| Objective:Due to the excessive use of chemical fertilizers and pesticides during the rapid development of intensive agriculture in China,a series of cultivated land problems have been caused,such as nutritional imbalance,destruction of microbial diversity and excessive accumulation of heavy metals.As a typical heavy metal pollution,chromium does great harm to plant growth and human health.Plant growth promoting bacteria（PGPBs）are very important microorganisms in soil,which have many positive effects on plant growth and disease prevention,and many PGPBs have certain capacity for heavy metal resistance and remediation.Based on this background,we aimed to develop immobilized microbial inoculant which can not only promote plant growth and prevent disease,but also remedy soil chromium pollution and improve soil microecology.In this thesis,the adhesion mechanism between bacteria and bentonite carrier,the chemotaxis response of immobilized inoculants to plant root exudates and their transport and diffusion,and the remediation effect of immobilized inoculants on hexavalent chromium[Cr（VI）]contaminated soil were discussed in depth,providing certain theoretical guidance for the development and utilization of microbial inoculants.Methods:Based on the previous studies on the growth promotion and disease prevention of Bacillus subtilis SL-44 in plants,the resistance and reduction mechanism of the strain to Cr（VI）was analyzed through the genome and transcriptome.Next,the interface adhesion mechanism of this strain on bentonite carrier and the migration and diffusion behavior of the immobilized microbial inoculant in Cr（OH）3 modified porous media were explored.In addition,through physical characterization and physiological analysis,a humic acid protectant with good performance was selected for SL-44 strain.Finally,the effects of SL-44 immobilized by carrier-protectant complex on chromium stability and microecology in Cr（VI）contaminated soil were evaluated.Results:1)In order to remedy the current chromium pollution in farmland and meet the Class II limit of heavy metals.In this study,Bacillus subtilis SL-44,previously screened in our laboratory,was found to adsorb Cr（VI）and Cr（III）reduction products,resulting in the wrinkled surface and the formation of Cr2O3nanoparticles inside cells.Chromate reductases such as nfs A,nfr A and yod C and chromate effluent-related resistance genes such as ywr A,ywr B and ywr C were found in the SL-44 genome.The results of transcriptome analysis showed that the genes involved in transcription,structural composition of ribosomes and cell membrane biogenesis were up-regulated in SL-44 cells under chromium stress,while genes involved in oxidoreductase activity,membrane transport and amino acid metabolism were down-regulated,which indicated that Cr（VI）had certain extent biological toxicity to SL-44 cells.2)Bentonite mineral can be used as the carrier of microorganisms,and the bacterial survival stability and Cr（VI）reduction rate can be improved after SL-44 is loaded by bentonite.In this study,the interfacial interactions of two bacteria（SL-44 and Rs-2）with five different bentonite carriers were investigated.The results showed that the hydrophobic stearic acid modified bentonite（SA-BENT）had the greatest bacterial adsorption capacity and affinity due to the maximum acid-base interaction.The stronger acid-base interaction between Rs-2 and the carriers promoted its adhesion,which may be attributed to its more hydrophobic surface structure acting as a broom to remove interfacial water.SL-44 had a stronger adsorption binding energy and smaller adsorption capacity since its EPS components（e.g.,low polarity,aromatic lipophilic protein macromolecules）were preferentially absorbed and occupied absorption sites based on the fluorescence excitation-emission matrix（EEM）and normalized volume integral of the fluorescence region.3)The transport of bacteria in porous media are essential for the fate of microorganism and bioremediation efficiency of soil contaminant.However,the impact of bacterial tactic response toward root secretions on cell dispersion remains unknown.Increasing the concentrations of the chemo-effectors（such as proline,sucrose,malic acid,and gallic acid）and addition of immobilization carrier（100 mg/L bentonite or biochar）under flow conditions significantly increased and reduced SL-44’s tactic response and transport in Cr（OH）3modified sand column,respectively.The decreased transport correspond to the increased hydrodynamic dispersion coefficient,an outcome which was obtained by fitting the experimental data with the advective dispersive equation.Great linear correlation were found among indexes quantifying SL-44’s tactic response ability and transport behavior（R2=0.8779-0.9248）.Real-time q PCR indicated that the bacteria immobilized by carrier could enhance the bacterial deposition in Cr（OH）3 contaminated soil,which provided theoretical guidance for the remediation of chromium pollutants by microbial inoculants.4)In this thesis,humic acid（MA-HA）and SL-44 were selected as research objects,the strengthening mechanism of MA-HA on Cr（VI）reduction by SL-44 was discussed by means of spectroscopy,electrochemical characterization and transcriptome analysis.The results showed that,the phenolic groups and carboxyl on the surface of MA-HA are firstly complex with Cr ions,and the fluorescent component with moreп-пconjugate structure in humic acid is the most sensitive species.Compared with single bacteria,the application of SL-MA complex not only enhanced the reduction efficiency of Cr（VI）and the generation of intermediate Cr（V）,but also reduced the electrochemical impedance and achieved good electron transfer.The addition of MA-HA can significantly reduce the toxicity of Cr（VI）to bacteria and reduce the accumulation of glutathione in bacterial extracellular polymers.Transcriptome analysis showed that the presence of MA-HA down-regulated genes involved in glutathione synthesis and polyhydroxybutyric acid（PHB）hydrolysis in SL-44 under chromium stress.Moreover,the addition of exogenous humic acid can enhance the reducing ability of bentonite immobilized SL-44 to Cr（VI）and shorten the time required for Cr（VI）reduction.5)Potting experiments were performed to evaluate the respective contributions of SL-44,humic acid protectants and microbial carriers in the remediation of chrome-contaminated soil.The results showed that the application of SL-MA complex increased the proportion of residual chromium in soil and decreased the chromium enrichment in the roots and leaves of Chinese cabbage.In addition,compared with the carrier immobilized bacteria treatment,the carrier-humic acid complex immobilized SL-44 treatment group significantly enhanced the activities of soil catalase and urease,and decreased the response of soil microbial community to changes in chromium content,thus enhancing the microbial community resistance to chromium toxicity.All treatments did not affect the original ecological structure of soil,and the OTU9 of Gemmatimonadota showed a significant negative correlation with chromic fractions（F1,F2 and F3）.Functional prediction analysis showed that exogenous biodegradation metabolism and terpene polyketone metabolism enzymes activities in the carrier-humic acid complex immobilized SL-44 treatment group were significantly higher than those in the treatment group without adding humic acid,which further confirmed the contribution of humic acid protectants.Conclusions:In conclusion,this study explored the Cr（VI）reduction mode and possible reducing genes of SL-44,and further analyzed the influence of chromium stress on its transcriptional regulation.In addition,the interface adhesion behavior between SL-44 and bentonite carrier was elucidated,and the diffusion deposition law of the carrier immobilized SL-44 in Cr（OH）3 modified porous media was explored.The internal strengthening mechanism of Cr（VI）reduction by SL-44 with humic acid protectant was revealed.The effects of carrier-protectant complex immobilized SL-44 on the stability of chromium and microecology in soil were analyzed comprehensively,which provided theoretical guidance for the preparation and rational application of microbial inoculant. |
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