Study On The Remediation Mechanism Of Arsenic Contaminated Soil In The Mining Area By Microalgae-bacteria Coupling

Arsenic（As）has attracted much attention as a toxic and carcinogenic substance.Once As enters the soil,it will increase the risk of soil arsenic pollution.Microbial remediation of As-contaminated soil is a widely used bioremediation method.At present,there are relatively many studies on the remediation of As-contaminated soil by bacteria and fungi,while there are relatively few studies on the remediation of As-contaminated soil by algae.In this study,the remediation mechanism and effect of As contaminated soil were studied by using soil microalgae in Shimen tailings area.Firstly,the biotransformation,accumulation and detoxification mechanism of As by indigenous microalgae were explored.Secondly,pot experiments were conducted to explore the remediation mechanism of indigenous cyanobacteria on As-contaminated soil.Thirdly,the algae-bacteria composite crust was constructed by functional bacteria simulation to study the mechanism of functional bacteria strengthening the formation of biological soil crust and repairing As-contaminated soil.Finally,sunflower was used as the test plant,and the algae-bacteria-plant ternary system was constructed by single inoculation of soil cyanobacteria,bacteria and combined inoculation to further explore the remediation mechanism of microalgae-bacteria coupling on As-contaminated soil in mining areas.The main conclusions are as follows:（1）Biotransformation,accumulation and detoxification mechanism of arsenic by indigenous microalgaeIndigenous soil microalgae（Chlorella thermophila SM01 and Leptolyngbya sp.XZMQ）were isolated from BSCS in Shimen mining area.When the concentration of As（Ⅲ）in the culture medium was 0.5,1 and 10 mg L^-1,the growth inhibition rates of C.thermophila SM01 were 20.03%,24.96%and 34.62%after 15 days of culture,while those of Leptolyngbya sp.XZMQ were 21.50%,23.65%and 31.09%respectively.The increase of As（Ⅲ）concentration showed a significant negative gradient effect on the growth（Biomass）of the two microalgae.It took 2 days for C.thermophila SM01 to completely oxidize 10 mg L^-1 As（Ⅲ）in the culture medium,while it took 2 days,3 days and 9 days for Leptolyngbya sp.XZMQ to completely oxidize 0.5,1,10 mg L^-1 As（Ⅲ）,respectively.On the whole,the oxidation efficiency of As（Ⅲ）by C.thermophila SM01was higher than that by Leptolyngbya sp.XZMQ.However,the As accumulation by C.thermophila SM01(1586.65μg g^-1DW)was lower than that by Leptolyngbya sp.XZMQ(2429.90μg g^-1DW).The two microalgae carried out arsenic detoxification through the synergistic effect between antioxidant enzymes（SOD and CAT）and GSH.The-COOH,-OH and-NH₂ were involved in As（Ⅲ）detoxification.Principal component analysis（PCA）and Pearson correlation analysis showed that the antioxidant system and accumulation of C.thermophila SM01 were negatively correlated with OD,biomass and Chl a.There was no correlation between MDA and OD,biomass and Chl a after 15 days of culture.Therefore,the detoxification mechanism of C.thermophila SM01 and Leptolyngbya sp.XZMQ on arsenic is mainly through extracellular oxidation,adsorption,intracellular absorption,and the synergistic effect between antioxidant enzymes（SOD and CAT）and GSH,and has different arsenic toxicity response and bioaccumulation ability in time.（2）Remediation of arsenic contaminated soil by Leptolyngbya sp.XZMQCyanobacterial crust can be obviously formed by inoculating native Leptolyngbya sp.XZMQ on the surface of farmland soil and tailings soil.And it has high As accumulation.Cyanobacteria alter biotic and abiotic synergism to immobilize As.Non-EDTA-exchangeable As is the main part of As accumulation in cyanobacterial crust.Cyanobacterial crust reduced the bioavailability of As in the micro-scale soil layer.There was no significant difference in the mineral composition of cyanobacterial crust between the two soil types.Clay minerals and rock-forming minerals are distributed in cyanobacterial crusts,and Si O₂ is the main crystalline mineral.The-OH,-COOH,-CO and iron oxides were involved in the accumulation of arsenic in cyanobacterial crust.After inoculation with cyanobacteria,Cyanobacteria,Proteobacteria and Actinobacteriota were the dominant bacteria in the sub-crust soil.The arsenic biotransformation genes aio A,arr A,ars C,and ars M were identified.Among them,aio A had the highest abundance in the two types of soil,followed by ars M,while the average relative abundance of arr A and ars C was low.This is conducive to reducing the content and toxicity of soil As in the microenvironment.In addition,cyanobacterial crust can increase soil organic matter and available nitrogen content,improve soil p H and EC,and increase soil fertility in the microenvironment.In summary,indigenous cyanobacteria（Leptolyngbya sp.XZMQ）can enhance remediation of As-contaminated soil by regulating the physical and chemical properties,microbial community structure and function in soil microenvironment.（3）Synergistic effect of Bacillus XZM on remediation of arsenic-contaminated soil by microalgaeCo-inoculation of microalgae（C.thermophila SM01,Leptolyngbya sp.XZMQ）and plant growth-promoting bacteria（Bacillus XZM）can rapidly form BSC_S,especially the BSC_S formed by Leptolyngbya sp.XZMQ and Bacillus XZM,which is the most significant.Compared with the control group,the co-inoculation group（L+B）could increase the soil moisture content by 21.28%,and increase the soil catalase,alkaline phosphatase,sucrase and urease activities by 30.54%,96.26%,60.58%and 272.17%,respectively.On the contrary,the soil available As content decreased by 25.71%.In addition,the co-inoculation group（L+B）increased the extracellular polymer（EPS）content by 47.22%,and induced the formation of BSC_Sand the accumulation of As.Three-dimensional fluorescence spectroscopy（3D-EEM）analysis showed that BSC_Sinduced by co-inoculation of native microalgae and Bacillus XZM could enhance the arsenic immobilization by increasing the contents of tryptophan and tyrosine substances,fulvic acid,and humic acid in extracellular proteins.Moreover,the distribution of the ions（e.g.,Si,Al,and Fe）adsorbed by the induced BSC_S was consistent with the distribution of arsenic,indicating that these ions could participate in the accumulation of arsenic by BSC_S by coprecipitation.BSC_S formed by co-inoculation of Leptolyngbya sp.XZMQ and Bacillus XZM had the highest enrichment of As.In summary,inoculating microorganisms into the surface layer of As-contaminated soil has the effect of rapidly constructing BSCs and reducing the toxicity of As in the surface soil.These findings are useful for enhancing the remediation of As-contaminated soil by using the BSCs approach.（4）Improvement of arsenic-contaminated soil by coupling Leptolyngbya sp.XZMQ with Bacillus XZMLeptolyngbya sp.XZMQ,Bacillus XZM,and their mixture were inoculated in the sunflower root zone for the pot experiment.The results showed that both Leptolyngbya sp.XZMQ and Bacillus XZM could survive in the rhizosphere soil of sunflower,and co-inoculation had a certain growth-promoting effect.The single inoculation of Leptolyngbya sp.XZMQ（A）and Bacillus XZM（B）increased the EPS content of sunflower rhizosphere soil by 21.99%and 14.36%,respectively,while the content of EPS increased by 35%under the co-inoculation condition（A+B）.Besides,co-inoculation could significantly increase the enzyme activity（catalase,sucrase,and urease）of sunflower rhizosphere soil,reduce the toxic effect of arsenic in plants,and reduce the activities of catalase,peroxidase,and superoxide dismutase in sunflower plants.Co-inoculation enabled cyanobacteria and bacteria to attach and entangle in the root area of the plant to form a biological protective film,reducing the effective content and toxicity of arsenic and protecting the plant roots.Co-inoculation could regulate the structure of rhizosphere soil microbial community,and increase the abundance of Leptolyngbya and Bacillus.High-throughput sequencing and q PCR showed that co-inoculation increased the abundance of aio A,arr A,ars C,and ars M genes in soil,especially the abundance of microorganisms with aio A and ars M,which was beneficial to reduce the mobility and bioavailability of arsenic in soil,further reduce the absorption of arsenic by plants,and reduce the toxic effects of arsenic.Inoculation of Leptolyngbya sp.XZMQ significantly increased the abundance of nif H in soil,while co-inoculation significantly increased the abundance of cbb L,indicating that the coupling of Leptolyngbya sp.XZMQ and Bacillus XZM could stimulate the activity of nitrogen-fixing and carbon-fixing microorganisms in soil and had a certain improvement effect on arsenic-contaminated soil.In summary,this study used soil microalgae（especially Leptolyngbya sp.XZMQ）to couple with bacteria to form BSC_S,revealing the As immobilization mechanism of BSC_S.In addition,Leptolyngbya sp.XZMQ enhanced remediation of As-contaminated soils by regulating the physicochemical properties,microbial community structure and function of soil microenvironment.The coupling of Leptolyngbya sp.XZMQ and Bacillus XZM also reduced As absorption in sunflower rhizosphere,increased sunflower biomass,and stimulated the activity of arsenic oxidizing bacteria and methylation microorganisms in rhizosphere.This study provides a new method for the remediation of As-contaminated soil in mining areas.