Dynamic Changes of Soil Parameters and Bacterial Communities during Bioremediation of Multiple Heavy Metals Contaminated Tailings by Compound Bacterial Flora

Dynamic Changes of Soil Parameters and Bacterial Communities during Bioremediation of Multiple Heavy Metals Contaminated Tailings by Compound Bacterial Flora | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dynamic Changes of Soil Parameters and Bacterial Communities during Bioremediation of Multiple Heavy Metals Contaminated Tailings by Compound Bacterial Flora Huan Wang, Lingui Xue, Yanli Huo, Yecheng Ma, Jiahui Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2144977/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Many researchers have paid attention to solving the problem of multiple heavy metals pollution in the tailings area. However, the studies only stay at the laboratory level, which cannot completely demonstrate an efficient approach to remediate polluted environment due to tailings. This study aimed at investigating the effects of compound bacterial flora we constructed to remediate the tailings area of Linze County, Zhangye City, Gansu Province due to the accumulation of numerous heavy metals such as Pb, Cd and Mn. The remediation effects of different treatment groups were evaluated by monitoring the physical and chemical properties of soil and studying the bacterial community structure during the remediation process. Compared with the control (TCK), after 84 days of restoration, parameters of the tailings soil inoculated with the compound bacterial flora were improved, pH was decreased from 7.74 to 7.02, EC (electrical conductivity) was decreased by 21.15% ~70.49%, and activities of alkaline phosphatase, sucrase and urease were increased by 95.94%~99.64%、88.42%~98.43%、83.28%~86.95% respectively. Among the 43 identified core optional taxonomic units (OTUs), Proteobacteria (44.9%), Actinobacteriota (21.7%), Bacteroidota (17.9%) were the dominant bacterial phyla, and Sphingobacterium (20.2%), Arthrobacter (8.5%), Sphingomonas (6.1%) were the dominant bacterial genus. The results of alpha diversity showed that the bacterial diversity and richness of the treatment groups inoculated with compound bacterial flora were significantly higher than controls (TCK and HCK). The correlation analysis of RDA, VPA and Spearman showed that soil pH, WHC, EC, POR, TEMP and heavy metals had significant effects on the composition and distribution of bacterial communities. Our study monitored the changes of soil physical and chemical properties and bacterial community structure during the whole remediation process, which will provide a reference basis for the remediation approach to the soil polluted by multiple heavy metals. Heavy metals Outfield Site remediation Compound bacterial flora Bacterial community structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction The increasing demand for mineral resources has led to large-scale mining, beneficiation and smelting of mine resources all over the country. The development of mineral resources not only brings economic benefits, but also causes serious environmental pollution. High concentrations of heavy metals in water or soil environments, affect human health and safety (Mengbo Liu et al., 2021 ). Tailings are substances that can be excavated again after the extraction and beneficiation of ores in the mining industry (Hai-Hong Gu et al., 2017 ), but tailings often contain a variety of high concentrations of heavy metals (Hanno Matthaei et al., 2020; Sha Li et al., 2021 ). With the circulation of the biological chain, a large number of tailings are accumulated in the iron ore reservoir of Linze County, Zhangye City, Gansu Province, in which the contents of Pb, Cd and Mn seriously exceed the local soil background value, and these heavy metals are very easy to migrate and cause pollution of the surrounding environment and harm to human health. Studies have shown that long-term exposure to lead and cadmium will cause acute or chronic damage to the human nervous system, renal insufficiency and respiratory system (Paul B. Tchounwou et al., 2012 ). For years, people have been looking for ways to get rid of heavy metal pollution in the environment that are both safe and long-lasting. The research showed that heavy metal pollution is hard to get rid of because it stays in the environment and is toxic to organisms (Siallelli et al., 2011; Fan Du et al., 2018 ; Liu et al., 2018). Many researchers have favored bioremediation technology in the past few years because of its eco-friendliness and cost-effectiveness. To remediate heavy metal-contaminated soil, bioremediation uses bacteria and/or plants (Biao Song et al., 2017 ). Many studies have shown that the combined repair technology of compound bacterial flora and plants can shorten the repair time and improve the repair effect, which is significantly better than that of inoculating only single strain or compound bacterial flora or plants (Hassan Etesami, 2018; Akhtar et al., 2018 ; Arora et al., 2016 ; Utobo, E.B. et al., 2015). After the compound bacterial flora is inoculated into the soil polluted by heavy metals, they can not only interact with heavy metals, but also stimulate the adjustment of soil bacterial community structure, promote plants growth and development, further absorb heavy metals in the soil and repair the soil polluted by heavy metals more effectively. However, so far, most studies have stayed at the laboratory level, and tailings are large and open areas. The impact of environmental factors on their remediation technology cannot be solved in laboratory research, and more practical remediation data cannot be obtained. Since the relevant personnel of our laboratory have screened out the strains with high tolerance to multiple heavy metals and the hyperaccumulation plant suitable for the growth of this region ( Peganum harmala ) in the preliminary study, the purpose of this test is to (a) construct the best ratio of compound bacterial flora and apply it to the outdoor site remediation test to evaluate its impact on the parameters of tailings soil; (b) explore the changes of the microbial diversity and community structure of tailings soil in different periods after inoculation with compound bacterial flora. The test applies laboratory level research to outdoor site remediation, in order to provide more practical reference data for remediation of multiple heavy metals contaminated soil in Northwest China. 2 Materials And Method 2.1 Reagents and metal standard solutions NaCl, yeast extract, tryptone, and agar used for cell culture were purchased from Sinopharm Chemical Reagent Co., Ltd and Guangdong Huankai Microbial Technology Co., Ltd, respectively. The following chemicals were obtained from Tianjin Damao Chemical Reagent Factory: urea, methylbenzene, phenol, methanol, acetone, citric acid, ammonium sulphate, disodium phenyl phosphate, borax, 4-aminoantipyrine, K [Fe (CN)], glucose, 3,5-Dinitrosalicylic acid, and potassium sodium tartrate. Cd (NO 3 ) 2 ·4H 2 O, Pb (NO 3 ) 2, and MnCl 2 ·4H 2 O used for metal standard solutions were separately dissolved in sterile water supplied by Sinopharm Chemical Reagent Co., Ltd. All solutions were reserved in the dark at 4°C. The chemical reagents used in this experiment were analytical grade. 2.2 Selection and characterization of metal-tolerant strains According to the types of excessive heavy metals in the tail mining area, the strains used in this experiment were based on our previous studies which were C1-h, C4-h, C8-h, P2-h, P3-h, M1-h, M3-h (I renumbered them for memory) (Lu Zhang et al., 2022 ). Cadmium-tolerant strains were C1-h, C4-h, and C8-h; lead-tolerant strains were P2-h and P3-h; and manganese-tolerant strains were M1-h and M3-h. Relevant personnel in the laboratory had identified the seven strains in the previous study (Lu Zhang et al., 2022 ; Sha Li et al., 2021 ). Each strain belongs to the following genus: C1-h ( Klebsiella pneumoniae ), C4-h ( Bacillus ), C8-h ( Pseudomonas ), M1-h ( Bacillus ), M3-h ( Bacillus pumilus ), P2-h ( Klebsiella pneumoniae ) and P3-h ( Enterobacter Xiangfang ). The Agar Diffusion Method (Parisot H J., 2008) was used to observe whether there was mutual antagonism between them. 2.3 Selection of ratios and action conditions by growing cells The strains were cultured in LB liquid media at 150 rpm and 30℃ for 12 h and harvested, respectively. Take 5 ml culture solution of each strain and centrifugate them at 6000 r/min for 5 minutes, the supernatant was discarded, the cells were collected and washed them with sterile water for 2 ~ 3 times. Finally, resuspended the cells with sterile water and adjusted the optical density (OD) to about 1.0 at 600 nm. The prepared cells with eight different ratios were inoculated into 100 mL of liquid sterile growth media containing 15 mg/L Cd (II), 2500 mg/L Pb (II) and 1000 mg/L Mn (II), and an inoculum size of 2% (v/v) was used. Then the removal efficiency of heavy metals at 12 h, 24 h, and 48 h were measured to determine the best ratio. Eight different ratios were determined according to the growth curve of each strain (the growth curve of each strain was basically the same), which were 1) 1:1:1:1:1:1:1; 2) 2:1:1:1:1:1:1; 3) 1:2:1:1:1:1:1; 4) 1:1:2:1:1:1:1; 5) 1:1:1:2:1:1:1; 6) 1:1:1:1:2:1:1; 7) 1:1:1:1:1:2:1 and 8) 1:1:1:1:1:1:2. The content of heavy metals in the supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent Technologies Co. Ltd., ICP-MS 7900). Set three parallels and the media without inoculating cells and only adding metal were used as the blank control. After determining the best ratio, the prepared cells were inoculated into the media containing 15 mg/L Cd (II), 2500 mg/L Pb (II) and 1000 mg/L Mn (II) under different conditions in a ratio of 1:1:1:1:1:1:1 (the best ratio), cultured for 12 h, and the supernatant was taken after centrifugation to determine the heavy metals, so as to explore the optimal action conditions of compound bacterial flora. Different action conditions include different gradients of pH, temperature, salt concentration and inoculation amount (Table S1). Similarly, set three parallel and the media in which the prepared cells were not inoculated but only the metals were added were used as the blank control. 2.4 Outfield site test The tailings soil was taken from the iron ore reservoir in Pingchuan Town, Linze County, Zhangye City, Gansu Province, China (100 o 27’21” E, 38 o 56’11” N). The soil samples were obtained after screening, and their main characteristics were as follows: pH 7.73, WHC (water holding capacity) 6.28%, EC (conductivity) 772.449 us/cm, POR (porosity) 28.91%, Pb 2194 mg/kg, Cd 11.8 mg/kg, Mn 989 mg/kg (heavy metal content was higher than soil background value in Gansu Province (Pb 20.83 mg/kg, Cd 0.13 mg/kg, Mn 769.6 mg/kg)) (Qiong Liang et al., 2017 ). The tailings soil samples were taken back to the laboratory and then separately packed into round cloth bags (diameter × height: 30 cm×25 cm), and each bag containing 14 kg soil samples for disposal. The liquid compound bacterial flora with the best ratio above were cultured under the optimal conditions for 12 h, then the proportion of 15% (Lu Zhang et al., 2022 ) sterilized bran were added to make them become semi-solid compound bacterial flora with the moisture content of 60%~70%. Bran played a role in loosening soil and providing nutrients for microorganisms. The semi-solid compound bacterial flora was shaken at 30℃ for 1 h to make them mixed evenly, and then were inoculated into different treatment groups. The large and plump seeds of Peganum harmala were selected and washed with sterile water for 3 ~ 5 times for standby. The treatment groups for outfield site test were set as follows: (a) TCK (CK, total tailings soil, no-inoculated compound bacterial flora and no-inoculated seeds); (b) HCK (CK, added yellow cinnamon soil, no-inoculated compound bacterial flora and no-inoculated seeds); (c) TM (total tailings soil, inoculated compound bacterial flora but no-inoculated seeds); (d) TMP (total tailings soil, inoculated compound bacterial flora and inoculated seeds); (e) HM (add yellow cinnamon soil, inoculated compound bacterial flora but no-inoculated seeds); (f) HMP (add yellow cinnamon soil, inoculated compound bacterial flora and inoculated seeds). Each group was set up with three parallels. For the above groups added with yellow cinnamon soil (total tailings soil: yellow cinnamon soil = 3:1), this can dilute heavy metals in the tailings soil and also simulate the soil type heavily polluted by multiple heavy metals, which was conducive to the growth of compound bacterial flora and plants. This was also studied by relevant personnel in the early stage of the laboratory. The compound bacterial flora weas inoculated into the corresponding groups of cloth bags at 20 g/m 2 (Hao Xu et al., 2020 ) and the seeds inoculated in the cloth bags of the above-mentioned corresponding groups (50 seeds/bag). Then choose a wider outfield to place all the cloth bags. After the plants grow, 20 plants with similar growth would be left for follow-up study. 2.5 Analysis of soil parameters and metals content The whole outfield site test was 84 days. Soil samples were taken every two weeks to analyze their physical and chemical properties, including enzyme activities (sucrase, urease, alkaline phosphatase), soil (TMP) temperature, pH, (WHC) water holding capacity, (POR) porosity and (EC) electrical conductivity. The activities of urease, alkaline phosphatase and sucrase were determined according to the methods of R.P. Dick et al., 1996 , Xuedan Li et al., 2019 and Arsalan A. Quresh et al., 2020 . The (POR) porosity was measured by the bulk density specific gravity method, and the (EC) conductivity was measured by conductivity meter (Shanghai Leici dds-307, DDs-307A). The removal rate of heavy metals was calculated by measuring the total content of heavy metals before and after remediation by ICP-MS. Pretreatment of soil samples: weighed 0.1g of soil samples, added 5ml aqua regia, and conducted microwave digestion. After digestion, drove the acid to viscous state at 140℃, fixed the volume to 25ml, and tested on the machine (ICP-MS). Over the whole restoration, plant samples were collected to determine the enrichment factor. After drying the plant samples at 85℃, grinded and screened them, determined the content of heavy metals in the plants, and calculated the enrichment coefficient of heavy metals. Samples pretreatment method: weighed 0.5 g plant samples, added 5ml nitric acid and 1ml hydrogen peroxide, placed it overnight for microwave digestion. After digestion, drove the acid to 1 ml at 140℃, fixed the volume to 25 ml, and tested on the machine (ICP-MS). $$\text{H}\text{e}\text{a}\text{v}\text{y} \text{m}\text{e}\text{t}\text{a}\text{l} \text{e}\text{n}\text{r}\text{i}\text{c}\text{h}\text{m}\text{e}\text{n}\text{t} \text{f}\text{a}\text{c}\text{t}\text{o}\text{r}=\frac{\begin{array}{c}\\ Heavy metal content in plants\end{array}}{\text{H}\text{e}\text{a}\text{v}\text{y} \text{m}\text{e}\text{t}\text{a}\text{l} \text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t} \text{i}\text{n} \text{s}\text{o}\text{i}\text{l}}\times 100\%$$ 2.6 Analysis of microbial diversity and community structure The soil samples taken on the 28th, 42nd, 56th and 84th days of the repair process were stored in the refrigerator at -80℃ for subsequent delivery to the company (Shanghai Majorbio Bio-pharm Technology Co., Ltd) for high-throughput sequencing. The obtained data were used to analyze the changes in microbial diversity and community structure during the restoration process. 2.7 Data analysis All experiments were performed in triplicate. The experimental data were statistically analyzed in Microsoft Excel 2010 and SPSS 25.0, using Duncan's multiple comparative analysis method, and visualized in Origin 2019. The high-throughput sequencing data were analyzed on the free online platform of Majorbio Cloud Platform (majorbio.com). 3 Results 3.1 Construction of compound bacterial flora 3.1.1 Strains characteristics and antagonistic experiment The characteristics of the 7 strains were shown in Table 1 . It can be seen from the table that Bacillus has the highest proportion of them. Strain number resistance to major HMs* search number 16SrRNA identification Table 1 Characteristics of seven heavy metals tolerant strains. C1-h Cd MT973991 Klebsiella pneumoniae C4-h Cd MW812244 Bacillus sp. C8-h Cd MT967291 Pseudomonas fluorescens M1-h Mn MW652627 Bacillus altitudinis M3-h Mn MW485109 Bacillus pumilus P2-h Pb MT974007 Klebsiella pneumoniae P3-h Pb MT994615 Enterobacter sp. Strain number Colony morphology C1-h Yellow round colony, small and opaque, moist and smooth surface, raised center and neat edge, easy to provoke C4-h Yellow round colony, large colony, convex center, smooth surface, neat edge, easy to provoke C8-h The colony center is orange yellow, the colony is large, the surface is wet and smooth, the edge is neat, and it is easy to provoke M1-h White round colony, convex center, wet and smooth surface, neat edge, easy to provoke M3-h White round colony, convex center, wet and smooth surface, neat edge, easy to provoke P2-h Off white colony, round, moist surface, neat edge, easy to provoke P3-h Yellow round colony, smooth surface, neat edge, easy to provoke *HMs: heavy metals. Previous studies had shown that the 7 strains were resistant to variety of other heavy metals in addition to above major heavy metals. To ensure the best effect of the compound bacterial flora constructed subsequently, the antagonistic experiment was carried out on 7 strains in pairs (Fig. S1). If there was an antagonistic effect between strains, it would affect the effect of the compound bacterial flora. It can be seen from the figure that there was no bacteriostatic circle between the seven selected heavy metals tolerant strains, so there was no antagonism. Therefore, follow-up experiments can be continued. Fig S1. The antagonism among the seven strains was from left to right in each row: 1)C1-h + C4-h、C1-h + C8-h、C1-h + M1-h、C1-h + M3-h、C1-h + P2-h、C1-h + P3-h、P2-h + C8-h; 2) C4-h + C8-h、C4-h + M1-h、C4-h + M3-h、C4-h + P2-h、C4-h + P3-h、M3-h + P2-h、M3-h + P3-h; 3) C8-h + M1-h、C8-h + M3-h、C8-h + P3-h、M1-h + M3-h、M1-h + P2-h、M1-h + P3-h、P2-h + P3-h. 3.1.2 Determination of optimum ratio and action conditions To replicate the environment polluted by heavy metals in tailings, 15 mg/L Cd, 2500 mg/L Pb and 1000 mg/L Mn were added to LB medium. Compound bacterial flora were inoculated at the above 8 different ratios, cultured at 30 ℃ and 120 r/min, and then the heavy metals removal rate of 12 h, 24 h and 48 h were measured. The results showed that the removal rate of heavy metals in 12 h was higher than that in 24 h and 48 h. At 12 h, the removal rate of heavy metals in the compound bacterial agent with a ratio of 1:1:1:1:1:1:1 was higher than that in the other seven ratios (Fig. 1ABC; Table S2). The removal rates of Mn 2+ , Cd 2+ and Pb 2+ were 55.3%, 65.1% and 78.4% respectively. Therefore, the best ratio of compound bacterial agent was 1:1:1:1:1:1:1, and the removal rate of heavy metals under 12 h was selected in the subsequent optimization experiment of compound bacterial flora action conditions. Different pH (5, 6, 7, 8, and 9), temperature (25°C, 28°C, 30°C, 32°C, and 35°C), inoculation amount (1%, 2%, 3%, 4%, and 5%) and salt concentration (1%, 2%, 3%, 4%, and 5%) gradients were used to investigate the best conditions. From the experimental data, the results are as follows: (1) Compared with other pH values, the removal rate of heavy metals by compound bacterial flora at pH = 7 were higher, and the removal rate of Mn 2+ , Cd 2+ and Pb 2+ were 47.5%, 50.2% and 91.95% respectively; (2) When compared to other temperatures, the removal rate of heavy metals were higher at 28°C, with removal rate of Mn 2+ , Cd 2+ and Pb 2+ being 56.6%, 78.7% and 98.2%, respectively; (3) When the inoculation amount of compound bacterial flora was 3%, the removal rate of heavy metals was higher, with removal rate of Mn 2+ , Cd 2+ and Pb 2+ being 59.2%, 67.3% and 91.6%, respectively; (4) When the salt concentration was 3%, the removal rate of heavy metals was higher, with removal rate of Mn 2+ , Cd 2+ and Pb 2+ being 56.7%, 73% and 89.5%, respectively. To summarize, the best conditions for compound bacterial flora action were pH = 7, culture temperature of 28°C, inoculation amount of 3% and salt concentration of 3% (Fig. 1DEFG). Under heavy metals stress, the compound bacterial flora with the best ratio was cultured under optimal conditions, and the removal rate of heavy metals were measured. The removal rate of Mn 2+ , Cd 2+ and Pb 2+ could be as high as 60.7%, 71.6% and 90.5% respectively (Table S3). 3.2 Evaluation of outfield site test repair effect 3.2.1 Changes in physical and chemical properties of tailings soil during remediation To monitor the changes of soil physical and chemical properties during the remediation process, pH, WHC (water holding capacity), POR (porosity), EC (electric conductivity) were measured. The pH of tailings soil at the sampling point was alkaline, the pH of control group TCK is 7.74, and the pH of control group HCK is 7.24. In addition, compared with the control groups, during the repair process, the pH of different treatment groups gradually decreased and tended to be neutral (Fig. 2 A), especially in the treatment group of HMP. The pH of all T treatment groups decreased from 7.74 (TCK) to 7.18 (TM) and 7.11 (TMP) after 84 days, and the pH of all H treatment groups decreased from 7.24 (HCK) to 7.08 (HM) and 7.02 (HMP). The ability of plant-microbial combination technology to improve soil pH was higher than that only inoculated compound bacterial flora. Compared with the control groups, the soil WHC (water holding capacity) of tailings in different treatment groups increased (Fig. 2 B). Whether inoculated with flora or combined technology, there was little difference in the change in WHC between them, but in general, the WHC showed an upward trend after inoculation of compound bacterial flora with the increase of repair time. During the repair process, the POR (porosity) of the treatment groups inoculated with only compound bacterial flora and the control groups were measured. The results showed that with the increase of repair time, the POR of different treatment groups increased, and the treatment effect of HM was particularly obvious (Fig. 2 C). At 84 days, the POR of HM and TM were 49.54% and 41.91% respectively, which increased by 13.69% and 8.53% respectively compared with the controls (HCK and TCK). In the process of remediation, the changes of soil EC (electric conductivity) in different treatment groups fluctuated, but on the whole, the soil conductivity increased first and then decreased with the increase of remediation time (Fig. 2 D), and the EC of the treatment groups inoculated with both compound bacterial flora and Peganum harmala fluctuated significantly compared with the treatment groups inoculated with only compound bacterial flora, with an overall decrease of 21.15% ~ 70.49%. Compared with the 0-day controls, at 84 days of repair, the EC of HM, TM, HMP and TMP were 307 us/cm, 611 us/cm, 228 us/cm and 265 us/cm respectively. The decrease of EC in the treatment groups may related to the temperature changes and available state of heavy metals. These above results indicated that the physical and chemical properties of soil were different with the increase of remediation time in different treatment groups, and the soil improvement effects were also different. 3.2.2 Changes of enzyme activities in tailings soil during remediation To further monitor the soil quality improvement effect of different treatment groups during the restoration process, different treatment groups soil samples were taken every 14 days to determine the enzyme activities of alkaline phosphatase, sucrase and urease (Fig. 3ABC). The three enzyme activities of TCK and HCK were almost unchanged during the remediation process, while the enzyme activities of all H treatment groups (HM, HMP) were higher than those of the corresponding T treatment groups (TM, TMP). Compared with the controls (TCK and HCK), the activities of alkaline phosphatase, sucrase and urease in the four experimental groups increased significantly on the 14th day. From 14 to 42 days, the activity of alkaline phosphatase changed little and began to increase gradually after 42 days. Compared with the controls (TCK and HCK), the alkaline phosphatase activities of all treatment groups at 84 days were HMP (75.6 µg/g) > HM (44.6 µg/g) > TMP (43.8 µg/g) > TM (33.4 µg/g). Compared with the control groups, the activity of sucrase increased at 14 days. After 42 days, the enzyme activity of all T treatment groups increased slowly, and the enzyme activity of the corresponding H treatment groups increased rapidly. At 84 days, the sucrase activities of all treatment groups were HMP (35.6 mg/g) > HM (34.6 mg/g) > TMP (26.7 mg/g) > TM (25.5 mg/g). Compared with alkaline phosphatase and sucrase, urease activity fluctuated greatly in the repair process. The enzyme activity of different treatment groups had a trend of “first rising, then falling and then rising again”, but the repair time points corresponding to their decline and rise were different. Compared with the control groups, the urease activity of all treatment groups showed an upward trend as a whole. At 84 days, the urease activities of all treatment groups were HM (0.35 mg/g) > HMP (0.34 mg/g) > TMP (0.32 mg/g) > TM (0.3 mg/g). From above results, it can be seen that the inoculation of compound bacterial flora had significant impact on enzyme activities. 3.2.3 Removal rate of heavy metals in soil and enrichment coefficient of heavy metals in Peganum harmala The heavy metals content of TCK before remediation and each treatment group’s soil heavy metals content after remediation were measured to evaluate the heavy metals removal rate of different treatment groups in the whole remediation process (Table 1 ). It can be seen from the Table 1 that the repair effect of all H treatment groups was better than that of all T treatment groups, and the repair effect of only inoculating compound bacterial flora was better, while the microbial-plant joint repair effect was the best, and the removal rate of Pb, Cd and Mn were 54.74%, 57.46% and 24.97% respectively. The plant heavy metal enrichment coefficients (Table S4) of TMP and HMP treatment groups were measured. From the data in the table, it can be seen that Peganum harmala has the best enrichment effect on Mn. 3.3 Analysis of microbial diversity and community structure 3.3.1 Summary of sequences and annotation of taxa A total of 2,360,069 high-quality reads were obtained from 52 samples through Illumina MiSeq sequencing after data filtering (Table S5). Rarefaction curves showed that Shannon diversity index at the OTU level tended to be constant with the increase of sequence number, and sequencing almost reached saturation state (Fig. S2). A total of 5558 operational taxonomic units (OTUs) were obtained by cluster analysis at a 97% similarity level (Table S5), and were classed into 39 phyla, 124 classes, 285 orders, 485 families, 951 genera and 2019 species. Also, 97.4%, 93.5%, 92.3%, 90.1%, 87.6% and 72.6% of OTUs could be precisely annotated at the levels of phylum, class, order, family, genus and species, respectively. For the relative abundance of OTUs, Proteobacteria was uppermost (42.7%) at the phylum level, followed by Actinobacteriota (21.7%), Bacteroidota (16.4%), Chloroflexi (4.5%), Acidobacteriota (4.2%), Firmicutes (2.9%), Gemmatimonadota (2.1%), Myxococcota (1.9%) and Verrucomicrobiota (1.4%). The 3 dominant phyla (relative abundance > 5%) accounted for 80.8% of the total OTUs and the top 9 phyla made up 97.8% of all bacterial communities. FigureS2. Rarefaction curves. In order to explore adaptation characteristics of bacteria to the tailings soil habitats in different treatment groups in this study, we specifically analyzed the core OTUs. As shown in Fig. 4 , there were a total of 43 core OTUs (6.1%) in all samples, which were defined as the core microbiome, while the numbers of different OTUs in each sample were considered to be the unique microbiomes. At the phylum level, the predominant members (relative abundance > 3%) of 303 core OTUs belonged to Proteobacteria (44.9%), Actinobacteriota (21.7%), Bacteroidota (17.9%), Chloroflexi (4.3%) and Acidobacteriota (4.1%), among which OTU4751 (affiliated to genus Sphingobacterium ) was the most abundant (20.2%) followed by OTU2712 (8.5%), OTU2463 (6.1%), OTU3470 (5.9%), OTU2358 (4.6%) and OTU2378 (4.1%), affiliated to genus Arthrobacter , Sphingomonas , Brevundimonas , unclassified Rhizobiaceae and Blastococcus , respectively. It can be seen from the figure that these core OTUs had existed throughout the remediation process and were also the core OTUs of TCK. Therefore, these core OTUs represent the bacterial communities commonly existing in the iron tailings reservoir area and different treatment groups, and maybe we can call them “indigenous microbiome”. These “indigenous microbiome” may have an impact on the compound bacterial flora that was subsequently inoculated. 3.3.2 Soil bacterial community diversity and composition To characterize the soil bacterial community structure of different treatment groups, we conducted diversity analyses. The α (alpha) diversity index of different treatment groups were used for statistics (Table 2 ), including the richness index (Chao index and ACE index) and diversity index (Shannon index and Simpson index). Compared with all T treatment groups, the bacterial richness index and diversity index of all H treatment groups were improved. The ACE index of all T treatment groups was between 368.39 ~ 633.18, Shannon index was between 0.95 ~ 4.28, and Simpson index was between 0.028 ~ 0.627. And the ACE index was between 2811.34 ~ 3577.25, Shannon index was between 5.74 ~ 6.76, and Simpson index was between 0.003 ~ 0.013 of all H treatment groups. Whether T treatment groups or H treatment groups, the treatment groups inoculated compound bacterial flora showed higher α diversity for soil bacterial communities compared with control treatment groups (TCK and HCK), especially the treatment groups combined with compound bacterial flora and peganum harmala . The Chao index, Shannon index, Simpson index and ACE index showed that the richness and diversity of the treatment groups added with compound bacterial flora and the treatment groups with combined microbial-plant repair were significantly higher than those of the control groups ( p ＜0.05), and their richness index and diversity index were also increasing with the increase in repair time. These results indicated that the inoculation of compound bacterial flora and peganum harmala significantly affected the richness and diversity of soil bacterial communities in different treatment groups. Table 2 The Chao index, Shannon index, Simpson index and ACE index of different treatment groups. Group H Chao Shannon Simpson ACE HCK 2806.68 ± 212.98d 5.74 ± 0.12e 0.013 ± 0.005a 2942.58 ± 438.56bc HM1 2845.32 ± 71.08d 6.03 ± 0.02d 0.008 ± 0.001b 2811.34 ± 75.34c HM2 3029.69 ± 45.06bcd 6.21 ± 0.03bcd 0.007 ± 0.001b 3016.42 ± 33.37bc HM3 2908.67 ± 81.17cd 6.14 ± 0.27cd 0.007 ± 0.002b 2897.05 ± 115.14bc HM4 3102.09 ± 157.33bc 6.31 ± 0.09bc 0.007 ± 0.001b 3062.57 ± 118.80bc HMP1 3189.47 ± 38.43b 6.24 ± 0.05bc 0.007 ± 0.0001b 3133.89 ± 47.81bc HMP2 3177.37 ± 168.47b 6.3 ± 0.07bc 0.006 ± 0.0004b 3170.27 ± 196.29b HMP3 3238.94 ± 128.07b 6.36 ± 0.05b 0.006 ± 0.0006bc 3216.63 ± 59.18b HMP4 3571.99 ± 77.31a 6.76 ± 0.02a 0.003 ± 0.0001c 3577.25 ± 53.06a Group T TCK 444.32 0.95 0.627 475.84 TM1 400.84 ± 36.75b 3.85 ± 0.06de 0.055 ± 0.002a 409.75 ± 21.22b TM2 398.91 ± 19.80b 3.91 ± 0.03cde 0.045 ± 0.005ab 397.73 ± 28.88b TM3 476.53 ± 54.82ab 4.19 ± 0.04ab 0.034 ± 0.001bc 479.65 ± 44.04ab TM4 505.65 ± 98.75ab 4.28 ± 0.05a 0.028 ± 0.003c 522.33 ± 137.83ab TMP1 363.88 ± 39.53b 3.69 ± 0.17e 0.054 ± 0.005a 368.39 ± 38.92b TMP2 418.17 ± 62.24b 3.97 ± 0.11bcd 0.04 ± 0.005bc 441.94 ± 86.12ab TMP3 593.48 ± 77.93a 4.06 ± 0.24abcd 0.047 ± 0.016ab 633.18 ± 92.10a TMP4 616.23 ± 193.4a 4.13 ± 0.16abc 0.039 ± 0.006bc 612.22 ± 213.83a Data are shown as means ± standard deviations (n = 3). The different lowercase letters in the same column indicate significant differences ( p < 0.05) among samples. TCK was the control of all T treatment groups. HCK was the control of all H treatment groups. The number after each treatment group represented the sampling time. For example, HM1 represented the sample taken from HM treatment group on the 28th day. For the bacterial community composition, the proportion of Firmicutes and Bacteroidota in all other treatment groups (including HCK) decreased significantly compared with the control TCK, while the proportion of Proteobacteria increased (Fig. 5 A, S3A). Interestingly, the proportion of Actinobacteriota in the control TCK and all H treatment groups were higher than T treatment groups (TM, TMP) (Fig. 5 A, S3A). Furthermore, Chloroflexi at all H treatment groups were significantly more dominant than at all T treatment groups (p < 0.05) (Fig. 5 A). With the increase of sampling time, the changes of community structure in the same treatment groups were not very obvious, but their relative abundance were different (Fig. 5 A). At the genus level, the proportion of Phingobacterium in all T treatment groups first increased and then decreased with the increase of sampling time, compared with the control TCK, while the proportion in all H treatment groups were extremely low (Fig. 5 B, S3B). The proportion of Planococcus in control TCK were large but in all other treatment groups were quite low (Fig. 5 B, S3B). Compared with all T treatment groups, the bacterial communities of all H treatment groups were more complex and more abundant. In addition, the bacterial community structure after inoculation of compound bacterial flora was more complex and diverse compared with the reference controls (TCK and HCK) (Fig. 5 B, S3B). These results indicated that the inoculation of compound bacterial flora discharge remarkably shifts the composition of soil bacterial communities. FigureS3. Different proportion of soil bacterial community composition in different groups with relative abundance > 5% at the phylum ( A ) and genus ( B ) levels. 3.3.3 Effects of environmental factors on microbial communities To determine the relationship among samples, soil bacterial communities and environmental factors, all of the 8 measured environmental variables were conducted to redundancy analysis (RDA). Among these environmental variables, 7 had significant effects on bacterial communities, including pH, WHC (water holding capacity), TEMP (soil temperature), POR (porosity), Pb, Cd, Mn ( p < 0.05) (Table S6). At the phyla, the RDA1 and RDA2 explained 66.06% and 30.05% of the total variances of soil bacterial community composition, respectively (Fig. 6 A; Table S7). To further determine the impact of 8 environmental variables on bacterial communities, a variance partitioning analysis (VPA) was performed by dividing the environmental variables into two groups: heavy metals (Pb, Cd, Mn) and soil physical and chemical properties (pH, WHC, TEMP, EC, POR). It can be seen from Fig. 6 B that 97.17% of the bacterial community variance was explained by the two groups of environmental variables, and the contribution of soil physical and chemical properties (77.34%) to the variance was higher than that of heavy metals (69.29%). The above results indicated that these 8 environmental factors had significant effects on soil communities. Further, the Spearman correlation Heatmap showed that Pb and Cd significantly negative correlated with phyla Gemmatimonadota, Fibrobacterota, WPS-2, GAL15, Actinobacteriota, MBNT15, Methylomirabilota, Latescibacterota, Elusimicrobiota, Entotheonellaeota, Acidobacteriota, RCP2-54, unclassified_k_ norank_ d_Bacteria, Chloroflexi, Patescibacteria, Nitrospirota, Dependentiae, NB1-j, Planctomycetota and Desulfobacterota ( p < 0.001), while Mn only negatively with phyla WPS-2, GAL15, unclassified_k_ norank_ d_Bacteria, Chloroflexi, Patescibacteria ( p < 0.05 or p < 0.01) (Fig.S4A; Tables S8-S9).Moreover, there were negative correlations between EC and Spirochaetota ( p < 0.01), pH and Spirochaetota, Verrucomicrobiota, TXA1-33, Hydrogenedentes ( p < 0.05 or p < 0.01), TEMP and Spirochaetota ( p < 0.05) (Fig.S4A; Tables S8-S9). In addition, the POR significantly positive correlated with phyla Gemmatimonadota, Fibrobacterota, WPS-2, GAL15, Elusimicrobiota, unclassified_k_ norank_ d_Bacteria, Chloroflexi, Patescibacteria, Nitrospirota, Planctomycetota and Desulfobacterota ( p < 0.01 or p < 0.001) (Fig.S4A; Tables S8-S9). At the genus level, Pb and Cd contributed to the abundances of Sphingobacterium , Rhodopseudomonas , Algoriphagus , Pseudoxanthomonas , Pedobacter and Sphingopyxi ( p < 0.05 or p < 0.01), but significantly negative correlated with the proportions of genus norank_f_Gem matimonadaceae, Streptomyces , norank_f__JG30-KF-CM45 , norank_f_Xanthobacteraceae , Nocardioides , Gaiella , norank_f_Vicinamibacteraceae , RB41 , norank_f_norank_o_Vicinamibacterales , norank_ f_ norank_o_norank_c_KD4-96 , norank_f_norank_o_Microtrichales , norank_f_Ilumatobacteraceae , Solirubrobacter and norank_f_67 − 14 ( p < 0.001) (Fig.S4B; Tables S10-S11). Interestingly, the correlations between Mn and most of phyla or genus were not very significant (Fig.S4A; Tables S8-S9; Fig.S4B; Tables S10-S11). All above results indicated that the bacterial populations of different treatment groups had different responses to environmental variables. Figure S4. The effects of environmental factors (Pb, Cd, POR, WHC, Mn, EC, pH, TEMP) on bacterial community structure.( A ) at the Phylum level; ( B ) at the Genus level (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). 4 Discussion Bioremediation of waste tailings was a green and environmentally friendly remediation method with the concept of sustainable development, which mainly includes microbial remediation, phytoremediation and microbial plant combined remediation (Bin Xuan et al., 2017 ). Current studies had found that some functional groups on the surface of microbial cells can adsorb or precipitate heavy metals (Ying Wang et al., 2020 ), and microorganisms can also secrete some secondary metabolites, that would interact with heavy metals, such as mineralization (C.-H. Kang et al., 2015 ), dissolution of heavy metals by organic acids (N. Papassiopi et al., 2014 ), etc. However, the process of remediation after adding microorganisms to the soil was very complex, and the growth of microorganisms also had a certain period of time, so the fate of microorganisms in the later stage was also difficult to solve. Phytoremediation can remove heavy metals from soil, and plants were easy to collect in the later stage, so they can deal with heavy metals intensively. However, the efficiency of phytoremediation technology was often limited by the high concentration of heavy metals, coexistence of multiple heavy metals and low-level nutrition (Benidir L et al., 2020). In fact, there were few plants around the tailings reservoir area where we collected soil samples, and only a few drought-enduring plants were scattered. Therefore, before planting plants in heavy metals contaminated soil, inoculate microorganisms to reduce the soil toxicity of tailings, promote plants growth, reduce stress and chelate heavy metals (Nida Zainab et al., 2020), and give full play to the effect of combined microbial and plants remediation. In fact, the resistance of soil bacteria to heavy metals and their ability to promote plants growth make them the first choice for phytoremediation of heavy metals contaminated soil in previous studies (Tirry et al., 2018 ). It had been found that the inoculated compound bacterial flora resistant to heavy metals could affect the mobility and availability of heavy metals in plants through rhizosphere chelation, acidification and oxidation-reduction reactions, which was conducive to the absorption of heavy metals by plants, thus directly affecting the remediation effect (Ma et al., 2011 ) and further illustrated the important role of microorganisms in heavy metal contaminated soil. Therefore, in this study, compound bacterial flora resistant to heavy metals was constructed, and the two bioremediation methods (only inoculated compound bacterial flora and the combination of compound bacterial flora and Peganum harmala ) were set up in different groups for tailings remediation outfield test, so as to obtain more practical remediation schemes and reference data according to the changes of soil parameters and bacterial communities of different treatment groups during bioremediation process. A major feature of the tailings we studied was multiple heavy metals pollution, which was similar to the previous reports (Nouri and Haddioui, 2016 ; Huang et al., 2013 ). This also showed that no matter where the iron tailings and soil around the tailings pond were located, multiple heavy metals pollution often existed. Therefore, we selected strains that were resistant to multiple heavy metals to construct compound bacterial flora. Seven strains with no antagonistic effect between two were selected in the test (Fig. S1). The antagonistic effect between strains was that the life activities of one microorganism or its metabolites inhibit or interfere with the life activities of another microorganism, which would make the constructed compound bacterial flora unable to give full play to its efficacy and repair the tailings soil to the greatest extent. In this study, the antagonistic experiment results of seven strains showed that they had no antagonistic effect and could coexist under the same cultural conditions, so they could be used for the construction of subsequent compound bacterial flora. In addition, the ratio among the strains would also affect the effectiveness of compound bacterial flora, this study also explored the best ratio among the seven strains. It can be seen from the follow-up outdoor site remediation test results that the appropriate ratio of compound bacterial flora indeed played an important role in the remediation process of tailings soil. In our study, microorganisms played the most important role in different treatment groups, and soil quality determined the composition and distribution of microbial communities. Therefore, we monitored the changes of soil physical and chemical properties and enzyme activities during the whole remediation process. The existence and migration of heavy metals in soil were determined by many environmental factors, among which soil pH and EC (electric conductivity) play important roles. The change in pH would change the solubility of heavy metals in soil (Teng Xu et al., 2020 ; Alicja Kinska et al., 2022). In particular, the bacteria combined plant technology was adopted in this study, and the change of soil pH would affect the bioavailability of nutrients or heavy metals absorbed by plants in the rhizosphere (E. Oburger et al., 2020 ). In the process of remediation, the pH of different samples changes from alkaline to neutral. At the end of remediation, the pH of tailings soil of different treatment groups was stable at about 7.1. The neutral environment was conducive to the survival of microorganisms, and relevant studies had found that an appropriate pH can promote microbial activity (Myoung-Jin Kim et al., 2004). At the same time, soil pH was also considered to be the main driving force behind changes in the bacterial community (Xiaoxu Sun et al., 2020). Our results were consistent with previous studies. Redundancy analysis (RDA) and Spearman correlation Heatmap showed that pH was indeed an important environmental variable affecting bacterial communities. EC can indirectly reflect the concentration of metal ions in soil, while in soil polluted by multiple heavy metals such as tailings, EC can indirectly determine the concentration of heavy metal ions (Kasemodel et al., 2019 ). The test results showed that although EC fluctuated with the increase of remediation time, it may be due to the dynamic transformation of different forms of heavy metal ions during the remediation process, which still showed a downward trend as a whole (Fig. 2 D), indicating that the addition of compound bacterial flora or planting Peganum harmala would reduce the concentration of heavy metal ions in tailings soil, it may be due to the adsorption heavy metal ions on the cell surface of the compound bacterial flora and interaction of the secondary metabolites produced by the compound bacterial flora with heavy metal ions. But interestingly, the reduction of EC did not significantly affect the bacterial communities from the results of Redundancy analysis (RDA) and Spearman correlation Heatmap, which was inconsistent with previous studies (Wei Wu et al., 2022 ). The changing trend of soil POR was just the opposite to that of EC. It was well known that the size of soil porosity depends on soil quality. During the experimental remediation process, the soil POR of different treatment groups after adding compound bacterial flora gradually was increased (Fig. 2 C). This indicated that the quality of tailings soil had improved after adding compound bacterial flora. Soil enzyme activities were good indicator of soil quality improvement, and their changes can provide characteristics and relevant information about microbial community succession (Adewele T. Adetunji et al., 2017). The test results showed that the addition of compound bacterial flora would improve the enzyme activities of sucrase, urease and alkaline phosphatase in tailings soil (Fig. 3ABC), which was consistent with the results reported by Souhir Abdelkrim (2020) and others. When the soil inoculated with compound bacterial flora was compared with the soil without inoculation, the activities of β-glucosidase, urease and alkaline phosphatase increased significantly. In addition, other literatures had also reported the increase of soil enzyme activities after bacterial flora inoculation (Upadhyay et al., 2016 ; Utobo-Tewari, 2015; Wenliang Ju et al., 2019 ). The increase of soil enzyme activities was conducive to the growth and development of plant roots. For example, the increase of urease activity would promote the absorption of nitrogen by plant roots (Saadani, O. et al., 2019 ), which was very friendly to the microbial-plant joint remediation technology adopted in this experiment, so as to achieve the improvement of soil quality by microorganisms, and the improvement of soil quality promotes the growth and development of Peganum harmala , and finally achieve a mutually beneficial and win-win relationship. In general, the richness index and diversity index representing the bacterial communities showed that the Chao, Shannon, Simpson and ACE index of H treatment groups were higher than those T treatment groups, which may due to the dilution of the content of heavy metals in the whole tailings soil by adding exogenous yellow cinnamon soil, resulting in the decrease of soil toxicity. At the same time, the richness index and diversity index of treatment groups with compound bacterial flora and Peganum harmala were higher than controls (TCK and HCK), and with the increase of repair time, the richness index and diversity index of different treatment groups were also increasing. This result was consistent with the report of RAúl Zornoza (2017) and others. Previous studies have reported that Firmicutes, Actinobacteriota, Proteobacteria and Bacteroidota were dominant bacterial phyla in mining areas (Yuanqing Chaoet al., 2016 ; Jianwei Xinget al., 2017 ; Lur Epelde et al., 2015 ; Sibanda et al., 2019 ). These results were similar to the composition of the control groups (TCK, HCK). The most abundant phylum of the 43 core OTUs was Proteobacteria in our study. However, prior to inoculation of compound bacterial flora, the dominant bacterial phyla in TCK were Firmicutes and Actinobacteriota, and the dominant bacterial phyla of corresponding T treatment groups (TM, TMP) were Proteobacteria and Bacteroidota after the inoculation of compound bacterial flora. These results may be related to the compound bacterial flora we inoculated. And the bacterial community structure in the tailings area had changed after the compound bacterial flora was inoculated, which was consistent with the research results of Wood (2016) et al, Cd changed the competitive dynamics in the soil bacterial community, causing changes in the bacterial community. Interestingly, there was also a certain proportion of Proteobacteria and Bacteroidota in HCK, but it was not higher than the proportion of T treatment groups after inoculation of compound bacterial flora. This made us wonder about the bacterial community structure changes may not only be caused by the inoculation of compound bacterial flora, but also may be caused the inoculation of compound bacterial flora stimulated the reproduction of some “native bacterial phyla” and formed unique dominant bacterial phyla, similar results had been reported by M Shuaib (2018). Moreover, we determined 8 environmental variables that affect bacterial communities in different treatment groups, of which heavy metals were the main factors because of their greatest impact on the bacterial communities in our study. Although VPA analysis showed that the contribution of soil physical and chemical properties to the variance was higher than that of heavy metals. This may be due to the fact that soil physical and chemical property variables (five) were more than heavy metal variables (three). Our results were consistent with previous studies (A. Markowicz, 2016; Luo et al., 2018 ; Song et al., 2018 ) showing that heavy metals inhibited bacterial communities. However, some reports were inconsistent with our research results and their research showed that heavy metals would not inhibit or even promote the bacterial communities (Mohamed and Abdelmajid, 2017 ; Hong et al., 2015 ; Reis et al., 2013 ). These differences may due to the types, duration of heavy metals and various environmental factors in different mining areas. In fact, the impact of heavy metals on bacterial communities was extremely complex, and our research process had only gone through three months, because our main purpose was to gain the removal rate of heavy metals in different treatment groups and the restoration effect of soil quality. The results of heavy metals removal rate showed that our research could achieve the tentative goal. For this study, we only measured the relationship between the total amount of heavy metals and changes of bacterial community structure in a certain period of time. In addition, it was worth noting that we only studied the structural diversity of soil bacterial communities, and specific functional genes and related metabolic pathways in bacterial communities of different treatment groups need to be further explored. Moreover, for such complex environment in tailings area, further detailed and long-term research was needed to deeply understand the heavy metals pollution situation and the impact of various environmental factors on the bacterial communities. 5 Conclusions Our results showed that the constructed compound bacterial flora had significant effect after inoculation with different treatment groups, especially in the treatment group of combined remediation of compound bacterial flora and Peganum harmala . At the same time, we suggest that if we want to solve the problem of multiple heavy metals contaminated soil or tailings soil in a shorter time under the condition of resources, it is a good choice to add exogenous pollution-free yellow cinnamon soil, because our results showed that HMP treatment group had the most significant remediation effect in the same time. We removed most of the existing laboratory level research to outfield for site remediation test, obtained some valuable data and these data would provide a reference for researchers who conduct subsequent outfield tests. Declarations Acknowledgements We are grateful to the National Natural Science Foundation of China (Grant No. 31860163); Key Research and Development Projects of Gansu Province (Grant No. 20YF3NA018); Technology Departmen Youth Science Project of Gansu Provincial Science (Grant No.20JR10RA255. We appreciate the technical support from Shanghai Majorbio Bio-pharm Technology Co., Ltd. Ethical Approval and Consent to Participate The manuscript does not involve human or animal experiments. Consent to publish The Author confirms: · that the work described has not been published before; · that it is not under consideration for publication elsewhere; · that its publication has been approved by all co-authors, if any; · that its publication has been approved (tacitly or explicitly) by the responsible authorities at the institution where the work is carried out. The Author agrees to publication in the Journal indicated below and also to publication of the article in English by Springer in Springer’s corresponding English-language journal. The copyright to the English-language article is transferred to Springer effective if and when the article is accepted for publication, The author warrants that her contribution is original and that she was full power to make this grant. The author signs for and accepts responsibility for releasing this material on behalf of any and all co-authors. The copyright transfer covers the exclusive right to reproduce and distribute the article, including reprints, translations, photographic reproductions, microform, electronic form (offline, online) or any other reproductions of similar nature. After submission of the agreement signed by the corresponding author, changes of authorship or in the order of the authors listed will not be accepted by Springer. Authors Contributions Statement Huan Wang : Investigation, Formal analysis, Data curation, Writing-original draft. Lingui Xue : Conceptualization, Supervision, Funding acquisition, Writing - review & editing. Yanli Huo : Formal analysis, Data curation. Yecheng Ma : Investigation, Data curation. Jiahui Li : Methodology, Validation. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 31860163); Key Research and Development Projects of Gansu Province (Grant No. 20YF3NA018); Technology Departmen Youth Science Project of Gansu Provincial Science (Grant No.20JR10RA255). Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Availability of data and materials The datasets used and/or analyzed during the current study are available from the author on reasonable request. References Arsalan A. Quresh, Tasneem G. Kazi, Jameel A.Baig, et al (2020) Exposure of heavy metals in coal gangue soil, in and outside the mining area using BCR conventional and vortex assisted and single step extraction methods. Impact on orchard grass. Chemosphere 255-126960. https://doi.org/10.1016/j.chemosphere.2020.126960. Adewole T. Adetunji, Francis B. 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Sha Li, Juanli Wu, Yanli Huo et al (2021) Profiling multiple heavy metal contamination and bacterial communities surrounding an iron tailing pond in Northwest China. Science of the Total Environment 752-141827. https://doi.org/10.1016/j.scitotenv.2020.141827. Sibanda, T., Selvarajan, R., Msagati, T., Venkatachalam, S., Meddows-Taylor, S., 2019. Defunct gold mine tailings are natural reservoir for unique bacterial communities revealed by high-throughput sequencing analysis. Sci. Total Environ. 650, 2199–2209. https://doi.org/10.1016/j.scitotenv.2018.09.380. Song, J.W., Shen, Q.L., Wang, L., Qiu, G.Y., Shi, J.C., Xu, J.M., Brookes, P.C., Liu, X.M., 2018. Effects of Cd, Cu, Zn and their combined action on microbial biomass and bacterial community structure. Environ. Pollut. 243 (Pt A), 510–518. https://doi.org/10.1016/j.envpol.2018.09.011. Teng Xu, Feng Nan, Xiaofeng Jiang, et al (2020) Effect of soil pH on the transport, fractionation, and oxidation of chromium (III). Ecotoxicology and Environmental Safety 195-110459. https://doi.org/10.1016/j.ecoenv.2020.110459. Tirry, N., Tahri Joutey, N., Sayel, H., Kouchou, A., Bahafid, W., Asri, M., El Ghachtouli, N. (2018) Screening of plant growth promoting traits in heavy metals resistant bacteria: prospects in phytoremediation. Genet. Eng. Biotechnol. 16 (2):613–619. https://dio.org/10.1016/j.jgeb.2018.06.004. Utobo, E.B. ‒ Tewari. (2015) Soil enzymes as bioindicators of soil ecosystem status. Applied Ecology and Environmental Research 13(1): 147-169. https://dio.org/10.15666/aeer/1301_147169. Utobo, E.B., Tewari, L. (2015) Soil enzymes as bioindicators of soil ecosystem status. Applied Ecology and Environment Research 13(1): 147-169. https://doi.org/10.15666/aeer/1301_147169. Wenliang Ju, Lei Liu, Linchuan Fang et al (2019) Impact of co-inoculation with plant-growth-promoting rhizobacteria and rhizobium on the biochemical responses of alfalfa-soil system in copper contaminated soil. Ecotoxicology and Environmental Safety (167)218-226. https://doi.org/10.1016/j.ecoenv.2018.10.016. Wei Wu, Bo Yuan, Penghui Zou, Ruoting Yang and Xiaode Zhou (2022). Distribution characteristics of bacterial communities in photovoltaic industrial parks in Northwest China. Earth and Environmental Science 983-012093. https://dio.org/10.1088/1755-1315/983/1/012093. Xuedan Li, Kemeng Xiao, Hang Ma, et al (2019) Mechanisms into the removal and translocation of cadmium by Oudemansiella radicata in soil. Environmental Science and Pollution Research 26 (7) 6388–6398. https://dio.org/10.1007/s11356-018-4042-3. Xiaoxu Sun, Tianle Kong, Rui Xu et al (2020) Comparative characterization of microbial communities that inhabit arsenic-rich and antimony-rich contaminated sites: Responses to two different contamination conditions. Environmental Pollution 260-114052. https://doi.org/10.1016/j.envpol.2020.114052. Ying Wang, Yao Luo, Guoquan Zeng, et al (2020) Characteristics and in situ remediation effects of heavy metal immobilizing bacteria on cadmium and nickel co-contaminated soil. Ecotoxicology and Environmental Safety 192-110294. https://doi.org/10.1016/j.ecoenv.2020.110294. Yuanqing Chao, Wenshen Liu, Yanmei Chen et al (2016) Structure, Variation, and Co-occurrence of Soil Microbial Communities in Abandoned Sites of a Rare Earth Elements Mine. Environmental Science and Technology 50(21):11481-11490. https://dio.org/10.1021/acs.est.6b02284. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2144977","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":181840819,"identity":"6973c448-70cd-4afe-9e3b-12febe64ae67","order_by":0,"name":"Huan Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACeWbmg4//8Py342dvPkCcFsP2tmQDHhnmZMmeYwlEWnPmjJkEjw0z44YbOQbE6WCckZZsIJHDxgzU+/HGGwY7Od0GAlrYJZIPPjA4w8PH2N672XIOQ7Kx2QFibEnskWBm5jm7TZqH4UDiNkJaGG7kmEkc/GfA2CaR84xILUDvSzbwJDD2AD1EnBZQIBsz8BxIluA5Zmw5x4AIv4CjEqjFzv5488Mbbyrs5AhqQQESPERGDbIWUnWMglEwCkbBiAAAvudCdfa45FcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-0723-6414","institution":"Lanzhou Jiaotong University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Wang","suffix":""},{"id":181840820,"identity":"db8d21a9-3673-42e4-876d-b55e61095755","order_by":1,"name":"Lingui Xue","email":"","orcid":"https://orcid.org/0000-0001-5634-429X","institution":"lanzhou junqu zong yiyuan: Chinese People's Liberation Army Lanzhou General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lingui","middleName":"","lastName":"Xue","suffix":""},{"id":181840821,"identity":"90466a4d-ec17-4352-900d-2e7db04deb6e","order_by":2,"name":"Yanli Huo","email":"","orcid":"","institution":"Lanzhou Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Huo","suffix":""},{"id":181840822,"identity":"633e7c77-ff10-4b87-9331-90b7aec5e8b3","order_by":3,"name":"Yecheng Ma","email":"","orcid":"","institution":"Lanzhou Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yecheng","middleName":"","lastName":"Ma","suffix":""},{"id":181840823,"identity":"ec8a515c-d499-4a7c-920e-8af7ea4e919a","order_by":4,"name":"Jiahui Li","email":"","orcid":"","institution":"Lanzhou Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiahui","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2022-10-08 10:54:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2144977/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2144977/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34232770,"identity":"a4f5aab8-b5d6-4691-ac04-5bc7669fc7bd","added_by":"auto","created_at":"2023-03-14 14:19:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57519,"visible":true,"origin":"","legend":"\u003cp\u003eThe heavy metals removal rate of 8 different ratios of compound bacterial flora under three periods (\u003cstrong\u003eA\u003c/strong\u003e: 12 h; \u003cstrong\u003eB\u003c/strong\u003e: 24 h; \u003cstrong\u003eC\u003c/strong\u003e: 48 h); \u003cstrong\u003eD\u003c/strong\u003e: Determine the optimum pH of the compound bacterial agent; \u003cstrong\u003eE\u003c/strong\u003e: Determine the optimum culture temperature of the compound bacterial agent; \u003cstrong\u003eF\u003c/strong\u003e: Determine the optimum inoculation amount of the compound bacterial agent; \u003cstrong\u003eG\u003c/strong\u003e: Determine the optimum salt concentration of the compound bacterial agent.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2144977/v1/4b1824a20fa0f3320c73e15d.png"},{"id":34236235,"identity":"9919bec6-4721-40d2-b052-503c9b11e543","added_by":"auto","created_at":"2023-03-14 14:43:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54274,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e: Changes of pH of tailings soil in different time periods; \u003cstrong\u003eB\u003c/strong\u003e: Changes of soil WHC (water holding capacity) of tailings in different time periods; \u003cstrong\u003eC\u003c/strong\u003e: Changes of POR (porosity) in different time periods; \u003cstrong\u003eD\u003c/strong\u003e: Changes of soil EC (electric conductivity) of tailings in different time periods.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2144977/v1/a6c671ed80a22b39be577915.png"},{"id":34232769,"identity":"480a8b64-25e1-4412-aedc-64ba91b66fb9","added_by":"auto","created_at":"2023-03-14 14:19:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e: Changes of alkaline phosphatase during repair; \u003cstrong\u003eB\u003c/strong\u003e: Changes of sucrase during repair; \u003cstrong\u003eC\u003c/strong\u003e: Changes of urease during repair.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2144977/v1/cce7f070899cd0f910b05008.png"},{"id":34234695,"identity":"2edfe685-2cd9-4bcb-b5aa-8e8203cfcc17","added_by":"auto","created_at":"2023-03-14 14:35:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":78000,"visible":true,"origin":"","legend":"\u003cp\u003eFlower plot exhibiting the core and unique OTU numbers across all samples. (HM1, HM2, HM3, HM4 represent the samples taken from HM treatment group on 28\u003csup\u003eth\u003c/sup\u003e, 42\u003csup\u003end\u003c/sup\u003e, 56\u003csup\u003eth\u003c/sup\u003e and 84\u003csup\u003eth\u003c/sup\u003e day; HMP1, HMP2, HMP3, HMP4, TM1, TM2, TM3, TM4 and TMP1, TMP2, TMP3, TMP4 were the same as HM.)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2144977/v1/51572aef980a938bf874d4da.png"},{"id":34232775,"identity":"5206ffc6-ed03-497c-a94a-db232ff9e929","added_by":"auto","created_at":"2023-03-14 14:19:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":665676,"visible":true,"origin":"","legend":"\u003cp\u003eSoil bacterial community composition of different groups with relative abundance \u0026gt; 1% at the phylum (\u003cstrong\u003eA\u003c/strong\u003e) and genus (\u003cstrong\u003eB\u003c/strong\u003e) levels.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2144977/v1/c74f786312fa50aaecf8a0bd.png"},{"id":34233867,"identity":"d3ba8150-04bc-4298-a986-a13664c46f04","added_by":"auto","created_at":"2023-03-14 14:27:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":75370,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations of environmental factors and bacterial communities. (\u003cstrong\u003eA\u003c/strong\u003e) Redundancy analysis (RDA) shows the relationship between the environmental variables, bacterial community distribution and all treatment groups at the same time (the 84\u003csup\u003eth\u003c/sup\u003e day). The green arrow stands for the environmental variables Pb, Cd, Mn and the red arrow stands for pH, WHC (water holding capacity), TEMP (soil temperature), EC (electric conductivity) and POR (porosity). The blue arrow stands for the dominant bacterial phyla. (\u003cstrong\u003eB\u003c/strong\u003e) Variance partitioning analysis (VPA) illustrates the individual and combined explanation of the two groups of environmental variables on the bacterial community variations.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2144977/v1/632261594f8fccfd8236d39b.png"},{"id":38328228,"identity":"5e7df8aa-53ac-4d35-ba45-353678e848d0","added_by":"auto","created_at":"2023-06-10 10:11:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1268873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2144977/v1/901d255e-c1f5-46fa-85b3-3aae671bddb5.pdf"},{"id":34238395,"identity":"9c419943-c527-4989-acb5-213d56f661ab","added_by":"auto","created_at":"2023-03-14 14:51:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1716660,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-2144977/v1/1e8382fd5eb0935027516cad.docx"}],"financialInterests":"","formattedTitle":"Dynamic Changes of Soil Parameters and Bacterial Communities during Bioremediation of Multiple Heavy Metals Contaminated Tailings by Compound Bacterial Flora","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe increasing demand for mineral resources has led to large-scale mining, beneficiation and smelting of mine resources all over the country. The development of mineral resources not only brings economic benefits, but also causes serious environmental pollution. High concentrations of heavy metals in water or soil environments, affect human health and safety (Mengbo Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Tailings are substances that can be excavated again after the extraction and beneficiation of ores in the mining industry (Hai-Hong Gu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), but tailings often contain a variety of high concentrations of heavy metals (Hanno Matthaei et al., 2020; Sha Li et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). With the circulation of the biological chain, a large number of tailings are accumulated in the iron ore reservoir of Linze County, Zhangye City, Gansu Province, in which the contents of Pb, Cd and Mn seriously exceed the local soil background value, and these heavy metals are very easy to migrate and cause pollution of the surrounding environment and harm to human health. Studies have shown that long-term exposure to lead and cadmium will cause acute or chronic damage to the human nervous system, renal insufficiency and respiratory system (Paul B. Tchounwou et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For years, people have been looking for ways to get rid of heavy metal pollution in the environment that are both safe and long-lasting. The research showed that heavy metal pollution is hard to get rid of because it stays in the environment and is toxic to organisms (Siallelli et al., 2011; Fan Du et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Liu et al., 2018).\u003c/p\u003e \u003cp\u003eMany researchers have favored bioremediation technology in the past few years because of its eco-friendliness and cost-effectiveness. To remediate heavy metal-contaminated soil, bioremediation uses bacteria and/or plants (Biao Song et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Many studies have shown that the combined repair technology of compound bacterial flora and plants can shorten the repair time and improve the repair effect, which is significantly better than that of inoculating only single strain or compound bacterial flora or plants (Hassan Etesami, 2018; Akhtar et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Arora et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Utobo, E.B. et al., 2015). After the compound bacterial flora is inoculated into the soil polluted by heavy metals, they can not only interact with heavy metals, but also stimulate the adjustment of soil bacterial community structure, promote plants growth and development, further absorb heavy metals in the soil and repair the soil polluted by heavy metals more effectively. However, so far, most studies have stayed at the laboratory level, and tailings are large and open areas. The impact of environmental factors on their remediation technology cannot be solved in laboratory research, and more practical remediation data cannot be obtained.\u003c/p\u003e \u003cp\u003eSince the relevant personnel of our laboratory have screened out the strains with high tolerance to multiple heavy metals and the hyperaccumulation plant suitable for the growth of this region (\u003cem\u003ePeganum harmala\u003c/em\u003e) in the preliminary study, the purpose of this test is to (a) construct the best ratio of compound bacterial flora and apply it to the outdoor site remediation test to evaluate its impact on the parameters of tailings soil; (b) explore the changes of the microbial diversity and community structure of tailings soil in different periods after inoculation with compound bacterial flora. The test applies laboratory level research to outdoor site remediation, in order to provide more practical reference data for remediation of multiple heavy metals contaminated soil in Northwest China.\u003c/p\u003e"},{"header":"2 Materials And Method","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Reagents and metal standard solutions\u003c/h2\u003e\n \u003cp\u003eNaCl, yeast extract, tryptone, and agar used for cell culture were purchased from Sinopharm Chemical Reagent Co., Ltd and Guangdong Huankai Microbial Technology Co., Ltd, respectively. The following chemicals were obtained from Tianjin Damao Chemical Reagent Factory: urea, methylbenzene, phenol, methanol, acetone, citric acid, ammonium sulphate, disodium phenyl phosphate, borax, 4-aminoantipyrine, K [Fe (CN)], glucose, 3,5-Dinitrosalicylic acid, and potassium sodium tartrate. Cd (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO, Pb (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2,\u003c/sub\u003e and MnCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO used for metal standard solutions were separately dissolved in sterile water supplied by Sinopharm Chemical Reagent Co., Ltd. All solutions were reserved in the dark at 4\u0026deg;C. The chemical reagents used in this experiment were analytical grade.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Selection and characterization of metal-tolerant strains\u003c/h2\u003e\n \u003cp\u003eAccording to the types of excessive heavy metals in the tail mining area, the strains used in this experiment were based on our previous studies which were C1-h, C4-h, C8-h, P2-h, P3-h, M1-h, M3-h (I renumbered them for memory) (Lu Zhang et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Cadmium-tolerant strains were C1-h, C4-h, and C8-h; lead-tolerant strains were P2-h and P3-h; and manganese-tolerant strains were M1-h and M3-h. Relevant personnel in the laboratory had identified the seven strains in the previous study (Lu Zhang et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sha Li et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Each strain belongs to the following genus: C1-h (\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e), C4-h (\u003cem\u003eBacillus\u003c/em\u003e), C8-h (\u003cem\u003ePseudomonas\u003c/em\u003e), M1-h (\u003cem\u003eBacillus\u003c/em\u003e), M3-h (\u003cem\u003eBacillus pumilus\u003c/em\u003e), P2-h (\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e) and P3-h (\u003cem\u003eEnterobacter Xiangfang\u003c/em\u003e). The Agar Diffusion Method (Parisot H J., 2008) was used to observe whether there was mutual antagonism between them.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3 Selection of ratios and action conditions by growing cells\u003c/h2\u003e\n \u003cp\u003eThe strains were cultured in LB liquid media at 150 rpm and 30℃ for 12 h and harvested, respectively. Take 5 ml culture solution of each strain and centrifugate them at 6000 r/min for 5 minutes, the supernatant was discarded, the cells were collected and washed them with sterile water for 2\u0026thinsp;~\u0026thinsp;3 times. Finally, resuspended the cells with sterile water and adjusted the optical density (OD) to about 1.0 at 600 nm. The prepared cells with eight different ratios were inoculated into 100 mL of liquid sterile growth media containing 15 mg/L Cd (II), 2500 mg/L Pb (II) and 1000 mg/L Mn (II), and an inoculum size of 2% (v/v) was used. Then the removal efficiency of heavy metals at 12 h, 24 h, and 48 h were measured to determine the best ratio. Eight different ratios were determined according to the growth curve of each strain (the growth curve of each strain was basically the same), which were 1) 1:1:1:1:1:1:1; 2) 2:1:1:1:1:1:1; 3) 1:2:1:1:1:1:1; 4) 1:1:2:1:1:1:1; 5) 1:1:1:2:1:1:1; 6) 1:1:1:1:2:1:1; 7) 1:1:1:1:1:2:1 and 8) 1:1:1:1:1:1:2. The content of heavy metals in the supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent Technologies Co. Ltd., ICP-MS 7900). Set three parallels and the media without inoculating cells and only adding metal were used as the blank control.\u003c/p\u003e\n \u003cp\u003eAfter determining the best ratio, the prepared cells were inoculated into the media containing 15 mg/L Cd (II), 2500 mg/L Pb (II) and 1000 mg/L Mn (II) under different conditions in a ratio of 1:1:1:1:1:1:1 (the best ratio), cultured for 12 h, and the supernatant was taken after centrifugation to determine the heavy metals, so as to explore the optimal action conditions of compound bacterial flora. Different action conditions include different gradients of pH, temperature, salt concentration and inoculation amount (Table S1). Similarly, set three parallel and the media in which the prepared cells were not inoculated but only the metals were added were used as the blank control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 Outfield site test\u003c/h2\u003e\n \u003cp\u003eThe tailings soil was taken from the iron ore reservoir in Pingchuan Town, Linze County, Zhangye City, Gansu Province, China (100\u003csup\u003eo\u003c/sup\u003e27\u0026rsquo;21\u0026rdquo; E, 38\u003csup\u003eo\u003c/sup\u003e56\u0026rsquo;11\u0026rdquo; N). The soil samples were obtained after screening, and their main characteristics were as follows: pH 7.73, WHC (water holding capacity) 6.28%, EC (conductivity) 772.449 us/cm, POR (porosity) 28.91%, Pb 2194 mg/kg, Cd 11.8 mg/kg, Mn 989 mg/kg (heavy metal content was higher than soil background value in Gansu Province (Pb 20.83 mg/kg, Cd 0.13 mg/kg, Mn 769.6 mg/kg)) (Qiong Liang et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). The tailings soil samples were taken back to the laboratory and then separately packed into round cloth bags (diameter \u0026times; height: 30 cm\u0026times;25 cm), and each bag containing 14 kg soil samples for disposal. The liquid compound bacterial flora with the best ratio above were cultured under the optimal conditions for 12 h, then the proportion of 15% (Lu Zhang et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) sterilized bran were added to make them become semi-solid compound bacterial flora with the moisture content of 60%~70%. Bran played a role in loosening soil and providing nutrients for microorganisms. The semi-solid compound bacterial flora was shaken at 30℃ for 1 h to make them mixed evenly, and then were inoculated into different treatment groups. The large and plump seeds of \u003cem\u003ePeganum harmala\u003c/em\u003e were selected and washed with sterile water for 3\u0026thinsp;~\u0026thinsp;5 times for standby. The treatment groups for outfield site test were set as follows: (a) TCK (CK, total tailings soil, no-inoculated compound bacterial flora and no-inoculated seeds); (b) HCK (CK, added yellow cinnamon soil, no-inoculated compound bacterial flora and no-inoculated seeds); (c) TM (total tailings soil, inoculated compound bacterial flora but no-inoculated seeds); (d) TMP (total tailings soil, inoculated compound bacterial flora and inoculated seeds); (e) HM (add yellow cinnamon soil, inoculated compound bacterial flora but no-inoculated seeds); (f) HMP (add yellow cinnamon soil, inoculated compound bacterial flora and inoculated seeds). Each group was set up with three parallels. For the above groups added with yellow cinnamon soil (total tailings soil: yellow cinnamon soil\u0026thinsp;=\u0026thinsp;3:1), this can dilute heavy metals in the tailings soil and also simulate the soil type heavily polluted by multiple heavy metals, which was conducive to the growth of compound bacterial flora and plants. This was also studied by relevant personnel in the early stage of the laboratory. The compound bacterial flora weas inoculated into the corresponding groups of cloth bags at 20 g/m\u003csup\u003e2\u003c/sup\u003e (Hao Xu et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) and the seeds inoculated in the cloth bags of the above-mentioned corresponding groups (50 seeds/bag). Then choose a wider outfield to place all the cloth bags. After the plants grow, 20 plants with similar growth would be left for follow-up study.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5 Analysis of soil parameters and metals content\u003c/h2\u003e\n \u003cp\u003eThe whole outfield site test was 84 days. Soil samples were taken every two weeks to analyze their physical and chemical properties, including enzyme activities (sucrase, urease, alkaline phosphatase), soil (TMP) temperature, pH, (WHC) water holding capacity, (POR) porosity and (EC) electrical conductivity. The activities of urease, alkaline phosphatase and sucrase were determined according to the methods of R.P. Dick et al., \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e, Xuedan Li et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e and Arsalan A. Quresh et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e. The (POR) porosity was measured by the bulk density specific gravity method, and the (EC) conductivity was measured by conductivity meter (Shanghai Leici dds-307, DDs-307A). The removal rate of heavy metals was calculated by measuring the total content of heavy metals before and after remediation by ICP-MS. Pretreatment of soil samples: weighed 0.1g of soil samples, added 5ml aqua regia, and conducted microwave digestion. After digestion, drove the acid to viscous state at 140℃, fixed the volume to 25ml, and tested on the machine (ICP-MS).\u003c/p\u003e\n \u003cp\u003eOver the whole restoration, plant samples were collected to determine the enrichment factor. After drying the plant samples at 85℃, grinded and screened them, determined the content of heavy metals in the plants, and calculated the enrichment coefficient of heavy metals. Samples pretreatment method: weighed 0.5 g plant samples, added 5ml nitric acid and 1ml hydrogen peroxide, placed it overnight for microwave digestion. After digestion, drove the acid to 1 ml at 140℃, fixed the volume to 25 ml, and tested on the machine (ICP-MS).\u003c/p\u003e\n \u003cdiv class=\"Equation\" id=\"Equa\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\text{H}\\text{e}\\text{a}\\text{v}\\text{y} \\text{m}\\text{e}\\text{t}\\text{a}\\text{l} \\text{e}\\text{n}\\text{r}\\text{i}\\text{c}\\text{h}\\text{m}\\text{e}\\text{n}\\text{t} \\text{f}\\text{a}\\text{c}\\text{t}\\text{o}\\text{r}=\\frac{\\begin{array}{c}\\\\ Heavy metal content in plants\\end{array}}{\\text{H}\\text{e}\\text{a}\\text{v}\\text{y} \\text{m}\\text{e}\\text{t}\\text{a}\\text{l} \\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t} \\text{i}\\text{n} \\text{s}\\text{o}\\text{i}\\text{l}}\\times 100\\%$$\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6 Analysis of microbial diversity and community structure\u003c/h2\u003e\n \u003cp\u003eThe soil samples taken on the 28th, 42nd, 56th and 84th days of the repair process were stored in the refrigerator at -80℃ for subsequent delivery to the company (Shanghai Majorbio Bio-pharm Technology Co., Ltd) for high-throughput sequencing. The obtained data were used to analyze the changes in microbial diversity and community structure during the restoration process.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.7 Data analysis\u003c/h2\u003e\n \u003cp\u003eAll experiments were performed in triplicate. The experimental data were statistically analyzed in Microsoft Excel 2010 and SPSS 25.0, using Duncan\u0026apos;s multiple comparative analysis method, and visualized in Origin 2019. The high-throughput sequencing data were analyzed on the free online platform of Majorbio Cloud Platform (majorbio.com).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e3.1 Construction of compound bacterial flora\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec12\"\u003e\n \u003ch2\u003e3.1.1 Strains characteristics and antagonistic experiment\u003c/h2\u003e\n \u003cp\u003eThe characteristics of the 7 strains were shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. It can be seen from the table that \u003cem\u003eBacillus\u003c/em\u003e has the highest proportion of them.\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eresistance to major HMs*\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003esearch number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e16SrRNA identification\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCharacteristics of seven heavy metals tolerant strains.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC1-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMT973991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC4-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMW812244\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus sp.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC8-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMT967291\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas fluorescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM1-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMW652627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus altitudinis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM3-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMW485109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus pumilus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP2-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMT974007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP3-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMT994615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter sp.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eColony morphology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC1-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eYellow round colony, small and opaque, moist and smooth surface, raised center and neat edge, easy to provoke\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC4-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eYellow round colony, large colony, convex center, smooth surface, neat edge, easy to provoke\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC8-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eThe colony center is orange yellow, the colony is large, the surface is wet and smooth, the edge is neat, and it is easy to provoke\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM1-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eWhite round colony, convex center, wet and smooth surface, neat edge, easy to provoke\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM3-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eWhite round colony, convex center, wet and smooth surface, neat edge, easy to provoke\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP2-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eOff white colony, round, moist surface, neat edge, easy to provoke\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP3-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eYellow round colony, smooth surface, neat edge, easy to provoke\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e*HMs: heavy metals. Previous studies had shown that the 7 strains were resistant to variety of other heavy metals in addition to above major heavy metals.\u003c/p\u003e\n \u003cp\u003eTo ensure the best effect of the compound bacterial flora constructed subsequently, the antagonistic experiment was carried out on 7 strains in pairs (Fig. S1). If there was an antagonistic effect between strains, it would affect the effect of the compound bacterial flora. It can be seen from the figure that there was no bacteriostatic circle between the seven selected heavy metals tolerant strains, so there was no antagonism. Therefore, follow-up experiments can be continued.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFig S1.\u003c/strong\u003e The antagonism among the seven strains was from left to right in each row: 1)C1-h\u0026thinsp;+\u0026thinsp;C4-h、C1-h\u0026thinsp;+\u0026thinsp;C8-h、C1-h\u0026thinsp;+\u0026thinsp;M1-h、C1-h\u0026thinsp;+\u0026thinsp;M3-h、C1-h\u0026thinsp;+\u0026thinsp;P2-h、C1-h\u0026thinsp;+\u0026thinsp;P3-h、P2-h\u0026thinsp;+\u0026thinsp;C8-h; 2) C4-h\u0026thinsp;+\u0026thinsp;C8-h、C4-h\u0026thinsp;+\u0026thinsp;M1-h、C4-h\u0026thinsp;+\u0026thinsp;M3-h、C4-h\u0026thinsp;+\u0026thinsp;P2-h、C4-h\u0026thinsp;+\u0026thinsp;P3-h、M3-h\u0026thinsp;+\u0026thinsp;P2-h、M3-h\u0026thinsp;+\u0026thinsp;P3-h; 3) C8-h\u0026thinsp;+\u0026thinsp;M1-h、C8-h\u0026thinsp;+\u0026thinsp;M3-h、C8-h\u0026thinsp;+\u0026thinsp;P3-h、M1-h\u0026thinsp;+\u0026thinsp;M3-h、M1-h\u0026thinsp;+\u0026thinsp;P2-h、M1-h\u0026thinsp;+\u0026thinsp;P3-h、P2-h\u0026thinsp;+\u0026thinsp;P3-h.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec13\"\u003e\n \u003ch2\u003e3.1.2 Determination of optimum ratio and action conditions\u003c/h2\u003e\n \u003cp\u003eTo replicate the environment polluted by heavy metals in tailings, 15 mg/L Cd, 2500 mg/L Pb and 1000 mg/L Mn were added to LB medium. Compound bacterial flora were inoculated at the above 8 different ratios, cultured at 30 ℃ and 120 r/min, and then the heavy metals removal rate of 12 h, 24 h and 48 h were measured. The results showed that the removal rate of heavy metals in 12 h was higher than that in 24 h and 48 h. At 12 h, the removal rate of heavy metals in the compound bacterial agent with a ratio of 1:1:1:1:1:1:1 was higher than that in the other seven ratios (Fig.\u0026nbsp;1ABC; Table S2). The removal rates of Mn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e and Pb\u003csup\u003e2+\u003c/sup\u003e were 55.3%, 65.1% and 78.4% respectively. Therefore, the best ratio of compound bacterial agent was 1:1:1:1:1:1:1, and the removal rate of heavy metals under 12 h was selected in the subsequent optimization experiment of compound bacterial flora action conditions.\u003c/p\u003e\n \u003cp\u003eDifferent pH (5, 6, 7, 8, and 9), temperature (25\u0026deg;C, 28\u0026deg;C, 30\u0026deg;C, 32\u0026deg;C, and 35\u0026deg;C), inoculation amount (1%, 2%, 3%, 4%, and 5%) and salt concentration (1%, 2%, 3%, 4%, and 5%) gradients were used to investigate the best conditions. From the experimental data, the results are as follows: (1) Compared with other pH values, the removal rate of heavy metals by compound bacterial flora at pH\u0026thinsp;=\u0026thinsp;7 were higher, and the removal rate of Mn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e and Pb\u003csup\u003e2+\u003c/sup\u003e were 47.5%, 50.2% and 91.95% respectively; (2) When compared to other temperatures, the removal rate of heavy metals were higher at 28\u0026deg;C, with removal rate of Mn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e and Pb\u003csup\u003e2+\u003c/sup\u003e being 56.6%, 78.7% and 98.2%, respectively; (3) When the inoculation amount of compound bacterial flora was 3%, the removal rate of heavy metals was higher, with removal rate of Mn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e and Pb\u003csup\u003e2+\u003c/sup\u003e being 59.2%, 67.3% and 91.6%, respectively; (4) When the salt concentration was 3%, the removal rate of heavy metals was higher, with removal rate of Mn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e and Pb\u003csup\u003e2+\u003c/sup\u003e being 56.7%, 73% and 89.5%, respectively. To summarize, the best conditions for compound bacterial flora action were pH\u0026thinsp;=\u0026thinsp;7, culture temperature of 28\u0026deg;C, inoculation amount of 3% and salt concentration of 3% (Fig. 1DEFG).\u003c/p\u003e\n \u003cp\u003eUnder heavy metals stress, the compound bacterial flora with the best ratio was cultured under optimal conditions, and the removal rate of heavy metals were measured. The removal rate of Mn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e and Pb\u003csup\u003e2+\u003c/sup\u003e could be as high as 60.7%, 71.6% and 90.5% respectively (Table S3).\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.2 Evaluation of outfield site test repair effect\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.2.1 Changes in physical and chemical properties of tailings soil during remediation\u003c/h2\u003e\n \u003cp\u003eTo monitor the changes of soil physical and chemical properties during the remediation process, pH, WHC (water holding capacity), POR (porosity), EC (electric conductivity) were measured. The pH of tailings soil at the sampling point was alkaline, the pH of control group TCK is 7.74, and the pH of control group HCK is 7.24. In addition, compared with the control groups, during the repair process, the pH of different treatment groups gradually decreased and tended to be neutral (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), especially in the treatment group of HMP. The pH of all T treatment groups decreased from 7.74 (TCK) to 7.18 (TM) and 7.11 (TMP) after 84 days, and the pH of all H treatment groups decreased from 7.24 (HCK) to 7.08 (HM) and 7.02 (HMP). The ability of plant-microbial combination technology to improve soil pH was higher than that only inoculated compound bacterial flora.\u003c/p\u003e\n \u003cp\u003eCompared with the control groups, the soil WHC (water holding capacity) of tailings in different treatment groups increased (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Whether inoculated with flora or combined technology, there was little difference in the change in WHC between them, but in general, the WHC showed an upward trend after inoculation of compound bacterial flora with the increase of repair time.\u003c/p\u003e\n \u003cp\u003eDuring the repair process, the POR (porosity) of the treatment groups inoculated with only compound bacterial flora and the control groups were measured. The results showed that with the increase of repair time, the POR of different treatment groups increased, and the treatment effect of HM was particularly obvious (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). At 84 days, the POR of HM and TM were 49.54% and 41.91% respectively, which increased by 13.69% and 8.53% respectively compared with the controls (HCK and TCK).\u003c/p\u003e\n \u003cp\u003eIn the process of remediation, the changes of soil EC (electric conductivity) in different treatment groups fluctuated, but on the whole, the soil conductivity increased first and then decreased with the increase of remediation time (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD), and the EC of the treatment groups inoculated with both compound bacterial flora and \u003cem\u003ePeganum harmala\u003c/em\u003e fluctuated significantly compared with the treatment groups inoculated with only compound bacterial flora, with an overall decrease of 21.15% ~ 70.49%. Compared with the 0-day controls, at 84 days of repair, the EC of HM, TM, HMP and TMP were 307 us/cm, 611 us/cm, 228 us/cm and 265 us/cm respectively. The decrease of EC in the treatment groups may related to the temperature changes and available state of heavy metals. These above results indicated that the physical and chemical properties of soil were different with the increase of remediation time in different treatment groups, and the soil improvement effects were also different.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.2.2 Changes of enzyme activities in tailings soil during remediation\u003c/h2\u003e\n \u003cp\u003eTo further monitor the soil quality improvement effect of different treatment groups during the restoration process, different treatment groups soil samples were taken every 14 days to determine the enzyme activities of alkaline phosphatase, sucrase and urease (Fig.\u0026nbsp;3ABC). The three enzyme activities of TCK and HCK were almost unchanged during the remediation process, while the enzyme activities of all H treatment groups (HM, HMP) were higher than those of the corresponding T treatment groups (TM, TMP). Compared with the controls (TCK and HCK), the activities of alkaline phosphatase, sucrase and urease in the four experimental groups increased significantly on the 14th day. From 14 to 42 days, the activity of alkaline phosphatase changed little and began to increase gradually after 42 days. Compared with the controls (TCK and HCK), the alkaline phosphatase activities of all treatment groups at 84 days were HMP (75.6 \u0026micro;g/g)\u0026thinsp;\u0026gt;\u0026thinsp;HM (44.6 \u0026micro;g/g)\u0026thinsp;\u0026gt;\u0026thinsp;TMP (43.8 \u0026micro;g/g)\u0026thinsp;\u0026gt;\u0026thinsp;TM (33.4 \u0026micro;g/g).\u003c/p\u003e\n \u003cp\u003eCompared with the control groups, the activity of sucrase increased at 14 days. After 42 days, the enzyme activity of all T treatment groups increased slowly, and the enzyme activity of the corresponding H treatment groups increased rapidly. At 84 days, the sucrase activities of all treatment groups were HMP (35.6 mg/g)\u0026thinsp;\u0026gt;\u0026thinsp;HM (34.6 mg/g)\u0026thinsp;\u0026gt;\u0026thinsp;TMP (26.7 mg/g)\u0026thinsp;\u0026gt;\u0026thinsp;TM (25.5 mg/g).\u003c/p\u003e\n \u003cp\u003eCompared with alkaline phosphatase and sucrase, urease activity fluctuated greatly in the repair process. The enzyme activity of different treatment groups had a trend of \u0026ldquo;first rising, then falling and then rising again\u0026rdquo;, but the repair time points corresponding to their decline and rise were different. Compared with the control groups, the urease activity of all treatment groups showed an upward trend as a whole. At 84 days, the urease activities of all treatment groups were HM (0.35 mg/g)\u0026thinsp;\u0026gt;\u0026thinsp;HMP (0.34 mg/g)\u0026thinsp;\u0026gt;\u0026thinsp;TMP (0.32 mg/g)\u0026thinsp;\u0026gt;\u0026thinsp;TM (0.3 mg/g). From above results, it can be seen that the inoculation of compound bacterial flora had significant impact on enzyme activities.\u003c/p\u003e\n \u003cp\u003e3.2.3 Removal rate of heavy metals in soil and enrichment coefficient of heavy metals in \u003cem\u003ePeganum harmala\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eThe heavy metals content of TCK before remediation and each treatment group\u0026rsquo;s soil heavy metals content after remediation were measured to evaluate the heavy metals removal rate of different treatment groups in the whole remediation process (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). It can be seen from the Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e that the repair effect of all H treatment groups was better than that of all T treatment groups, and the repair effect of only inoculating compound bacterial flora was better, while the microbial-plant joint repair effect was the best, and the removal rate of Pb, Cd and Mn were 54.74%, 57.46% and 24.97% respectively.\u003c/p\u003e\n \u003cp\u003eThe plant heavy metal enrichment coefficients (Table S4) of TMP and HMP treatment groups were measured. From the data in the table, it can be seen that \u003cem\u003ePeganum harmala\u003c/em\u003e has the best enrichment effect on Mn.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e3.3 Analysis of microbial diversity and community structure\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003cdiv class=\"Section3\" id=\"Sec18\"\u003e\n \u003ch2\u003e3.3.1 Summary of sequences and annotation of taxa\u003c/h2\u003e\n \u003cp\u003eA total of 2,360,069 high-quality reads were obtained from 52 samples through Illumina MiSeq sequencing after data filtering (Table S5). Rarefaction curves showed that Shannon diversity index at the OTU level tended to be constant with the increase of sequence number, and sequencing almost reached saturation state (Fig. S2). A total of 5558 operational taxonomic units (OTUs) were obtained by cluster analysis at a 97% similarity level (Table S5), and were classed into 39 phyla, 124 classes, 285 orders, 485 families, 951 genera and 2019 species. Also, 97.4%, 93.5%, 92.3%, 90.1%, 87.6% and 72.6% of OTUs could be precisely annotated at the levels of phylum, class, order, family, genus and species, respectively. For the relative abundance of OTUs, Proteobacteria was uppermost (42.7%) at the phylum level, followed by Actinobacteriota (21.7%), Bacteroidota (16.4%), Chloroflexi (4.5%), Acidobacteriota (4.2%), Firmicutes (2.9%), Gemmatimonadota (2.1%), Myxococcota (1.9%) and Verrucomicrobiota (1.4%). The 3 dominant phyla (relative abundance\u0026thinsp;\u0026gt;\u0026thinsp;5%) accounted for 80.8% of the total OTUs and the top 9 phyla made up 97.8% of all bacterial communities.\u003c/p\u003e\n \u003cp\u003eFigureS2. Rarefaction curves.\u003c/p\u003e\n \u003cp\u003eIn order to explore adaptation characteristics of bacteria to the tailings soil habitats in different treatment groups in this study, we specifically analyzed the core OTUs. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, there were a total of 43 core OTUs (6.1%) in all samples, which were defined as the core microbiome, while the numbers of different OTUs in each sample were considered to be the unique microbiomes. At the phylum level, the predominant members (relative abundance\u0026thinsp;\u0026gt;\u0026thinsp;3%) of 303 core OTUs belonged to Proteobacteria (44.9%), Actinobacteriota (21.7%), Bacteroidota (17.9%), Chloroflexi (4.3%) and Acidobacteriota (4.1%), among which OTU4751 (affiliated to genus \u003cem\u003eSphingobacterium\u003c/em\u003e) was the most abundant (20.2%) followed by OTU2712 (8.5%), OTU2463 (6.1%), OTU3470 (5.9%), OTU2358 (4.6%) and OTU2378 (4.1%), affiliated to genus \u003cem\u003eArthrobacter\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003eBrevundimonas\u003c/em\u003e, unclassified Rhizobiaceae and \u003cem\u003eBlastococcus\u003c/em\u003e, respectively. It can be seen from the figure that these core OTUs had existed throughout the remediation process and were also the core OTUs of TCK. Therefore, these core OTUs represent the bacterial communities commonly existing in the iron tailings reservoir area and different treatment groups, and maybe we can call them \u0026ldquo;indigenous microbiome\u0026rdquo;. These \u0026ldquo;indigenous microbiome\u0026rdquo; may have an impact on the compound bacterial flora that was subsequently inoculated.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec19\"\u003e\n \u003ch2\u003e3.3.2 Soil bacterial community diversity and composition\u003c/h2\u003e\n \u003cp\u003eTo characterize the soil bacterial community structure of different treatment groups, we conducted diversity analyses. The \u0026alpha; (alpha) diversity index of different treatment groups were used for statistics (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), including the richness index (Chao index and ACE index) and diversity index (Shannon index and Simpson index). Compared with all T treatment groups, the bacterial richness index and diversity index of all H treatment groups were improved. The ACE index of all T treatment groups was between 368.39\u0026thinsp;~\u0026thinsp;633.18, Shannon index was between 0.95\u0026thinsp;~\u0026thinsp;4.28, and Simpson index was between 0.028\u0026thinsp;~\u0026thinsp;0.627. And the ACE index was between 2811.34\u0026thinsp;~\u0026thinsp;3577.25, Shannon index was between 5.74\u0026thinsp;~\u0026thinsp;6.76, and Simpson index was between 0.003\u0026thinsp;~\u0026thinsp;0.013 of all H treatment groups. Whether T treatment groups or H treatment groups, the treatment groups inoculated compound bacterial flora showed higher \u0026alpha; diversity for soil bacterial communities compared with control treatment groups (TCK and HCK), especially the treatment groups combined with compound bacterial flora and \u003cem\u003epeganum harmala\u003c/em\u003e. The Chao index, Shannon index, Simpson index and ACE index showed that the richness and diversity of the treatment groups added with compound bacterial flora and the treatment groups with combined microbial-plant repair were significantly higher than those of the control groups (\u003cem\u003ep\u003c/em\u003e＜0.05), and their richness index and diversity index were also increasing with the increase in repair time. These results indicated that the inoculation of compound bacterial flora and \u003cem\u003epeganum harmala\u003c/em\u003e significantly affected the richness and diversity of soil bacterial communities in different treatment groups.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab8\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe Chao index, Shannon index, Simpson index and ACE index of different treatment groups.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup H\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChao\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eShannon\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSimpson\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eACE\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2806.68\u0026thinsp;\u0026plusmn;\u0026thinsp;212.98d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.013\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2942.58\u0026thinsp;\u0026plusmn;\u0026thinsp;438.56bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2845.32\u0026thinsp;\u0026plusmn;\u0026thinsp;71.08d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.008\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2811.34\u0026thinsp;\u0026plusmn;\u0026thinsp;75.34c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3029.69\u0026thinsp;\u0026plusmn;\u0026thinsp;45.06bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3016.42\u0026thinsp;\u0026plusmn;\u0026thinsp;33.37bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2908.67\u0026thinsp;\u0026plusmn;\u0026thinsp;81.17cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2897.05\u0026thinsp;\u0026plusmn;\u0026thinsp;115.14bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3102.09\u0026thinsp;\u0026plusmn;\u0026thinsp;157.33bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3062.57\u0026thinsp;\u0026plusmn;\u0026thinsp;118.80bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHMP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3189.47\u0026thinsp;\u0026plusmn;\u0026thinsp;38.43b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0001b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3133.89\u0026thinsp;\u0026plusmn;\u0026thinsp;47.81bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHMP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3177.37\u0026thinsp;\u0026plusmn;\u0026thinsp;168.47b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0004b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3170.27\u0026thinsp;\u0026plusmn;\u0026thinsp;196.29b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHMP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3238.94\u0026thinsp;\u0026plusmn;\u0026thinsp;128.07b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0006bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3216.63\u0026thinsp;\u0026plusmn;\u0026thinsp;59.18b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHMP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3571.99\u0026thinsp;\u0026plusmn;\u0026thinsp;77.31a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0001c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3577.25\u0026thinsp;\u0026plusmn;\u0026thinsp;53.06a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup T\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e444.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e475.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e400.84\u0026thinsp;\u0026plusmn;\u0026thinsp;36.75b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.055\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e409.75\u0026thinsp;\u0026plusmn;\u0026thinsp;21.22b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e398.91\u0026thinsp;\u0026plusmn;\u0026thinsp;19.80b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.045\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e397.73\u0026thinsp;\u0026plusmn;\u0026thinsp;28.88b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e476.53\u0026thinsp;\u0026plusmn;\u0026thinsp;54.82ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.034\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e479.65\u0026thinsp;\u0026plusmn;\u0026thinsp;44.04ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTM4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e505.65\u0026thinsp;\u0026plusmn;\u0026thinsp;98.75ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.028\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e522.33\u0026thinsp;\u0026plusmn;\u0026thinsp;137.83ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTMP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e363.88\u0026thinsp;\u0026plusmn;\u0026thinsp;39.53b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.054\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e368.39\u0026thinsp;\u0026plusmn;\u0026thinsp;38.92b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTMP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e418.17\u0026thinsp;\u0026plusmn;\u0026thinsp;62.24b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e441.94\u0026thinsp;\u0026plusmn;\u0026thinsp;86.12ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTMP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e593.48\u0026thinsp;\u0026plusmn;\u0026thinsp;77.93a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24abcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.047\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e633.18\u0026thinsp;\u0026plusmn;\u0026thinsp;92.10a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTMP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e616.23\u0026thinsp;\u0026plusmn;\u0026thinsp;193.4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e612.22\u0026thinsp;\u0026plusmn;\u0026thinsp;213.83a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eData are shown as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (n\u0026thinsp;=\u0026thinsp;3). The different lowercase letters in the same column indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) among samples. TCK was the control of all T treatment groups. HCK was the control of all H treatment groups. The number after each treatment group represented the sampling time. For example, HM1 represented the sample taken from HM treatment group on the 28th day.\u003c/p\u003e\n \u003cp\u003eFor the bacterial community composition, the proportion of Firmicutes and Bacteroidota in all other treatment groups (including HCK) decreased significantly compared with the control TCK, while the proportion of Proteobacteria increased (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, S3A). Interestingly, the proportion of Actinobacteriota in the control TCK and all H treatment groups were higher than T treatment groups (TM, TMP) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, S3A). Furthermore, Chloroflexi at all H treatment groups were significantly more dominant than at all T treatment groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). With the increase of sampling time, the changes of community structure in the same treatment groups were not very obvious, but their relative abundance were different (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). At the genus level, the proportion of \u003cem\u003ePhingobacterium\u003c/em\u003e in all T treatment groups first increased and then decreased with the increase of sampling time, compared with the control TCK, while the proportion in all H treatment groups were extremely low (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, S3B). The proportion of \u003cem\u003ePlanococcus\u003c/em\u003e in control TCK were large but in all other treatment groups were quite low (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, S3B). Compared with all T treatment groups, the bacterial communities of all H treatment groups were more complex and more abundant. In addition, the bacterial community structure after inoculation of compound bacterial flora was more complex and diverse compared with the reference controls (TCK and HCK) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, S3B). These results indicated that the inoculation of compound bacterial flora discharge remarkably shifts the composition of soil bacterial communities.\u003c/p\u003e\n \u003cp\u003eFigureS3. Different proportion of soil bacterial community composition in different groups with relative abundance\u0026thinsp;\u0026gt;\u0026thinsp;5% at the phylum (\u003cstrong\u003eA\u003c/strong\u003e) and genus (\u003cstrong\u003eB\u003c/strong\u003e) levels.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec20\"\u003e\n \u003ch2\u003e3.3.3 Effects of environmental factors on microbial communities\u003c/h2\u003e\n \u003cp\u003eTo determine the relationship among samples, soil bacterial communities and environmental factors, all of the 8 measured environmental variables were conducted to redundancy analysis (RDA). Among these environmental variables, 7 had significant effects on bacterial communities, including pH, WHC (water holding capacity), TEMP (soil temperature), POR (porosity), Pb, Cd, Mn (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table S6). At the phyla, the RDA1 and RDA2 explained 66.06% and 30.05% of the total variances of soil bacterial community composition, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA; Table S7). To further determine the impact of 8 environmental variables on bacterial communities, a variance partitioning analysis (VPA) was performed by dividing the environmental variables into two groups: heavy metals (Pb, Cd, Mn) and soil physical and chemical properties (pH, WHC, TEMP, EC, POR). It can be seen from Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB that 97.17% of the bacterial community variance was explained by the two groups of environmental variables, and the contribution of soil physical and chemical properties (77.34%) to the variance was higher than that of heavy metals (69.29%). The above results indicated that these 8 environmental factors had significant effects on soil communities.\u003c/p\u003e\n \u003cp\u003eFurther, the Spearman correlation Heatmap showed that Pb and Cd significantly negative correlated with phyla Gemmatimonadota, Fibrobacterota, WPS-2, GAL15, Actinobacteriota, MBNT15, Methylomirabilota, Latescibacterota, Elusimicrobiota, Entotheonellaeota, Acidobacteriota, RCP2-54, unclassified_k_ norank_ d_Bacteria, Chloroflexi, Patescibacteria, Nitrospirota, Dependentiae, NB1-j, Planctomycetota and Desulfobacterota (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while Mn only negatively with phyla WPS-2, GAL15, unclassified_k_ norank_ d_Bacteria, Chloroflexi, Patescibacteria (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.S4A; Tables S8-S9).Moreover, there were negative correlations between EC and Spirochaetota (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), pH and Spirochaetota, Verrucomicrobiota, TXA1-33, Hydrogenedentes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), TEMP and Spirochaetota (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.S4A; Tables S8-S9). In addition, the POR significantly positive correlated with phyla Gemmatimonadota, Fibrobacterota, WPS-2, GAL15, Elusimicrobiota, unclassified_k_ norank_ d_Bacteria, Chloroflexi, Patescibacteria, Nitrospirota, Planctomycetota and Desulfobacterota (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.S4A; Tables S8-S9). At the genus level, Pb and Cd contributed to the abundances of \u003cem\u003eSphingobacterium\u003c/em\u003e, \u003cem\u003eRhodopseudomonas\u003c/em\u003e, \u003cem\u003eAlgoriphagus\u003c/em\u003e, \u003cem\u003ePseudoxanthomonas\u003c/em\u003e, \u003cem\u003ePedobacter\u003c/em\u003e and \u003cem\u003eSphingopyxi\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), but significantly negative correlated with the proportions of genus \u003cem\u003enorank_f_Gem\u003c/em\u003e\u003cem\u003ematimonadaceae, Streptomyces\u003c/em\u003e, \u003cem\u003enorank_f__JG30-KF-CM45\u003c/em\u003e, \u003cem\u003enorank_f_Xanthobacteraceae\u003c/em\u003e, \u003cem\u003eNocardioides\u003c/em\u003e, \u003cem\u003eGaiella\u003c/em\u003e, \u003cem\u003enorank_f_Vicinamibacteraceae\u003c/em\u003e, \u003cem\u003eRB41\u003c/em\u003e, \u003cem\u003enorank_f_norank_o_Vicinamibacterales\u003c/em\u003e, \u003cem\u003enorank_ f_ norank_o_norank_c_KD4-96\u003c/em\u003e, \u003cem\u003enorank_f_norank_o_Microtrichales\u003c/em\u003e, \u003cem\u003enorank_f_Ilumatobacteraceae\u003c/em\u003e, \u003cem\u003eSolirubrobacter\u003c/em\u003e and \u003cem\u003enorank_f_67\u0026thinsp;\u0026minus;\u0026thinsp;14\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.S4B; Tables S10-S11). Interestingly, the correlations between Mn and most of phyla or genus were not very significant (Fig.S4A; Tables S8-S9; Fig.S4B; Tables S10-S11). All above results indicated that the bacterial populations of different treatment groups had different responses to environmental variables.\u003c/p\u003e\n \u003cp\u003eFigure S4. The effects of environmental factors (Pb, Cd, POR, WHC, Mn, EC, pH, TEMP) on bacterial community structure.(\u003cstrong\u003eA\u003c/strong\u003e) at the Phylum level; (\u003cstrong\u003eB\u003c/strong\u003e) at the Genus level (* \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eBioremediation of waste tailings was a green and environmentally friendly remediation method with the concept of sustainable development, which mainly includes microbial remediation, phytoremediation and microbial plant combined remediation (Bin Xuan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Current studies had found that some functional groups on the surface of microbial cells can adsorb or precipitate heavy metals (Ying Wang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and microorganisms can also secrete some secondary metabolites, that would interact with heavy metals, such as mineralization (C.-H. Kang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), dissolution of heavy metals by organic acids (N. Papassiopi et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), etc. However, the process of remediation after adding microorganisms to the soil was very complex, and the growth of microorganisms also had a certain period of time, so the fate of microorganisms in the later stage was also difficult to solve. Phytoremediation can remove heavy metals from soil, and plants were easy to collect in the later stage, so they can deal with heavy metals intensively. However, the efficiency of phytoremediation technology was often limited by the high concentration of heavy metals, coexistence of multiple heavy metals and low-level nutrition (Benidir L et al., 2020). In fact, there were few plants around the tailings reservoir area where we collected soil samples, and only a few drought-enduring plants were scattered. Therefore, before planting plants in heavy metals contaminated soil, inoculate microorganisms to reduce the soil toxicity of tailings, promote plants growth, reduce stress and chelate heavy metals (Nida Zainab et al., 2020), and give full play to the effect of combined microbial and plants remediation. In fact, the resistance of soil bacteria to heavy metals and their ability to promote plants growth make them the first choice for phytoremediation of heavy metals contaminated soil in previous studies (Tirry et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It had been found that the inoculated compound bacterial flora resistant to heavy metals could affect the mobility and availability of heavy metals in plants through rhizosphere chelation, acidification and oxidation-reduction reactions, which was conducive to the absorption of heavy metals by plants, thus directly affecting the remediation effect (Ma et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and further illustrated the important role of microorganisms in heavy metal contaminated soil. Therefore, in this study, compound bacterial flora resistant to heavy metals was constructed, and the two bioremediation methods (only inoculated compound bacterial flora and the combination of compound bacterial flora and \u003cem\u003ePeganum harmala\u003c/em\u003e) were set up in different groups for tailings remediation outfield test, so as to obtain more practical remediation schemes and reference data according to the changes of soil parameters and bacterial communities of different treatment groups during bioremediation process.\u003c/p\u003e \u003cp\u003eA major feature of the tailings we studied was multiple heavy metals pollution, which was similar to the previous reports (Nouri and Haddioui, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This also showed that no matter where the iron tailings and soil around the tailings pond were located, multiple heavy metals pollution often existed. Therefore, we selected strains that were resistant to multiple heavy metals to construct compound bacterial flora. Seven strains with no antagonistic effect between two were selected in the test (Fig. S1). The antagonistic effect between strains was that the life activities of one microorganism or its metabolites inhibit or interfere with the life activities of another microorganism, which would make the constructed compound bacterial flora unable to give full play to its efficacy and repair the tailings soil to the greatest extent. In this study, the antagonistic experiment results of seven strains showed that they had no antagonistic effect and could coexist under the same cultural conditions, so they could be used for the construction of subsequent compound bacterial flora. In addition, the ratio among the strains would also affect the effectiveness of compound bacterial flora, this study also explored the best ratio among the seven strains. It can be seen from the follow-up outdoor site remediation test results that the appropriate ratio of compound bacterial flora indeed played an important role in the remediation process of tailings soil.\u003c/p\u003e \u003cp\u003eIn our study, microorganisms played the most important role in different treatment groups, and soil quality determined the composition and distribution of microbial communities. Therefore, we monitored the changes of soil physical and chemical properties and enzyme activities during the whole remediation process. The existence and migration of heavy metals in soil were determined by many environmental factors, among which soil pH and EC (electric conductivity) play important roles. The change in pH would change the solubility of heavy metals in soil (Teng Xu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Alicja Kinska et al., 2022). In particular, the bacteria combined plant technology was adopted in this study, and the change of soil pH would affect the bioavailability of nutrients or heavy metals absorbed by plants in the rhizosphere (E. Oburger et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the process of remediation, the pH of different samples changes from alkaline to neutral. At the end of remediation, the pH of tailings soil of different treatment groups was stable at about 7.1. The neutral environment was conducive to the survival of microorganisms, and relevant studies had found that an appropriate pH can promote microbial activity (Myoung-Jin Kim et al., 2004). At the same time, soil pH was also considered to be the main driving force behind changes in the bacterial community (Xiaoxu Sun et al., 2020). Our results were consistent with previous studies. Redundancy analysis (RDA) and Spearman correlation Heatmap showed that pH was indeed an important environmental variable affecting bacterial communities.\u003c/p\u003e \u003cp\u003eEC can indirectly reflect the concentration of metal ions in soil, while in soil polluted by multiple heavy metals such as tailings, EC can indirectly determine the concentration of heavy metal ions (Kasemodel et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The test results showed that although EC fluctuated with the increase of remediation time, it may be due to the dynamic transformation of different forms of heavy metal ions during the remediation process, which still showed a downward trend as a whole (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating that the addition of compound bacterial flora or planting \u003cem\u003ePeganum harmala\u003c/em\u003e would reduce the concentration of heavy metal ions in tailings soil, it may be due to the adsorption heavy metal ions on the cell surface of the compound bacterial flora and interaction of the secondary metabolites produced by the compound bacterial flora with heavy metal ions. But interestingly, the reduction of EC did not significantly affect the bacterial communities from the results of Redundancy analysis (RDA) and Spearman correlation Heatmap, which was inconsistent with previous studies (Wei Wu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The changing trend of soil POR was just the opposite to that of EC. It was well known that the size of soil porosity depends on soil quality. During the experimental remediation process, the soil POR of different treatment groups after adding compound bacterial flora gradually was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This indicated that the quality of tailings soil had improved after adding compound bacterial flora.\u003c/p\u003e \u003cp\u003eSoil enzyme activities were good indicator of soil quality improvement, and their changes can provide characteristics and relevant information about microbial community succession (Adewele T. Adetunji et al., 2017). The test results showed that the addition of compound bacterial flora would improve the enzyme activities of sucrase, urease and alkaline phosphatase in tailings soil (Fig.\u0026nbsp;3ABC), which was consistent with the results reported by Souhir Abdelkrim (2020) and others. When the soil inoculated with compound bacterial flora was compared with the soil without inoculation, the activities of β-glucosidase, urease and alkaline phosphatase increased significantly. In addition, other literatures had also reported the increase of soil enzyme activities after bacterial flora inoculation (Upadhyay et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Utobo-Tewari, 2015; Wenliang Ju et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The increase of soil enzyme activities was conducive to the growth and development of plant roots. For example, the increase of urease activity would promote the absorption of nitrogen by plant roots (Saadani, O. et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which was very friendly to the microbial-plant joint remediation technology adopted in this experiment, so as to achieve the improvement of soil quality by microorganisms, and the improvement of soil quality promotes the growth and development of \u003cem\u003ePeganum harmala\u003c/em\u003e, and finally achieve a mutually beneficial and win-win relationship.\u003c/p\u003e \u003cp\u003eIn general, the richness index and diversity index representing the bacterial communities showed that the Chao, Shannon, Simpson and ACE index of H treatment groups were higher than those T treatment groups, which may due to the dilution of the content of heavy metals in the whole tailings soil by adding exogenous yellow cinnamon soil, resulting in the decrease of soil toxicity. At the same time, the richness index and diversity index of treatment groups with compound bacterial flora and \u003cem\u003ePeganum harmala\u003c/em\u003e were higher than controls (TCK and HCK), and with the increase of repair time, the richness index and diversity index of different treatment groups were also increasing. This result was consistent with the report of RA\u0026uacute;l Zornoza (2017) and others.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that Firmicutes, Actinobacteriota, Proteobacteria and Bacteroidota were dominant bacterial phyla in mining areas (Yuanqing Chaoet al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jianwei Xinget al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lur Epelde et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sibanda et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These results were similar to the composition of the control groups (TCK, HCK). The most abundant phylum of the 43 core OTUs was Proteobacteria in our study. However, prior to inoculation of compound bacterial flora, the dominant bacterial phyla in TCK were Firmicutes and Actinobacteriota, and the dominant bacterial phyla of corresponding T treatment groups (TM, TMP) were Proteobacteria and Bacteroidota after the inoculation of compound bacterial flora. These results may be related to the compound bacterial flora we inoculated. And the bacterial community structure in the tailings area had changed after the compound bacterial flora was inoculated, which was consistent with the research results of Wood (2016) et al, Cd changed the competitive dynamics in the soil bacterial community, causing changes in the bacterial community. Interestingly, there was also a certain proportion of Proteobacteria and Bacteroidota in HCK, but it was not higher than the proportion of T treatment groups after inoculation of compound bacterial flora. This made us wonder about the bacterial community structure changes may not only be caused by the inoculation of compound bacterial flora, but also may be caused the inoculation of compound bacterial flora stimulated the reproduction of some \u0026ldquo;native bacterial phyla\u0026rdquo; and formed unique dominant bacterial phyla, similar results had been reported by M Shuaib (2018).\u003c/p\u003e \u003cp\u003eMoreover, we determined 8 environmental variables that affect bacterial communities in different treatment groups, of which heavy metals were the main factors because of their greatest impact on the bacterial communities in our study. Although VPA analysis showed that the contribution of soil physical and chemical properties to the variance was higher than that of heavy metals. This may be due to the fact that soil physical and chemical property variables (five) were more than heavy metal variables (three). Our results were consistent with previous studies (A. Markowicz, 2016; Luo et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) showing that heavy metals inhibited bacterial communities. However, some reports were inconsistent with our research results and their research showed that heavy metals would not inhibit or even promote the bacterial communities (Mohamed and Abdelmajid, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hong et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Reis et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These differences may due to the types, duration of heavy metals and various environmental factors in different mining areas. In fact, the impact of heavy metals on bacterial communities was extremely complex, and our research process had only gone through three months, because our main purpose was to gain the removal rate of heavy metals in different treatment groups and the restoration effect of soil quality. The results of heavy metals removal rate showed that our research could achieve the tentative goal. For this study, we only measured the relationship between the total amount of heavy metals and changes of bacterial community structure in a certain period of time.\u003c/p\u003e \u003cp\u003eIn addition, it was worth noting that we only studied the structural diversity of soil bacterial communities, and specific functional genes and related metabolic pathways in bacterial communities of different treatment groups need to be further explored. Moreover, for such complex environment in tailings area, further detailed and long-term research was needed to deeply understand the heavy metals pollution situation and the impact of various environmental factors on the bacterial communities.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eOur results showed that the constructed compound bacterial flora had significant effect after inoculation with different treatment groups, especially in the treatment group of combined remediation of compound bacterial flora and \u003cem\u003ePeganum harmala\u003c/em\u003e. At the same time, we suggest that if we want to solve the problem of multiple heavy metals contaminated soil or tailings soil in a shorter time under the condition of resources, it is a good choice to add exogenous pollution-free yellow cinnamon soil, because our results showed that HMP treatment group had the most significant remediation effect in the same time. We removed most of the existing laboratory level research to outfield for site remediation test, obtained some valuable data and these data would provide a reference for researchers who conduct subsequent outfield tests.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We are grateful to the National Natural Science Foundation of China (Grant No. 31860163); Key Research and Development Projects of Gansu Province (Grant No. 20YF3NA018); Technology Departmen Youth Science Project of Gansu Provincial Science (Grant No.20JR10RA255. We appreciate the technical support from Shanghai Majorbio Bio-pharm Technology Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript does not involve human or animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Author confirms:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026middot;\u003c/strong\u003ethat the work described has not been published before;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026middot;\u003c/strong\u003ethat it is not under consideration for publication elsewhere;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026middot;\u003c/strong\u003ethat its publication has been approved by all co-authors, if any;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026middot;\u003c/strong\u003ethat its publication has been approved (tacitly or explicitly) by the responsible authorities at the institution where the work is carried out.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The Author agrees to publication in the Journal indicated below and also to publication of the article in English by Springer in Springer\u0026rsquo;s corresponding English-language journal.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The copyright to the English-language article is transferred to Springer effective if and when the article is accepted for publication, The author warrants that her contribution is original and that she was full power to make this grant. The author signs for and accepts responsibility for releasing this material on behalf of any and all co-authors. The copyright transfer covers the exclusive right to reproduce and distribute the article, including reprints, translations, photographic reproductions, microform, electronic form (offline, online) or any other reproductions of similar nature.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;After submission of the agreement signed by the corresponding author, changes of authorship or in the order of the authors listed will not be accepted by Springer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuan Wang\u003c/strong\u003e: Investigation, Formal analysis, Data curation, Writing-original draft. \u003cstrong\u003eLingui Xue\u003c/strong\u003e: Conceptualization, Supervision, Funding acquisition, Writing - review \u0026amp; editing. \u003cstrong\u003eYanli Huo\u003c/strong\u003e: Formal analysis, Data curation. \u003cstrong\u003eYecheng Ma\u003c/strong\u003e: Investigation, Data curation. \u003cstrong\u003eJiahui Li\u003c/strong\u003e: Methodology, Validation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 31860163); Key Research and Development Projects of Gansu Province (Grant No. 20YF3NA018); Technology Departmen Youth Science Project of Gansu Provincial Science (Grant No.20JR10RA255).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the author on reasonable request.\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArsalan A. Quresh, Tasneem G. Kazi, Jameel A.Baig, et al (2020) Exposure of heavy metals in coal gangue soil, in and outside the mining area using BCR conventional and vortex assisted and single step extraction methods. Impact on orchard grass. Chemosphere 255-126960. https://doi.org/10.1016/j.chemosphere.2020.126960.\u003c/li\u003e\n\u003cli\u003eAdewole T. Adetunji, Francis B. Lewu, Reckson Mulidzi, Bongani Ncube (2017) The biological activities of \u0026beta;-glucosidase, phosphatase and urease as soil quality indicators: a review. Journal of Soil Science and Plant Nutrition 17 (3):794-807. https://doi.org/10.4067/S0718- 95162017000300018.\u003c/li\u003e\n\u003cli\u003eAkhtar, M.J., Ullah et al (2018) Nickel phytoextraction through bacterial inoculation in Raphanus sativus. Chemosphere 190:234\u0026ndash;242. https://doi.org/10.1016/j.chemosphere.2017.09.136.\u003c/li\u003e\n\u003cli\u003eArora, Naveen Kumar, Mehnaz et al (2016) Bioformulations: for Sustainable Agriculture. 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Pereira et al (2020) Synergistic effect of organo-mineral amendments and plant growth promoting rhizobacteria (PGPR) on the establishment of vegetation cover and amelioration of mine tailings Chemosphere. https://doi.org/10.1016/j.chemosphere.2020.127803.\u003c/li\u003e\n\u003cli\u003eChang-Ho Kang, Soo Ji Oh, YuJin Shin, et al (2015) Bioremediation of lead by ureolytic bacteria isolated from soil at abandoned metal mines in South Korea Ecological Engineering 74:402-407. http://dx.doi.org/10.1016/j.ecoleng.2014.10.009.\u003c/li\u003e\n\u003cli\u003eE. Oburger, C.V. Cid, D. Schwertberger, et al (2020) Response of tungsten (W) solubility and chemical fractionation to changes in soil pH and soil aging. Science of the Total Environment 3(5). https://doi.org/10.1016/j.scitotenv.2020.139224.\u003c/li\u003e\n\u003cli\u003eFan Du, Zhaoguang Yang, Peng Liu et al (2018) Accumulation, translocation, and assessment of heavy metals in the soil-rice systems near a mine-impacted region. Environmental Science and Pollution Research 25:32221-32230. https://doi.org/10.1007/s11356-018-3184-7.\u003c/li\u003e\n\u003cli\u003eHao Xu, Jian Sun, Liansheng Xu, Hong Zhao, Xiaohan Chen, Zhaojie Cui, Rui Zhao (2020) The invention relates to a microbial agent and a method for planting plants to improve tailings sand. P. China, CN 112410265 A. \u003c/li\u003e\n\u003cli\u003eHai-Hong Gu, Zheng Zhou, Yu-Qian Gao et al (2017) The Influences of Arbuscular Mycorrhizal Fungus on Phytostabilization of Lead/Zinc Tailings Using of Four Plant species International Journal of Phytoremediation 19:739-745. https://doi.org/10.1080/15226514.2017.1284751.\u003c/li\u003e\n\u003cli\u003eHassan Etesami (2018) Bacterial mediated alleviation of heavy metal stress and decreased accumulation of metals in plant tissues: Mechanisms and future prospects. 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Environmental Science and Technology 50(21):11481-11490. https://dio.org/10.1021/acs.est.6b02284.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Heavy metals, Outfield, Site remediation, Compound bacterial flora, Bacterial community structure","lastPublishedDoi":"10.21203/rs.3.rs-2144977/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2144977/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMany researchers have paid attention to solving the problem of multiple heavy metals pollution in the tailings area. However, the studies only stay at the laboratory level, which cannot completely demonstrate an efficient approach to remediate polluted environment due to tailings. This study aimed at investigating the effects of compound bacterial flora we constructed to remediate the tailings area of Linze County, Zhangye City, Gansu Province due to the accumulation of numerous heavy metals such as Pb, Cd and Mn. The remediation effects of different treatment groups were evaluated by monitoring the physical and chemical properties of soil and studying the bacterial community structure during the remediation process. Compared with the control (TCK), after 84 days of restoration, parameters of the tailings soil inoculated with the compound bacterial flora were improved, pH was decreased from 7.74 to 7.02, EC (electrical conductivity) was decreased by 21.15% ~70.49%, and activities of alkaline phosphatase, sucrase and urease were increased by 95.94%~99.64%、88.42%~98.43%、83.28%~86.95% respectively. Among the 43 identified core optional taxonomic units (OTUs), Proteobacteria (44.9%), Actinobacteriota (21.7%), Bacteroidota (17.9%) were the dominant bacterial phyla, and \u003cem\u003eSphingobacterium\u003c/em\u003e (20.2%), \u003cem\u003eArthrobacter\u003c/em\u003e (8.5%), \u003cem\u003eSphingomonas\u003c/em\u003e (6.1%) were the dominant bacterial genus. The results of alpha diversity showed that the bacterial diversity and richness of the treatment groups inoculated with compound bacterial flora were significantly higher than controls (TCK and HCK). The correlation analysis of RDA, VPA and Spearman showed that soil pH, WHC, EC, POR, TEMP and heavy metals had significant effects on the composition and distribution of bacterial communities. Our study monitored the changes of soil physical and chemical properties and bacterial community structure during the whole remediation process, which will provide a reference basis for the remediation approach to the soil polluted by multiple heavy metals.\u003c/p\u003e","manuscriptTitle":"Dynamic Changes of Soil Parameters and Bacterial Communities during Bioremediation of Multiple Heavy Metals Contaminated Tailings by Compound Bacterial Flora","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-14 14:19:38","doi":"10.21203/rs.3.rs-2144977/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3faf9726-8cce-4ee4-a8d9-9d6b8a1df09e","owner":[],"postedDate":"March 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-06-10T10:11:31+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-14 14:19:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2144977","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2144977","identity":"rs-2144977","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}