Remediation Of Dibutyl Phthalate Contaminated Black Soil By Biochar Coupled Functional Microorganisms

Dibutyl phthalate（DBP）is one of the most common phthalates（PAEs）compounds.As one of the main components of agricultural plastic films,its pollution in black soil is serious.Potassium plays an important role in the activation of some metabolic processes in plants.However,soil potassium utilization is often adversely affected by soil DBP pollution and declines,resulting in limited crop yield and quality.Bioremediation is widely recognized for its thorough degradation and minimal impact on humans and the environment,and is considered the best way to remove DBP from soil.Potassium-solubilizing bacteria can convert insoluble potassium in the soil into available potassium that can be absorbed and utilized by plants,so as to alleviate the limitation of the polluted environment on the utilization of soil potassium.The combined use of DBP-degrading bacteria and potassium-solubilizing bacteria to prepare a composite inoculant can solve the problems of soil DBP pollution and fertility decline at the same time.However,pollutants in soil can easily lead to the inactivation of some functional strains.Therefore,protecting the strains from DBP stress and affecting their functions is a key problem to be solved in the preparation of compound inoculants.Polydopamine（PDA）has good biocompatibility,is easy to coat on various substrates and does not cause secondary pollution to the environment,and can be used as a coating material for introducing organic shells into living cells.In this study,a recyclable composite bacterial agent（KPSB）was prepared by using Fe₃O₄modified biochar loaded with PDA immobilized potassium solubilizing bacteria Paenibacillus sp.KT（inside）and DBP degrading bacteria Enterobacterium sp.DNB-S2（outside）.The optimal ratio of degrading bacteria and potassium-solubilizing bacteria was explored,the conditions for coating KT with PDA were optimized,and the coating mechanism of the strain was clarified by means of characterization.The mechanism of DBP degradation and the effect of compound inoculants under different environmental conditions were studied to achieve the purpose of removing DBP and increasing soil available potassium content.The main results of this study are as follows:（1）Fe₃O₄modified biochar MBC-CMC was successfully prepared and characterized.The straw-based biochar was prepared by pyrolysis at 500℃ using typical agricultural waste corn straw as raw material.The modified biochar MBC-CMC was prepared by loading Fe₃O₄and CMC on BC.Materials were characterized by SEM,BET,FTIR,XRD,XPS and VSM.The study found that the surface of MBC-CMC was rough and there were fine particles.The specific surface area,pore volume and average pore size were significantly higher than those of the original biochar.The adsorption and desorption isotherms are consistent with type IV isotherms,showing H3 type hysteresis loop isotherms.Various functional groups exist on MBC-CMC,including C=C,C-O,and-OH.The saturation magnetization ratio of MBC-CMC was 7.51 emu/g,and the magnetic separation efficiency was 92.8%,showing a good recovery potential.（2）A new compound bacterial agent was created to explore its pollution removal efficiency and nutrient synergistic mechanism.Response surface methodology was used to optimize the coating conditions of PDA for potassium-solubilizing bacteria,and the coating time,PDA concentration and inoculation amount were optimized.The optimal coating conditions were 2%KT inoculum,1.50 mg/mL dopamine monomer content,and 0.5 h coating time.The DBP-degrading bacteria and PDA-coated potassium-solubilizing bacteria were immobilized on the modified biochar MBC-CMC to create a new functional bacterial agent KPSB.At pH 5,7 and 9,the DBP degradation rates of KPSB reached 98.50%,98.72%,and 98.41%,respectively,and the potassium dissolution efficiency increased by 1.58,2.24,and 2.73 times,respectively.Through the observation of the microstructure and the determination of the IAA-producing ability,the organic acid-producing ability and the gene expression,the synergistic mechanism of the compound bacterial agent was clarified.The microstructure map showed that the immobilized KT strain had more complete cell membrane and cell structure,while the free KT strain was easily damaged or even ruptured by DBP.Compared with pure bacteria,potassium-solubilizing ability of KPSB was significantly improved,the total amount of small molecular organic acids produced by KPSB increased,and the types of small molecular organic acids were changed to some extent.In the presence of various ions(Fe³⁺and Zn²⁺),the strains coated with PDA showed good growth ability.DBP degradation-related enzymes（oph A4）,glutathione S-transferase（junni GL003465）and membrane integrity（junni GL003628）genes were significantly increased in DBP-degrading bacteria,which promoted DBP degradation.（3）The compound bacterial agent KPSB was applied to 20 mg/kg DBP-contaminated black soil,and its effects on soil microbial community at different temperatures（15℃ and 30℃）were investigated.The degradation efficiencies of DBP were 79.17%and 96.45%after 7 days of application of the inoculum.Compared with the original soil,the content of available potassium increased by 25.22%and 27.92%respectively after 28 days.High-throughput sequencing results showed that DBP contamination would disrupt the soil ecosystem,resulting in a decrease in the abundance and diversity of microbial communities in soil,compared with pristine soil.The alpha diversity index of the polluted soil decreased,and the relative contents of Acidobacteria,Chloroflexbacteria and Bacillus were lower than those of the original soil.The addition of KPSB at 30℃ was helpful for soil microbial recovery.Both Shannon index and Simpson index were up-regulated to a certain extent,and at the same time,it mediated the dynamic changes of bacterial phyla and increased the relative abundance of Proteobacteria and Firmicutes.The addition of KPSB also increased the abundance of some DBP degradation-related and plant growth-promoting-related genera,such as Sphingomonas,Bacillus,and Streptomyces.In conclusion,in this study,a new type of compound inoculant with good polluted soil remediation performance was prepared,and the mechanism of action of the compound inoculant was further explained.This study has important theoretical significance and application value for the removal of DBP pollutants in cold black soil,and provides a new idea for the development of environmentally friendly remediation technology for polluted black soil.