The activation and extraction systems using organic acids and Lentinus edodes to remediate cadmium contaminated soil

Environmental Pollution 255 (2019) 113252

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The activation and extraction systems using organic acids and Lentinus edodes to remediate cadmium contaminated soil* Hang Ma 1, Xuedan Li 1, Siyu Hou, Dinghua Peng, Ying Wang, Fei Xu, Heng Xu* Key Laboratory of Bio-resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, Sichuan, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 May 2019 Received in revised form 23 August 2019 Accepted 12 September 2019 Available online 17 September 2019

To develop a high efficient and eco-friendly approach to remediate cadmium (Cd) contaminated soil, we designed the activation and extraction systems, on the basis of combined effects between the ability of organic acids to activate Cd and the ability of mushroom accumulator (Lentinus edodes) to extract Cd. The results showed that the proportion of acetic acid-extractable Cd significant increased with the application of exogenous organic acids. Additionally, soil microecology analysis indicated that exogenous organic acids evidently enhanced the numbers of microbial cells and the activities of soil enzymes. Besides, high throughput sequencing analysis revealed exogenous organic acids improved the diversity and structure of soil bacterial community after remediation. Particularly, the combination application of mushroom and exogenous citric acid had highest accumulation efficiency of Cd, and its efficiency was 59.19% higher than single mushroom treatment. Hence, exogenous organic acids could alleviate soil microecology and increase mycoextraction efficiency, which suggested it was a feasible route to remediate Cd contaminated soil by the activation and extraction systems. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Mycoextraction Organic acids Cadmium Bacterial community Microecology

1. Introduction Heavy metal pollution has become an eye-catching environmental issue, which seriously threatened safety of ecological environment over the past few decades (Jin et al., 2011; Taiwo et al., 2016). Particularly, the agricultural land around mining and smelting site has been seriously polluted by heavy metals, on account of long-term sewage irrigation and atmospheric sedimentation (Timofeev et al., 2018; Zeng et al., 2011). Cadmium (Cd) is one of the most harmful contaminants for human body, due to its high € leli et al., 2004). To remediate Cd polluted soil, vaecotoxicity (Ko rieties of physicochemical and biological methods have been widely applied in recent years, of which bioremediation has been recognized as a cost-effective and environment-friendly route (Mani et al., 2015). Mycoextraction has been reported as a promising bioremediation method to extract heavy metals from polluted soil, as

* €rg Rinklebe. This paper has been recommended for acceptance by Dr. Jo * Corresponding author. E-mail address: [email protected] (H. Xu). 1 The first two authors contributed equally to this work and should be considered co-first authors.

https://doi.org/10.1016/j.envpol.2019.113252 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

macrofungi are potent metal accumulators that have low cost, aggressive growth and high biomass (Jiang et al., 2015a; Lipka and Falandysz, 2017; Xiao et al., 2017). Lentinus edodes (L. edodes) is the second most cultivated edible mushroom worldwide (Gao et al., 2015), and it mainly was used as a biosorbent to remove pollutants from wastewater according to previous researches (Chen et al., 2006; Zhang et al., 2014), duo to its wide distribution, rapid growth, and capacity to absorb heavy metals. However, there is lacking of research in removal of Cd from polluted soil by L. edodes. In view of this, L. edodes was chosen as a new mushroom accumulator material in this study. Low-molecular-weight organic acids (such as oxalic acid, malic acid and citric acid) are important components of root exudates, and they can increase the bioavailability of heavy metals in soil to promote the uptake of heavy metals into accumulator (Kutrowska and Szelag, 2014; Montiel-Rozas et al., 2016). Moreover, organic acids can take part in the transportation and intracellular detoxification of Cd through formation different Cd-ligand complexes and sequestration of those complexes into vacuole (Dresler et al., 2014; Li et al., 2019a). And it is reported that exogenous organic acids can improve the removal efficiency of Cd from soil to hyperaccumulator and mitigate the phytotoxicity of Cd (Li et al., 2014; Liu et al., 2015b). Nonetheless, there is little information about

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combination of macrofungi and organic acids to remove Cd from polluted soil. Previous researchers mainly focused on the phytoremediation efficiency enhanced by exogenous organic acids or physiological and biochemical alterations of plants (Farid et al., 2017; Hawrylak-Nowak et al., 2015; Liu et al., 2015b; Wang et al., 2017a), however, it was also necessary to evaluate the alteration of soil biochemical properties as well as soil bacterial community, which were used as sensitive indicators of heavy metal ecotoxicity, and also could reflect the remediation effects (Abdu et al., 2017). In addition, microbiome in natural environment is hypothesized to play a crucial role in impacting the ecosystem's composition, productivity, function as well as recoverability against disturbance and stress (Wu et al., 2019). Hence, the study for indigenous microbe activity is of ecological importance. On this context, we designed the bottom up activation and extraction systems using organic acids and L. edodes, of which organic acids were used to activate Cd, improve soil biochemical qualities and interact with accumulator, and L. edodes was applied as accumulator material to extract and accumulate Cd from soil. The objectives of this study were to evaluate the remediation performances of the activation and extraction systems by focusing on soil microecology (numbers of microbial cells, activities of soil enzymes, and bacterial community) and Cd accumulation in mushroom.

presented as mg triphenylformazan (TPF) per g soil per h (Benefield et al., 1977). 2.3. Bacterial community analysis Total community genomic DNA of rhizospheric soil samples (10 g) was extracted by using a Soil DNA Kit (Omega Bio-tek Inc., Norcross, GA, USA) according to the operation instruction, and 16S rRNA gene of the soil bacterial DNA was amplified with a pair of primers (338F, 50 -ACTCCTACGGGAGGCAGCAG-30 ; 806R, 50 -GGACTACHVGGGTWTCTAAT-30 ) by polymerase chain reaction (PCR) system (GeneAmp9700, ABI, USA) for further bacterial community analysis (Li et al., 2016). Then, the 16S rRNA tag-encoded highthroughput sequencing was analyzed on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Sequences were clustered into operational taxonomic units (OTUs) at 97% similarity by using the Usearch program (version 7.1 http:// drive5.com/uparse/) (Wang et al., 2016). Then, systematic classification at multiple levels was carried on based on the biological information through ribosomal database project (RDP). Based on taxonomic information, statistics of community structure could be analyzed on various classification levels.

2. Materials and methods

2.4. Analysis of Cd

2.1. Soil preparation and pot experiment

The dried samples (1 g) of mushroom from all the treatments were powdered in a mortar. Specifically, the dried samples (1 g) of mushroom were placed in a mortar (diameter: 8 cm), then, 5 ml of liquid nitrogen was added to the mortar, and immediately grinding the sample with the mortar rod. Depending on the degree of grinding, a little more liquid nitrogen was added into the mortar, continuing grinding until the samples were fully ground into powder. The powdered samples were digested by using HNO3 and H2O2 (5:2, v/v) in a microwave oven (SINEO, MDS-6) according to the procedure of Tuzen et al. (2007), then determined by inductively coupled plasma mass spectrometry (ICP-MS, aurora M90). The operating conditions were as follows: cooling air flow rate, 18 L/ min; auxiliary gas flow rate, 1.8 L/min; atomizing gas flow rate, 1 L/ min; sample depth, 6.5 mm; radio frequency (RF) power, 1400 W. The isotopes selected for measurement were 113Cd with internal standard 103Rh (Li et al., 2019a). Cadmium speciation in soil were separately extracted using the modified European Community Bureau of Reference (BCR) procedure (Junhui et al., 2010)，and operationally defined as acetic acid-extractable (HOAc-extractable), reducible, oxidizable and residual fractions. The residual fraction of soil samples was digested by using a mixture of HNO3: HCl: HF (5:2:2, v:v:v) in a microwave oven as described above. The concentrations of Cd in the four stages were also determined by ICP-MS.

Soil used in our study was collected from the base of heavy metal remediation in Sichuan University, Chengdu, China. And basic characteristics of the soil were presented in Table S1. Firstly, CdCl2$2.5H2O solution was uniformly sprayed to soil to acquire 10 mg/kg Cd spiked soil. Then the spiked soil was equilibrated for four months. The experiments were conducted in plastic pots (Diameter ¼ 20 cm, High ¼ 18 cm), and each plastic pot contained 2.0 kg polluted soil. Different organic acids were added to the Cd polluted soil to obtain 10 mmol/kg oxalic acid (T1), 10 mmol/kg malic acid (T2) and 10 mmol/kg citric acid (T3), respectively. And Cd polluted soil without organic acids was marked as control experiment (CK). There were three replications in every group. After incubation 3 d, 150 g mycelia bag of L. edodes (purchased from Sichuan Academy of Agricultural Sciences, China) was put into soil in each pot, and all groups were carried out unified water and temperature management (Li et al., 2019a). About 30 d later, the mature fruiting bodies of L. edodes were successively collected. After washed with deionized water, those samples were dried at 60  C for 4 days in an oven, then their dry weights (DW) were measured. 2.2. The numbers of microbial cells and the activities of soil enzymes

2.5. Analysis of organic acids contents in soil and soil pH Mushroom rhizospheric soil samples were collected to analysis the numbers of microbial cells and the activities of soil enzymes to reflect soil biochemical qualities. The numbers of colony forming units (CFU) of fungi and bacteria were counted by the spread plate count method as described by Vieira and Nahas (2005). The activities of acid phosphatase and urease were measured referring to previous study (Dick, 1994). And the activities of acid phosphatase and urease were expressed individually as the production of pnitrophenol (pNP) per g soil and mg NHþ 4 -N per g soil per 24 h, and spectrophotometrically determined at 410 nm and 578 nm, respectively. Dehydrogenase activity was determined by using 2, 3, 5-triphenyl tetrazolium chloride (TTC) as reaction substrate and

Mushroom rhizospheric soil samples (5 g) were extracted by 10 mL of H3PO4 (0.1%) solution. Then the mixture was centrifuged at 5000 g for 10 min after shaking for 1 min. The supernatant was filtered and stored at 4  C for further analysis (Chen et al., 2014). The concentrations of organic acids were measured by highperformance liquid chromatography (HPLC) with a SWELL Chromplus C18 column (250 mm  4.6 mm  5 mm). The temperature and wavelength were at 30  C and 210 nm. The mobile phase consisted of 25 mM KH2PO4 (pH 2.5) and methanol (98:2, v/v) at a flow rate of 1 mL/min (Mleczek et al., 2016). Soil pH was measured in a soil/water slurry at a 1:5 (w/v) ratio.

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2.6. Statistical analyses The experimental results were evaluated by SPSS 18.0 package, and means values of groups were deemed to be different when p < 0.05 using least significant difference (LSD). All statistics were performed using the Origin 9.0 (USA). 3. Results and discussion 3.1. The distribution of Cd speciation in soil Not only the content but also the extractability and mobility of metals in soil influenced the accumulation of metals in macrofungi (Li et al., 2009). And the extractability and mobility of metals mostly depended on the their chemical form and binding state (Lu et al., 2007). The distribution of Cd speciation in soil was analyzed by BCR method (Fig. 1). Relative to single accumulator treatment (CK), the proportion of HOAc-extractable Cd increased by 3.91%, 5.71% and 5.96%, but the proportion of reducible Cd decreased by 11.21%, 12.19% and 14.57%, when added with exogenous oxalic acid, malic acids and citric acid, respectively. The contents of oxidizable Cd and residual Cd in soil were relatively few (less than 5%), and their contents were also descended in exogenous organic acids assisted groups compared to single mushroom group. It suggested that the increase of HOAc-extractable Cd was resulted from the transformation of oxidizable, reducible and residual Cd (Liu et al., 2015a). The most proportion of Cd speciation in soil was the HOAcextractable Cd, which was the most soluble, mobile and available fraction of the metal (Zhang et al., 2016a). In the activation and extraction systems, the synergy of exogenous organic acids and mushroom notably increased the content of HOAc-extractable Cd than application of individual mushroom. The result was probably duo to the following reasons. On the one hand, the addition of organic acids could increase the Cd solubility and availability directly via release of Hþ and strong organic ligands (Li et al., 2014). Previous research also demonstrated that the increased mobility of metals was caused by the variability of soil pH (Turgut et al., 2004). Application of 10 mmol/kg exogenous organic acids, the soil pH decreased modestly (Table S2), which could increase the proportion of HOAc-extractable Cd in soil. And the organic ligands with active functional groups (e.g. hydroxyl and carboxyl) could form Cd-organic acids complexes with 5 or 6 membered ring structures to prevent Cd2þ from binding to soil particles (Jiang et al., 2012). On

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the other hand, soil microbes could affect Cd binding state to enhance the extractability of the metal by secreting metabolites such as organic acids, biosurfactants, siderophores and polysaccharides (Ashraf et al., 2017; Liang et al., 2014). In the present study, the activities of microbes were more active in groups added with exogenous organic acids than CK (see “Soil microecology analysis” section). Hence, the increase in the activities of indigenous microbes resulted in an increase in the content of HOAcextractable Cd in groups applied with exogenous organic acids. The content of residual Cd was very lower than the contents of other three Cd fractions in all samples, and it was investigated to relate to the Cd binding capacities and soil basic properties including soil pH, cation exchange capacity, organic matter, etc. The low content of residual Cd might be the result of the low organic matter content and weak Cd binding ability of soil used in this study (Wang et al., 2017b; Yin et al., 2016). To discuss the mechanism that the proportion of HOAcextractable Cd was the most in citric acid assisted treatment, soil pH and contents of three organic acids in soil were examined. As showed in Table S2, with the addition of exogenous organic acids, soil pH decreased significantly in the first few days. Particularly, the lowest pH value was in the citric acid groups. However, as the experiment went on, soil pH went back up. At the end of the experiment, there were no obvious changes of soil pH in organic acid assisted treatments compared to CK, with the lack of statistical significance. Based on the above phenomena, we found that the acidification of organic acids occurred in the early stage of the experiment. And the possible reason was that the Hþ released by organic acids would be neutralized by some substances in soil (e.g. calcium carbonate) (Qin et al., 2004; Zhang et al., 2011). After the experiment, the concentrations of three organic acids in soil were shown in Table S3. The contents of the organic acids secreted by mushrooms and soil microorganisms were far lower than the contents of the exogenous organic acids added in organic acids assisted treatments. The results indicated that it was still the exogenous organic acids that played the main role in the activation of Cd, rather than the organic acids secreted by mushrooms and soil microorganisms. In exogenous organic acids assisted treatments, the exogenous organic acids added were degraded in various degree. As presented in Table S3, the residual amount of exogenous citric acid was the higher than the residual amount of other two exogenous organic acids, which might be one of the reasons why the proportion of HOAc-extractable Cd was the most in citric acid assisted treatment. Besides, the difference in the effect on Cd desorption by organic acids was also related to their chemical structures. On the one hand, citric acid had the highest molecular weight among the three types of organic acids (the relative molecular weights of malic acid, citric acid and oxalic acid were 134.09, 192.14 and 90.04, respectively), thus citric acid possessed more surface area and more negative charge, and it could chelate more metals (Jing et al., 2007). On the other hand, citric acid possessed three carboxyl and one hydroxyl groups, malic acids possessed two carboxylic and one hydroxyl groups, and oxalic acids only possessed two carboxylic groups, accordingly, citric acids could desorb more Cd2þ (Lu et al., 2007), which was also the possible mechanism why the proportion of HOAs-extractable Cd was the most in citric acid assisted treatment among the three organic acids assisted treatments. 3.2. The soil microecology analysis

Fig. 1. The distribution of Cd speciation in soil with different treatments.

3.2.1. The numbers of microbial cells and the activities of soil enzymes The numbers of microbial cells and the activities of soil enzymes were as sensitive indicators of soil biochemical qualities (Chopra

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et al., 2007; Zhang et al., 2015), therefore, they were measured to evaluate the soil microecology. Both the numbers of fungal and bacterial cells were evidently increased (Fig. 2a). When added with exogenous oxalic acid, malic acid and citric acid, the numbers of fungal cells were individually 152%, 136% and 148% of CK, and the numbers of bacterial cells were individually 162%, 154% and 151% of CK. Similar results were presented while assaying the activities of soil enzymes. The activity of acid phosphatase was 8.47e21.04% higher than CK (Fig. 2b), whereas the activity of urease was 4.85e6.63% higher than CK (Fig. 2c). In addition, the dehydrogenase activity was highest in oxalic acid assisted treatment and lowest in malic acid assisted treatment (Fig. 2d). Those results indicated that the impacts of different organic acids on enzyme activities were varied. Previous researchers had confirmed that lipid peroxidation of organisms would be ameliorated by exogenous organic acids in Cd polluted soil (Li et al., 2014; Liu et al., 2015b; Mahmud et al., 2018). In addition, Li et al. (2019b) reported that organic acids could combine with free Cd ions to form Cd-organic acids coordination compound, and could reduce the numbers of free Cd ions in soil, thereby mitigating Cd toxicity to microorganisms and promoting microbial growth. On the other hand, the degradation of organic acids could form labile carbon (C), which could provide C source for microbes (Bowers et al., 1996). Besides, other nutrients, such as nitrogen (N), phosphorus (P) and iron (Fe), could be activated directly or indirectly by organic acids to feed microbes (Mihoub et al., 2016). What's more, mushroom could provide large surfaces for microorganisms to colonize, and excrete extracellular

metabolic substances (enzymes, proteins, saccharide, etc.) to nourish microorganisms and alleviate Cd toxicity for microorganisms (Qian et al., 2015; Xiao et al., 2016). In general, for the interaction of exogenous organic acids and L. edodes, the numbers of microbial cells were substantially increased in this study. The activities of enzymes could further reflect the qualities of soil microecology, and the acid phosphatase and the urease played key roles in the geochemical process of N and P cycle (Wang et al., 2017b), while the activity of dehydrogenase was an important index of soil microbial activities and their oxidative capacity (Burns, 2000). Soil enzymes were secreted by indigenous microbes and mushroom (Chen et al., 2015), thus, the increase in activities of enzymes might be related to the augment of microbial biomass. Moreover, organic acids prevented Cd from binding to sulfhydryl groups of enzymes via forming Cd-organic acids complex (Sanadi, 1982), which might also be the reason for an increase in enzymes activities. All in all, an increase in microbial activities could enhance the activities of soil enzymes. In return, soil enzymes could promote the geochemical cycle of elements to offer nutrition to soil microbes and macrofungi. Accordingly, mycoextraction assisted with exogenous organic acids provided a virtuous circle in Cdpolluted soil microecology, thereby doing a favor to metal removal as well as soil reutilization after remediation. 3.2.2. Bacterial community structure Since bacterial community was also highly sensitive to environmental stress including heavy metal pollution, it was also considered an index for soil microecology (Zhang et al., 2016b).

Fig. 2. Numbers of microbial cells (a), acid phosphatase activity (b), urease activity (c) and dehydrogenase activity (d) in soil with different treatments. Error bars represented the standard deviation of three replicates. Different letters indicated significant (p < 0.05) difference among different treatments.

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After mushrooms were harvested, rhizospheric soil samples from all treatments were collected for bacterial community analysis. A total of 160072 valid sequences were obtained from different soil samples, and were clustered into 1762 OTUs. Alpha-diversity indexes calculated from OTU level were analyzed and showed in Table S4, of which Chao1 was commonly used as an index of microbial community richness (Chao, 1984), and Shannon and Simpson indexes were also widely used to analyze microbial community diversity (Gotelli, 1995). Results showed that higher bacterial community richness and higher bacterial community diversity as well as more OTUs were detected in organic acids assisted treatments than CK, which further indicated that exogenous organic acid could mitigate the toxicity of Cd to soil microecology. No significant difference between different organic acids assisted treatments was observed for Simpson index values. However, Shannon index of citric acid treatment groups was higher than oxalic acid and malic acid treatment groups (p < 0.05), and the reason might be that citric acid could chelate more metals to decrease Cd toxicity for bacteria, compared to oxalic acid and malic acid. The relative abundance of bacteria on genus level in different treatments was illustrated in Fig. 3. Genera with low abundance (less than 1%) were assigned as others. For bacteria distribution, the dominant genera across all samples were Bacillus (2.91%e17.30%), Arthrobacter (2.40e11.74%), Streptomyces (0.09e9.02%) and so on. Furthermore, the top 50 abundance genera of bacteria in soil samples were chosen to analysis bacterial community through the community heatmap of relative abundance (Fig. 4), which revealed that there was similar composition but different abundance of bacterial community in these four groups, and the bacterial richness in exogenous organic acids treatments was higher than CK. The results that exogenous organic acids improved the diversity and richness of soil bacterial community, fully proved the bacterial community structure was more adapted to the Cd polluted soil with the combination utilization of exogenous organic acids and mushroom. In return, the diversity of soil bacterial community could exert a crucial role in maintaining the functionality and stability of heavy metal polluted soil (Huang et al., 2019). In the present study, the highest percentage of Bacillus abundance was detected in CK (17.30%). And the percentage of Bacillus abundance decreased to 2.91e3.32% in exogenous organic acids treatments, however, there was no significant difference in the

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three organic acid treatments. It was reported that Bacillus was tolerant to heavy metal pollutant, and Bacillus was quite abundant in various heavy metal polluted environments including mine sediment, mine drainage, and heavy metal contaminated farmland (Babu et al., 2013; Li et al., 2017; Pugazhendhi et al., 2018). Treatments by exogenous organic acids could ameliorate heavy metal toxicity to soil microorganisms, which would explain why the abundance of Bacillus was less with treatments. And other studies also reported the same phenomena that the abundance of Bacillus would decrease duo to the reduction of heavy metal toxicity (Li et al., 2016). Compared with CK, the abundance of Arthrobacter and Streptomyces dramatically increased by1.74e8.96% and 5.04e8.93% respectively in organic acids treatments, but there was no significant difference in the three organic acid treatments. It was often reported that Arthrobacter and Streptomyces were related to degradation of organic matters (Chikhi et al., 2016; Jiang et al., 2015b). Therefore, the increase of the relative abundance of Arthrobacter and Streptomyces might be caused by the degradation of organic acids and the reduction of heavy metal toxicity after remediation in contaminated soil. And some researches also reported that the abundance of Arthrobacter and Streptomyces all increased after remediation in heavy metal polluted soil (Luo et al., 2018). 3.3. The biomass of mushroom and Cd content in accumulator Effect of Cd on the biomass of stipe and pileus was illustrated in Fig. 5a, which presented that evident changes of biomass were not existed in exogenous organic acids assisted groups, compared to CK. As depicted in Fig. 5b, Cd concentration of stipe in exogenous organic acids addition treatments slightly increased than CK. Whereas, Cd concentration of pileus in exogenous organic acids assisted groups substantially increased by 25.68e64.29% related to CK. Consequently, compared with CK, the accumulation of Cd in fruiting body of L. edodes was enhanced by 21.45%, 25.64%, and 59.19% in exogenous oxalic acid, malic acid, and citric acid assisted groups, respectively (Fig. 5c). It had been reported that the biomass of mushroom would be reduced with an increase of Cd accumulation (Li et al., 2019b). However, exogenous organic acids could relieve the toxicity of Cd to organisms via forming Cd-organic acids complex (Lu et al., 2007). And the addition of exogenous organic acids would also improve

Fig. 3. Relative abundance of bacterial genus with different treatments.

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Fig. 4. The community heatmap of relative abundance of bacterial genus with different treatments.

soil microecology (Figs. 2 and 3). Good soil microecology was beneficial to mushroom growth. Herein, the biomass of L. edodes was not inhibited with the increase of Cd accumulation in this research. Nonetheless, the increase in Cd concentration of stipe and pileus contributed to the augment of Cd accumulation in L. edodes.

Organic acids could improve mycoextraction efficiency through various ways. Firstly, organic acids were able to promote the growth of accumulator to facilitate the accumulation of Cd. Secondly, organic acids could directly increase Cd availability via acidification, chelation and precipitation to increase the

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Fig. 5. Biomass of mushroom (a), Cd concentration in mushroom (b), and Cd accumulation in mushroom (c) with different treatments. Error bars represented the standard deviation of three replicates. Different letters indicated significant (p < 0.05) difference among different treatments.

accumulation of Cd in accumulator (Dong et al., 2007). Besides, organic acids could improve the activities of indigenous microbes and mushroom through mitigating soil microecology, and microbes and mushroom could activate Cd by secreting metabolites (siderophores, surfactants, enzymes, polysaccharides, etc.) (Liu et al., 2015a). Additionally, organic acids, which were with high polarity, were apt to interact with polar groups of cell membranes and insert into cell membranes, thereby increasing the penetrability of cell membranes and promoting Cd across cell membranes (Liao and Huang, 2002). The removal efficiency of Cd from soil not only depended on the availability and mobility of metal in soil, but also was related to the ability of accumulator to uptake and transport metals from soil (Wu et al., 2016). The mycelia of mushroom could spread over large areas and effectively accumulate heavy metals from rhizospheric soil to the fruiting bodies (Li et al., 2019a). Exploration on physiological mechanisms for mushroom to accumulation heavy metals showed that mushroom had an excellent performance to transport heavy metals to the aboveground tissue (Li et al., 2016). And the Cd concentration of pileus increased than CK by addition of exogenous organic acids (Fig 5b), indicating that organic acids were conducive to Cd migration from soil to the aboveground tissue of mushroom. The highest Cd accumulation was in exogenous citric acid assisted group, which was 23.71% and 21.08% higher than exogenous oxalic acid and malic acid addition groups, duo to the better ability of citric acid to activate Cd relative

to oxalic acid and malic acid (Fig. 1). 4. Conclusions In conclusion, the activation and extraction systems using L. edodes and exogenous organic acids performed excellently in remediating Cd contaminated soil. The mycoextraction efficiency was improved by exogenous organic acids. At the same time, the numbers of soil microbial cells and the activities of soil enzymes were enhanced. In particularly, the community of indigenous bacteria was ameliorated with the activation and extraction systems. In consideration of the low cost, easy accessibility and high efficiency of those materials, the activation and extraction systems were technically feasible to remediate Cd contaminated soil. Conflicts of interest There was no conflict of interest. Acknowledgements This study was financially supported by the National Key Research and Development Program (2018YFC1802605), the Science and Technology Project of Sichuan Province (2019YFS0506), the Key Research and Development Program of Sichuan Province

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