Screening Of Mn-tolerant Bacteria And Their Effects On Mn-contaminated Soil Rehabilitation By Broussonetia Papyrifera

Microbial enhanced phytoremediation has become one of the important ways to control heavy metal contaminated soil all over the world.Rhizosphere microorganisms are closely related to soil environment and plant growth,and play an important role in promoting plant growth and mineral uptake.In this study,three Mn-tolerant bacteria were screened from Xiangtan Mn mining area in Hunan Province,and the candidate strains for plant-microbial combined remediation were identified by testing their physiological characteristics,Mn adsorption characteristics and plant growth promotion ability.After investigating the physiological response mechanism and accumulation characteristics of B.papyrifera under Mn stress,the effects of Mn-tolerant strains with inoculation on root development,plant growth,physiological response,photosynthesis and Mn accumulation characteristics of B.papyrifera under different Mn pollution conditions were studied by pot experiment.Finally,a combined remediation system of B.papyrifera and B.cereus HM5/B.thuringiensis HM7 was constructed to provide theoretical basis and technical approaches for the remediation of heavy metal contaminated soil.The main results are as follows:?1?Three Mn-tolerant bacteria isolated from Xiangtan Mn slag in Hunan province were identified as Bacillus cereus HM5,Bacillus thuringiensis HM7 and Ralstonia picketti HM8by 16S rDNA sequencing analysis.In the biosorption experiments,the maximum removal rates of B.cereus HM5,B.thuringiensis HM7 and R.picketti HM8 reached 99.51%,95.04%and 46.02%at 400 mg/L of Mn²⁺in solution,respectively.While,R.picketti HM8 could still grow at very high Mn concentration?10000 mg/L?,and the removal amount of Mn²⁺was1002.83 mg/L.Single factor experiments under different culture conditions showed that initial Mn²⁺concentration,temperature,time,and pH had significant effects on the growth of the strain and the absorption of Mn²⁺.FTIR and SEM analysis showed that the functional groups?carboxyl,hydroxyl,carbonyl,amide,phosphoric acid and alkyl groups?on the cell surface of the strain participated in the process of Mn²⁺biosorption.After adsorption,the size of the strain changed,the surface folded and flocculent substances appeared between the strains.These phenomena may be related to the redox of Mn?II?by bacteria.In plant growth promotion ability of the strains,B.cereus HM5,B.thuringiensis HM7 and R.picketti HM8 all had the ability to produce IAA and dissolve phosphorus,and B.cereus HM5 and B.thuringiensis HM7 had the ability to produce iron carrier.However,because R.picketti HM8might be pathogenic to plants,B.cereus HM5 and B.thuringiensis HM7 were finally identified as candidate strains for combined phytoremediation.?2?Soil pot experiments showed that low concentration of Mn could promote root development?0-5 mmol/L?and plant growth?0-2 mmol/L?of B.papyrifera.Mn could be accumulated in B.papyrifera,and the distribution of Mn content in different tissues was leaf>root>stem.With the increase of Mn concentration in soil,the accumulation of Mn in plant tissues also increased.The maximum values of root,stem and leaf appeared at 50 mmol/L,compared with the control group?0 mmol/L?,the Mn content increased by 177.37%,124.88%and 511.86%respectively.?3?The accumulation of Mn resulted in lipid peroxidation of cell membranes and accumulation of reactive oxygen species?ROS?free radicals,leading to a significant increase of MDA in B.papyrifera.with a short time?3 h?.Then,proline,soluble protein and soluble sugar increased,which may reduce osmotic pressure caused by Mn stress and alleviate the toxic effect of Mn on B.papyrifera.On the other hand,B.papyrifera can effectively alleviate oxidative stress by maintaining high activity of antioxidant enzymes?SOD,POD,CAT?to scavenge ROS in plants.In the range of 0-2 mmol/L Mn concentration,SOD and POD played a major regulatory role.At 2-50 mmol/L,the change range of CAT was much larger than that of SOD and POD.That is to say,CAT played a major regulatory role in the high concentration.However,due to the accumulation of time and concentration,SOD,POD and CAT were still needed to co-regulate and reduce lipid peroxidation.Therefore,the antioxidant enzymes system of B.papyrifera showed high resistance to Mn,which indicated that B.papyrifera had strong adaptability to oxidative stress and had a good antioxidant enzymes system.This characteristic was quiet imperative to the restoration and application of heavy metal contaminated soil of B.papyrifera.?4?Pot experiments on B.papyrifera were carried out by adding B.cereus HM5 or B.thuringiensis HM7 into roots of Mn contaminated soil?5 mmol/L,50 mmol/L and Mn slag?.The results showed that inoculation of B.cereus HM5 and B.thuringiensis HM7 improved soil composition and TOC and TP contents.At the same time,the biomass,root structure and activity of B.papyrifera inoculated with B.cereus HM5 or B.thuringiensis HM7 were significantly higher than those of the control group,indicating that B.cereus HM5 and B.thuringiensis HM7 promoted root development and plant growth.In terms of physiological response,microorganisms reduced MDA content,CAT,SOD and POD activity in leaves,indicating that B.cereus HM5 and B.thuringiensis HM7 could reduce oxidative stress which induced by Mn,and alleviated lipid peroxidation of cell membrane.In terms of heavy metal accumulation,B.cereus HM5 and B.thuringiensis HM7 both enhanced the Mn accumulation ability of B.papyrifera and the ability of Mn²⁺transporting from roots to aboveground parts,thus reducing the Mn content in polluted soil.In the joint B.papyrifera-microbial remediation system,B.cereus HM5 and B.thuringiensis HM7 could improve the polluted soil environment and plant growth through the ability of phosphorus solubility and the production of IAA and iron carrier.Finally,B.cereus HM5 and B.thuringiensis HM7 would strengthen the remediation of Mn-contaminated soil by promoting the growth of B.papyrifera and the accumulation of Mn by the plant.