Application of wetland waste plant biochar in combination with arbuscular mycorrhizal fungi on immobilization of Cd in contaminated soil

Application of wetland waste plant biochar in combination with arbuscular mycorrhizal fungi on immobilization of Cd in contaminated soil | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Application of wetland waste plant biochar in combination with arbuscular mycorrhizal fungi on immobilization of Cd in contaminated soil Liang Qiu, Qi lin Zhu, Qing hua Luo, Hai lan Li, Jia li Yan, Tao Liao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2176911/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 In this study, biochar pyrolyzed from different wetland waste plants ( Reed , Typha , Canna ) was used to immobilize Cd in contaminated soil. The results showed that biochar could all enhance the residual Cd 2+ in soil. Moreover, Ca500 (pyrolyzed at 500 ℃ from Canna ) showed the best immobilization capacity due to its larger surface, higher number of pores and its regular, smooth structure. Optimum conditions for Ca-500 immobilization to Cd 2+ in soil were: amount 6% (wt%) and contacting time 16 weeks, the exchangeable and the state of bound to carbonates of heavy metals reduced 92.08% and 67.64%, respectively. After combining biochar with arbuscular mycorrhizal, the results showed that the combined system could more effectively reduce the transportable Cd content in soil, increase the proportion of residual Cd, effectively reduce the migration of Cd, improve the biomass and root length of vegetables, reduce Cd absorption by vegetables and the ability of vegetables to transfer Cd from root to the upper part, and reduce the edible safety risk. Simultaneously, biochar with arbuscular mycorrhizal can improve the antioxidant capacity of vegetables and their resistance to heavy metals, optimize the composition of soil microbial community, and improve soil microbial abundance and community β-diversity. It can also significantly change the functional gene expression of soil microorganisms, soil fertility, and the self-healing ability to heavy metals. wetland plant biochar heavy metals arbuscular mycorrhiza immobilization performance amaranth Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 1. Introduction With the growing shortage of global resources, together with the industrial production and various chemical products application, heavy metals have entered the environment in various ways(Honma et al., 2016 ). Over ten million polluted sites were can be found worldwide, and more than 50% of these sites contain heavy metals(Tunēns et al., 2022 ). Most of these polluted sites are found in farmland. From 2015 to 2020 in China, researchers found that 21.49% of the samples collected from sites across 3000 surveyed areas were polluted, which was approximately 5% higher than in 2014. The copper pollution in Hunan, Guangxi province is nearly two times higher than background values; the lead content in Guanxi and Sichuan Provinces is about 2.5 times higher than background values; the cadmium content in most provinces is more than double the background values, and in some area is even ten times higher(Bank, 2014 ). These heavy metals contaminate sediments and are the main long-term source in the soil(Chlopecka&Adriano, 1996 ; Cao&Harris, 2010 ), Especially copper, lead, and cadmium. Therefore, controlling heavy metal pollution and reducing its impact on agricultural products has the potential to minimize the food safety risk and improve the utilization of farmland(Cao et al., 2011 ). There are two methods known to control the soil pollution from heavy metals: one is to change the existing forms of heavy metals in the soil to reduce their migration and bioavailability in the environment(Shaheen&Rinklebe, 2015 ); the other is to remove heavy metals from the soil(Ahmad et al., 2017 ). Regarding these methods, researchers suggest physical, chemical, and biological treatment methods. The conventional methods employed includes precipitation, ion extraction, dilution, and planting enriched plants(Abel et al., 2017 ; Amstaetter et al., 2012 ; Bagreev et al., 2001 ; Beckingham et al., 2013 ). However, these methods usually have some limitations to their practical application like high costs, difficult operation, and possible secondary pollution to the natural environment(Arán et al., 2017 ). Recently, many studies proved that biochar had a certain immobilizing ability for heavy metals in the soil due to its available pore structure, large surface area, and high pH(Bandara et al., 2017 ; Liu et al., 2018 ; Libralato et al., 2018 ; Goswami et al., 2016 ). Biochar immobilization of the heavy metals in soil is done by reducing the bioavailability of heavy metal through methods like cation exchange, complexation(Jia et al., 2017 ). However, the pyrolytic temperature and feedstock of biochar has been reported to have a large effect on its properties(Goswami et al., 2016 ). Researchers speculate that higher temperature results in a higher pH and larger surface area of the biochar, and higher heavy metals adsorption ability(Gong et al., 2018 ). Niazi et al. reported that biochar produced from perilla leaf at 700 ℃ removed more As at pH 7–9 than at 300 ℃(Niazi et al., 2018 ). Moreover, Shen et al. found that at higher temperatures, the surface area of the biochar decreased, and contributed to the lower Cr(VI) removal ability(Shen et al., 2012 ). However, there is a lack of studies concerning the pyrolytic temperature-mediated immobilization effect of biochar in soil polluted by different kinds of heavy metals(Chen et al., 2018 ). It is important to conduct studies regarding the pyrolytic temperature effect of biochar on the immobilization of different kinds of heavy metals. Such studies will promote the application of biochar in heavy metal polluted soil treatment; however, excessive use of biochar could be harmful to plants due to the high pH. Combining biochar with other strategies has become a new method of soil remediation(Gul et al., 2015 ). Mycorrhizal fungi are closely associated with plants and can reduce the mobility of heavy metals and improve the plants resistance to heavy metals(Cao&Harris, 2010 ). A number of studies have proved that microorganisms have strong adsorption on a variety of heavy metals, including Cd, and can significantly reduce their mobility. As a common soil microorganism, arbuscular mycorrhiza is a parasitic fungus and has a close relationship with plant roots, can promote plant growth, improve plant resistance to heavy metals, and reduce heavy metal transfer to the upper part of plants(Xu et al., 2020 ). However, when the concentration of pollutants in the soil is too high or the environmental conditions of the soil are not suitable for microbial growth, the repair efficiency of arbuscular mycorrhizal is significantly reduced and the repair cycle will be prolonged(Jeffery et al., 2011 ). Biochar can supply a good protection to arbuscular mycorrhizal since its large pore size and carbon source can provide an ideal growth environment for arbuscular mycorrhiza, greatly improving its remediation efficiency. Thus, biochar combined with arbuscular mycorrhiza can be a promising way to solve soil pollution by heavy metals(Liu et al., 2019 ). In this study, biochar pyrolyzed from wetland waste plants ( Reed (Re), Typha (Ty), Canna (Ca)) at temperatures of 400, 500, and 600 ℃. These productions were employed as representatives to evaluate the immobilization ability on Cd contaminated soil by conducting comprehensive studies. The effects of different biochar on Cd immobilization and its potential mechanism in the soil were also studied by distribution of heavy metals fraction in soil. Based on these experiments, the best biochar and arbuscular mycorrhiza combination was used to study the solidification properties on Cd in soil. Meanwhile, pot experiments were used to explore the effects of the combined system on the accumulation of heavy metals in plants, antioxidant indicators, and soil microorganisms. 2. Materials And Methods 2.1. Soil samples and reagents The incubation soil experiments were divided into two parts due to the amount of time needed. The first part was dedicated to analyzing the immobilization ability of different biochar. The second part functioned to explore the best conditions for biochar application. Soil samples for incubation experiments were obtained from Fuzhou, Jiangxi Province (Fz) with little heavy metal polluted soil. Fz samples were taken to the laboratory, air-dried, homogenized, and sifted with a 2 mm sieve. Deionized water was used to keep the soil moisture content at 70%. Then the samples were aged for 30 days at room temperature. Samples for part two were collected from a farmland in Xiangjiang Reiver (Xj). Heavy metals were analyzed by ICP optical emission spectroscopy (ICP-MS; PerkinElmer NexION 300X, USA) after soil digestion via HF + HNO 3 + H 2 SO 4 . The heavy metals concentration of Fz and Xj are shown in the supporting information. The distribution of heavy metals was determined using glacial acetic acid (HOAc), sodium hydroxide (NaOH), hydrogen peroxide (H 2 O 2 ), ammonium acetate (NH 4 OAc), sodium acetate (NaOAc), nitric acid (HNO 3 ), calcium chloride (CaCl 2 ), and hydroxylammonium chloride (OH-NH 2 ·HCl). The Cd soil was prepared using cadmium nitrate Cd(NO 3 ) 2 . All the reagents were purchased from CNW (Shanghai, China) and GL Biochem Ltd. (Shanghai, China). 2.2. Preparation of biochar Biochar was pyrolyzed from wetland waste plants ( Reed (Re), Typha (Ty), Canna (Ca)) from the experiment base (Wuhan, Hubei Province; Institute of Hydrobiology, Chinese Academy of Sciences). Each sample was air-dried, crushed, and sifted with 40 mesh sieves. The temperatures of the pyrolysis process were 400, 500, and 600 ℃ (each biochar from here referred to as Re400, Re500, Re600, Ty400, Ty500, Ty600, Ca400, Ca500, and Ca600). The biochar was under a continuous stream of N for 4 h to keep the temperature constant. At last, biochar was crushed and passed through 100 mesh sieves. 2.3. Characterization and analysis methods Fourier transform infrared spectroscopy of the biochar was obtained using Shimadzu-IRaffinity-1 equipment (Shimadzu Corporation, Tokyo, Japan) along with an ATR module (FTIR-ATR), under a nitrogen purge. Measurements were recorded in the range 500–4000 cm − 1 with 4.0 resolution, obtaining 256 scans while applying atmosphere and background correction. X-ray diffraction (XRD, 5–70 °) was measured on a D8-Focus X-ray diffractometer (Bruker Optics, Germany) with a test rate of 10 °ꞏmin − 1 . The results were analyzed by using the Jade version (9.0) and Origin version (9.0) programs. The specific surface area( S BET )and micropore volume (W 0 ) were determined by nitrogen adsorption isotherms, acquired at 77 K using a Micromeritics Instrument, Gemini VII 2380 (USA), after outgassing the materials overnight at 120 ºC. S BET was calculated from the Brunauer–Emmett–Teller equation under the relative pressure range 0.01–0.1. Pore volume (Vp) was estimated from the amount of nitrogen adsorbed at a relative pressure of 0.99. The surface morphology of the materials was analyzed using scanning electron microscopy (SEM; Hitachi S4100, Japan). The images were obtained at magnifications of 800 and 5000. 2.4. Incubation soil experiment To analyze the immobilization of each biochar, each biochar was applied to 500 g of Cd-contaminated soil (from Fz). Heavy metal contaminated soil without biochar was used as the control experiment (CK) and three groups of parallel experiments were set per process. Each biochar treatment was done in triplicate. To explore the relationship between time and distribution of heavy metals, and to investigate the effect of different biochar weights, Ca500 was applied to 500 g of soil from Xj. The time of contact was 14 weeks. The samples of soil were removed and Cd determined at 2, 6, 10, and 14 weeks. Biochar was applied at 10, 20, 30, 40, and 50 g (2, 4, 6, 8, and 10%, respectively). The amount of heavy metal was determined by ICP-MS. 2.5. Experiment of arbuscular mycorrhizal infection Arbuscular mycorrhiza ( G. etunicatum , G. intraradices , and G. mosseae ) was purchased from the Institute of Nutrition and Resources, Beijing Academy of Agricultural and Forestry Sciences. The infection rate of arbuscular mycorrhiza on the roots of Chinese cabbage and amaranth was studied using the acetic acid ink method. To start, 20% KOH solution (v:v) was added to the cleaned 1–1.5 cm root segment. The segment was heated in a water bath for 60 min at 60 ℃. It was then removed and washed under running water for 5 min, then soaked in acetic acid for five min. An acid ink solution (5% solution, acetic acid: ink = 95 ml:5 ml) was used for dyeing in a 60 ℃ water bath for 30 min. After the water bath, the segments were soaked in water for 12 h to decolorize. Sudan IV staining solution (3 g Sudan IV solid plus 1000 ml of 70% ethanol solution) was used for re-staining in a 60 ℃ water bath for 60 min. Segments were then rinsed with running water for 3 min, and finally soaked in 70% ethanol solution for 5 min. The dyed root segments were observed under an optical microscope and the proportion of dyed fibrous roots was calculated. 2.6. Pot experiment on the combination of biochar and arbuscular mycorrhiza Before the start of the experiment, the bacterial agent was mixed with the soil (20 g bacterial agent: 1kg soil) and the soil moisture was determined after a week when the field water holding rate was about 60%. This was to ensure the smooth germination of arbuscular mycorrhizal spores. The experiment comprised three groups: CK (without adding biochar and arbuscular mycorrhiza), AMF (adding arbuscular mycorrhiza fungi alone), and AMF + BC (adding arbuscular mycorrhiza fungi and Ca500), and each group was done in triplicate. In the pre-treatment before planting, biochar was added to the soil in a ratio of 2% and mixed evenly. Compound chemical fertilizer was added in a ratio of 0.3 g fertilizer to ꞏ1kg soil to ensure the normal plant growth. After one day of pretreatment, several vegetable (Chinese cabbage and amaranth ) seeds were added to each container. after Seven days later, seedlings were thinned down to ten seedlings in each incubator (50 cm long, 30 cm wide, and 20 cm high plastic planting pots were used for this purpose). The culture period was 42 days. After harvest, Cd content in soil, plant biomass, Cd content in different parts of the plant and antioxidant indices (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA)) were determined. The bioconcentration factor (BF) and transfer factor (TF) were then calculated. 2.7. DNA analysis of soil microbial samples Soil samples were collected from the experimental soil after the vegetables were harvested in the pot experiment involving combining biochar and arbuscular mycorrhiza. A soil sample without treatment was collected as the blank group. The samples were stored a at -20°C until use. The experimental process was implemented in accordance with the standard protocol provided by Illumina, including sample quality detection, library construction, library quality detection, library sequencing, and other processes. The specific operations were as follows: after the genomic DNA extracted by CTAB method was tested and qualified, the DNA was fragmented by mechanical interruption (ultrasound). The fragmented DNA was purified, the end was repaired and the 3′ end was added, and a sequencing connector was connected. The fragment size was selected by agarose gel electrophoresis. Sequencing Library (nebnext ® Ultra ™ DNALibraryPrepKitforIllumina ®) were amplified by PCR according to the manufacturer's instructions. The established libraries were inspected first, and the qualified libraries were sequenced using Illumina NovaSeq. After sequencing, MEGAHIT assembly software was used for analysis (version 1.2.9), and the default parameters of MEGAHIT (generated contigs N50 with high quality) were selected for assembly to obtain the sample contigs. Prodigal was used to predict the gene of the assembly results. After the prediction was completed, CD-HIT (version 4.6) was used to remove the redundant annotation genes. The longer one in each class was selected as a unique gene. The similarity threshold and coverage threshold were set to 95% and 90%, respectively. 2.8. Results and discussion 2.9. Characterization of different biochar FTIR spectrometry of the different biochar were used to shed light on the structures (Fig. 1 ). Our results showed that regardless of which wetland plant was observed, they did not appear or reduce any functional group, but the temperatures of the pyrolysis process influenced the peaks. The peaks in the range of 3800–3516 cm − 1 were mainly caused by the –OH stretching vibration associated with intermolecular hydrogen bonding(Ghani et al., 2013 ). As the temperature increased, the intensity of the peaks for Ty and Ca decreased and Re increased. This may be because Re is a monocts while Ty and Ca are dicots. The stems of grass plants are more fragile than those of herbs(Igalavithana et al., 2017 ). Thus, during the pyrolysis, it could have been easier for Re to lose the –OH group due to dehydration and the dehydrogenation reactions. The peaks at around 2927 cm − 1 and 2877 cm − 1 represent asymmetric and symmetric vibration adsorption of lipoid (-CH 2 ), respectively(Zhai et al., 2020 ). There are no differences in our results, which may be because the peaks observed belong to carbon dioxide. The peaks at 1621 cm − 1 were identified as C = C. Our results reflected that these peaks shifted to a blue wavelength in the high temperature tests, suggesting that carbonization is better at high temperature(Gong et al., 2021 ). The other peaks in the spectra could be assigned to C \(-\) O \(-\) (1099 cm − 1 ), CO 3 (1430 cm − 1 ), and C \(-\) H (748 cm − 1 ) as their positions did not obviously change(Graber et al., 2010 ). These results suggested that herbaceous plants were more stable than grass and the production temperature of the biochar used had a great influence on their surface functional group. The S BET results are shown in Table 1. The S BET of each biochar was as follows: Re:1.05–69.7 m 2 ꞏg − 1 ; Ca: 1.46–87.3 m 2 ꞏg − 1 ; Ty: 1.39–5.49 m 2 ꞏg − 1 , similar S BET values were determined for biochar in other studies. Our results suggested that with higher pyrolysis temperatures, the surface area was larger and the volume of the pores was smaller. This might be contributed to volatile substance release and appearance of vascular bundle structures at high temperatures(Lu et al., 2017 ). From Table 1, we observed that in comparing these feedstocks that Ca had a larger surface area and pore size, supporting the finding that Ca had a better immobilization capacity comparing to Re and Ty. Therefore, the pyrolysis temperature is an important factor for biochar since biochar produced at higher temperature has a larger surface area, which influences its bonding to heavy metals(Lyu et al., 2018 ). In this study, herbaceous plant biochar had a larger surface area than grass biochar. Consequently, it is important to select suitable pyrolysis temperatures and feedstock before applying the biochar. Table.1 Physical characterization of the biochars Biochars S BET (m 2 g − 1 ) V p (cm 3 g − 1 ) D (nm) Ty-4 1.05 0.005 6.63 Ty-5 4.90 0.010 5.71 Ty-6 69.7 0.005 3.29 Ca-4 1.46 0.005 7.42 Ca-5 3.17 0.009 7.21 Ca-6 87.3 0.05 2.48 Re-4 1.39 0.04 7.23 Re-5 2.61 0.01 6.95 Re-6 5.49 0.04 4.47 The XRD spectrum of different biochar in the range of 20 to 75 ° is shown in Fig. 3 , all biochar samples presented a broad diffraction peak at 23–25 °, belonging to the amorphous carbon property(Bashir et al., 2018a ). Moreover, our findings suggested that at higher temperatures, the observed carbon peak and content of carbon would be higher. The XRD results indicated that the ratio of crystallite carbon in biochar was mainly determined by the feedstock, and the content of carbon was higher at high temperatures (400–600 ℃). The same results were found by previous studies(Zhang et al., 2020 ). The surface of the biochar was examined using SEM (Fig. 4 , 5 , 6 ). The results suggested that at lower temperature, the productions had a vascular bundle structure, due to the use of leaves of the wetland waste plants. With increased temperature, their structure changed to blocks and Ca appeared more regularly, which might be the reason why Ca had the largest surface area. There were many micropores observed on the surface and as the temperature increased, the volume of the pores became larger and new pores appeared. Compared to Re and Ty, biochar pyrolyzed from Ca showed more developed macro-porosity, since Ca reflected more pores and the structure was relatively regular and smooth. Other studies also showed that with as temperature increased, the volume of the pores became larger and their structure mainly depended on the cellular structure of original feedstock(Bandara et al., 2017 ; Meier et al., 2017 ). 2.10. Immobilization performance of Re, Ty, and Ca The speciation of Cd in soil was analyzed by sequential extraction procedures proposed by Tessier. According to this method, The distribution of Cd in different states: exchangeable (Ex), bound to carbonates (Bc), bound to iron and manganese oxide (Bo), bound to organic matter (Bm), and residual (Re) in the soil were determined using the Tessier methods. Among those fractions of heavy metals, exchangeability, and the state of bondage to carbonates can be released in cationic solutions, and can be easily utilized by organisms or plants. The states of bound to iron, manganese oxide, and organic could be converted to exchangeable under appropriate conditions, which can be indirectly utilized by plants. The residual state is mostly stable but cannot be directly utilized by plants(Jun et al., 2020 ). Therefore, the preliminary goal of this study is to decrease the exchangeable and CB-bound fraction. Before the soil incubation (Fig. 6 ), the exchangeable fraction was dominant (77.15%), followed by carbonate-bound fraction (14.92%), and the rest of fractions occupying a small proportion, especially the percentage of OM-bound fraction (2.27%). After 42 d of incubation, the proportion of exchangeable fractions in all experimental groups declined significantly and settled in range of 60.62 to 73.72% while other fractions increased. In particular, the fraction of Fe-Mn-bound increased sharply and surpassed the CB-bound fraction, reaching 8.43 to 12.42%. The Canna biochar treatments showed a great ability to immobilize Cd in soil, especially the biochar prepared using 500 ℃. This may be due to the high specific surface area as well as the better ability of ion exchange. Many studies have proved that biochar can immobilize heavy metals through ion exchange. The current results were similar to those of most studies(Kiran&Prasad, 2019 ). Conversely, all the reed biochar treatments (Re400, Re500, and Re600) showed poor immobilization performance. Compared with CK, the proportion of easily labile fractions decreased by no more than 5%. This might be because feedstock is one of the largest factors determining the immobilization ability of biochar(Lahori et al., 2017 ). The specific surface area of the reed biochar was also noticeably lower than other tested biochar. 2.11. Experimental results of arbuscular mycorrhizal infection The experimental results are shown in Table 2. The infection of all AMF to Chinese cabbage was very low, this may because that arbuscular mycorrhiza seldomly invades cruciferous plants. While, the infection rate of G. intraradices in the root to amaranth plant is known to be most common, therefore, the arbuscular mycorrhiza selected in the pot experiment of the combination of biochar and arbuscular mycorrhiza was G. intraradices , and the tested plant was amaranth. Table.2 Colonization rate of arbuscular mycorrhiza fungi (AMF) to different vegetables AMF Chinese cabbage (%) Amaranth (%) G. etunicatum 3.53 11.25 G. intraradices 6.32 25.01 G. mosseae 3.22 9.24 2.12. Solidification effect of combined system on soil Cd The difference of Cd extracted state is shown in Fig. 7. In the CK group, exchangeable Cd (62.9%), carbonate bound (1.03%), iron manganese bound (24.76%), organic bound (4.17%), and residual (7.14%). The proportion of exchangeable Cd in the AMF and AMF+BC group decreased to about 45%, and the lowest proportion of exchangeable Cd in the AMF+BC group was 42.22%. The carbonate bound state increased slightly. The proportion of residual states increased from 7.14% in the CK group to 14.41% and 15.61% in the AMF and AMF+BC group, respectively. The results showed that the use of AMF or AMF+BC has the potential to reduce the proportion of exchangeable Cd in soil, thereby effectively increasing the content of residual Cd, with good fixation performance. The main reasons for these results are due to biochar changing the form of heavy metals or adsorbing on the surface of biochar to reduce its mobility through its own cation exchange, electrostatic adsorption, and surface oxidation-reduction reaction. The glycoproteins rich in aliphatic hydrocarbons and methylene secreted by arbuscular mycorrhiza have a high affinity for metals and can effectively combine with heavy metals to form stable structures. The reason for the small difference between the AMF and AMF+BC group may be that AMF plays a major role in Cd solidification and biochar provides suitable growth conditions for arbuscular mycorrhiza. 2.13. Effect of combined system on plants 2.13.1 Effects on biomass and root The purpose of determining the biomass and taproot length of plants was to observe the effects of the combined system of biochar and arbuscular mycorrhiza on heavy metal migration and plant physiology in Cd-contaminated soil. The biomass and root length of amaranth are shown in Fig. 8 . The biomass and root length in the CK group were 0.20 g and 3.33 cm respectively. The biomass in the AMF group increased significantly, but the root length was not significant. The biomass and root length in the AMF + BC group increased significantly to 0.81 g and 5.11 cm respectively. Similarly, found that the use of AMF and biochar could increase the biomass, root length, root diameter, and root volume of maize, and improve its nutrient absorption. Singh et al. found that arbuscular mycorrhizal colonization in asparagus roots doubled after adding biochar(Singh et al., 2021 ). Similarly, they showed that the addition of biochar could promote the growth of arbuscular mycorrhiza. Biochar itself is a rich carbon source, which can improve soil cation exchange capacity and redox reactions, improving the soil microbial community abundance and soil enzyme activity, enhancing soil nutrients and improving nutrient utilization400(Houben et al., 2013 ). 2.13.2 Distribution of Cd content, bioconcentration coefficient, and transfer coefficient in plants The content of Cd in roots, stems, and leaves of plants is shown in Fig. 9 . The Cd content of roots, stems, and leaves in CK group was 1.213 mgꞏkg − 1 , 0.642 mgꞏkg − 1 , and 0.623 mgꞏkg − 1 , respectively. This suggests that plants can easily absorb heavy metals from the environment and transfer them to their edible upper parts. Compared with CK, Cd content in roots, stems, and leaves of the AMF group decreased significantly, (15.7, 31.6, and 22.6%, respectively). When combined with biochar, the lowest content of Cd in roots, stems, and leaves were recorded (0.953 mgꞏkg − 1 , 0.243 mgꞏkg − 1 , and 0.28 mgꞏkg − 1 , respectively). It can be seen from the transfer coefficient that the proportion of Cd transferred from the underground root to the upper part of the plant has greatly decreased and Cd tends to accumulate in the roots (Table.S1). Guo Xiongfei explored the effect of biochar and arbuscular mycorrhizal application on the absorption of heavy metals in Cassia occidentalis (Houben et al., 2013 ). The results showed that the use of a combined system could reduce the absorption of heavy metals in plants, promote the accumulation of heavy metals in their roots, and reduce the concentration and transfer coefficient of heavy metals in stems and leaves above the ground. A similar conclusion was also made in this study, possibly because the addition of biochar increased the infection rate of arbuscular mycorrhiza. The negative ions such as hydroxyl and carboxyl groups on the mycelium surface of arbuscular mycorrhiza combine easily with heavy metal cations, resulting in a large number of heavy metals being fixed to the root system. In addition, it can promote the increase of woodiness of plant root cell wall and root branching, change the root structure, and enhance the ability of plant root to combine heavy metals(Avio et al., 2017 ). 2.13.3 Antioxidant activity of plants After absorbing excessive heavy metals and other harmful substances, plants will display a variety of adaptive biochemical reactions, among which the most significant changes are the changes of antioxidant enzymes (SOD, POD, and CAT) and malondialdehyde (MDA). Therefore, measuring the antioxidant indices in different treatment groups can clearly show the health status of plants under heavy metal stress. The contents of SOD, POD, CAT, and MDA in plants are shown in Fig. 10. SOD is a ubiquitous antioxidant metal enzyme in organisms which can catalyze superoxide free radicals to produce hydrogen peroxide and oxygen(Bashir et al., 2018b ). It plays a vital role in the antioxidant balance of the plants and is positively correlated with the stress resistance of plants(Cui et al., 2016 ). In this study, SOD enzyme activity of amaranth in the CK group was 604 Uꞏg − 1 fresh tissue weight. SOD activity in the AMF and AMF + BC group increased by 12.9 and 2.9%, respectively, compared with CK. SOD activity in the AMF + BC group was lower than that the AMF group. This is similar to Elmer W H research results(Elmer&Pignatello, 2011 ). After adding biochar, the SOD content of ryegrass did not change significantly but the SOD in alfalfa increased significantly. The reason may be that the root nodules of leguminous plants can more effectively combine with biochar, enhancing the SOD activity in plants. The effect on plants without root nodules is not as obvious(Li et al., 2012 ). The addition of biochar can significantly change the physical and chemical properties of soil, especially the pH value. SOD in plants is sensitive to soil pH value, which may affect the activity of SOD in vegetables to a certain extent. POD is an enzyme with hydrogen peroxide and phenolic amines as substrates and is an important part of the body's antioxidant system. In this study, POD activity in the CK group was 777 Uꞏg − 1 fresh tissue weight. The AMF and AMF + BC group increased by 11.1 and 27.5%, respectively, compared with the CK group, and POD enzyme activity was 863 Uꞏg − 1 and 991 Uꞏg − 1 fresh tissue weight, respectively. The results showed that arbuscular mycorrhiza could improve the POD activity of vegetables under Cd stress, and the effect was greater when combined with biochar. Li et al. found that the POD activity of Suaeda salsa under salt stress increased significantly after adding arbuscular mycorrhiza. Harindintwali J D et al. found that the combination of biochar and arbuscular mycorrhiza can significantly increase plant POD activity but the effect of adding biochar has different results(Harindintwali et al., 2020 ). CAT is an enzyme that catalyzes the decomposition of hydrogen peroxide into water and oxygen in cells and is the enzyme in the final stage of the body's antioxidant system. In this study, CAT activity in CK group was 69.9 Uꞏg − 1 tissue fresh weight, and CAT activity in AMF group was 67.3 Uꞏg − 1 tissue fresh weight, which decreased by 3.5%. Similarly, Kang et al found that the CAT activity of alfalfa was significantly reduced after inoculation with arbuscular mycorrhiza, which may be due to the intervention of arbuscular mycorrhiza reducing the chemical stimulation effect of antioxidant enzymes(Kang et al., 2020 ). However, when AMF + BC were combined, CAT activity reached 125.2 Uꞏg − 1 tissue fresh weight, an increase of 79.1% compared with CK. Results show that the combination of biochar and arbuscular mycorrhiza can greatly improve CAT activity in vegetables; it may be that the biochar can significantly improve the soil environment, promote the growth of arbuscular mycorrhiza, indirectly promote the growth of plants, enhance resistance of plants, and improve the antioxidant enzymes of heavy metals by plants, leading to increased CAT activity. In addition, Igalavithana et al. believe that CAT is more sensitive to biochar than POD and SOD, which may also be the reason why CAT activity changes more than the other two antioxidant enzymes(Igalavithana et al., 2017 ). However, some scholars believe that POD and CAT show a complementary relationship in plants, but this phenomenon was not observed in this study. The specific reasons and the action mechanism of CAT needs to be examined further(Graber et al., 2010 ). MDA is an important index reflecting the level of membrane lipid peroxidation in plants. The increase of membrane lipid peroxidation is usually caused by environmental damage, which leads to the increase of reactive oxygen content and the destruction of cell membrane structures, thus inhibiting plant growth. In this study, the MDA content of plants in the CK group was 157.6 nmolꞏg-1, while MDA content in the AMF and AMF + BC group decreased from 26.7 and 47.3%, to 115.4 nmolꞏg-1 and 83.1 nmolꞏg-1, respectively. This indicates that the combination of arbuscular mycorrhiza and biochar could reduce the level of membrane lipid peroxidation and damage to plants. These results support the findings that CAT and POD enzyme activity changes. This may be because biochar can transform the exchangeable Cd in soil into its stable residual state and reduce the amount of Cd transported by the cell membranes, allowing more Cd to accumulate in AMF, thus reducing the level of leaf membrane lipid peroxidation(Bashir et al., 2018b ). 2.14. Analysis of soil microbial community 2.14.1. Composition and analysis of species diversity In this study, Karken2 + Bracken was used for species annotation, and clean reads were compared directly with marker gene sets. Bacteria, archaea, eukaryotes, and viruses in the samples were accurately annotated to the species level based on a metagenomic sample bank. The comparison results in species classification are shown in Table.S2. Principal coordinate analysis (PCA) results for community β-diversity based on Bray–Curtis distance are shown in Fig. 11 . The first two PCA ordination axes explain 68 and 29% of the β-diversity difference, respectively. The first axis distinguishes the blank group from the soil samples of other plants, and the second axis distinguishes the experimental group with biochar from the other two groups. The results indicate that arbuscular mycorrhizal combined with biochar could significantly change the microbial community in soil β-diversity. The relative abundance of species in various samples is shown in Fig. S.12. At the phylum level, except for the blank group, the group with the highest abundance in all the samples was Proteobacteria. The other dominant groups were Actinobacteria and Chordata. At the genus level, the abundance of Chlamydia in the AMF + BC group was much lower than that in the CK and AMF groups, indicating that the addition of biochar could inhibit the growth of this group. Streptomyces was the dominant group with the highest relative abundance in CK and the two experimental groups. The other dominant groups were Pseudomonas and Chlamydia . Compared with CK, the relative abundance of Bradyrhizobium and Micromonospora in the two experimental groups decreased. The relative abundance of Variovorax , Sphingopyxis , Azoarcus , and Hydrogenophaga increased, and the proportion in the AMF + BC group was higher. Some studies have shown that Pseudomonas can regulate auxin concentrations to maintain plant root health and promote plant growth. Azotobacter and Hydrogenophaga can improve the utilization of soil nutrients. In addition, studies have shown that Sphingomonas can degrade a variety of heavy metals and organic compounds including Cd. The results indicated that the addition of arbuscular mycorrhiza could increase the number of microorganisms related to soil fertility enhancement and heavy metal degradation, and the effect was significant when combined with biochar. In addition, the number and abundance of species in the sample α-diversity index (Table 3) indicated that the Berger, Parker, Simpson, and Shannon index were lower in the two experimental groups than the control group. Berger and Parker index indicate that the proportion of dominant microbial species in the soil has decreased after inoculating arbuscular mycorrhiza. Simpson and Shannon index indicate that the microbial diversity in the soil has decreased after inoculating arbuscular mycorrhiza. This is consistent with the results of other studies. The reason may be that the addition of arbuscular mycorrhiza can promote the growth of microorganisms closely related to arbuscular mycorrhiza, enhancing their competitive ability and inhibiting the growth of other microorganisms(Lu et al., 2017 ). The Chao 1 index was the largest in AMF + BC group, indicating that the number of species in this group was the largest. Table.3 The α-diversity index of community Sample Berger-parker chao1 simpson shannon_e Blank 0.0364 4525 0.00294 7.23 Control 0.0434 4582 0.00359 7.3 AMF + BC 0.0406 5005 0.00341 7.28 AMF 0.041 4561 0.0033 7.29 2.14.2. Gene function annotation KEGG is a major public database for the systematic analysis of the pathways of gene products in cells and the functions of these gene products, which are used to study the complex biological behavior of genes(Ma et al., 2020 ). After the KEGG annotation, genes were classified according to the KEGG metabolic pathway they participated in, as shown in Fig. 13 . It can be seen from the figure that there were 19,8443 functional genes annotated in the four samples. The metabolic pathways include four cell process pathways, three environmental information processing pathways, four gene information processing pathways, 12 metabolic pathways, and nine systemic pathways. The current three pathways of gene number in biological process are DNA transcription regulation, phosphorylation signal transduction system and methylation (Fig. 13 ). At present, the three pathways of gene number in cell components are membrane component, cytoplasm, and plasma membrane and the three pathways of gene number in molecular function are ATP synthesis, metal ion binding, and hydrolase activity(Ren et al., 2020 ). The Gene Ontology (GO) results are shown in Fig. 14 . It can be seen from the figure that the current three pathways of gene number in biological process are DNA transcription regulation, phosphorylation signal transduction system and methylation; At present, the three pathways of gene number in cell components are membrane component, cytoplasm and plasma membrane(Singh et al., 2021 ). 2.14.3. Gene abundance estimation and difference analysis The gene abundance density distribution map after standardization and PCA result of transcript expression is shown in Fig. 15 . According to the figure (Fig. 15 A), the gene abundance of soil treated with biochar and arbuscular mycorrhiza was slightly higher than that of CK and arbuscular mycorrhiza treatment group alone and was significantly higher than that of the blank group. In addition, it can be seen from the result Fig. S.15 of principal component analysis (PCA) on gene data of different samples. The first two PCA axes accounted for 50 and 26% of the gene differences in different samples, indicating that there were significant differences between different samples. It can also be seen that the gene composition of the two experimental groups (AMF, AMF + BC) treated with arbuscular mycorrhizal fungi was very similar. There was a significant difference in gene composition between the control group and the blank group. It may be that the experimental groups all used arbuscular mycorrhizal as a remediation agent, which had similar effects on the soil microbial communities. The GO term enrichment analysis results are shown in Fig.S3. Compared with CK, the significant metabolic pathways of AMF were macromolecular biosynthesis and cyanobacterin synthase activity (Q < 0.05), followed by phosphotransferase activity (Q < 0.1). The results showed that adding arbuscular mycorrhiza to soil could promote soil microbial activity, produce nutrients, and improve soil use value. The comparison of AMF + BC and CK indicates that the Q value of the first 20 metabolic processes with significant differences is 0.12, indicating that the combination of biochar and arbuscular mycorrhiza can significantly change the gene expression in soil(Ren et al., 2020 ). The metabolic pathways with the largest number of differential genes were abnormal proteolysis, RNA helicase activity, cell response to heat, and sulfate transmembrane transport ATP hydrolase activity(Singh et al., 2021 ). The results showed that the resistance of the soil microbial community to external stress was improved after adding biochar. When AMF + BC was compared with AMF, the significant gene expression item was ethanol metabolism (Q < 0.05). Macromolecule biosynthesis, virus, and cyanobacteria synthase activity were the next (Q = 0.055). The number of differential genes in bacterial flagella assembly was the largest. 3. Conclusion Biochar pyrolyzed from wetland waste plants: Reed , Typha , and Canna were employed to comparatively evaluate the immobilization performance on heavy metal-contaminated soil. The results showed that biochar could all immobilize the Cd. Biochar with different feedstocks could be ranked in the following order based on comprehensive immobilization performance: Ca༞Ty༞Re, the best weight and temperature of pyrolysis process was 6 and 500 ℃, respectively. The combination of biochar and arbuscular mycorrhiza can significantly reduce the content of Cd transportable states, the mobility of Cd, and increase the proportion of residual Cd states. Furthermore, this can also improve the biomass and root length of vegetables, reduce the absorption of Cd by vegetables, and enhance the ability of vegetables to transfer Cd from the roots to upper parts, reducing the edible safety risk. Moreover, the combination of biochar and arbuscular mycorrhiza could effectively improve the antioxidant capacity of vegetables and their resistance to heavy metals. Soil microbial community composition, soil microbial abundance, and β-diversity was optimized by its presence. Functional gene expression, soil fertility, and self-repair ability to heavy metals were significantly changed. Declarations Ethical Approval Not applicable Consent to Participate Not applicable Consent to Publish Not applicable Authors Contributions Formal analysis, Qing.hua.Luo.; investigation, Liang Qiu.; data curation, Xin Li.; writing—original draft preparation, Jiali Yan and Qi lin Zhu.; writing—review and editing, Liang Qiu and Qi lin Zhu.; project administration, Hailan Li. and Xin Li.; funding acquisition, Tao Liao, L.Q. and Qi lin Zhu. did the same contribution to this work. All authors have read and agreed to the published version of the manuscript. Funding This study was supported in part by grants from National Key R&D Program of China (2019YFD0902000)； Key R&D Program of Guangzhou City (202103000087)；Guangzhou people's Livelihood Science and technology project（202002020087） Competing Interests The authors declare that they have no competing interests Availability of data and materials Not applicable References Abel, S., Nybom, I., Mäenpää, K., Hale, S.E., Cornelissen, G., and Akkanen, J. (2017). Mixing and Capping Techniques for Activated Carbon Based Sediment Remediation – Efficiency and Adverse Effects for Lumbriculus Variegatus. Water Research. 114 104-112 Ahmad, M., Lee, S.S., Lee, S.E., Al-Wabel, M.I., Tsang, D.C.W., and Ok, Y.S. (2017). Biochar-Induced Changes in Soil Properties Affected Immobilization/Mobilization of Metals/Metalloids in Contaminated Soils. Journal of Soils and Sediments. 17 (3) 717-730 Amstaetter, K., Eek, E., and Cornelissen, G. (2012). 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Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3PrwrCUBTH8XMRloarW9FXuCCYZD6Cr3CGYBMWFwyOwTWIPoOPMN/gzgNaJlbDwix2i2gRN//U7UbB+02/cD7hAOh0P5jVACbxNRthjkGvnjgRwIewiOfpqJ5wWZD3ZMI5iY0C2Zlc5pPMjQ+JCNCQYM3mWE2oILg9D2OZiCOaGdjpPlYgBg15EhbEPgO3x9Wk+yKPghATPnKqJ52SeIJcvmUCEBWIExm+9JaETsoiG+XIrP3FGtD6dLtSv3mg5HJ/9FrWbFFNvnkr+R6m0nlZvz1VvtXpdLp/6wn97FYPExd9qgAAAABJRU5ErkJggg==","orcid":"","institution":"Hubei Academy of Agricultural Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2022-10-18 02:13:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2176911/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2176911/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29791613,"identity":"c336acad-e772-4fd3-9cf7-4b9ac4c667fc","added_by":"auto","created_at":"2022-12-01 20:20:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92771,"visible":true,"origin":"","legend":"\u003cp\u003eThe FTIR results of different biochar (A:Re, B:Ty,C:Ca)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/5154ff31a560c556a26c0939.png"},{"id":29791615,"identity":"b234ee46-b041-47db-ab67-beb91d7acb64","added_by":"auto","created_at":"2022-12-01 20:20:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68350,"visible":true,"origin":"","legend":"\u003cp\u003eThe XRD results of different biochar (A:Re, B:Ty,C:Ca)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/1e5b981dd5b6cc55d57f2cd1.png"},{"id":29791791,"identity":"83c96b7a-bb16-4b5d-a0a5-fd15b3b3e03f","added_by":"auto","created_at":"2022-12-01 20:28:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":967526,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM results of Re (A:400℃, B: 500℃,C: 600℃)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/a331ce3eb121e82275b0d9fe.png"},{"id":29791617,"identity":"7b338c41-6040-4780-87a6-e6cd6874e2ae","added_by":"auto","created_at":"2022-12-01 20:20:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1043795,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM results of Ty (A:400℃, B: 500℃, C: 600℃)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/b2d89e367398e5c4ee042a28.png"},{"id":29791619,"identity":"cf3eed0a-477f-4894-9699-c876c32b5208","added_by":"auto","created_at":"2022-12-01 20:20:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":959089,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM results of Ca (A:400℃, B: 500℃, C: 600℃)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/0ee64fc835b0a0f9ca0b0303.png"},{"id":29791620,"identity":"f16d4e81-a50e-4bb6-ba35-0c890a7f38b9","added_by":"auto","created_at":"2022-12-01 20:20:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14377,"visible":true,"origin":"","legend":"\u003cp\u003eImmobilization performance of Cd by different biochar\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/7f11a2978e2c9d4b7eef96e7.png"},{"id":29791624,"identity":"37fac2e9-52fa-43ea-adf5-66ce04899f19","added_by":"auto","created_at":"2022-12-01 20:20:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10657,"visible":true,"origin":"","legend":"\u003cp\u003eImmobilization performance of Cd by combined system\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/c779972942c6fc0c112810e9.png"},{"id":29791614,"identity":"0c5887a5-c42a-4df7-9797-ccc47f459f0b","added_by":"auto","created_at":"2022-12-01 20:20:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":15107,"visible":true,"origin":"","legend":"\u003cp\u003eThe biomass and root of amaranth by combined system\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/056c7c3cf86c87068b6abdd0.png"},{"id":29791628,"identity":"3141ebf9-cb83-443e-8f5b-50a8deca4df9","added_by":"auto","created_at":"2022-12-01 20:20:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":61868,"visible":true,"origin":"","legend":"\u003cp\u003eThe Cd content in different part of amaranth\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/daead115e5fec38670f26366.png"},{"id":29791623,"identity":"b46bd816-b52b-4d66-bf9e-b67e239ad6a2","added_by":"auto","created_at":"2022-12-01 20:20:45","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":90644,"visible":true,"origin":"","legend":"\u003cp\u003eThe antioxidant enzymes of different groups\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/e1c7e3abb09a31276ad14820.png"},{"id":29791621,"identity":"5a9960f9-d13f-490a-883f-75257ebede66","added_by":"auto","created_at":"2022-12-01 20:20:45","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":7483,"visible":true,"origin":"","legend":"\u003cp\u003ePCoA plot based on bray-curtis distances\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/9f7bd3534c05793d9f901a3b.png"},{"id":29791629,"identity":"6d68760f-c385-4153-93e0-e692f49c6960","added_by":"auto","created_at":"2022-12-01 20:20:48","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":35148,"visible":true,"origin":"","legend":"\u003cp\u003eThe diagram of soil microbial abundance\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/86bd7e6fe0864a7eab06e94a.png"},{"id":29791627,"identity":"f408ae8b-6426-40de-ad60-aade052188e2","added_by":"auto","created_at":"2022-12-01 20:20:46","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":22345,"visible":true,"origin":"","legend":"\u003cp\u003eResult classification diagram of KEGG pathway\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/d495146470ad81dc824b27f6.png"},{"id":29791792,"identity":"7bc75172-9a42-463c-b662-513f89e98673","added_by":"auto","created_at":"2022-12-01 20:28:45","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":39791,"visible":true,"origin":"","legend":"\u003cp\u003eResult classification diagram of GO term\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/ccd090179f94380d80852868.png"},{"id":29791616,"identity":"ad0d78ec-e6e8-425d-8dd0-3390ae3c8437","added_by":"auto","created_at":"2022-12-01 20:20:43","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":26803,"visible":true,"origin":"","legend":"\u003cp\u003eA:Density distribution of transcript expression (A: blank, B: control, C: AMF+BC, D: AMF );B: PCoA result of transcript expression ( Group1：AMF+BC；Group2：AMF )\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/9a75e52176a72a63dd6fd320.png"},{"id":30472460,"identity":"ce5b2573-56b2-4381-ab37-ff796dfe0df4","added_by":"auto","created_at":"2022-12-17 20:35:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5094739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/cd08a9a9-f1c4-4cb1-8cf9-dbdb6d7e2f4a.pdf"},{"id":29791630,"identity":"1de65afa-53d6-4271-b9d0-06c750db72be","added_by":"auto","created_at":"2022-12-01 20:20:48","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":268923,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2176911/v1/565ea0de1f5dfff22939a4a0.docx"}],"financialInterests":"","formattedTitle":"Application of wetland waste plant biochar in combination with arbuscular mycorrhizal fungi on immobilization of Cd in contaminated soil","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWith the growing shortage of global resources, together with the industrial production and various chemical products application, heavy metals have entered the environment in various ways(Honma et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Over ten million polluted sites were can be found worldwide, and more than 50% of these sites contain heavy metals(Tunēns et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Most of these polluted sites are found in farmland. From 2015 to 2020 in China, researchers found that 21.49% of the samples collected from sites across 3000 surveyed areas were polluted, which was approximately 5% higher than in 2014. The copper pollution in Hunan, Guangxi province is nearly two times higher than background values; the lead content in Guanxi and Sichuan Provinces is about 2.5 times higher than background values; the cadmium content in most provinces is more than double the background values, and in some area is even ten times higher(Bank, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These heavy metals contaminate sediments and are the main long-term source in the soil(Chlopecka\u0026amp;Adriano, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Cao\u0026amp;Harris, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), Especially copper, lead, and cadmium. Therefore, controlling heavy metal pollution and reducing its impact on agricultural products has the potential to minimize the food safety risk and improve the utilization of farmland(Cao et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are two methods known to control the soil pollution from heavy metals: one is to change the existing forms of heavy metals in the soil to reduce their migration and bioavailability in the environment(Shaheen\u0026amp;Rinklebe, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); the other is to remove heavy metals from the soil(Ahmad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Regarding these methods, researchers suggest physical, chemical, and biological treatment methods. The conventional methods employed includes precipitation, ion extraction, dilution, and planting enriched plants(Abel et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Amstaetter et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Bagreev et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Beckingham et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, these methods usually have some limitations to their practical application like high costs, difficult operation, and possible secondary pollution to the natural environment(Ar\u0026aacute;n et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Recently, many studies proved that biochar had a certain immobilizing ability for heavy metals in the soil due to its available pore structure, large surface area, and high pH(Bandara et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Libralato et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Goswami et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Biochar immobilization of the heavy metals in soil is done by reducing the bioavailability of heavy metal through methods like cation exchange, complexation(Jia et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the pyrolytic temperature and feedstock of biochar has been reported to have a large effect on its properties(Goswami et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Researchers speculate that higher temperature results in a higher pH and larger surface area of the biochar, and higher heavy metals adsorption ability(Gong et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Niazi et al. reported that biochar produced from perilla leaf at 700 ℃ removed more As at pH 7\u0026ndash;9 than at 300 ℃(Niazi et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, Shen et al. found that at higher temperatures, the surface area of the biochar decreased, and contributed to the lower Cr(VI) removal ability(Shen et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, there is a lack of studies concerning the pyrolytic temperature-mediated immobilization effect of biochar in soil polluted by different kinds of heavy metals(Chen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is important to conduct studies regarding the pyrolytic temperature effect of biochar on the immobilization of different kinds of heavy metals. Such studies will promote the application of biochar in heavy metal polluted soil treatment; however, excessive use of biochar could be harmful to plants due to the high pH. Combining biochar with other strategies has become a new method of soil remediation(Gul et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMycorrhizal fungi are closely associated with plants and can reduce the mobility of heavy metals and improve the plants resistance to heavy metals(Cao\u0026amp;Harris, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). A number of studies have proved that microorganisms have strong adsorption on a variety of heavy metals, including Cd, and can significantly reduce their mobility. As a common soil microorganism, arbuscular mycorrhiza is a parasitic fungus and has a close relationship with plant roots, can promote plant growth, improve plant resistance to heavy metals, and reduce heavy metal transfer to the upper part of plants(Xu et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, when the concentration of pollutants in the soil is too high or the environmental conditions of the soil are not suitable for microbial growth, the repair efficiency of arbuscular mycorrhizal is significantly reduced and the repair cycle will be prolonged(Jeffery et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Biochar can supply a good protection to arbuscular mycorrhizal since its large pore size and carbon source can provide an ideal growth environment for arbuscular mycorrhiza, greatly improving its remediation efficiency. Thus, biochar combined with arbuscular mycorrhiza can be a promising way to solve soil pollution by heavy metals(Liu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, biochar pyrolyzed from wetland waste plants (\u003cem\u003eReed\u003c/em\u003e (Re), \u003cem\u003eTypha\u003c/em\u003e (Ty), \u003cem\u003eCanna\u003c/em\u003e (Ca)) at temperatures of 400, 500, and 600 ℃. These productions were employed as representatives to evaluate the immobilization ability on Cd contaminated soil by conducting comprehensive studies. The effects of different biochar on Cd immobilization and its potential mechanism in the soil were also studied by distribution of heavy metals fraction in soil. Based on these experiments, the best biochar and arbuscular mycorrhiza combination was used to study the solidification properties on Cd in soil. Meanwhile, pot experiments were used to explore the effects of the combined system on the accumulation of heavy metals in plants, antioxidant indicators, and soil microorganisms.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Soil samples and reagents\u003c/h2\u003e \u003cp\u003eThe incubation soil experiments were divided into two parts due to the amount of time needed. The first part was dedicated to analyzing the immobilization ability of different biochar. The second part functioned to explore the best conditions for biochar application. Soil samples for incubation experiments were obtained from Fuzhou, Jiangxi Province (Fz) with little heavy metal polluted soil. Fz samples were taken to the laboratory, air-dried, homogenized, and sifted with a 2 mm sieve. Deionized water was used to keep the soil moisture content at 70%. Then the samples were aged for 30 days at room temperature. Samples for part two were collected from a farmland in Xiangjiang Reiver (Xj). Heavy metals were analyzed by ICP optical emission spectroscopy (ICP-MS; PerkinElmer NexION 300X, USA) after soil digestion via HF\u0026thinsp;+\u0026thinsp;HNO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The heavy metals concentration of Fz and Xj are shown in the supporting information.\u003c/p\u003e \u003cp\u003eThe distribution of heavy metals was determined using glacial acetic acid (HOAc), sodium hydroxide (NaOH), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), ammonium acetate (NH\u003csub\u003e4\u003c/sub\u003eOAc), sodium acetate (NaOAc), nitric acid (HNO\u003csub\u003e3\u003c/sub\u003e), calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e), and hydroxylammonium chloride (OH-NH\u003csub\u003e2\u003c/sub\u003e\u0026middot;HCl). The Cd soil was prepared using cadmium nitrate Cd(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. All the reagents were purchased from CNW (Shanghai, China) and GL Biochem Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of biochar\u003c/h2\u003e \u003cp\u003eBiochar was pyrolyzed from wetland waste plants (\u003cem\u003eReed\u003c/em\u003e (Re), \u003cem\u003eTypha\u003c/em\u003e (Ty), \u003cem\u003eCanna\u003c/em\u003e (Ca)) from the experiment base (Wuhan, Hubei Province; Institute of Hydrobiology, Chinese Academy of Sciences). Each sample was air-dried, crushed, and sifted with 40 mesh sieves. The temperatures of the pyrolysis process were 400, 500, and 600 ℃ (each biochar from here referred to as Re400, Re500, Re600, Ty400, Ty500, Ty600, Ca400, Ca500, and Ca600). The biochar was under a continuous stream of N for 4 h to keep the temperature constant. At last, biochar was crushed and passed through 100 mesh sieves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization and analysis methods\u003c/h2\u003e \u003cp\u003eFourier transform infrared spectroscopy of the biochar was obtained using Shimadzu-IRaffinity-1 equipment (Shimadzu Corporation, Tokyo, Japan) along with an ATR module (FTIR-ATR), under a nitrogen purge. Measurements were recorded in the range 500\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with 4.0 resolution, obtaining 256 scans while applying atmosphere and background correction.\u003c/p\u003e \u003cp\u003eX-ray diffraction (XRD, 5\u0026ndash;70 \u0026deg;) was measured on a D8-Focus X-ray diffractometer (Bruker Optics, Germany) with a test rate of 10 \u0026deg;ꞏmin\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The results were analyzed by using the Jade version (9.0) and Origin version (9.0) programs.\u003c/p\u003e \u003cp\u003eThe specific surface area( S\u003csub\u003e\u003cem\u003eBET\u003c/em\u003e\u003c/sub\u003e )and micropore volume (W\u003csub\u003e0\u003c/sub\u003e) were determined by nitrogen adsorption isotherms, acquired at 77 K using a Micromeritics Instrument, Gemini VII 2380 (USA), after outgassing the materials overnight at 120 \u0026ordm;C. S\u003csub\u003e\u003cem\u003eBET\u003c/em\u003e\u003c/sub\u003e was calculated from the Brunauer\u0026ndash;Emmett\u0026ndash;Teller equation under the relative pressure range 0.01\u0026ndash;0.1. Pore volume (Vp) was estimated from the amount of nitrogen adsorbed at a relative pressure of 0.99.\u003c/p\u003e \u003cp\u003eThe surface morphology of the materials was analyzed using scanning electron microscopy (SEM; Hitachi S4100, Japan). The images were obtained at magnifications of 800 and 5000.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Incubation soil experiment\u003c/h2\u003e \u003cp\u003eTo analyze the immobilization of each biochar, each biochar was applied to 500 g of Cd-contaminated soil (from Fz). Heavy metal contaminated soil without biochar was used as the control experiment (CK) and three groups of parallel experiments were set per process. Each biochar treatment was done in triplicate.\u003c/p\u003e \u003cp\u003eTo explore the relationship between time and distribution of heavy metals, and to investigate the effect of different biochar weights, Ca500 was applied to 500 g of soil from Xj. The time of contact was 14 weeks. The samples of soil were removed and Cd determined at 2, 6, 10, and 14 weeks. Biochar was applied at 10, 20, 30, 40, and 50 g (2, 4, 6, 8, and 10%, respectively). The amount of heavy metal was determined by ICP-MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Experiment of arbuscular mycorrhizal infection\u003c/h2\u003e \u003cp\u003eArbuscular mycorrhiza (\u003cem\u003eG. etunicatum\u003c/em\u003e, \u003cem\u003eG. intraradices\u003c/em\u003e, and \u003cem\u003eG. mosseae\u003c/em\u003e) was purchased from the Institute of Nutrition and Resources, Beijing Academy of Agricultural and Forestry Sciences. The infection rate of arbuscular mycorrhiza on the roots of Chinese cabbage and amaranth was studied using the acetic acid ink method. To start, 20% KOH solution (v:v) was added to the cleaned 1\u0026ndash;1.5 cm root segment. The segment was heated in a water bath for 60 min at 60 ℃. It was then removed and washed under running water for 5 min, then soaked in acetic acid for five min. An acid ink solution (5% solution, acetic acid: ink\u0026thinsp;=\u0026thinsp;95 ml:5 ml) was used for dyeing in a 60 ℃ water bath for 30 min. After the water bath, the segments were soaked in water for 12 h to decolorize. Sudan IV staining solution (3 g Sudan IV solid plus 1000 ml of 70% ethanol solution) was used for re-staining in a 60 ℃ water bath for 60 min. Segments were then rinsed with running water for 3 min, and finally soaked in 70% ethanol solution for 5 min. The dyed root segments were observed under an optical microscope and the proportion of dyed fibrous roots was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Pot experiment on the combination of biochar and arbuscular mycorrhiza\u003c/h2\u003e \u003cp\u003eBefore the start of the experiment, the bacterial agent was mixed with the soil (20 g bacterial agent: 1kg soil) and the soil moisture was determined after a week when the field water holding rate was about 60%. This was to ensure the smooth germination of arbuscular mycorrhizal spores. The experiment comprised three groups: CK (without adding biochar and arbuscular mycorrhiza), AMF (adding arbuscular mycorrhiza fungi alone), and AMF\u0026thinsp;+\u0026thinsp;BC (adding arbuscular mycorrhiza fungi and Ca500), and each group was done in triplicate. In the pre-treatment before planting, biochar was added to the soil in a ratio of 2% and mixed evenly. Compound chemical fertilizer was added in a ratio of 0.3 g fertilizer to ꞏ1kg soil to ensure the normal plant growth. After one day of pretreatment, several vegetable (Chinese cabbage and amaranth ) seeds were added to each container. after Seven days later, seedlings were thinned down to ten seedlings in each incubator (50 cm long, 30 cm wide, and 20 cm high plastic planting pots were used for this purpose). The culture period was 42 days. After harvest, Cd content in soil, plant biomass, Cd content in different parts of the plant and antioxidant indices (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA)) were determined. The bioconcentration factor (BF) and transfer factor (TF) were then calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. DNA analysis of soil microbial samples\u003c/h2\u003e \u003cp\u003eSoil samples were collected from the experimental soil after the vegetables were harvested in the pot experiment involving combining biochar and arbuscular mycorrhiza. A soil sample without treatment was collected as the blank group. The samples were stored a at -20\u0026deg;C until use. The experimental process was implemented in accordance with the standard protocol provided by Illumina, including sample quality detection, library construction, library quality detection, library sequencing, and other processes. The specific operations were as follows: after the genomic DNA extracted by CTAB method was tested and qualified, the DNA was fragmented by mechanical interruption (ultrasound). The fragmented DNA was purified, the end was repaired and the 3\u0026prime; end was added, and a sequencing connector was connected. The fragment size was selected by agarose gel electrophoresis. Sequencing Library (nebnext \u0026reg; Ultra \u0026trade; DNALibraryPrepKitforIllumina \u0026reg;) were amplified by PCR according to the manufacturer's instructions. The established libraries were inspected first, and the qualified libraries were sequenced using Illumina NovaSeq.\u0026nbsp;After sequencing, MEGAHIT assembly software was used for analysis (version 1.2.9), and the default parameters of MEGAHIT (generated contigs N50 with high quality) were selected for assembly to obtain the sample contigs. Prodigal was used to predict the gene of the assembly results. After the prediction was completed, CD-HIT (version 4.6) was used to remove the redundant annotation genes. The longer one in each class was selected as a unique gene. The similarity threshold and coverage threshold were set to 95% and 90%, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Results and discussion\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Characterization of different biochar\u003c/h2\u003e \u003cp\u003eFTIR spectrometry of the different biochar were used to shed light on the structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our results showed that regardless of which wetland plant was observed, they did not appear or reduce any functional group, but the temperatures of the pyrolysis process influenced the peaks. The peaks in the range of 3800\u0026ndash;3516 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were mainly caused by the \u0026ndash;OH stretching vibration associated with intermolecular hydrogen bonding(Ghani et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). As the temperature increased, the intensity of the peaks for Ty and Ca decreased and Re increased. This may be because Re is a monocts while Ty and Ca are dicots. The stems of grass plants are more fragile than those of herbs(Igalavithana et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, during the pyrolysis, it could have been easier for Re to lose the \u0026ndash;OH group due to dehydration and the dehydrogenation reactions. The peaks at around 2927 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2877 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent asymmetric and symmetric vibration adsorption of lipoid (-CH\u003csub\u003e2\u003c/sub\u003e), respectively(Zhai et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). There are no differences in our results, which may be because the peaks observed belong to carbon dioxide. The peaks at 1621 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were identified as C\u0026thinsp;=\u0026thinsp;C. Our results reflected that these peaks shifted to a blue wavelength in the high temperature tests, suggesting that carbonization is better at high temperature(Gong et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The other peaks in the spectra could be assigned to C\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(-\\)\u003c/span\u003e\u003c/span\u003eO\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(-\\)\u003c/span\u003e\u003c/span\u003e(1099 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CO\u003csub\u003e3\u003c/sub\u003e (1430 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and C\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(-\\)\u003c/span\u003e\u003c/span\u003eH (748 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as their positions did not obviously change(Graber et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These results suggested that herbaceous plants were more stable than grass and the production temperature of the biochar used had a great influence on their surface functional group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe S\u003csub\u003e\u003cem\u003eBET\u003c/em\u003e\u003c/sub\u003e results are shown in Table\u0026nbsp;1. The S\u003csub\u003e\u003cem\u003eBET\u003c/em\u003e\u003c/sub\u003e of each biochar was as follows: Re:1.05\u0026ndash;69.7 m\u003csup\u003e2\u003c/sup\u003eꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Ca: 1.46\u0026ndash;87.3 m\u003csup\u003e2\u003c/sup\u003eꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Ty: 1.39\u0026ndash;5.49 m\u003csup\u003e2\u003c/sup\u003eꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, similar S\u003csub\u003e\u003cem\u003eBET\u003c/em\u003e\u003c/sub\u003e values were determined for biochar in other studies. Our results suggested that with higher pyrolysis temperatures, the surface area was larger and the volume of the pores was smaller. This might be contributed to volatile substance release and appearance of vascular bundle structures at high temperatures(Lu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). From Table\u0026nbsp;1, we observed that in comparing these feedstocks that Ca had a larger surface area and pore size, supporting the finding that Ca had a better immobilization capacity comparing to Re and Ty. Therefore, the pyrolysis temperature is an important factor for biochar since biochar produced at higher temperature has a larger surface area, which influences its bonding to heavy metals(Lyu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, herbaceous plant biochar had a larger surface area than grass biochar. Consequently, it is important to select suitable pyrolysis temperatures and feedstock before applying the biochar.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.1\u003c/b\u003e Physical characterization of the biochars\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiochars\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS\u003c/em\u003e\u003csub\u003eBET\u003c/sub\u003e (m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e (cm\u003csup\u003e3\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eD\u003c/em\u003e (nm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTy-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTy-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTy-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e69.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e87.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRe-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRe-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRe-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003eThe XRD spectrum of different biochar in the range of 20 to 75 \u0026deg; is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, all biochar samples presented a broad diffraction peak at 23\u0026ndash;25 \u0026deg;, belonging to the amorphous carbon property(Bashir et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). Moreover, our findings suggested that at higher temperatures, the observed carbon peak and content of carbon would be higher. The XRD results indicated that the ratio of crystallite carbon in biochar was mainly determined by the feedstock, and the content of carbon was higher at high temperatures (400\u0026ndash;600 ℃). The same results were found by previous studies(Zhang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe surface of the biochar was examined using SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results suggested that at lower temperature, the productions had a vascular bundle structure, due to the use of leaves of the wetland waste plants. With increased temperature, their structure changed to blocks and Ca appeared more regularly, which might be the reason why Ca had the largest surface area. There were many micropores observed on the surface and as the temperature increased, the volume of the pores became larger and new pores appeared. Compared to Re and Ty, biochar pyrolyzed from Ca showed more developed macro-porosity, since Ca reflected more pores and the structure was relatively regular and smooth. Other studies also showed that with as temperature increased, the volume of the pores became larger and their structure mainly depended on the cellular structure of original feedstock(Bandara et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Meier et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Immobilization performance of Re, Ty, and Ca\u003c/h2\u003e \u003cp\u003eThe speciation of Cd in soil was analyzed by sequential extraction procedures proposed by Tessier. According to this method, The distribution of Cd in different states: exchangeable (Ex), bound to carbonates (Bc), bound to iron and manganese oxide (Bo), bound to organic matter (Bm), and residual (Re) in the soil were determined using the Tessier methods. Among those fractions of heavy metals, exchangeability, and the state of bondage to carbonates can be released in cationic solutions, and can be easily utilized by organisms or plants. The states of bound to iron, manganese oxide, and organic could be converted to exchangeable under appropriate conditions, which can be indirectly utilized by plants. The residual state is mostly stable but cannot be directly utilized by plants(Jun et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, the preliminary goal of this study is to decrease the exchangeable and CB-bound fraction. Before the soil incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), the exchangeable fraction was dominant (77.15%), followed by carbonate-bound fraction (14.92%), and the rest of fractions occupying a small proportion, especially the percentage of OM-bound fraction (2.27%). After 42 d of incubation, the proportion of exchangeable fractions in all experimental groups declined significantly and settled in range of 60.62 to 73.72% while other fractions increased. In particular, the fraction of Fe-Mn-bound increased sharply and surpassed the CB-bound fraction, reaching 8.43 to 12.42%. The \u003cem\u003eCanna\u003c/em\u003e biochar treatments showed a great ability to immobilize Cd in soil, especially the biochar prepared using 500 ℃. This may be due to the high specific surface area as well as the better ability of ion exchange. Many studies have proved that biochar can immobilize heavy metals through ion exchange. The current results were similar to those of most studies(Kiran\u0026amp;Prasad, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Conversely, all the reed biochar treatments (Re400, Re500, and Re600) showed poor immobilization performance. Compared with CK, the proportion of easily labile fractions decreased by no more than 5%. This might be because feedstock is one of the largest factors determining the immobilization ability of biochar(Lahori et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The specific surface area of the reed biochar was also noticeably lower than other tested biochar.\u003c/p\u003e \u003ch2\u003e2.11. Experimental results of arbuscular mycorrhizal infection\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe experimental results are shown in Table\u0026nbsp;2. The infection of all AMF to Chinese cabbage was very low, this may because that arbuscular mycorrhiza seldomly invades cruciferous plants. While, the infection rate of \u003cem\u003eG. intraradices\u003c/em\u003e in the root to amaranth plant is known to be most common, therefore, the arbuscular mycorrhiza selected in the pot experiment of the combination of biochar and arbuscular mycorrhiza was \u003cem\u003eG. intraradices\u003c/em\u003e, and the tested plant was amaranth.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.2\u003c/b\u003e Colonization rate of arbuscular mycorrhiza fungi (AMF) to different vegetables\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChinese cabbage (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmaranth (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eG. etunicatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eG. intraradices\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eG. mosseae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003ch2\u003e2.12. Solidification effect of combined system on soil Cd\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe difference of Cd extracted state is shown in Fig. 7. In the CK group, exchangeable Cd (62.9%), carbonate bound (1.03%), iron manganese bound (24.76%), organic bound (4.17%), and residual (7.14%). The proportion of exchangeable Cd in the AMF and AMF+BC group decreased to about 45%, and the lowest proportion of exchangeable Cd in the AMF+BC group was 42.22%. The carbonate bound state increased slightly. The proportion of residual states increased from 7.14% in the CK group to 14.41% and 15.61% in the AMF and AMF+BC group, respectively. The results showed that the use of AMF or AMF+BC has the potential to reduce the proportion of exchangeable Cd in soil, thereby effectively increasing the content of residual Cd, with good fixation performance. The main reasons for these results are due to biochar changing the form of heavy metals or adsorbing on the surface of biochar to reduce its mobility through its own cation exchange, electrostatic adsorption, and surface oxidation-reduction reaction. The glycoproteins rich in aliphatic hydrocarbons and methylene secreted by arbuscular mycorrhiza have a high affinity for metals and can effectively combine with heavy metals to form stable structures. The reason for the small difference between the AMF and AMF+BC group may be that AMF plays a major role in Cd solidification and biochar provides suitable growth conditions for arbuscular mycorrhiza.\u003c/p\u003e\u003ch2\u003e2.13.\tEffect of combined system on plants\u003c/h2\u003e\u003ch3\u003e2.13.1 Effects on biomass and root\u003c/h3\u003e \u003cp\u003eThe purpose of determining the biomass and taproot length of plants was to observe the effects of the combined system of biochar and arbuscular mycorrhiza on heavy metal migration and plant physiology in Cd-contaminated soil. The biomass and root length of amaranth are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The biomass and root length in the CK group were 0.20 g and 3.33 cm respectively. The biomass in the AMF group increased significantly, but the root length was not significant. The biomass and root length in the AMF\u0026thinsp;+\u0026thinsp;BC group increased significantly to 0.81 g and 5.11 cm respectively. Similarly, found that the use of AMF and biochar could increase the biomass, root length, root diameter, and root volume of maize, and improve its nutrient absorption. Singh et al. found that arbuscular mycorrhizal colonization in asparagus roots doubled after adding biochar(Singh et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, they showed that the addition of biochar could promote the growth of arbuscular mycorrhiza. Biochar itself is a rich carbon source, which can improve soil cation exchange capacity and redox reactions, improving the soil microbial community abundance and soil enzyme activity, enhancing soil nutrients and improving nutrient utilization400(Houben et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003ch3\u003e2.13.2 Distribution of Cd content, bioconcentration coefficient, and transfer coefficient in plants\u003c/h3\u003e \u003cp\u003eThe content of Cd in roots, stems, and leaves of plants is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. The Cd content of roots, stems, and leaves in CK group was 1.213 mgꞏkg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.642 mgꞏkg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 0.623 mgꞏkg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. This suggests that plants can easily absorb heavy metals from the environment and transfer them to their edible upper parts. Compared with CK, Cd content in roots, stems, and leaves of the AMF group decreased significantly, (15.7, 31.6, and 22.6%, respectively). When combined with biochar, the lowest content of Cd in roots, stems, and leaves were recorded (0.953 mgꞏkg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.243 mgꞏkg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 0.28 mgꞏkg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively). It can be seen from the transfer coefficient that the proportion of Cd transferred from the underground root to the upper part of the plant has greatly decreased and Cd tends to accumulate in the roots (Table.S1). Guo Xiongfei explored the effect of biochar and arbuscular mycorrhizal application on the absorption of heavy metals in \u003cem\u003eCassia occidentalis\u003c/em\u003e(Houben et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The results showed that the use of a combined system could reduce the absorption of heavy metals in plants, promote the accumulation of heavy metals in their roots, and reduce the concentration and transfer coefficient of heavy metals in stems and leaves above the ground. A similar conclusion was also made in this study, possibly because the addition of biochar increased the infection rate of arbuscular mycorrhiza. The negative ions such as hydroxyl and carboxyl groups on the mycelium surface of arbuscular mycorrhiza combine easily with heavy metal cations, resulting in a large number of heavy metals being fixed to the root system. In addition, it can promote the increase of woodiness of plant root cell wall and root branching, change the root structure, and enhance the ability of plant root to combine heavy metals(Avio et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003ch3\u003e2.13.3 Antioxidant activity of plants\u003c/h3\u003e \u003cp\u003eAfter absorbing excessive heavy metals and other harmful substances, plants will display a variety of adaptive biochemical reactions, among which the most significant changes are the changes of antioxidant enzymes (SOD, POD, and CAT) and malondialdehyde (MDA). Therefore, measuring the antioxidant indices in different treatment groups can clearly show the health status of plants under heavy metal stress. The contents of SOD, POD, CAT, and MDA in plants are shown in Fig.\u0026nbsp;10.\u003c/p\u003e \u003cp\u003eSOD is a ubiquitous antioxidant metal enzyme in organisms which can catalyze superoxide free radicals to produce hydrogen peroxide and oxygen(Bashir et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e). It plays a vital role in the antioxidant balance of the plants and is positively correlated with the stress resistance of plants(Cui et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this study, SOD enzyme activity of amaranth in the CK group was 604 Uꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh tissue weight. SOD activity in the AMF and AMF\u0026thinsp;+\u0026thinsp;BC group increased by 12.9 and 2.9%, respectively, compared with CK. SOD activity in the AMF\u0026thinsp;+\u0026thinsp;BC group was lower than that the AMF group. This is similar to Elmer W H research results(Elmer\u0026amp;Pignatello, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). After adding biochar, the SOD content of ryegrass did not change significantly but the SOD in alfalfa increased significantly. The reason may be that the root nodules of leguminous plants can more effectively combine with biochar, enhancing the SOD activity in plants. The effect on plants without root nodules is not as obvious(Li et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The addition of biochar can significantly change the physical and chemical properties of soil, especially the pH value. SOD in plants is sensitive to soil pH value, which may affect the activity of SOD in vegetables to a certain extent.\u003c/p\u003e \u003cp\u003ePOD is an enzyme with hydrogen peroxide and phenolic amines as substrates and is an important part of the body's antioxidant system. In this study, POD activity in the CK group was 777 Uꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh tissue weight. The AMF and AMF\u0026thinsp;+\u0026thinsp;BC group increased by 11.1 and 27.5%, respectively, compared with the CK group, and POD enzyme activity was 863 Uꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 991 Uꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh tissue weight, respectively. The results showed that arbuscular mycorrhiza could improve the POD activity of vegetables under Cd stress, and the effect was greater when combined with biochar. Li et al. found that the POD activity of \u003cem\u003eSuaeda salsa\u003c/em\u003e under salt stress increased significantly after adding arbuscular mycorrhiza. Harindintwali J D et al. found that the combination of biochar and arbuscular mycorrhiza can significantly increase plant POD activity but the effect of adding biochar has different results(Harindintwali et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCAT is an enzyme that catalyzes the decomposition of hydrogen peroxide into water and oxygen in cells and is the enzyme in the final stage of the body's antioxidant system. In this study, CAT activity in CK group was 69.9 Uꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tissue fresh weight, and CAT activity in AMF group was 67.3 Uꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tissue fresh weight, which decreased by 3.5%. Similarly, Kang et al found that the CAT activity of alfalfa was significantly reduced after inoculation with arbuscular mycorrhiza, which may be due to the intervention of arbuscular mycorrhiza reducing the chemical stimulation effect of antioxidant enzymes(Kang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, when AMF\u0026thinsp;+\u0026thinsp;BC were combined, CAT activity reached 125.2 Uꞏg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tissue fresh weight, an increase of 79.1% compared with CK.\u003c/p\u003e \u003cp\u003eResults show that the combination of biochar and arbuscular mycorrhiza can greatly improve CAT activity in vegetables; it may be that the biochar can significantly improve the soil environment, promote the growth of arbuscular mycorrhiza, indirectly promote the growth of plants, enhance resistance of plants, and improve the antioxidant enzymes of heavy metals by plants, leading to increased CAT activity. In addition, Igalavithana et al. believe that CAT is more sensitive to biochar than POD and SOD, which may also be the reason why CAT activity changes more than the other two antioxidant enzymes(Igalavithana et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, some scholars believe that POD and CAT show a complementary relationship in plants, but this phenomenon was not observed in this study. The specific reasons and the action mechanism of CAT needs to be examined further(Graber et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMDA is an important index reflecting the level of membrane lipid peroxidation in plants. The increase of membrane lipid peroxidation is usually caused by environmental damage, which leads to the increase of reactive oxygen content and the destruction of cell membrane structures, thus inhibiting plant growth. In this study, the MDA content of plants in the CK group was 157.6 nmolꞏg-1, while MDA content in the AMF and AMF\u0026thinsp;+\u0026thinsp;BC group decreased from 26.7 and 47.3%, to 115.4 nmolꞏg-1 and 83.1 nmolꞏg-1, respectively. This indicates that the combination of arbuscular mycorrhiza and biochar could reduce the level of membrane lipid peroxidation and damage to plants. These results support the findings that CAT and POD enzyme activity changes. This may be because biochar can transform the exchangeable Cd in soil into its stable residual state and reduce the amount of Cd transported by the cell membranes, allowing more Cd to accumulate in AMF, thus reducing the level of leaf membrane lipid peroxidation(Bashir et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e). \u003c/p\u003e \u003ch2\u003e2.14. Analysis of soil microbial community\u003c/h2\u003e \u003ch3\u003e2.14.1. Composition and analysis of species diversity\u003c/h3\u003e \u003cp\u003eIn this study, Karken2\u0026thinsp;+\u0026thinsp;Bracken was used for species annotation, and clean reads were compared directly with marker gene sets. Bacteria, archaea, eukaryotes, and viruses in the samples were accurately annotated to the species level based on a metagenomic sample bank. The comparison results in species classification are shown in Table.S2.\u003c/p\u003e \u003cp\u003ePrincipal coordinate analysis (PCA) results for community β-diversity based on Bray\u0026ndash;Curtis distance are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003e. The first two PCA ordination axes explain 68 and 29% of the β-diversity difference, respectively. The first axis distinguishes the blank group from the soil samples of other plants, and the second axis distinguishes the experimental group with biochar from the other two groups. The results indicate that arbuscular mycorrhizal combined with biochar could significantly change the microbial community in soil β-diversity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relative abundance of species in various samples is shown in Fig. S.12. At the phylum level, except for the blank group, the group with the highest abundance in all the samples was Proteobacteria. The other dominant groups were Actinobacteria and Chordata. At the genus level, the abundance of \u003cem\u003eChlamydia\u003c/em\u003e in the AMF\u0026thinsp;+\u0026thinsp;BC group was much lower than that in the CK and AMF groups, indicating that the addition of biochar could inhibit the growth of this group. \u003cem\u003eStreptomyces\u003c/em\u003e was the dominant group with the highest relative abundance in CK and the two experimental groups. The other dominant groups were \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eChlamydia\u003c/em\u003e. Compared with CK, the relative abundance of \u003cem\u003eBradyrhizobium\u003c/em\u003e and \u003cem\u003eMicromonospora\u003c/em\u003e in the two experimental groups decreased. The relative abundance of \u003cem\u003eVariovorax\u003c/em\u003e, \u003cem\u003eSphingopyxis\u003c/em\u003e, \u003cem\u003eAzoarcus\u003c/em\u003e, and \u003cem\u003eHydrogenophaga\u003c/em\u003e increased, and the proportion in the AMF\u0026thinsp;+\u0026thinsp;BC group was higher. Some studies have shown that \u003cem\u003ePseudomonas\u003c/em\u003e can regulate auxin concentrations to maintain plant root health and promote plant growth. \u003cem\u003eAzotobacter\u003c/em\u003e and \u003cem\u003eHydrogenophaga\u003c/em\u003e can improve the utilization of soil nutrients. In addition, studies have shown that \u003cem\u003eSphingomonas\u003c/em\u003e can degrade a variety of heavy metals and organic compounds including Cd. The results indicated that the addition of arbuscular mycorrhiza could increase the number of microorganisms related to soil fertility enhancement and heavy metal degradation, and the effect was significant when combined with biochar.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the number and abundance of species in the sample α-diversity index (Table\u0026nbsp;3) indicated that the Berger, Parker, Simpson, and Shannon index were lower in the two experimental groups than the control group. Berger and Parker index indicate that the proportion of dominant microbial species in the soil has decreased after inoculating arbuscular mycorrhiza. Simpson and Shannon index indicate that the microbial diversity in the soil has decreased after inoculating arbuscular mycorrhiza. This is consistent with the results of other studies. The reason may be that the addition of arbuscular mycorrhiza can promote the growth of microorganisms closely related to arbuscular mycorrhiza, enhancing their competitive ability and inhibiting the growth of other microorganisms(Lu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The Chao 1 index was the largest in AMF\u0026thinsp;+\u0026thinsp;BC group, indicating that the number of species in this group was the largest.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.3\u003c/b\u003e The α-diversity index of community\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBerger-parker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003echao1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003esimpson\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eshannon_e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0434\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMF\u0026thinsp;+\u0026thinsp;BC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cbr\u003e\u003ch3\u003e2.14.2. Gene function annotation\u003c/h3\u003e\u003cp\u003eKEGG is a major public database for the systematic analysis of the pathways of gene products in cells and the functions of these gene products, which are used to study the complex biological behavior of genes(Ma et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). After the KEGG annotation, genes were classified according to the KEGG metabolic pathway they participated in, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e13\u003c/span\u003e. It can be seen from the figure that there were 19,8443 functional genes annotated in the four samples. The metabolic pathways include four cell process pathways, three environmental information processing pathways, four gene information processing pathways, 12 metabolic pathways, and nine systemic pathways. The current three pathways of gene number in biological process are DNA transcription regulation, phosphorylation signal transduction system and methylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e13\u003c/span\u003e). At present, the three pathways of gene number in cell components are membrane component, cytoplasm, and plasma membrane and the three pathways of gene number in molecular function are ATP synthesis, metal ion binding, and hydrolase activity(Ren et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Gene Ontology (GO) results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e14\u003c/span\u003e. It can be seen from the figure that the current three pathways of gene number in biological process are DNA transcription regulation, phosphorylation signal transduction system and methylation; At present, the three pathways of gene number in cell components are membrane component, cytoplasm and plasma membrane(Singh et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003ch3\u003e2.14.3. Gene abundance estimation and difference analysis\u003c/h3\u003e \u003cp\u003eThe gene abundance density distribution map after standardization and PCA result of transcript expression is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e15\u003c/span\u003e. According to the figure (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e15\u003c/span\u003eA), the gene abundance of soil treated with biochar and arbuscular mycorrhiza was slightly higher than that of CK and arbuscular mycorrhiza treatment group alone and was significantly higher than that of the blank group. In addition, it can be seen from the result Fig. S.15 of principal component analysis (PCA) on gene data of different samples. The first two PCA axes accounted for 50 and 26% of the gene differences in different samples, indicating that there were significant differences between different samples. It can also be seen that the gene composition of the two experimental groups (AMF, AMF\u0026thinsp;+\u0026thinsp;BC) treated with arbuscular mycorrhizal fungi was very similar. There was a significant difference in gene composition between the control group and the blank group. It may be that the experimental groups all used arbuscular mycorrhizal as a remediation agent, which had similar effects on the soil microbial communities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe GO term enrichment analysis results are shown in Fig.S3. Compared with CK, the significant metabolic pathways of AMF were macromolecular biosynthesis and cyanobacterin synthase activity (Q\u0026thinsp;\u0026lt;\u0026thinsp;0.05), followed by phosphotransferase activity (Q\u0026thinsp;\u0026lt;\u0026thinsp;0.1). The results showed that adding arbuscular mycorrhiza to soil could promote soil microbial activity, produce nutrients, and improve soil use value. The comparison of AMF\u0026thinsp;+\u0026thinsp;BC and CK indicates that the Q value of the first 20 metabolic processes with significant differences is 0.12, indicating that the combination of biochar and arbuscular mycorrhiza can significantly change the gene expression in soil(Ren et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The metabolic pathways with the largest number of differential genes were abnormal proteolysis, RNA helicase activity, cell response to heat, and sulfate transmembrane transport ATP hydrolase activity(Singh et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The results showed that the resistance of the soil microbial community to external stress was improved after adding biochar. When AMF\u0026thinsp;+\u0026thinsp;BC was compared with AMF, the significant gene expression item was ethanol metabolism (Q\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Macromolecule biosynthesis, virus, and cyanobacteria synthase activity were the next (Q\u0026thinsp;=\u0026thinsp;0.055). The number of differential genes in bacterial flagella assembly was the largest.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eBiochar pyrolyzed from wetland waste plants: \u003cem\u003eReed\u003c/em\u003e, \u003cem\u003eTypha\u003c/em\u003e, and \u003cem\u003eCanna\u003c/em\u003e were employed to comparatively evaluate the immobilization performance on heavy metal-contaminated soil. The results showed that biochar could all immobilize the Cd. Biochar with different feedstocks could be ranked in the following order based on comprehensive immobilization performance: Ca༞Ty༞Re, the best weight and temperature of pyrolysis process was 6 and 500 ℃, respectively. The combination of biochar and arbuscular mycorrhiza can significantly reduce the content of Cd transportable states, the mobility of Cd, and increase the proportion of residual Cd states. Furthermore, this can also improve the biomass and root length of vegetables, reduce the absorption of Cd by vegetables, and enhance the ability of vegetables to transfer Cd from the roots to upper parts, reducing the edible safety risk. Moreover, the combination of biochar and arbuscular mycorrhiza could effectively improve the antioxidant capacity of vegetables and their resistance to heavy metals. Soil microbial community composition, soil microbial abundance, and β-diversity was optimized by its presence. Functional gene expression, soil fertility, and self-repair ability to heavy metals were significantly changed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormal analysis, Qing.hua.Luo.; investigation, Liang Qiu.; data curation, Xin Li.; writing\u0026mdash;original draft preparation,\u0026nbsp;Jiali Yan\u0026nbsp;and Qi lin Zhu.; writing\u0026mdash;review and editing, Liang Qiu and Qi lin Zhu.; project administration, Hailan Li. and Xin Li.; funding acquisition, Tao Liao, L.Q. and Qi lin Zhu. did the same contribution to this work. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported in part by grants from National Key R\u0026amp;D Program of China (2019YFD0902000)；\u0026nbsp;Key R\u0026amp;D Program of Guangzhou City (202103000087)；Guangzhou people\u0026apos;s Livelihood Science and technology project（202002020087）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbel, S., Nybom, I., M\u0026auml;enp\u0026auml;\u0026auml;, K., Hale, S.E., Cornelissen, G., and Akkanen, J. (2017). 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Rice Waste Biochars Produced at Different Pyrolysis Temperatures for Arsenic and Cadmium Abatement and Detoxification in Sediment. \u003cem\u003eChemosphere.\u0026nbsp;\u003c/em\u003e250 \u0026nbsp;126268\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":"wetland plant, biochar, heavy metals, arbuscular mycorrhiza, immobilization performance, amaranth","lastPublishedDoi":"10.21203/rs.3.rs-2176911/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2176911/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, biochar pyrolyzed from different wetland waste plants (\u003cem\u003eReed\u003c/em\u003e, \u003cem\u003eTypha\u003c/em\u003e, \u003cem\u003eCanna\u003c/em\u003e) was used to immobilize Cd in contaminated soil. The results showed that biochar could all enhance the residual Cd\u003csup\u003e2+\u003c/sup\u003e in soil. Moreover, Ca500 (pyrolyzed at 500 ℃ from \u003cem\u003eCanna\u003c/em\u003e) showed the best immobilization capacity due to its larger surface, higher number of pores and its regular, smooth structure. Optimum conditions for Ca-500 immobilization to Cd\u003csup\u003e2+\u003c/sup\u003e in soil were: amount 6% (wt%) and contacting time 16 weeks, the exchangeable and the state of bound to carbonates of heavy metals reduced 92.08% and 67.64%, respectively. After combining biochar with arbuscular mycorrhizal, the results showed that the combined system could more effectively reduce the transportable Cd content in soil, increase the proportion of residual Cd, effectively reduce the migration of Cd, improve the biomass and root length of vegetables, reduce Cd absorption by vegetables and the ability of vegetables to transfer Cd from root to the upper part, and reduce the edible safety risk. Simultaneously, biochar with arbuscular mycorrhizal can improve the antioxidant capacity of vegetables and their resistance to heavy metals, optimize the composition of soil microbial community, and improve soil microbial abundance and community β-diversity. It can also significantly change the functional gene expression of soil microorganisms, soil fertility, and the self-healing ability to heavy metals.\u003c/p\u003e","manuscriptTitle":"Application of wetland waste plant biochar in combination with arbuscular mycorrhizal fungi on immobilization of Cd in contaminated soil","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-01 20:20:34","doi":"10.21203/rs.3.rs-2176911/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":"9d58ae6f-fea8-43b3-a1da-65c80f0e0f53","owner":[],"postedDate":"December 1st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-17T20:35:35+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-01 20:20:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2176911","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2176911","identity":"rs-2176911","version":["v1"]},"buildId":"qQ7_6M8ijIrYJ9CiyUnPg","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}