biochemical characteristics, and 16S rDNA sequence. The research results from the optimization of culture conditions are as follows: This organism can grow well by using the special carbon source, such as polyethylene glycol (PEG), hexane, cyclohexane which belong to organic contaminants; The optimal concentration of the glucose confirmed as the suitable carbon source is 2% for the strain growth and production of bioflocculant with higher flocculating activity, (NH4)₂SO₄ is the optimal nitrogen source; It efficiently produces bioflocculant in liquid culture with glucose and ammonia sulphate in the optimal carbon/nitrogen ratio of 6:1; The optimal pH and pitching rate is 7.0, and 1,0× 10⁵ cells per milliliter culture solution, respectively; In the inorganic salts tested CaCl₂ and ZnCl₂ are very effective for the formation of flocculating activity in the culture process, the flocculating rate (FR)increases 5.93% and 5.92%, respectively when the initial concentration is as 4 mmol/L; The optimal culture temperature for LF-Tou2 is 27 ℃ among the temperature researched, and the completely parallel relationship do not exist between the formation of flocculating activity and the cell biomass; Vitamin B can promote the cells to formbioflocculant, and the corresponding flocculating activity increases 5.68% and 3.80% respectively while 40 mg/L B9 or 6 mg/L Bn is added into the culture system. The data from 1L of shaking flasks indicates that the cells reache the later logarithmic phase at 12 h and stationary phase at 18h. Before 24h the production of polysaccharide is parallel to cell growth and at this time the production reaches the highest level, and the content of polysaccharide decreased reversely with prolonging cultivation o The production of glucosyltransferase (GTase) forms wave-like curve and reaches maximum at 18h. The content of polysaccharide comes to maximum at 24h, i.e. 98.08%. The reducing sugar use up 91% at 12h and at the same time the FR achieves the supreme, i.e., 98.08%. The results of batch culture is that LF-Tou2 produces 2.78g/ of polysaccharide by using lOg/L of sugar. The fed-batch culture actually supplemented 0.8% of carbon source at 12h in the culture period of the batch. Although in the culture of batch-complemented culture the peak time of production of polysaccharide substance and formation of flocculating activity put off about 6h, the polysaccharide substance formed from 18g/L of carbon sugar reaches 6.06 g, and forming rate increases to 33.7% from 27.8% of batch.3. For probing into flocculating mechanism of microbial flocculant produced by Arthrobacter sp.LF-Tou2 the following experiments have been done, such as the effects of environmental factors for flocculant formation, e.g. metal cations > If1" concentration % charge neutralization and style, the affecting factors for flocculating function, e.g. protein ingredients^ -OKL cationsN absorption action of heavy metal ions, e.g. absorption during the cell culture^ absorption by flocculatiom the relationship between -OH and absorption and so on. The experimental results prove that metal cations can promote the flocculant formation, and the order for promoting action is Fe2+>Mn2+>Ca2+o Among the cations tested Ca2+ , which performed the charge neutralization action in the flocculating formation is the safest and has the wide applicability; On the condition of alkalinity pH the flocculating activity increases gradually with the pH value getting a raise, this fact reflects that -OH can assist the stretch of inner structure from polymer, thereby the display of flocculating activity is enhanced; On the basis of bridging mechanism in the designed three styles of charge neutralization, the best result is obtained for chargeneutralization by adding Ca2+ before and after the flocculating formation. The first time is to neutralize the small molecular particles, for the second time the charge neutralization is for the linked complex of particles and large molecular polymers, and at last the flocculating rate attains 98.35%. For the flocculating system, heavy metal ions are opposite ones also. The absorption for bioflocculant to heavy metal ions reflects the charge neutralization. The data researched by the experiments prove: the absorption rate of cells in the culture to Fe2"\ Mn2+> Hg2+ and Ni2+ under the concentration of test are all 100%, Cu2+ has a obviously inhibition for the initial cell growth and flocculant formation. The absorpting rates of bioflocculant broth for Fe2+ and Mn2+ are very high, i.e. 100%, 93.86% respectively. The testimonies from the research of effects of the factors to flocculating function are: protein is the functional ingredient of bioflocculant; DEAE is with a deflocculating action for the floe formed by bioflocculant and metal ions; -OH is functional group for taking place of flocculation; For the suitable particle concentration the ideal flocculation is with a optimal polymer concentration, and on the contrary for certain concentration of bioflocculant the optimal corresponding concentration of particles exists also. In short, bioflocculating model for bioflocculant produced by Arthrobacter sp. LF-Tou2: in the -OH environment just right quantity of cations (e.g. Ca2+) neutralized with the negative charged particles in flocculating system, after mixing the polymer bioflocculant carried net negative charge links by bridging with neutralizing or destabilizing particles, then adding another suitable quantity of cations to neutralize the charges carried by polymer itself, at last the desired flocculating result may be produced.4. The fermentation broth of Arthrobacter sp. LF-Tou2 cultured on the optimized conditions is purified through CTAB reaction â– > ion-exchange chromatograph of DEAE-Sepharose Fast Flow^ gel chromatograph of Sephadex G-200, and the samples obtained are further analyzed for their series structural characterization. The results prove: the purified sample possesses characterized absorption at 215 nm wavelength by series wavelength measurement with UNICO UV-2000 Ultraviolet Spectrophotometer and continuous scanning with UV02100 SHIMADZU Ultraviolet Spectrophotometer. It canbe seen from the scanning of NEXUS 470 Infared Spectrograph that the purified samples have a strong -OH stretching vibration at 3423.21 cm"1, and weak C-H stretching vibration at 3000-2800 cm"1, a turn vibration of C-H bonds at 1400.76 cm"1, and C-O-C stretching vibration of sugar rings at 1627.02 cm"1 and 1136.44 cm"1; There are p-glycosidic bonds on the tested sample which represent a typical polysaccharide substance; ^-NMR spectrum exhibit the linked bonds among the single sugars mainly are |}-glycosidic bonds , there are small amounts of a-glycosidic bonds in side chains parts. The 4 resonance signals of I3C-NMR spectrum in the range of 107-105ppm represent that the sample is miscellaneous polysacchride composed of 4 kinds of single sugar. Therefore there exist two kinds of glycosidic bonds with a or p configuration respectively, and with substitution of methyl and acetylamino; The polysacchride molecular weight is 3.89><106 Da measured by Sephadex G-200 Gel chromatograph; The single sugar analyses by TLC chromatograph and capillary elecotrophoresis for hydrolyzate of polysaccharide prove that the sample contains Gnu Rha- Gak and Man, and which have the flowing molar ratio: Glu:Rha:Man:Gal=9:6:l:lo The analyses of protein hydrolyzate of sample by automatic amino acids analyzer shows that the content of amino acids in the purified sample is 4.849%, and their weight ratio as following: aliphatic: hydrophobic: aromatic =30.51:7.08:1.00.It can be concluded from the analyses of purification and structural characterization that the bioflocculant produced by Arthrobacter sp. LT-Tou2 is protein polysaccharide Macromolecules, and among of which the polysaccharide is miscellaneous ones.5. Glucosyltransferase(Gtase) from Arthrobacter sp. LF-Tou2 is the enzyme for controlling synthesis of flocculant polysaccharide. The enzyme was released by sonicating the cells and purified through Sephadex G-200 gel chromatography and DEAE-Sepharose Fast Flow ion chromatography .The enzymatic characteristics were analyzed ,and the relationship between catalytic activity and 5 different amino acid residues were studied by 5 chemical modified reagents .The results shows that the molecular mass is 14315 Da analysed by MALDI-TOF-MS; The sample contains 31.7% of a-helix, 34.1% of P-turn and 34.2% of random-coil conformation measured by CD;Km value and Fmax value is 2.752Mraol and 58.056umol/min, respectively. Mn2+ is the strongest activator, then Fe2+. The tyrosyl residues and disulfide bonds are the structural groups in enzymatic molecular groups to catalytic activity. The tryptophan% mercapto and histidine are the essential groups, furthermore, tryptophan is the key group in the essential ones.6. In chapter 6 the experiments deal with the studies on mid-scale trail for bioflocculant fermentation process, optimized extractive conditions of downstream process? the flocculating results compared with chemical flocculant , the parameters for production compared with some reported bioflocculant and the effective test on the spots. The dynamic analyses of 2000L fermentation illustrates: in constant pH culture, polysaccharide and flocculating activity in the culture system reached maximum at 24h, i.e. 0.90% and 96.1% respectively, and the culture time shortened 6h compared with the natural pH culture of fed-batch culture. Optimized research of downstream conditions shows: on the one hand, when the broth which initial carbon source is 18g/L is extracted with ethyl alcohol the optimal addition fold is 2.4 and optimal pH value for precipitation of fermentation broth is 7.00. On the other hand inorganic cations used have a obvious effect on the extracting rate, and the order is Fe3+>Al3+.Mn2+>Ca2+, The effect of Fe3+ is prominent. The extracting rate of protein polysaccharide flocculant reaches the highest, i.e. 99.75% when the concentration of Fe3+ is 0.6 mmol/L, and it increase 21.02% than that of control. The flocculating effect for Yellow River water is very obvious. By treating the removing rate are : turbidity, 99.7%; SS, 100%; COD, 87%; Fe3+, 99.7%; Mn2+, 98.7%; Cu2+, 100%; Zn2+,91.8%; Ni2+, 100%; Hg2+, over 75%; FR, 98.92%. It can be found from the flocculating treatment of Domestic Sewage that the removing rate is respectively: COD, 93.5%; FR, 94.8%; SS, 100%; heavy metals, such as Fe3\ Ni2+ and Hg2+ all >90%. The removing rate of the Coal-Washing Waste Water by bioflocculant are: turbidity, 97%; SS, 100%; COD, 99%; Fe3\ Mn2\ Ni2+>92%; The removing rate for Ca2+ and Zn2+ are only about 60%, which is not better than that of above heavy metals; FR, 97.65%. The treatment cost for the water sample on the spots only is between 0.2 and 0.325 Yuan RMB/ton water. It is investigated from the comparison with chemicalflocculant products that the dosage for researched bioflocculant is only 4g/ton water, and FR for Kaolin clay suspension is as high as 98.9%. It is found from the comprehensive comparison with the reported bioflocculant on strains, culture period > productive yield > medium component etc. that the several targets of researched bioflocculant produced by Arthrobacter sp. LF-Tou2 are on the international advanced level no matter do the medium composition (sugar> ammonium sulphate)^ culture period (ld)N productive yield (6.67g/L), or macromolecular weight (3.8x106 Da).7. Toxicological characterizations tested by rats explain: the acute toxicity test through mouth the samples belongs actually to nontoxic; Ames test is negative and the sample is without micronuleus-producing action for the addicted multistaining erythrocyte from rats. The microbial flocculant researched is a kind of safe> nontoxic natural fermentation preparation.