Study On The Secretion Characteristics And Flocculation Mechanism Of Flocculating Substances Of Pseudomonas Sp.XD-3

Flocculation is a simple and effective method to remove the non-degradable solid suspended particles,bacterial cells and colloidal particles in water.It is widely used in drinking water,sewage and other industrial wastewater treatment.The selection and use of flocculants are the key technology of flocculation method.Microbial flocculant is a type of biodegradable polymer flocculant secreted by microorganisms and can be used as a potential substitute for traditional chemical flocculants.However,compared with the huge market demand,the flocculant products obtained by the current research are still insufficient.The low output and high cost have been the limitation factors restricting the practical application of flocculants.Therefore,screening new flocculent strains,optimizing culture conditions,and in-depth study of their flocculation mechanism will help enrich strain resources,reduce production costs,and provide theoretical guidance for the development of more targeted new high-efficiency microbial flocculants.In this paper,a functional strain XD-3 with flocculating properties was screened from activated sludge,the species of strain was identified and its growth characteristics were explored through morphological analysis,16S rDNA,and Logistic equation.Through a single-factor experiment,six factors of inoculum volume,culture time,carbon source,nitrogen source,C/N and initial p H value were optimized to determine the best culture conditions for strain XD-3 to produce flocculating substances.Qualitative and quantitative analysis of the flocculating substances distribution and composition characteristics of strain XD-3 by means of chemical analysis and 3D-EEM.FTIR,XPS and other methods were used to determine the functional groups and structures in the flocculating substances,and discuss the flocculation mechanism of the flocculating active substance in combination with zeta potential and force analysis.The research conclusions obtained in the thesis are as follows:A functional strain XD-3 with good flocculation performance was obtained through screening and isolation.The flocculating substances secreted by strain XD-3 were able to achieve good flocculation efficiency for kaolin suspension in the presence of a small amount of Ca²⁺.The strain was named Pseudomonas sp.XD-3 according to the strain colony,cell morphology and 16S rDNA identification results.The logistic equation could be used to fit the growth curve of strain XD-3 well,and the fitting correlation coefficient R² was 0.991.After the strain was inoculated into medium,it would enter the logarithmic growth phase directly and rapidly grow,and there was no adaptation period.At the same time,the optimal inoculation volume of strain XD-3 to produce flocculant was obtained by single factor experiment optimization as 5%,the optimal cultivation time was 12 h,the optimal carbon source was trisodium citrate with a content of 18.5 g/L,the optimal nitrogen source is urea with a content of 0.63 g/L,the optimal C/N is 15,and the optimal initial p H is 7.0.The flocculating active material of strain XD-3 could be divided into two parts:flocculating active material I and flocculating active material II,which were respectively distributed in the supernatant obtained by centrifugation of the fermentation broth and the bacterial cell suspension.They had numerous pore structures on the surface,which were conducive to the combination and adsorption of kaolin or other pollutantion particles.And after added into the mixed system of kaolin and Ca Cl₂,many larger flocs would be formed.Under the optimal conditions,the extractable flocculating substance I and flocculating substance II of the fermentation broth were 142.0 mg and 104.7 mg per liter,respectively.All of them contained proteins and polysaccharides.In addition,the flocculating substance I also contained humic substances.Further tests showed that protein was the most important component in both two types of flocculating substances,and its content is far greater than that of polysaccharides.The protein content of the flocculating substance I and the flocculating substance II were 265.26 mg/g CDW and191.59 mg/g CDW,accounting for 78.46%and 76.64%,respectively.The polysaccharides content was18.34 mg/g CDW and 17.62 mg/g CDW,accounting for 5.43%and 7.05%,respectively.The ratio of protein to polysaccharide in flocculating substance I and flocculating substance II was 14:1 and 11:1,respectively.For the flocculation system with the addition of flocculating substance I,the zeta potential of the supernatant after flocculation changed significantly,and it dropped directly to around 0 m V,reflecting the existence of electrical neutralization.While for the flocculating system with the addition of flocculating substance II,the zeta potential of the system was basically the same as the zeta potential of the system without flocculating substance addition,reflecting that electrical neutralization is not the main mechanism for the flocculation of active component II.In addition,Ca²⁺could compress the electrical double layer of kaolin particles to reduce the absolute value of the zeta potential on the surface of the particles,and played an indispensable role in the flocculation process.Analysis of the structure and functional group characteristics showed that the flocculating active substance I and the flocculating active substance II both contained amino,carboxyl,and hydroxyl groups which were beneficial to flocculation.And the protein secondary structure that beneficial to flocculation occupied a high proportion in both flocculating active substance I and flocculating active substance II,both accounted for a high proportion of 65.9%and 81.9%,respectively.The structure of the flocculating substance I was looser,which was conducive to exposing more ion binding sites.In addition,the flocculating substance I contained large number of positively charged protonated amino groups,which could neutralize the negative charge in the system,and had more-OH and C=OO^-,which could provide more cation binding than the flocculating substance II.Flocculating substance II contained more hydrophobic groups.Preliminary analysis of the flocculation mechanism of flocculating substance I and flocculating substance II,inferred that electrical neutralization played a major role in the flocculation process of flocculating active substance I.At the same time,there was also the existence of adsorption-bridging effect,and the adsorption force was mainly ionic bond and hydrogen bond.The flocculation process of flocculating substance II was dominated by adsorption-bridging,and the adsorption force was ionic bonds and hydrophobic forces.