| Phytoremediation is a sustainable and eco-friendly technology that employs plants to remove pollutants from contaminated soil or water.Although it has gained wide attention in recent years,the majority of identified cadmium（Cd）hyperaccumulators have slow growth rates and low biomass,which limit the efficiency of remediation.Therefore,there is a need to optimize and improve the efficiency of phytoremediation by identifying more suitable hyperaccumulators or high-biomass plants with strong metal-accumulative capacity.Moreover,Cd-contaminated soil is often associated with soil acidification,and the selected plants must cope with the dual stress of Cd and acid,thereby increasing the challenge of soil remediation.In this study,firstly,through the field investigation of acid and Cd co-contaminated mining areas,the plant with strong Cd-accumulative ability,Ageratum conyzoides L.,was screened out.Then,the tolerance and accumulation ability of this plant to Cd was verified by indoor pot experiment,and the physiological and biochemical mechanism of tolerance to Cd and acid was analyzed.Secondly,the content and composition of low molecular weight organic acid（LMWOA）secreted by roots of A.conyzoides under different Cd concentrations were studied by hydroponics and soil culture experiments,and the effects of organic acid secreted by roots on Cd accumulation and physiological and biochemical indexes of leaves by A.conyzoides were verified by adding exogenous oxalic acid.Thirdly,the bacteria strains resistant to Cd and acid were screened out from the soil contaminated by acid and Cd in the mining area.Through the single factor and orthogonal experiments of acid and Cd stress,the tolerance of the two strains to different concentrations of acid and Cd was tested,and then the adsorption characteristics of Cd were evaluated by isothermal adsorption model and adsorption kinetics model,and the changes of cell morphology and surface functional groups before and after Cd adsorption were analyzed by scanning electron microscopy（SEM）,transmission electron microscopy（TEM）and Fourier transform infrared spectroscopy（FTIR）.Finally,the selected bacterial strains were inoculated into the potted soil with A.conyzoides to evaluate its effectiveness on phytoremediation and related mechanisms.The main results of this paper were as follows:（1）A.conyzoides has strong tolerance to dual stress of Cd and acid in soilThirty-five wild plants and their surrounding soils were collected from the farmland near the heavy metal polluted mining area.Among them,the Cd concentrations in A.conyzoides and B.pilosa were relatively high,but B.pilosa was reported in many literatures,so this study mainly focused on A.conyzoides.In pot experiment,A.conyzoides grew normally at 100 mg kg-1Cd in soil,and the Cd concentrations in its roots,stems and leaves were 75.37±7.37,31.01±3.76 and 53.92±10.05 mg kg-1,respectively.In the case of acid tolerance experiment,all plant individuals of A.conyzoides grew normally when soil p H was over 3.5.Under strong acid conditions,the activity of SOD in leaves of A.conyzoides increased significantly.When the Cd concentration was 10 mg kg-1and the soil p H was 5.5 or 3.5,the activities of POD and CAT significantly increased.In addition,based on stepwise regression analysis,the Cd concentration in leaves was significantly positive correlated with the activities of SOD and POD in leaves of A.conyzoides.Therefore,A.conyzoides not only had a strong tolerance for Cd and acid pollution in soil,but also had a strong Cd-accumulative ability.As a common plant in the mining area,it had a great potential for the phytoremediation of Cd and acid co-contaminated soil.（2）Oxalic acid plays an important role in promoting the Cd accumulationIn the hydroponic culture experiment,when the concentration of the culture medium was 10 mg L-1,the Cd concentrations in the roots,stems and leaves of A.conyzoides reached the maximum,and Cd concentrations were 452.17±82.5,179.9±30and 291.95±76 mg kg-1,respectively.In addition,oxalic acid and malic acid could be detected in the roots,stems and leaves of A.conyzoides under hydroponic conditions,but only oxalic acid could be detected in the root exudates,and the amount of oxalic acid secretion increased with the increase of Cd concentration.In the soil culture experiment,when the soil concentration reached 30 mg kg-1,the Cd concentrations in the roots,stems and leaves of A.conyzoides reached the maximum,and Cd concentration were 16.8±4.2,17.5±3.5,26.0±5.7 mg kg-1,respectively.In addition,different concentrations of oxalic acid and citric acid were detected in the roots,stems and leaves of A.conyzoides under soil culture conditions,while only oxalic acid was detected in its root exudates.In addition,by adding exogenous oxalic acid,the Cd concentration in the stems of A.conyzoides was significantly higher than that in the control when the concentration of oxalic acid reached 2.5 g L-1.When the concentration of oxalic acid reached 1.0 g L-1and 2.5 g L-1,the Cd concentration in the leaves of A.conyzoides was significantly higher than that in the control.In addition,when the content of exogenous oxalic acid reached 1.0 g L-1,the SOD activity in the leaves of A.conyzoides was significantly higher than that of the control.When the concentration of exogenous oxalic acid was 0.25,0.5 and 1.0 g L-1,the POD activity in the leaves of A.conyzoides was significantly higher than that of the control.Therefore,oxalic acid was considered to be the main component of organic acid secreted by the roots of A.conyzoides,and oxalic acid played a role in promoting the Cd accumulation in A.conyzoides.（3）Strains B-1 and B-7 have high tolerance to Cd and adsorption performanceTwo strains（B-1 and B-7）with high tolerance to Cd were screened out from heavy metal polluted soil in a mining area,and they were identified as Burkholderia contaminans HA09 and Arthrobacter humicola,respectively.Two strains could tolerate500 mg L-1Cd,and both strains grew normally when soil p H was over 4.In addition,compared with strain B-7,strain B-1 had a higher OD600value under strong acid（p H=4）and high Cd(300 mg L-1)conditions.When the content of biosorbents was 4 g L-1,the Cd removal rates by dead cells B-1,dead cells B-7,living cells B-1 and living B-7were 779.7%,81.5%,76.8%and 80.5%,respectively.Moreover,Freundlich isothermal adsorption model and Pseudo-second-order kinetic model were more suitable to describe the Cd adsorption by four biosorbents.In addition,the Cd adsorption by both bacteria was mainly extracellular.SEM and TEM images showed that the cells were significantly damaged after Cd adsorption.FTIR showed that there were a large number of functional groups on the surface of the bacteria,such as carbonyl,acyl,alkyl,amino and hydroxyl groups,which played an important role in the adsorption of Cd.Therefore,strain B-1 and B-7 had a high tolerance and good adsorption for Cd,and could be used as a potential biosorption materials for the bioremediation of Cd polluted soil.（4）Inoculation with strain B-1 significantly improved the extraction efficiency of Cd from A.conyzoidesInoculated strain B-1 significantly increased the plant height and biomass of A.conyzoides,while inoculated strain B-7 significantly increased the plant height of A.conyzoides.Inoculation with strain B-1 significantly increased the Cd concentration in the roots and leaves of A.conyzoides to 39.4±3.06 and 35.68±0.26 mg kg-1,respectively,with the percentages of 30.9%and 27.9%higher than the control group.Inoculation with strain B-7 also significantly increased the concentration of Cd in roots and leaves,reaching 38.19±2.22 and 37.07±6.25 mg kg-1respectively,with the percentages of 26.8%and 37.07%higher than the control group.After the strains B-1and B-7 were inoculated,the extraction efficiency of Cd by A.conyzoides was increased by 114%and 45%,respectively.The reasons were as follows.Firstly,both strain B-1 and strain B-7 could significantly improve the dissolution of Cd CO3.Secondly,the two strains could significantly affect the availability of plant nutrients in the roots of A.conyzoides.In the experiment of dissolving soil nitrogen（N）,phosphorus（P）and potassium（K）,strains B-1 and B-7 had obvious effect of dissolving P,and the amount of dissolving P was 5.63 times and 2.76 times higher than that of the control group,respectively.The effect of strain B-7 on the dissolution of K was significant,and the dissolution of K was 1.79 times higher than that of the control group.Strains B-1 and B-7 had significant nitrogen fixation effect,and their nitrogen fixation amount was 29.53 times and 44.39 times higher than that of the control group,respectively.Thirdly,both strains B-1 and B-7 could produce a large amount of indoleacetic acid（IAA）.Under 100 mg L-1Cd stress,the concentration of IAA production was 43.24±6.3 and 14.39±4.03 mg L-1,respectively.Finally,compared with the control group,inoculated strains B-1 and B-7 could significantly increase the activities of CAT and SOD in the leaves of A.conyzoides,increased the content of chlorophyll a and total chlorophyll,and then promoted the photosynthesis of plants.In summary,A.conyzoides could tolerate the dual stress of Cd and acid and could accumulate Cd,making it a suitable plant for the remediation of acid Cd-polluted soil.Two acid and Cd-tolerant soil bacteria（B-1 and B-7）had a good Cd adsorption.The bacterial cells promoted Cd adsorption by the secretion of extracellular EPS and the participation of functional groups on the cell surface.The two strains could assist with A.conyzoides to remediate the acid and Cd co-contaminated soil by promoting the dissolution of Cd in the soil,improving the availability of plant nutrients,producing IAA and enhancing the antioxidant capacity of plants. |
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