Proteomic Of Extracellular Polymer Synthesis Of Paenibacillus Mucilaginosus And Flocculation Characteristics Of Heavy Metals

Paenibacillus mucilaginosus has the characteristics of strong stress resistance,multi-function and release of insoluble mineral elements.These characteristics make it possible for it to be used as biofertilizer and bioflocculant in agriculture and wastewater treatment,which has broad application prospects.These functions of Paenibacillus mucilaginosus are closely related to its ability to produce hypertrophic capsules and secrete a large number of extracellular polymers（EPS）,so the study of EPS is of great value.In order to further understand the molecular basis and mechanism of EPS function of Paenibacillus mucilaginosus,the fermentation conditions were optimized,the adsorption characteristics of microbial flocculants to heavy metal ions were studied,the proteome sequencing was performed and the identification of differentially expressed proteins by real-time quantitative fluorescence PCR to reveal the mechanism of increased EPS.The main results are as follows:（1）The optimum levels of yeast extract,mineral powder,sucrose and pH value were deter mined by orthogonal test.Compared with the basic medium,the optimized medium formulation with yeast extract（0.1 g/100 mL）,mineral powder（0.4 g/100mL）,pH value（8）and sucrose（2 g/100 mL）could significantly increase the polysaccharide content in fermentation broth.（2）The differentially expressed proteins of cultured bacteria with and without mineral powder were analyzed.We used iTRAQ technology to sequence the proteomes of different cultured strains and found that the total number of differentially expressed proteins was 963,of which the number of up-regulated and down-regulated proteins were 605 and 358,respectively.These differential proteins are mainly involved in cell components（cell）,biological processes（metabolic processes）,and molecular functions（catalytic activities）in GO functional classification.KEGG Pathway analysis showed that differentially expressed proteins were mainly concentrated in metabolic pathways,environmental information processing pathways and cellular processes,such as purine and pyrimidine metabolism,beta-alanine metabolism,arginine and proline metabolism,starch and sucrose metabolism,a minose and ribose metabolism,ABC transport system,quorum sensing and biofilm formation pathway.The candidate genes were identified by qRT-PCR,and the reliability of proteomic sequencing data was evaluated.It was found that the quantitative results of qRT-PCR were basically consistent with the sequencing results.（3）The adsorption of heavy metals by polysaccharide flocculant of Paenibacillus mucilaginosus was studied,and the experimental data were fitted with Langmuir isothermal adsorption model and quasi-second-order kinetic equation.The results showed that the flocculant could adsorb Cu²⁺,Cd²⁺,Cr³⁺and Pb²⁺at low concentration quickly and reach equilibrium within 1 minute.The adsorption effect was good.The adsorption process accords with quasi-second-order kinetics and Langmuir isothermal adsorption model.In this study,iTRAQ proteome sequencing technology was used to obtain enzymes involved in the up-regulation of starch and sucrose metabolism,a mino sugar and ribose metabolism and ABC transport system in bacteria stimulated by mineral powder culture medium,so as to reasonably explain the biological process of bacteria synthesizing a large number of polysaccharides and secreting them into extracellular EPS.At the same time,discovered the biological processes associated with resistance,such as antioxidant and biofilm formation,the enzymes involved were significantly higher expression,speculated that the process of shake flask culture of bacteria,mineral powder cause mechanical damage to bacteria,causing them to secrete a large number of EPS and produce other protect themselves through a series of stress reaction,thus preli minary clarified the Paenibacillus mucilaginosus under certain conditions the molecular mechanisms of EPS regulation pathway.