Insights Into The Biosynthesis Of Polysaccharide-based Microbial Flocculant By Bacillus Subtilis ZHX3 And Its Gene Regulation

Microbial flocculant（MBF）is a kind of non-toxic,harmless,easy to degrade,and non-secondary pollution green water treatment agent.However,at present,the research on MBF mostly stays at the level of screening flocculating bacteria and optimizing culture parameters,and lacks deep research on flocculent bacteria at the molecular level.In this thesis,the MBF-producing strain Bacillus subtilis ZHX3 was used as the research object and its characteristics in MBF-ZHX3 production were explored,and the structure of polysaccharide component in MBF-ZHX3was revealed.Based on genomics and transcriptomics technology,some key genes related to the biosynthesis of polysaccharide were diged out and its biosynthetic pathway was predicted.Finally,the biosynthesis process of polysaccharide was genetically regulated by constructing key genes overexpression strains and the treatment of wastewater was conducted.The detail research results are as follows:（1）MBF production characteristics of strain ZHX3 were analyzed comprehensively.Culture conditions of strain ZHX3 were optimized by single factor experiment as follows:soluble starch 10g/L,yeast extract5g/L,Na Cl 5g/L,3.25 mg/L Fe Cl_3,natural initial p H,inoculum 1%（v/v）and cultivation at 30°C.Under the optimal culture conditions,the maximum flocculating rate reached 95.5%,and the substances with flocculating activity mainly came from polysaccharide component in the fermentation supernatant.Total 3.14g/L MBF-ZHX3 was obtained,and it consisted of 77.2%polysaccharide and 14.8%protein.The molecular weight was 10,028 Da,and it contained multiple functional groups such as O-H,C-H,C=O,>P=O and C-O-C,and behaved good p H stability and thermal stability.The optimal kaolin suspension test conditions were adding 0.05g/L Ca²⁺,0.4mg/L MBF-ZHX3 and natural p H.By measuring the zeta potential,observing the morphology of the flocs under scanning electron microscope,and identifying the types of binding bonds by chemical methods,it was proposed that the flocculation mechanism of MBF-ZHX3 was mainly adsorption bridging.The simulated wastewater treatment experiment was carried out.The results showed that the maximum decolorization rates of MBF-ZHX3 for methylene blue and crystal violet were 76.3%and 89.8%,respectively,which were significantly better than that of polyacrylamide under the same conditions.The maximum removal rate of Pb²⁺and Cu²⁺by MBF-ZHX3 were 51.1%and 55.9%,respectively.（2）Separation,purification and structure analysis of polysaccharide component in MBF-ZHX3 were carried out.Thermal gravimetric analysis showed that the weight loss was most obvious when the temperature reached 275.3-332.8°C,indicating the decomposition reaction of polysaccharide was severe at this stage.Preliminarily purified polysaccharide was further separated and purified by DEAE cellulose DE-52 and Sephadex G-100 column chromatography.Two components named PS1-1 and PS2-1 were obtained with high purity,and their molecular weights were 5982Da and 17,577Da,respectively.Both of PS1-1 and PS2-1 were mainly composed of glucose（Glc）.According to infrared spectrum test,both PS1-1 and PS2-1 contained rich groups with polysaccharide characteristics andα-type glycosidic bond was detected.Through methylation reaction and gas chromatography-mass spectrometry,it was deduced that PS1-1 and PS2-1 made up of three glycosidic bonds of Glcp-（1→,[→4）Glcp-（1→]and[→4,6）Glcp-(1→],with the ratio of 1.1:6.0:1 and 1:8:1,respectively.The branch occured at the C₆ position of 1→4 linked Glc,and the branch was composed of Glcp-(1→.Combining with the molecular weight,it was deduced that PS1-1 contained four repeating units,while PS2-1 contained ten repeating units.（3）The whole genome sequencing of strain ZHX3 was conducted by the third-generation sequencing technology of Oxford Nanopore.The results showed that the genome of strain ZHX3 was composed of a chromosome and two plasmids with a size of 4,203,149 bp,a G+C content of 43.37%,4404 coding sequences,88 t RNAs and 30 r RNAs.COG,GO and KEGG database annotations showed that strain ZHX3contained a large number of functional genes related to carbohydrate metabolism,amino acid metabolism and transport.By comparative genome analysis,the close relationship between strain ZHX3 and B.subtilis DM2、B.subtilis QB928 and B.subtilis 168 was confirmed.The pan-genome modeling results of 28 Bacillus species showed that it was an open pan-genome.Many genes related to carbohydrate substrate absorption and polysaccharide synthesis have been found in the genome of strain ZHX3,such as coding genes responsible for the uptake of glucose,maltose and maltodextrin et al.,the synthesis of nucleotide sugars such as UDP-glucose,UDP-galactose,et al.,and eps gene cluster responsible for the assembly,modification,polymerization and output of sugar repetitive unit.According to the CAZymes database,there were 127genes encoding carbohydrate active enzymes,which could be used for the degradation of complex carbohydrates such as cellulose,hemicellulose,pectin,et al..According to Anti SMASH v6.1.1 database,strain ZHX3 had gene clusters for the synthesis of bacillibactin and surfactin,and their corresponding nonribosomal peptide synthetase（NRPS）modules with catalytic function were also complete.（4）The samples of strain ZHX3 in different culture conditions were applied for transcriptome sequencing.The results showed that the quality of sequencing data was high,as well as the repeatability of intra-group and the difference of inter-group.Besides,the reliability of transcriptome data was verified by real-time quantitative PCR（q PCR）.By analyzing the transcriptional level of related biosynthesis genes of polysaccharide,the key differentially expressed genes were screened,and the biosynthetic pathway was deduced as follows:extracellularα-amylase encoded by amy E was responsible for the hydrolysis of starch in the MBF production medium;enzymes encoded by pts G,mal P and mdx EFG-msm X were responsible for the transport of glucose,maltose and maltodextrin,respectively,which were the products of starch hydrolysis;glv A、bbm A、mdx K、mal L and yvd M were responsible the synthesis of glucose-6-phosphate;As an important precursor of nucleotide sugar synthesis,glucose-6-phosphate was converted to UDP-glucose,UDP-galactose,UDP-glucuronic acid and UDP-galactouronic acid under the catalysis of a series of enzymes encoded by pgc A,gta B,gal E,tua D and ytc B;UDP-glucose,as the main component of the polysaccharide chain,entered the assembly,polymerization and output of repeat units.This process was mainly participated by eps L,eps F,eps K,eps G,eps A and eps B.（5）Overexpression of some key genes in the biosynthetic pathway of polysaccharide was studied by gene recombination technology.By Xba I/Sma I double restriction enzyme digestion and T4 ligase connection,nine recombinant plasmids,such as p HT01-pgc A,et al.were constructed.Subsequently,these recombinant plasmids were introduced into strain ZHX3 by electric transformation method,respectively,and nine target genes overexpression strains such as ZHX3-p HT01-pgc A,et al.was constructed successfully.The flocculating performance and transcription level of genes in each overexpression strain were tested.The results indicated that pgc A,eps D,pts G and mal L overexpression strains still maintained relatively high flocculating activity,and the transcription level of pgc A,eps D,pts G and mal L was increased by 5.4-,5.84-,5.13-and3.27-fold,respectively.The cultivation of pgc A,eps D,pts G and mal L overexpressing strains in 1L system suggested that the highest flocculating rate appeared on the 4th day（97.3%）,5th day（98.4%）,7th day（92.7%）and 5th day（97.6%）,respectively.MBF yield,proportion of polysaccharide component and the content of uronic acid were all improved comparing with the wild strain.Under the treatment of MBF produced by pgc A overexpression strain,the decolorization efficiency of methylene blue and crystal violet were both significantly increased,and the removal rates of Pb²⁺and Cu²⁺were also rised by 31.54%and 16.47%,respectively.（6）The bioflocculant produced by pgc A overexpression strain,named MBF-ZHX3-pgc A was applied in the treatment of actual wastewater.The results showed that the best coagulants for the treatment of laboratory wastewater,printing and dyeing wastewater,and mineral processing wastewater were polyaluminum chloride,polyaluminum ferric sulfate and polymeric ferric sulfate,respectively.The addition of MBF-ZHX3-pgc A as a flocculant promoted the formation of larger and denser flocs,accelerated the sedimentation rate of the flocs,shortened the treatment time,and improved the removal rate of COD_Cr,suspended solids,chromaticity and heavy metals to varying degrees.Especially,the residual concentration of suspended solids and heavy metals could meet the emission standard.In terms of wastewater treatment efficiency,MBF-ZHX3-pgc A was equivalent to that of anionic polyacrylamide,and took an advantage of no secondary pollution.Through the above research,the flocculation characteristics and mechanism of B.subtilis ZHX3 were elucidated,and the structure of the key component polysaccharides in MBF-ZHX3 was revealed,and the biosynthetic pathway of polysaccharide-based MBF was constructed,and the regulation of key genes responsible for the biosynthesis of polysaccharide-based MBF by overexpression and the application of overexpressed products in wastewater treatment were achieved.This can provide a solid theoretical basis and method reference for the basic theoretical research and industrial application of microbial flocculant.