| Microbial flocculant(MBF)is a kind of flocculating substance produced by kinds of microorganisms at a stage of growth and metabolism under a certain culture conditions.With the advantages of high efficiency,safety and no residue,and as a water treatment agent,it has been widely used in the research and application of sewage treatment,such as living,printing and dyeing,dairy and so on.However,the microbial flocculant treatment of wheat alcohol wastewater with high acidity,concentration,viscosity and temperature has not been reported.At present,Chemical Flocculant(polyacrylamide)is often used to treat this kind of wastewater.Although the treatment cost is not high and its treatment effect is also better,but the residual acrylamide monomer is easy to appear.It is a nerve poison to human body,which can cause neurotoxicity and cancer effect.After poisoning,it shows symptoms such as muscle weakness and dyskinesia.Therefore,it is very important to develop microbial flocculants suitable for the treatment of wheat distillery wastewater.Through primary screening and re-screening flocculant-producing bacteria for efficient treatment of wheat distillery wastewater,and physiological and biochemical identification,it was systematically studied the fermentation characteristics,flocculation conditions,extraction and optimization conditions of microbial flocculant-producing bacteria,and the structure characteristics of flocculantand.The main research contents and results were as follows:(1)Screening and identification of flocculating bacteria for wheat distillery wastewater.More than 30 strains of bacteria were isolated from precipitated sludge of Anhui Ruifuxiang Food Co.Ltd.by dilution coating method.According to the results of preliminary screening and re-screening,M1 had higher flocculation activity to wheat distillery wastewater,and the initial flocculation activity was 72.09%.This strain has low nutritional requirements,and its colonies were transparent,sticky,and slimy.According to morphology,physiology and biochemistry,16Sr DNA molecular biology identification,identified as Klebsiella pneumoniae,and named M1.The strain is now stored in China's typical culture preservation center,the preservation number is CCTCC M 2018098,GenBank login number is:MG987011,it can be used as a high-efficiency flocculation strain of wheat alcohol wastewater for follow-up study.(2)The culture conditions of flocculant producing bacteria were optimized.Single factor and orthogonal design were used for studying flocculant conditions,M l culture conditions and the optimum medium composition.Based on single-factor tests,the optimum flocculent conditions were a resting time of 30 min,8%(v/v)culture medium dosage,and 3%(v/v)CaCl2.The optimum culture conditions were an incubation temperature of 30?for 48 h at pH 4.5 and rotation speed of 150 rpm.Under the optimum conditions,the flocculating degree was up to 82%.The best fermentation medium components were 15 g/L glucose,2 g/L peptone,1 g/L KH2PO4,and 2.5 g/L K2HPO4.A high flocculating degree was also achieved with the low-cost medium.The Klebsiella pneumoniae M1 bacteria strain can be used as a good bioflocculant produced bacteria for wheat distillery wastewater.(3)Optimization of extraction process for flocculants produced by M1 bacteria.The extraction technology of Klebsiella pneumoniae.M1 flocculant was studied,which could provide reference for the practical application of microbial flocculant.Single factor and response surface tests were used to optimize the extraction process of flocculants.The optimum extraction conditions of flocculant-producting bacteria M1 were as follow:The extracting agent was anhydrous ethanol,the material-liquid ratio was 1.54:1,the extractant pH was 9.06,the extraction time was 12 h,and the yield was up to 3.914 g/L.Model validation was adopted,under this condition,the flocculant extraction test was repeated 3 times,and the average yield of flocculant was 3.998 g/L.The relative error is about 2.1%,which showed that the model can predict the response surface value of flocculant very well.(4)It was study of physicochemical properties,purification and structure elucidation of flocculants produced by M1 bacteria.Phenol-sulfuric acid method,Coomassie brilliant blue method,free radical scavenging method were used to determine polysaccharide,protein content,antioxidant properties and so on.Sevage deproteinized and gel permeation chromatography were used as purify.Physical and chemical tests were done to qualitatively and quantitatively determine the components of the bioflocculant.Its molecular structure was analyzed by UV spectrum scanning,Fourier transform infrared spectrometer(FTIR),gas chromatography,gel chromatography,nuclear magnetic resonance spectroscopy(NMR)and scanning electron microscopy.The bioflocculant was composed mainly of polysaccharide and protein,whose content was 65.9%and 19.74%respectively.The molecular weight of polysaccharide was 4.784 × 106 D,and it comprised of L-rhamnose,L-Arabia sugar,L-fucose,D-xylose,D-mannose,D-glucose and D-galactose.Their molecular ratio was 0.29:0.36:1:0.31:0.47:0.57:1.01 and the polysaccharide contains hydroxyl,carbon and oxygen bonds,saturated hydrocarbon bonds and so on.It also contained Oxygen-containing groups(NMR spectrum 3.5?4.5 ppm)and aromatic groups(NMR spectrum 7 ppm).(5)Study on flocculation mechanism.Strong polar and non-polar substances were used to test the binding bonds between flocculants and particles in solution,the film-forming properties of flocculants,and advanced sequencing techniques of Illumina and PacBio were used to determine the whole genome sequence of M1 strain.With the analysing the structural characteristics of flocculants in the previous chapter,then the possible flocculation mechanism of flocculant produced by M1 was be certain.The results of binding bond test showed that the flocculant produced by M1 bacterium combined with suspended matter in wastewater mainly through ion bond,which might be the "adsorption bridge" effect of ion bond formation.The whole genome sequencing results showed that the genome of M1 bacterium was 5,511,794 bp in length and 58.39%in GC content,and the content distribution was normal,showing similar to Poisson's fraction.The genome contained about 5383 genes.By comparing the professional databases related to carbohydrate enzymes in functional gene database,it was found that there were five kinds of carbohydrate-related enzymes coding genes in the genome,indicating that there were functional genes closely related to polysaccharide production in M1 genome. |
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