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Elucidation of the mechanisms into effects of organic acids on soil fertility, cadmium speciation and ecotoxicity in contaminated soil
Chemosphere 239 (2020) 124706
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Elucidation of the mechanisms into effects of organic acids on soil fertility, cadmium speciation and ecotoxicity in contaminated soil Hang Ma 1, Xuedan Li 1, Mingyang Wei 1, Guoquan Zeng 1, Siyu Hou 1, Dan Li 1, Heng Xu*, 1 Key Laboratory of Bio-resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, Sichuan, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Soil fertility was improved with the addition of organic acids. The availability of Cd could increase with the application of organic acids. Organic acids were effective in soil bacterial community structure improvement. Citric acid was most effective on improving soil micro-ecology.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2019 Received in revised form 25 August 2019 Accepted 28 August 2019 Available online 31 August 2019
The remediation effect of organic acids in heavy metal contaminated soil was widely studied. However, the comprehensive evaluation of organic acids on micro-ecological environment in heavy metal contaminated soil was less known. Herein, this experiment was conducted to investigate the impact of malic acid, citric acid and oxalic acid on soil fertility, cadmium (Cd) speciation and ecotoxicity in contaminated soil. Especially, to evaluate the ecotoxicity of Cd, high-throughput sequencing was used to investigate the soil bacterial community structure and diversity after incubation with organic acids. The results showed that obvious changes in soil pH were not observed. Whereas, the contents of available phosphorus (Olsen-P) and alkali hydrolysable nitrogen (Alkeline-N) evidently increased with a significant difference. Furthermore, compared to control, the proportion of acetic acid-extractable Cd increased by 3.06e6.63%, 6.11e9.43% and 1.91e6.22% respectively in the groups amended with malic acid, citric acid and oxalic acid, which indicated that citric acid did better in improving the availability of Cd than malic acid and oxalic acid. In terms of biological properties, citric acid did best in bacteria count increase, enzyme activities and bacterial community structure improvement. Accordingly, these results provided a better understanding for the influence of organic acids on the micro-ecological environment in Cd contaminated soil, based on physicochemical and biological analysis. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: X. Cao Keywords: Organic acids Micro-ecology Soil Cadmium Biological properties
1. Introduction
* 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.chemosphere.2019.124706 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
With the development of industry and agriculture, heavy metal pollution is becoming one of the most widespread and serious environmental problem among various soil pollution due to their concealment and high ecotoxicity (Wu et al., 2019b). Heavy metal pollution accounts for 82.8% of soil contamination types in China,
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among which cadmium (Cd) is up to 7.0%, posing an acute threat to human health and the ecosystem (Xiao et al., 2017). To remediate Cd contaminated soil, various amendments have been performed, including heavy metal removal and immobilization (Liu et al., 2018; Rajendran et al., 2019; Wang et al., 2017b; Wu et al., 2016). There are so many kinds of soluble organic acids in the soil environment, among which malic acid, citric acid and oxalic acid etc., a class of small molecular organic compounds with one or more carboxyl groups, are one of the most important components of soluble organic acids (Chen et al., 2015; Emmanuel et al., 2009; Strobel, 2001). Organic acids are mainly produced from the decomposition of plant and animal residues, the secretion of plant roots and the microbial metabolism in soil. Moreover, the conversion of fertilizer and organic matter is also an important source for organic acids in soil (Jones et al., 2003; Strobel, 2001). Given the diversity of sources, the types and quantities of organic acids in soil are in the dynamic process of synthesis and decomposition, and their concentrations are generally not high (Jones, 1998). However, due to the large amount of organic acids secreted by roots and acidproducing bacteria, the contents of organic acids in the rhizosphere can generally be as high as 1e10 mmol/kg, even higher contents, and the contents of organic acids in the rhizosphere are significantly higher than those in the non-rhizosphere soil (Jones et al., 2003; Strobel, 2001). Biogeochemical processes that involve organic acids (such as malic acid, citric acid and oxalic acid) can be of considerable ecological importance (Hees et al., 2003). Some of the key roles of organic acids in the soil micro-ecological environment are thought to include the detoxification of heavy metals, such as copper (Cu), lead (Pb) and Cd (Arabi et al., 2017; Fei et al., 2004), and the mobilization of nutrients, such as iron (Fe), nitrogen (N), phosphorus (P) and potassium (K) (Bais et al., 2006). There are numerous researches about detoxification of heavy metal contaminated soils with the application of organic acids (Onireti et al., 2017; Wang et al., 2016b; Wu et al., 2007; Yang and Liao, 2010). In addition, organic acids can be used in phytoremediation process to enhance the bioaccumulation of metals into plants through the increase of heavy metal extractability (Liu et al., 2015; Mahmud et al., 2018). Nevertheless, their ecological security as soil conditioners has been rarely reported, and few reports are available concerning soil bacterial community structure and diversity as ecotoxicity indicators in contaminated soil remediated by organic acids. In addition to ecological security, soil fertility is also an important reference factor for soil remediation. And the contents of available phosphorus (Olsen-P) and alkali hydrolysable nitrogen (Alkeline-N) are significant indicators of soil fertility. Soil Olsen-P content restricted the efficient P fertilizer management strategies, and its deficiency was one of the main constraints limiting agricultural crop production (Marschner, 2012). Soil Alkeline-N was the most active component of soil N and an important source of soil available nutrients (Chen et al., 2016). Thus, a series of experiments using malic acid, citric acid and oxalic acid were carried out to (1) investigate the improvement of soil fertility; (2) compare the distribution Cd chemical speciation; (3) assess the toxicity to soil micro-ecology, including bacteria count, soil acid phosphatase and urease activities, bacterial community structures at the genus level. Furthermore, details of possible mechanistic insight into the remediation effects were carefully discussed. 2. Materials and methods 2.1. Soil preparation and experiment design The high concentration of Cd contaminated soil used in this
study was collected from a Cd contaminated site in Sichuan University, Chengdu, China. All of the soil samples were air-dried and passed through a 2-mm sieve to wipe out extraneous matters in soil. The main properties of the soil were presented in Table 1. As shown in Table 2, 10 treatments were set, including 1 control (CK), 9 treatments amended with different concentrations of malic acid, citric acid and oxalic acid, respectively. The concentrations of each type of organic acids were set to 10, 20 and 30 mmol/kg in soil, respectively. And each treatment was replicated for four times. The study was conducted in plastic pots individually, which contained sieved soil (1 kg) mixed with different contents of organic acids. During the period of incubation, all treatments were conducted unified water management and kept a constant temperature (25 C). In the remediation process, all treatments were sampled at 60 d for soil pH, available nutrient contents, heavy metal speciation distribution, bacteria count, enzyme activities, and bacterial community structure analysis. 2.2. Soil pH, Olsen-P and Alkeline-N analysis Soil pH was measured by the method of Corey described (Corey, 1971). The Olsen-P in soil was extracted with NaHCO3 solution for 30 min at 25 ± 1 C and its content was determined by the spectrophotometry at 880 nm (NY/T 1121.7e2014). The content of Alkeline-N in soil was measured by the method as described by previous studies (Wang et al., 2017b). In which the diffusion plate contained 2.0 g of air-dried soil samples and 1.0 g of ZneFeSO4 reducing agent in the outer chamber, 2.0 mL 20 g/L of boric acid indicator in the inner chamber, and the diffusion plate was incubated for 24 h at 40 C. Then, the NH3 absorbed by boric acid was titrated by 0.005 M H2SO4 solution. 2.3. Cadmium speciation distribution analysis Cadmium fractions in soil were separately extracted using the
Table 1 The main properties of soil samples. Properties
Value
pH Electrical conductivity (EC, mS/cm) Water holding capacity (%) Sand (%) Silt (%) Clay (%) CaCO3 (g/kg) Organic C (g/kg) Total N (g/kg) Total P (g/kg) Cd (mg/kg)
7.70 264 60.6 63.4 24.1 12.5 91.4 9.2 1.5 1.1 10.12
Table 2 The design of all the treatments. Treatments
Amendment
CK T1 T2 T3 T4 T5 T6 T7 T8 T9
Control, without amendment 10 mmol/kg malic acid 20 mmol/kg malic acid 30 mmol/kg malic acid 10 mmol/kg citric acid 20 mmol/kg citric acid 30 mmol/kg citric acid 10 mmol/kg oxalic acid 20 mmol/kg oxalic acid 30 mmol/kg oxalic acid
There were 4 replicates for each treatment.
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modified European Community Bureau of Reference (BCR) procedure, and operationally defined as HOAc-extractable (acetic acidextractable), reducible, oxidizable and residual fractions (Nemati et al., 2011; Wang et al., 2017b). The residual fractionation of soil samples was digested using a mixture of HNO3:HCl:HF (5:2:2, v:v:v) in a microwave oven (SINEO,MDS-6, Shanghai, China), then the concentrations of different heavy metal fractions in samples were detected by Flame Atomic Absorption Spectrometry (FAAS: VARIAN, SpectrAA 220FS, USA) (Recovery: 95.7%e105.0%; RSD ¼ 1.0%) (Wu et al., 2016). 2.4. Cadmium toxicity to soil micro-ecology analysis Bacteria count was counted according to the spread-plate method that described by Liu et al. (2018). Briefly speaking, aqueous extracts of 3 g fresh soil samples were serially diluted to spread on beef extract-peptone. Then, the numbers of colony forming units (CFU) of bacteria were recorded after incubation at suitable circumstance for 2e5 d. Acid phosphatase and urease activities were measured referring to previous study (Pan and Yu, 2011; Zhan et al., 2010). The activity of acid phosphatase was expressed as p-nitrophenol (pNP) mg per g soil per h, and spectrophotometrically determined at 410 nm. While, urease activity was expressed as NHþ 4 -N mg per g soil per 24 h and spectrophotometrically determined at 578 nm. For further bacterial community analysis, the bacterial DNA was extracted using a Soil DNA Kit (Omega Biotek Inc., Norcross, GA) and amplified. The bacterial community was investigated on Illumina MiSeq platform, which was conducted by Majorbio BioPharm Technology Co., Ltd (Shanghai, China). Sequences were clustered into operational taxonomic units (OTUs) and the biological information analysis was conducted on the Usearch program (version 7.1) at 97% similarity. 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 (Wang et al., 2016a). 2.5. Statistical analysis In our study, all results were presented by value ± standard deviation. Statistical significance was performed using SPSS 18.0 package and statistics were performed using the Origin 8.0 (USA).
Fig. 1. Soil pH (a) in Cd contaminated soil amended with different organic acids. Error bars represented the standard deviation of four replicates. Different letters (a, b, c ….) indicated significant (P < 0.05) difference among different treatments.
be taken into account. For example, some scholars had studied that the change of rhizosphere soil pH was not observed in the release of organic acids under environmental stress (LV, 1995). Especially for calcareous soil, there was a large amount of free calcium carbonate in calcareous soil. After adding organic acids into the soil, Hþ would be neutralized by calcium carbonate and greatly reduced, presumably due to the release of CO2 3 from CaCO3 and the formation of HCO 3 (Fei et al., 2004; Zhang et al., 2011). Therefore, in this experiment, when calcareous soil (91.4 g/kg CaCO3, as shown in Table 1) was treated with different types of organic acids at different concentrations, obvious changes in soil pH were not observed, and there was no significant difference in treatments.
3.2. The contents of Olsen-P and Alkeline-N analysis The contents of Olsen-P and Alkeline-N significantly increased (p < 0.05) in all treatments than CK (Fig. 2). It was obvious that the
3. Results and discussion 3.1. Soil pH analysis After 60 d incubation, the soil pH values of all the treatments were weakly changed compared to control (Fig. 1). When malic acid, citric acid and oxalic acid in different concentrations were added, the pH values slightly increased by 0.185e0.25, 0.275e0.41 and 0.15e0.28 than CK, respectively. However, there was no significant difference among the treatments. Soil pH was known as a major factor that regulated the availability of nutrients, distribution of heavy metal, and indigenous microbial activity (Liu et al., 2018). Thus, it was extremely important to investigate the changes of soil pH after incubation process. Hþ could be dissociated from the carboxyl group of the organic acids, which undoubtedly had a certain impact on soil pH, soil environment alteration and rhizosphere biochemical process € ttger, 1991). As evaluating the impact of organic (Petersen and Bo acids on soil pH factors, such as soil buffering, soil structure, clay composition, organic matter content, microbial activity etc., should
Fig. 2. Olsen-P and Alkeline-N (b) in Cd contaminated soil amended with different organic acids. Error bars represented the standard deviation of four replicates. Different letters (a, b, c ….) indicated significant (P < 0.05) difference among different treatments.
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highest content of Olsen-P was presented in the treatment groups amended with oxalic acid, and the content of Olsen-P in oxalic acid treatment groups increased by 272.55e353.68% compared with CK. Besides, in the treatments amended with citric acid and malic acid, the contents of Olsen-P increased by 234.64e329.74% and 157.76e248.28% compared to CK, respectively. Meanwhile, the content of alkaline-N increased by 153.08e229%, 193.5e341.48% and 105.62e209.5% respectively compared to CK, with the addition of malic acid, citric acid and oxalic acid. Generally, as the higher concentrations of the organic acids were applied in soil, the more contents of Olsen-P and Alkeline-N were extracted from the soil. As illustrated in Fig. 2a, the soil Olsen-P content was evidently enhanced with the application of the organic acids compared with the control treatment. The application of organic acids had potential to improve the mobilization of P through a series of measures including solubilizing minerals and desorbing P from mineral surfaces (Rasouli-Sadaghiani, 2014). Moreover, the distribution of heavy metal speciation in soil could be effected with the addition of organic acids (Lu et al., 2007). As numerous heavy metal releasing, the active adsorption sites for P were added, leading to the coordination adsorption addition and the augment of the Olsen-P (Wang et al., 2017b). In calcareous soil, oxalate might primarily release P held in CaeP minerals through the formation and precipitation of Ca-oxalate. In contrast, citrate acids had a relatively € m et al., 2001). This was supported to poor affinity for Ca2þ (Stro some extent by our experimental results, which showed that the activation effect of P of oxalic acid was better than citric and malic acids at the same molar concentration. The availability of some elements (e.g. P and Fe) were directly related to organic acids, while the availability of some other elements (e.g. N and K) might also be directly or indirectly related to organic acids (Jones and Darrah, 1995). Organic acids could also adsorb N and K through tight connections, such as hydrogen bonds and ionic bonds. Besides, soil Alkeline-N was related to indigenous microbial activity, which had capacity to influence the processes of N recycling (Saha et al., 2012). The addition of amendments not only increased the nutrient contents but also stimulated the microbial activity, and in return, the enhancement of microbial activity affected the activation of nutrient in soil (Saha et al., 2012). The addition of organic acids not only increased the nutrient contents but also stimulated the microbial activity, and in return, the enhancement of microbial activity could affect the activation of nutrient in soil. The microbial activity was more energetic in treatment groups added with citric acid than malic acid and oxalic acid (see “Soil bacteria count and enzyme activities analysis” section), which might be the possible reason why the highest content of Alkeline-N was presented in the treatment groups amended with citric acid.
3.3. Cadmium speciation distribution analysis In the present work, BCR extraction was used to investigate chemical form variation after incubation, and the results were described as Fig. 3. As shown by the results, the speciation distribution of Cd in soil was dramatically influenced by the addition of organic acids. Compared with the CK, the proportion of HOAcextractable Cd evidently increased by 3.06e6.63%, 6.11e9.43% and 1.91e6.22% in the treatment groups amended with malic acid, citric acid and oxalic acid, respectively. Obviously, the treatment groups amended with citric acid were presented the best activation effect on Cd, and their contents of HOAc-extractable Cd were more than the treatment groups amended with malic acid and oxalic acid. When the proportion of HOAc-extractable Cd increased in soil, the proportion of reducible Cd decreased by 2.13e4.15%, 4.875e6.22%
Fig. 3. Metal speciation distribution in Cd contaminated soil amended with different organic acids.
and 1.165e3.78% in the treatments amended with malic acid, citric acid and oxalic acid, respectively. In addition, the contents of oxidizable and residual Cd showed a decreasing tendency in general, and the content of residual Cd was the lowest compared with the content of HOAc-extractable, reducible, and oxidizable fractions. Cadmium binding capacities and soil properties (e.g. soil pH, CEC and organic matter) were reported to have relationships with the content of residual Cd in soil, the low and even none content of residual fraction of Cd in soil could be caused by the weak Cd binding capacity of soil and the low content of organic matter (Wang et al., 2014; Yin et al., 2016). It was widely recognized that the extractability and mobility of heavy metals in soil depended strongly on their speciation distribution and binding state (Lu et al., 2007). As described in the previous researches (Xiao et al., 2017), the HOAc-extractable Cd was strongly positive correlated with heavy metal extractability. Nevertheless, the reducible, oxidizable and residual fractions of Cd speciation were negatively correlated with heavy metal bioavailability, among which residual Cd had the biggest inhibition effect on the extractability of heavy metal. In present work, the proportion of HOAc-extractable Cd evidently increased and the proportion the remaining three forms of Cd speciation decreased. Therefore, the application of organic acids significantly improved the extractability of Cd. And some researches had reported that the bioaccumulation of Cd in plants were obvious enhanced with the addition of organic acids (Mahmud et al., 2018; Wang et al., 2017a). These works could also verify that organic acids could increase the availability of Cd. Previous studies suggested that organic acids, especially malic acid, citric acid and oxalic acid, could not only acidize soil, but also be able to form soluble complexes and chelates with metal ions (Liu et al., 2012, 2015). The availability of heavy metals would be increased with the decreasing of soil pH (Wang et al., 2017b). However, obvious changes in calcareous soil pH were not observed after amended with organic acids, indicating that pH was not the significant factor governing the Cd speciation distribution in this study. Therefore, the effect of Cd speciation distribution was mainly dependent on ionic form and concentration of organic acids. Malic acid, citric acid and oxalic acid that carried two or three carboxylic groups could form Cd-organic acids complexes with 5 or 6 membered ring structures, which increased mobility of Cd in soil and plant (Fei et al., 2004). Previous studies had found that free
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Cd2þ was more toxic to crops than Cd-organic acids (Aravind and Prasad, 2005; Wu et al., 2003). Accordingly, metal chelates formed by organic acids and Cd not only increased the mobility of Cd in plants, but also alleviated the toxicity of Cd to plants. For same type of organic acids, the activation effect on Cd increased with the increasing of organic acids concentration. While At the same molar concentration, the ability of organic acids to influence Cd speciation distribution might depend on the molecular weight and structure of organic acids. On one hand, citric acid had a 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 it carried more surface area and more negative charge (Jing et al., 2007). It was one of possible reasons that citric acid could chelate more metals than malic acid and oxalic acid. On the other hand, the functional groups of organic acids (e.g. carboxylic and hydroxyl groups) were important binding sites for metals. Citric acid had three carboxylic and one hydroxyl groups, malic acid had two carboxylic and one hydroxyl groups, and oxalic acid only had two carboxylic groups, accordingly citric acids could desorb more Cd2þ. In present study, we got the same results with other studies that citric acids did better than malic and oxalic acids in increasing the availability Cd in soil (Liu et al., 2015). 3.4. Cadmium toxicity to soil micro-ecology analysis 3.4.1. Soil bacteria count and enzyme activities analysis Microbial biomass and enzyme activities are important biochemical indexes of soil micro-ecology (Wu et al., 2019a). The impact of different organic acids on bacteria count and enzyme activities in Cd-polluted soil were depicted in Fig. 4. As shown in Fig. 4a, the bacteria count in treatments amended with organic acids dramatically increased after incubation, and there was a significant difference among treatments. The bacteria count increased by 36.23e158.94%, 45.41e192.27% and 17.39e88.35% than CK, when added with malic acid, citric acid and oxalic acid, respectively. Similar to the bacteria number, the activities of soil enzyme also had significant enhancements, and the highest activities of acid phosphatase and urease were all presented in the treatments with the application of citric acid (239.805e415 mg pNP/g soil/h and 0.706e0.934 mg NHþ 4 -N/g soil/24 h, respectively). Whereas the lowest acid phosphatase and urease activities were found in the treatments added with malic acid (182.775e333.19 mg pNP/g soil/h) and oxalic acid (0.3925e0.622 mg NHþ 4 -N/g soil/24 h), respectively. These results showed that different types of organic acids had different impacts on enzyme activities. What's more, the bacteria number increased and the activities of soil enzymes improved with an increase of the concentrations of amendments in Cd-polluted soil. Many previous studies had revealed that excessive heavy metals were toxic to soil microorganisms, through their capability of hindering enzyme activity, making DNA mutation and compromising cell membrane integrity (Poli et al., 2009; Wu et al., 2016). Fortunately, the toxicity of Cd to microorganisms in soil could be relieved by organic acids through a variety of direct or indirect strategies. On the one hand, organic acids could reduce the toxicity of Cd directly, by forming chelates with Cd2þ to prevent Cd2þ from reacting with bacterial active groups (Li et al., 2019). On the other hand, organic acids could provide C elements for microbes. As reported in the works of previous researchers (Bowers et al., 1996), organic acids (e.g. malic acid, citric acid and oxalic acid) were short chain organic acids with low molecular weight (less than 200), which could easily pass through the thinner cell walls and cell membranes of bacteria, and bacteria were able to use them as energy materials directly. In addition, some nutrient elements could be activated by organic
Fig. 4. Soil bacteria count (a), acid phosphatase activity (b) and urease activity (c) in Cd contaminated soil amended with different organic acids. Error bars represented the standard deviation of four replicates. Different letters (a, b, c ….) indicated significant (P < 0.05) difference among different treatments.
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Fig. 5. Bacterial community structures (a) and Shannon curves (b) at the genus level in Cd contaminated soil amended with different organic acids.
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acids, and it was verified in present study (Fig. 2). Moreover, organic acids were related to Fe absorption of organisms, and they could alleviate Fe deficiency induced by Cd to promote the growth of organisms (Sebastian and Prasad, 2018). When the three types of organic acids were added at the same molecular concentration, the groups added by citric acid did better in increasing the bacteria number than malic acid and oxalic acid. The reasons might be that citric acid had the better ability to chelate Cd2þ and activate nutrients (Fei et al., 2004; Khademi et al., 2009), and the results of this experiment also supported this conclusion. A large number of studies had shown that soil enzyme activities were sensitive to environmental stress and could be used to indicate the pollution status of soil micro-ecological environment, including for heavy metal pollution (Pascual et al., 2000; Wu et al., 2018). Therefore, the determination of activities of acid phosphatase and urease was of great significance. The enzyme activities integrally increased after incubation, and that might be related to the augment of soil microbial activity. In addition, the increasing of enzyme activities might be partly caused by functional groups of the organic acids, because these active functional groups could react with Cd2þ in soil by forming metal-organic acids complexes to prevent Cd2þ from binding to sulphydryl groups of enzymes (Sanadi, 1982). The activities of urease and acid phosphatase were the highest in the groups added with citric acid, and the possible reasons were related to the better abilities of citric acid to chelate Cd2þ and improve the microbial activity than malic acid and oxalic acid. Moreover, soil enzymes played important roles in the geochemical process of elements, and they could activate nutrients (e.g. N, P and Fe) to promote microbes growth (Lebrun et al., 2012). In return, microbes secreted more enzymes. Therefore, the addition of organic acids provide a virtuous circle in Cd-polluted soil microecology.
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Nocardioides increased were the degradation of organic acids and the reduction of Cd toxicity after remediation in contaminated soil. Furthermore, the treatments with the addition of organic acids showed higher Shannon indexes than CK (Fig. 5b), and the maximum value was 3.29 in the citric acid treatment groups. Shannon index was always widely used to analyze microbial community diversity (Wu et al., 2018). These results also implied the superiority of the application of the amendments for improving the soil bacterial diversity. 4. Conclusions Our results showed that the micro-ecological environment of Cd-polluted soil significantly improved after treatment with organic acids. With the application of organic acids, the contents of Olsen-P and Alkeline-N evidently increased. In addition, the availability of heavy metals greatly increased while the ecotoxicity decreased. Especially, bacteria count and enzyme activities, were more strongly influenced by organic acids addition. In this experiment, the higher the concentration of amendments was, and the better the repair effect was. Based on this, high-throughput sequencing was used to investigate the soil bacterial community structure after incubation with organic acids at the concentration of 30 mmol/kg. Briefly, citric acid had the best repairing effect among the three types of organic acids, taking the impacts on nutrient contents, heavy metal bioavailability, bacteria count, enzyme activities and bacterial community structure into consideration. Our results suggested that organic acids could be used as activators to increase the extractability of Cd and as ameliorants to improve micro-ecological environment in contaminated soil. Conflicts of interest There was no conflict of interest.
3.4.2. Soil bacterial community structure and diversity analysis As previously mentioned, the higher concentration of the organic acids were added in soil, and the greater repairing effects were presented in each type of organic acids treatment groups. Hence, three treatment groups and CK were investigated to analysis the diversity of bacterial community structure through highthroughput sequencing. The structures of bacterial community under different treatments on genus level were depicted as Fig. 5a, and sequences with low abundance (less than 1%) were assigned as others. The compositions of soil bacteria genera in control and other treatments were similar, whereas the abundance of each bacteria genus in every treatment was different. On genus level, the highest abundance genus in CK was Sphingomonas (22.60%), and the abundance of Sphingomonas decreased to 16.01%, 6.35% and 13.69% respectively in the treatments added with malic acid, citric acid and oxalic acid. Sphingomonas bacteria were proven as Cd resistant bacteria and promising applications for enhancing bioremediation in Cd-contaminated sites (Pan et al., 2016; Tangaromsuk et al., 2002). Treatment with organic acids could relieve the Cd stress to soil microorganisms, which would explain why the abundance of Sphingomonas decreased in organic acids treatment groups compared to CK. Moreover, the lowest abundance of Sphingomonas was in the citric acid group, which further indicated that citric acid possessed better detoxification ability than malic acid and oxalic acid in Cd-contaminated soil. The abundance of Nocardioides was also different in CK (2.27%) and organic acids treatment groups (5.82e9.73%). There was scarcely any research about the relationship between Nocardioides bacteria and heavy metals, but Nocardioides bacteria were reported to own the ability to degrade organic matters (Morrissey et al., 2015; Shrestha et al., 2013). Accordingly, the reasons why the relative abundance of
Acknowledgments 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 (2017SZ0181, 2018NZ0008), and the Fundamental Research Funds for the Central Universities (SCU2019D013). The authors also wish to thank Professor Guanglei Cheng from Sichuan University for the technical assistance. References Arabi, Z., et al., 2017. Cadmium removal from Cd-contaminated soils using some natural and synthetic chelates for enhancing phytoextraction. Chem. Ecol. 1e14. Aravind, P., Prasad, M.N.V., 2005. Cadmium-induced toxicity reversal by zinc in Ceratophyllum demersum L. (a free floating aquatic macrophyte) together with exogenous supplements of amino- and organic acids. Chemosphere 61, 1720e1733. Bais, H.P., et al., 2006. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu. Rev. Plant Biol. 57, 233e266. Bowers, J.H., et al., 1996. Infection and colonization of potato roots by Verticillium dahliae as affected by Pratylenchus penetrans and P. Crenatus. Phytopathology 86, 614e621. Chen, B., et al., 2016. Effect of N fertilization rate on soil alkali-hydrolyzable N, subtending leaf N concentration, fiber yield, and quality of cotton. Crop J. 4, 323e330. Chen, Z., et al., 2015. Confirmation and determination of carboxylic acids in root exudates using LC-ESI-MS. J. Sep. Sci. 30, 2440e2446. Corey, R.B., 1971. A Textbook of Soil Chemical Analysis. Emmanuel, D., et al., 2009. Spectroscopic characterization of organic matter of a soil and vinasse mixture during aerobic or anaerobic incubation. Waste Manag. 29, 1929e1935. Fei, Q., et al., 2004. Effects of low-molecular-weight organic acids and residence time on desorption of Cu, Cd, and Pb from soils. Chemosphere 57, 253e263.
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Hees, P.A.W.V., et al., 2003. Low molecular weight organic acid adsorption in forest soils: effects on soil solution concentrations and biodegradation rates. Soil Biol. Biochem. 35, 1015e1026. Jing, Y.D., et al., 2007. Effects of pH, organic acids, and competitive cations on mercury desorption in soils. Chemosphere 69, 1662e1669. Jones, D.L., 1998. Organic acids in the rhizosphere e a critical review. Plant Soil 205, 25e44. Jones, D.L., Darrah, P.R., 1995. Influx and efflux of organic-acids across the soil-root interface of Zea-Mays L and its implications in rhizosphere C flow. Plant Soil 173, 103e109. Jones, D.L., et al., 2003. Organic acid behavior in soils e misconceptions and knowledge gaps. Plant Soil 248, 31e41. Khademi, Z., et al., 2009. Organic acid mediated nutrient extraction efficiency in three calcareous soils. Aust. J. Soil Res. 47, 213e220. Lebrun, J.D., et al., 2012. Assessing impacts of copper on soil enzyme activities in regard to their natural spatiotemporal variation under long-term different land uses. Soil Biol. Biochem. 49, 150e156. Li, X., et al., 2019. Mechanisms into the removal and translocation of cadmium by Oudemansiella radicata in soil. Environ. Sci. Pollut. Control Ser. 1e11. Liu, H., et al., 2015. Mitigation mechanism of Cd contaminated soils by different levels of exogenous low-molecular-weight organic acids with Phytolacca americana. RSC Adv. 5, 47e49. Liu, H., et al., 2018. Effect of modified coconut shell biochar on availability of heavy metals and biochemical characteristics of soil in multiple heavy metals contaminated soil. Sci. Total Environ. 645, 702e709. Liu, M.Y., et al., 2012. Research on Differences of Component and Quantity of Organic Acids in the Root Exudates Among the Three Green Manures. World Automation Congress, pp. 1e4. Lu, H., et al., 2007. Low-molecular-weight organic acids exuded by Mangrove (Kandelia candel (L.) Druce) roots and their effect on cadmium species change in the rhizosphere. Environ. Exp. Bot. 61, 159e166. LV, K., 1995. Cellular mechanisms of aluminum toxicity and resistance in plants [review]. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 237e260. Mahmud, J.A., et al., 2018. Insights into citric acid-induced cadmium tolerance and phytoremediation in Brassica juncea L.: coordinated functions of metal chelation, antioxidant defense and glyoxalase systems. Ecotoxicol. Environ. Saf. 147, 990e1001. Marschner, H., 2012. Mineral nutrition of higher plants/Horst Marschner. J. Ecol. 76, 1250. Morrissey, E.M., et al., 2015. Dynamics of extracellular DNA decomposition and bacterial community composition in soil. Soil Biol. Biochem. 86, 42e49. Nemati, K., et al., 2011. Speciation of heavy metals by modified BCR sequential extraction procedure in different depths of sediments from Sungai Buloh, Selangor, Malaysia. J. Hazard Mater. 192, 402e410. Onireti, O.O., et al., 2017. Combined effects of low-molecular-weight organic acids on mobilization of arsenic and lead from multi-contaminated soils. Chemosphere 170, 161e168. Pan, F., et al., 2016. Endophytic bacterium Sphingomonas SaMR12 promotes cadmium accumulation by increasing glutathione biosynthesis in Sedum alfredii Hance. Chemosphere 154, 358e366. Pan, J., Yu, L., 2011. Effects of Cd or/and Pb on soil enzyme activities and microbial community structure. Ecol. Eng. 37, 1889e1894. Pascual, J.A., et al., 2000. Soil microbial activity as a biomarker of degradation and remediation processes. Soil Biol. Biochem. 32, 1877e1883. €ttger, M., 1991. Contribution of organic acids to the acidification of Petersen, W., Bo the rhizosphere of maize seedlings. Plant Soil 132, 159e163. Poli, A., et al., 2009. Heavy metal resistance of some thermophiles: potential use of a-amylase from Anoxybacillus amylolyticus as a microbial enzymatic bioassay. Res. Microbiol. 160, 99e106. Rajendran, M., et al., 2019. Effect of sulfur and sulfur-iron modified biochar on cadmium availability and transfer in the soil-rice system. Chemosphere 222,
314e322. Rasouli-Sadaghiani, M.H., 2014. Effects of low-molecular-weight organic acids on phosphorus sorption characteristics in some calcareous soils. Turk. J. Agric. For. 36, 459e468. Saha, S., et al., 2012. Vermicompost and Soil Quality. Springer, Netherlands. Sanadi, D.R., 1982. Mitochondrial coupling factor-B - properties and role in Atp synthesis. Biochim. Biophys. Acta 683, 39e56. Sebastian, A., Prasad, M.N.V., 2018. Exogenous citrate and malate alleviate cadmium stress in Oryza sativa L.: probing role of cadmium localization and iron nutrition. Ecotoxicol. Environ. Saf. 166, 215e222. Shrestha, B., et al., 2013. An evaluation of the impact of multiwalled carbon nanotubes on soil microbial community structure and functioning. J. Hazard Mater. 261, 188e197. Strobel, B.W., 2001. Influence of vegetation on low-molecular-weight carboxylic acids in soil solutionda review. Geoderma 99, 169e198. €m, L., et al., 2001. Organic acid behaviour in a calcareous soil: sorption reactions Stro and biodegradation rates. Soil Biol. Biochem. 33, 2125e2133. Tangaromsuk, J., et al., 2002. Cadmium biosorption by Sphingomonas paucimobilis biomass. Bioresour. Technol. 85, 103e105. Wang, C., et al., 2016a. Application of acclimated sewage sludge as a bioaugmentation/bio-stimulation strategy for remediating chlorpyrifos contamination in soil with/without cadmium. Sci. Total Environ. 579, 657e666. Wang, S., et al., 2017a. Differential effects of citric acid on cadmium uptake and accumulation between tall fescue and Kentucky bluegrass. Ecotoxicol. Environ. Saf. 145, 200e206. Wang, S., et al., 2016b. Effect of low-molecular-weight organic acids on nanohydroxyapatite adsorption of cadmium and lead. J. Biomater. Tissue Eng. 6, 433e439. Wang, T., et al., 2014. Immobilization of Cd in soil and changes of soil microbial community by bioaugmentation of UV-mutated Bacillus subtilis 38 assisted by biostimulation. Eur. J. Soil Biol. 65, 62e69. Wang, Y., et al., 2017b. Vermicompost and biochar as bio-conditioners to immobilize heavy metal and improve soil fertility on cadmium contaminated soil under acid rain stress. Sci. Total Environ. 621. Wu, B., et al., 2016. Mycoextraction by Clitocybe maxima combined with metal immobilization by biochar and activated carbon in an aged soil. Sci. Total Environ. 562, 732e739. Wu, B., et al., 2018. Response of soil micro-ecology to different levels of cadmium in alkaline soil. Ecotoxicol. Environ. Saf. 166, 116e122. Wu, B., et al., 2019a. The performance of biochar-microbe multiple biochemical material on bioremediation and soil micro-ecology in the cadmium aged soil. Sci. Total Environ. 686, 719e728. Wu, C., et al., 2019b. Effect of sulfur-iron modified biochar on the available cadmium and bacterial community structure in contaminated soils. Sci. Total Environ. 647, 1158e1168. Wu, L., et al., 2007. Manipulating soil metal availability using EDTA and lowmolecular-weight organic acids. Phytoremediation 23, 291e303. Wu, L.H., et al., 2003. Effects of EDTA and low molecular weight organic acids on soil solution properties of a heavy metal polluted soil. Chemosphere 50, 819e822. Xiao, K., et al., 2017. Remediation performance and mechanism of heavy metals by a bottom-up activation and extraction system using multiple biochemical materials. ACS Appl. Mater. Interfaces 9, 30448. Yang, H.L., Liao, B.H., 2010. Extraction condition for heavy metals from contaminated soils by using low molecular weight organic acids. J. Agro-Environ. Sci. 29, 2330e2337. Yin, D., et al., 2016. Varying effect of biochar on Cd, Pb and as mobility in a multimetal contaminated paddy soil. Chemosphere 152, 196e206. Zhan, X., et al., 2010. Interactive effect of dissolved organic matter and phenanthrene on soil enzymatic activities. J. Environ. Sci. 22, 607e614. Zhang, G.Z., et al., 2011. Effects of exogenous citric on nutrients of old manured loessal soil. J. Northwest For. Univ. 26, 47e51.
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Elucidation of the mechanisms into effects of organic acids on soil fertility, cadmium speciation and ecotoxicity in contaminated soil Hang Ma 1, Xuedan Li 1, Mingyang Wei 1, Guoquan Zeng 1, Siyu Hou 1, Dan Li 1, Heng Xu*, 1 Key Laboratory of Bio-resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, Sichuan, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Soil fertility was improved with the addition of organic acids. The availability of Cd could increase with the application of organic acids. Organic acids were effective in soil bacterial community structure improvement. Citric acid was most effective on improving soil micro-ecology.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2019 Received in revised form 25 August 2019 Accepted 28 August 2019 Available online 31 August 2019
The remediation effect of organic acids in heavy metal contaminated soil was widely studied. However, the comprehensive evaluation of organic acids on micro-ecological environment in heavy metal contaminated soil was less known. Herein, this experiment was conducted to investigate the impact of malic acid, citric acid and oxalic acid on soil fertility, cadmium (Cd) speciation and ecotoxicity in contaminated soil. Especially, to evaluate the ecotoxicity of Cd, high-throughput sequencing was used to investigate the soil bacterial community structure and diversity after incubation with organic acids. The results showed that obvious changes in soil pH were not observed. Whereas, the contents of available phosphorus (Olsen-P) and alkali hydrolysable nitrogen (Alkeline-N) evidently increased with a significant difference. Furthermore, compared to control, the proportion of acetic acid-extractable Cd increased by 3.06e6.63%, 6.11e9.43% and 1.91e6.22% respectively in the groups amended with malic acid, citric acid and oxalic acid, which indicated that citric acid did better in improving the availability of Cd than malic acid and oxalic acid. In terms of biological properties, citric acid did best in bacteria count increase, enzyme activities and bacterial community structure improvement. Accordingly, these results provided a better understanding for the influence of organic acids on the micro-ecological environment in Cd contaminated soil, based on physicochemical and biological analysis. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: X. Cao Keywords: Organic acids Micro-ecology Soil Cadmium Biological properties
1. Introduction
* 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.chemosphere.2019.124706 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
With the development of industry and agriculture, heavy metal pollution is becoming one of the most widespread and serious environmental problem among various soil pollution due to their concealment and high ecotoxicity (Wu et al., 2019b). Heavy metal pollution accounts for 82.8% of soil contamination types in China,
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among which cadmium (Cd) is up to 7.0%, posing an acute threat to human health and the ecosystem (Xiao et al., 2017). To remediate Cd contaminated soil, various amendments have been performed, including heavy metal removal and immobilization (Liu et al., 2018; Rajendran et al., 2019; Wang et al., 2017b; Wu et al., 2016). There are so many kinds of soluble organic acids in the soil environment, among which malic acid, citric acid and oxalic acid etc., a class of small molecular organic compounds with one or more carboxyl groups, are one of the most important components of soluble organic acids (Chen et al., 2015; Emmanuel et al., 2009; Strobel, 2001). Organic acids are mainly produced from the decomposition of plant and animal residues, the secretion of plant roots and the microbial metabolism in soil. Moreover, the conversion of fertilizer and organic matter is also an important source for organic acids in soil (Jones et al., 2003; Strobel, 2001). Given the diversity of sources, the types and quantities of organic acids in soil are in the dynamic process of synthesis and decomposition, and their concentrations are generally not high (Jones, 1998). However, due to the large amount of organic acids secreted by roots and acidproducing bacteria, the contents of organic acids in the rhizosphere can generally be as high as 1e10 mmol/kg, even higher contents, and the contents of organic acids in the rhizosphere are significantly higher than those in the non-rhizosphere soil (Jones et al., 2003; Strobel, 2001). Biogeochemical processes that involve organic acids (such as malic acid, citric acid and oxalic acid) can be of considerable ecological importance (Hees et al., 2003). Some of the key roles of organic acids in the soil micro-ecological environment are thought to include the detoxification of heavy metals, such as copper (Cu), lead (Pb) and Cd (Arabi et al., 2017; Fei et al., 2004), and the mobilization of nutrients, such as iron (Fe), nitrogen (N), phosphorus (P) and potassium (K) (Bais et al., 2006). There are numerous researches about detoxification of heavy metal contaminated soils with the application of organic acids (Onireti et al., 2017; Wang et al., 2016b; Wu et al., 2007; Yang and Liao, 2010). In addition, organic acids can be used in phytoremediation process to enhance the bioaccumulation of metals into plants through the increase of heavy metal extractability (Liu et al., 2015; Mahmud et al., 2018). Nevertheless, their ecological security as soil conditioners has been rarely reported, and few reports are available concerning soil bacterial community structure and diversity as ecotoxicity indicators in contaminated soil remediated by organic acids. In addition to ecological security, soil fertility is also an important reference factor for soil remediation. And the contents of available phosphorus (Olsen-P) and alkali hydrolysable nitrogen (Alkeline-N) are significant indicators of soil fertility. Soil Olsen-P content restricted the efficient P fertilizer management strategies, and its deficiency was one of the main constraints limiting agricultural crop production (Marschner, 2012). Soil Alkeline-N was the most active component of soil N and an important source of soil available nutrients (Chen et al., 2016). Thus, a series of experiments using malic acid, citric acid and oxalic acid were carried out to (1) investigate the improvement of soil fertility; (2) compare the distribution Cd chemical speciation; (3) assess the toxicity to soil micro-ecology, including bacteria count, soil acid phosphatase and urease activities, bacterial community structures at the genus level. Furthermore, details of possible mechanistic insight into the remediation effects were carefully discussed. 2. Materials and methods 2.1. Soil preparation and experiment design The high concentration of Cd contaminated soil used in this
study was collected from a Cd contaminated site in Sichuan University, Chengdu, China. All of the soil samples were air-dried and passed through a 2-mm sieve to wipe out extraneous matters in soil. The main properties of the soil were presented in Table 1. As shown in Table 2, 10 treatments were set, including 1 control (CK), 9 treatments amended with different concentrations of malic acid, citric acid and oxalic acid, respectively. The concentrations of each type of organic acids were set to 10, 20 and 30 mmol/kg in soil, respectively. And each treatment was replicated for four times. The study was conducted in plastic pots individually, which contained sieved soil (1 kg) mixed with different contents of organic acids. During the period of incubation, all treatments were conducted unified water management and kept a constant temperature (25 C). In the remediation process, all treatments were sampled at 60 d for soil pH, available nutrient contents, heavy metal speciation distribution, bacteria count, enzyme activities, and bacterial community structure analysis. 2.2. Soil pH, Olsen-P and Alkeline-N analysis Soil pH was measured by the method of Corey described (Corey, 1971). The Olsen-P in soil was extracted with NaHCO3 solution for 30 min at 25 ± 1 C and its content was determined by the spectrophotometry at 880 nm (NY/T 1121.7e2014). The content of Alkeline-N in soil was measured by the method as described by previous studies (Wang et al., 2017b). In which the diffusion plate contained 2.0 g of air-dried soil samples and 1.0 g of ZneFeSO4 reducing agent in the outer chamber, 2.0 mL 20 g/L of boric acid indicator in the inner chamber, and the diffusion plate was incubated for 24 h at 40 C. Then, the NH3 absorbed by boric acid was titrated by 0.005 M H2SO4 solution. 2.3. Cadmium speciation distribution analysis Cadmium fractions in soil were separately extracted using the
Table 1 The main properties of soil samples. Properties
Value
pH Electrical conductivity (EC, mS/cm) Water holding capacity (%) Sand (%) Silt (%) Clay (%) CaCO3 (g/kg) Organic C (g/kg) Total N (g/kg) Total P (g/kg) Cd (mg/kg)
7.70 264 60.6 63.4 24.1 12.5 91.4 9.2 1.5 1.1 10.12
Table 2 The design of all the treatments. Treatments
Amendment
CK T1 T2 T3 T4 T5 T6 T7 T8 T9
Control, without amendment 10 mmol/kg malic acid 20 mmol/kg malic acid 30 mmol/kg malic acid 10 mmol/kg citric acid 20 mmol/kg citric acid 30 mmol/kg citric acid 10 mmol/kg oxalic acid 20 mmol/kg oxalic acid 30 mmol/kg oxalic acid
There were 4 replicates for each treatment.
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modified European Community Bureau of Reference (BCR) procedure, and operationally defined as HOAc-extractable (acetic acidextractable), reducible, oxidizable and residual fractions (Nemati et al., 2011; Wang et al., 2017b). The residual fractionation of soil samples was digested using a mixture of HNO3:HCl:HF (5:2:2, v:v:v) in a microwave oven (SINEO,MDS-6, Shanghai, China), then the concentrations of different heavy metal fractions in samples were detected by Flame Atomic Absorption Spectrometry (FAAS: VARIAN, SpectrAA 220FS, USA) (Recovery: 95.7%e105.0%; RSD ¼ 1.0%) (Wu et al., 2016). 2.4. Cadmium toxicity to soil micro-ecology analysis Bacteria count was counted according to the spread-plate method that described by Liu et al. (2018). Briefly speaking, aqueous extracts of 3 g fresh soil samples were serially diluted to spread on beef extract-peptone. Then, the numbers of colony forming units (CFU) of bacteria were recorded after incubation at suitable circumstance for 2e5 d. Acid phosphatase and urease activities were measured referring to previous study (Pan and Yu, 2011; Zhan et al., 2010). The activity of acid phosphatase was expressed as p-nitrophenol (pNP) mg per g soil per h, and spectrophotometrically determined at 410 nm. While, urease activity was expressed as NHþ 4 -N mg per g soil per 24 h and spectrophotometrically determined at 578 nm. For further bacterial community analysis, the bacterial DNA was extracted using a Soil DNA Kit (Omega Biotek Inc., Norcross, GA) and amplified. The bacterial community was investigated on Illumina MiSeq platform, which was conducted by Majorbio BioPharm Technology Co., Ltd (Shanghai, China). Sequences were clustered into operational taxonomic units (OTUs) and the biological information analysis was conducted on the Usearch program (version 7.1) at 97% similarity. 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 (Wang et al., 2016a). 2.5. Statistical analysis In our study, all results were presented by value ± standard deviation. Statistical significance was performed using SPSS 18.0 package and statistics were performed using the Origin 8.0 (USA).
Fig. 1. Soil pH (a) in Cd contaminated soil amended with different organic acids. Error bars represented the standard deviation of four replicates. Different letters (a, b, c ….) indicated significant (P < 0.05) difference among different treatments.
be taken into account. For example, some scholars had studied that the change of rhizosphere soil pH was not observed in the release of organic acids under environmental stress (LV, 1995). Especially for calcareous soil, there was a large amount of free calcium carbonate in calcareous soil. After adding organic acids into the soil, Hþ would be neutralized by calcium carbonate and greatly reduced, presumably due to the release of CO2 3 from CaCO3 and the formation of HCO 3 (Fei et al., 2004; Zhang et al., 2011). Therefore, in this experiment, when calcareous soil (91.4 g/kg CaCO3, as shown in Table 1) was treated with different types of organic acids at different concentrations, obvious changes in soil pH were not observed, and there was no significant difference in treatments.
3.2. The contents of Olsen-P and Alkeline-N analysis The contents of Olsen-P and Alkeline-N significantly increased (p < 0.05) in all treatments than CK (Fig. 2). It was obvious that the
3. Results and discussion 3.1. Soil pH analysis After 60 d incubation, the soil pH values of all the treatments were weakly changed compared to control (Fig. 1). When malic acid, citric acid and oxalic acid in different concentrations were added, the pH values slightly increased by 0.185e0.25, 0.275e0.41 and 0.15e0.28 than CK, respectively. However, there was no significant difference among the treatments. Soil pH was known as a major factor that regulated the availability of nutrients, distribution of heavy metal, and indigenous microbial activity (Liu et al., 2018). Thus, it was extremely important to investigate the changes of soil pH after incubation process. Hþ could be dissociated from the carboxyl group of the organic acids, which undoubtedly had a certain impact on soil pH, soil environment alteration and rhizosphere biochemical process € ttger, 1991). As evaluating the impact of organic (Petersen and Bo acids on soil pH factors, such as soil buffering, soil structure, clay composition, organic matter content, microbial activity etc., should
Fig. 2. Olsen-P and Alkeline-N (b) in Cd contaminated soil amended with different organic acids. Error bars represented the standard deviation of four replicates. Different letters (a, b, c ….) indicated significant (P < 0.05) difference among different treatments.
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highest content of Olsen-P was presented in the treatment groups amended with oxalic acid, and the content of Olsen-P in oxalic acid treatment groups increased by 272.55e353.68% compared with CK. Besides, in the treatments amended with citric acid and malic acid, the contents of Olsen-P increased by 234.64e329.74% and 157.76e248.28% compared to CK, respectively. Meanwhile, the content of alkaline-N increased by 153.08e229%, 193.5e341.48% and 105.62e209.5% respectively compared to CK, with the addition of malic acid, citric acid and oxalic acid. Generally, as the higher concentrations of the organic acids were applied in soil, the more contents of Olsen-P and Alkeline-N were extracted from the soil. As illustrated in Fig. 2a, the soil Olsen-P content was evidently enhanced with the application of the organic acids compared with the control treatment. The application of organic acids had potential to improve the mobilization of P through a series of measures including solubilizing minerals and desorbing P from mineral surfaces (Rasouli-Sadaghiani, 2014). Moreover, the distribution of heavy metal speciation in soil could be effected with the addition of organic acids (Lu et al., 2007). As numerous heavy metal releasing, the active adsorption sites for P were added, leading to the coordination adsorption addition and the augment of the Olsen-P (Wang et al., 2017b). In calcareous soil, oxalate might primarily release P held in CaeP minerals through the formation and precipitation of Ca-oxalate. In contrast, citrate acids had a relatively € m et al., 2001). This was supported to poor affinity for Ca2þ (Stro some extent by our experimental results, which showed that the activation effect of P of oxalic acid was better than citric and malic acids at the same molar concentration. The availability of some elements (e.g. P and Fe) were directly related to organic acids, while the availability of some other elements (e.g. N and K) might also be directly or indirectly related to organic acids (Jones and Darrah, 1995). Organic acids could also adsorb N and K through tight connections, such as hydrogen bonds and ionic bonds. Besides, soil Alkeline-N was related to indigenous microbial activity, which had capacity to influence the processes of N recycling (Saha et al., 2012). The addition of amendments not only increased the nutrient contents but also stimulated the microbial activity, and in return, the enhancement of microbial activity affected the activation of nutrient in soil (Saha et al., 2012). The addition of organic acids not only increased the nutrient contents but also stimulated the microbial activity, and in return, the enhancement of microbial activity could affect the activation of nutrient in soil. The microbial activity was more energetic in treatment groups added with citric acid than malic acid and oxalic acid (see “Soil bacteria count and enzyme activities analysis” section), which might be the possible reason why the highest content of Alkeline-N was presented in the treatment groups amended with citric acid.
3.3. Cadmium speciation distribution analysis In the present work, BCR extraction was used to investigate chemical form variation after incubation, and the results were described as Fig. 3. As shown by the results, the speciation distribution of Cd in soil was dramatically influenced by the addition of organic acids. Compared with the CK, the proportion of HOAcextractable Cd evidently increased by 3.06e6.63%, 6.11e9.43% and 1.91e6.22% in the treatment groups amended with malic acid, citric acid and oxalic acid, respectively. Obviously, the treatment groups amended with citric acid were presented the best activation effect on Cd, and their contents of HOAc-extractable Cd were more than the treatment groups amended with malic acid and oxalic acid. When the proportion of HOAc-extractable Cd increased in soil, the proportion of reducible Cd decreased by 2.13e4.15%, 4.875e6.22%
Fig. 3. Metal speciation distribution in Cd contaminated soil amended with different organic acids.
and 1.165e3.78% in the treatments amended with malic acid, citric acid and oxalic acid, respectively. In addition, the contents of oxidizable and residual Cd showed a decreasing tendency in general, and the content of residual Cd was the lowest compared with the content of HOAc-extractable, reducible, and oxidizable fractions. Cadmium binding capacities and soil properties (e.g. soil pH, CEC and organic matter) were reported to have relationships with the content of residual Cd in soil, the low and even none content of residual fraction of Cd in soil could be caused by the weak Cd binding capacity of soil and the low content of organic matter (Wang et al., 2014; Yin et al., 2016). It was widely recognized that the extractability and mobility of heavy metals in soil depended strongly on their speciation distribution and binding state (Lu et al., 2007). As described in the previous researches (Xiao et al., 2017), the HOAc-extractable Cd was strongly positive correlated with heavy metal extractability. Nevertheless, the reducible, oxidizable and residual fractions of Cd speciation were negatively correlated with heavy metal bioavailability, among which residual Cd had the biggest inhibition effect on the extractability of heavy metal. In present work, the proportion of HOAc-extractable Cd evidently increased and the proportion the remaining three forms of Cd speciation decreased. Therefore, the application of organic acids significantly improved the extractability of Cd. And some researches had reported that the bioaccumulation of Cd in plants were obvious enhanced with the addition of organic acids (Mahmud et al., 2018; Wang et al., 2017a). These works could also verify that organic acids could increase the availability of Cd. Previous studies suggested that organic acids, especially malic acid, citric acid and oxalic acid, could not only acidize soil, but also be able to form soluble complexes and chelates with metal ions (Liu et al., 2012, 2015). The availability of heavy metals would be increased with the decreasing of soil pH (Wang et al., 2017b). However, obvious changes in calcareous soil pH were not observed after amended with organic acids, indicating that pH was not the significant factor governing the Cd speciation distribution in this study. Therefore, the effect of Cd speciation distribution was mainly dependent on ionic form and concentration of organic acids. Malic acid, citric acid and oxalic acid that carried two or three carboxylic groups could form Cd-organic acids complexes with 5 or 6 membered ring structures, which increased mobility of Cd in soil and plant (Fei et al., 2004). Previous studies had found that free
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Cd2þ was more toxic to crops than Cd-organic acids (Aravind and Prasad, 2005; Wu et al., 2003). Accordingly, metal chelates formed by organic acids and Cd not only increased the mobility of Cd in plants, but also alleviated the toxicity of Cd to plants. For same type of organic acids, the activation effect on Cd increased with the increasing of organic acids concentration. While At the same molar concentration, the ability of organic acids to influence Cd speciation distribution might depend on the molecular weight and structure of organic acids. On one hand, citric acid had a 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 it carried more surface area and more negative charge (Jing et al., 2007). It was one of possible reasons that citric acid could chelate more metals than malic acid and oxalic acid. On the other hand, the functional groups of organic acids (e.g. carboxylic and hydroxyl groups) were important binding sites for metals. Citric acid had three carboxylic and one hydroxyl groups, malic acid had two carboxylic and one hydroxyl groups, and oxalic acid only had two carboxylic groups, accordingly citric acids could desorb more Cd2þ. In present study, we got the same results with other studies that citric acids did better than malic and oxalic acids in increasing the availability Cd in soil (Liu et al., 2015). 3.4. Cadmium toxicity to soil micro-ecology analysis 3.4.1. Soil bacteria count and enzyme activities analysis Microbial biomass and enzyme activities are important biochemical indexes of soil micro-ecology (Wu et al., 2019a). The impact of different organic acids on bacteria count and enzyme activities in Cd-polluted soil were depicted in Fig. 4. As shown in Fig. 4a, the bacteria count in treatments amended with organic acids dramatically increased after incubation, and there was a significant difference among treatments. The bacteria count increased by 36.23e158.94%, 45.41e192.27% and 17.39e88.35% than CK, when added with malic acid, citric acid and oxalic acid, respectively. Similar to the bacteria number, the activities of soil enzyme also had significant enhancements, and the highest activities of acid phosphatase and urease were all presented in the treatments with the application of citric acid (239.805e415 mg pNP/g soil/h and 0.706e0.934 mg NHþ 4 -N/g soil/24 h, respectively). Whereas the lowest acid phosphatase and urease activities were found in the treatments added with malic acid (182.775e333.19 mg pNP/g soil/h) and oxalic acid (0.3925e0.622 mg NHþ 4 -N/g soil/24 h), respectively. These results showed that different types of organic acids had different impacts on enzyme activities. What's more, the bacteria number increased and the activities of soil enzymes improved with an increase of the concentrations of amendments in Cd-polluted soil. Many previous studies had revealed that excessive heavy metals were toxic to soil microorganisms, through their capability of hindering enzyme activity, making DNA mutation and compromising cell membrane integrity (Poli et al., 2009; Wu et al., 2016). Fortunately, the toxicity of Cd to microorganisms in soil could be relieved by organic acids through a variety of direct or indirect strategies. On the one hand, organic acids could reduce the toxicity of Cd directly, by forming chelates with Cd2þ to prevent Cd2þ from reacting with bacterial active groups (Li et al., 2019). On the other hand, organic acids could provide C elements for microbes. As reported in the works of previous researchers (Bowers et al., 1996), organic acids (e.g. malic acid, citric acid and oxalic acid) were short chain organic acids with low molecular weight (less than 200), which could easily pass through the thinner cell walls and cell membranes of bacteria, and bacteria were able to use them as energy materials directly. In addition, some nutrient elements could be activated by organic
Fig. 4. Soil bacteria count (a), acid phosphatase activity (b) and urease activity (c) in Cd contaminated soil amended with different organic acids. Error bars represented the standard deviation of four replicates. Different letters (a, b, c ….) indicated significant (P < 0.05) difference among different treatments.
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Fig. 5. Bacterial community structures (a) and Shannon curves (b) at the genus level in Cd contaminated soil amended with different organic acids.
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acids, and it was verified in present study (Fig. 2). Moreover, organic acids were related to Fe absorption of organisms, and they could alleviate Fe deficiency induced by Cd to promote the growth of organisms (Sebastian and Prasad, 2018). When the three types of organic acids were added at the same molecular concentration, the groups added by citric acid did better in increasing the bacteria number than malic acid and oxalic acid. The reasons might be that citric acid had the better ability to chelate Cd2þ and activate nutrients (Fei et al., 2004; Khademi et al., 2009), and the results of this experiment also supported this conclusion. A large number of studies had shown that soil enzyme activities were sensitive to environmental stress and could be used to indicate the pollution status of soil micro-ecological environment, including for heavy metal pollution (Pascual et al., 2000; Wu et al., 2018). Therefore, the determination of activities of acid phosphatase and urease was of great significance. The enzyme activities integrally increased after incubation, and that might be related to the augment of soil microbial activity. In addition, the increasing of enzyme activities might be partly caused by functional groups of the organic acids, because these active functional groups could react with Cd2þ in soil by forming metal-organic acids complexes to prevent Cd2þ from binding to sulphydryl groups of enzymes (Sanadi, 1982). The activities of urease and acid phosphatase were the highest in the groups added with citric acid, and the possible reasons were related to the better abilities of citric acid to chelate Cd2þ and improve the microbial activity than malic acid and oxalic acid. Moreover, soil enzymes played important roles in the geochemical process of elements, and they could activate nutrients (e.g. N, P and Fe) to promote microbes growth (Lebrun et al., 2012). In return, microbes secreted more enzymes. Therefore, the addition of organic acids provide a virtuous circle in Cd-polluted soil microecology.
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Nocardioides increased were the degradation of organic acids and the reduction of Cd toxicity after remediation in contaminated soil. Furthermore, the treatments with the addition of organic acids showed higher Shannon indexes than CK (Fig. 5b), and the maximum value was 3.29 in the citric acid treatment groups. Shannon index was always widely used to analyze microbial community diversity (Wu et al., 2018). These results also implied the superiority of the application of the amendments for improving the soil bacterial diversity. 4. Conclusions Our results showed that the micro-ecological environment of Cd-polluted soil significantly improved after treatment with organic acids. With the application of organic acids, the contents of Olsen-P and Alkeline-N evidently increased. In addition, the availability of heavy metals greatly increased while the ecotoxicity decreased. Especially, bacteria count and enzyme activities, were more strongly influenced by organic acids addition. In this experiment, the higher the concentration of amendments was, and the better the repair effect was. Based on this, high-throughput sequencing was used to investigate the soil bacterial community structure after incubation with organic acids at the concentration of 30 mmol/kg. Briefly, citric acid had the best repairing effect among the three types of organic acids, taking the impacts on nutrient contents, heavy metal bioavailability, bacteria count, enzyme activities and bacterial community structure into consideration. Our results suggested that organic acids could be used as activators to increase the extractability of Cd and as ameliorants to improve micro-ecological environment in contaminated soil. Conflicts of interest There was no conflict of interest.
3.4.2. Soil bacterial community structure and diversity analysis As previously mentioned, the higher concentration of the organic acids were added in soil, and the greater repairing effects were presented in each type of organic acids treatment groups. Hence, three treatment groups and CK were investigated to analysis the diversity of bacterial community structure through highthroughput sequencing. The structures of bacterial community under different treatments on genus level were depicted as Fig. 5a, and sequences with low abundance (less than 1%) were assigned as others. The compositions of soil bacteria genera in control and other treatments were similar, whereas the abundance of each bacteria genus in every treatment was different. On genus level, the highest abundance genus in CK was Sphingomonas (22.60%), and the abundance of Sphingomonas decreased to 16.01%, 6.35% and 13.69% respectively in the treatments added with malic acid, citric acid and oxalic acid. Sphingomonas bacteria were proven as Cd resistant bacteria and promising applications for enhancing bioremediation in Cd-contaminated sites (Pan et al., 2016; Tangaromsuk et al., 2002). Treatment with organic acids could relieve the Cd stress to soil microorganisms, which would explain why the abundance of Sphingomonas decreased in organic acids treatment groups compared to CK. Moreover, the lowest abundance of Sphingomonas was in the citric acid group, which further indicated that citric acid possessed better detoxification ability than malic acid and oxalic acid in Cd-contaminated soil. The abundance of Nocardioides was also different in CK (2.27%) and organic acids treatment groups (5.82e9.73%). There was scarcely any research about the relationship between Nocardioides bacteria and heavy metals, but Nocardioides bacteria were reported to own the ability to degrade organic matters (Morrissey et al., 2015; Shrestha et al., 2013). Accordingly, the reasons why the relative abundance of
Acknowledgments 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 (2017SZ0181, 2018NZ0008), and the Fundamental Research Funds for the Central Universities (SCU2019D013). The authors also wish to thank Professor Guanglei Cheng from Sichuan University for the technical assistance. References Arabi, Z., et al., 2017. Cadmium removal from Cd-contaminated soils using some natural and synthetic chelates for enhancing phytoextraction. Chem. Ecol. 1e14. Aravind, P., Prasad, M.N.V., 2005. Cadmium-induced toxicity reversal by zinc in Ceratophyllum demersum L. (a free floating aquatic macrophyte) together with exogenous supplements of amino- and organic acids. Chemosphere 61, 1720e1733. Bais, H.P., et al., 2006. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu. Rev. Plant Biol. 57, 233e266. Bowers, J.H., et al., 1996. Infection and colonization of potato roots by Verticillium dahliae as affected by Pratylenchus penetrans and P. Crenatus. Phytopathology 86, 614e621. Chen, B., et al., 2016. Effect of N fertilization rate on soil alkali-hydrolyzable N, subtending leaf N concentration, fiber yield, and quality of cotton. Crop J. 4, 323e330. Chen, Z., et al., 2015. Confirmation and determination of carboxylic acids in root exudates using LC-ESI-MS. J. Sep. Sci. 30, 2440e2446. Corey, R.B., 1971. A Textbook of Soil Chemical Analysis. Emmanuel, D., et al., 2009. Spectroscopic characterization of organic matter of a soil and vinasse mixture during aerobic or anaerobic incubation. Waste Manag. 29, 1929e1935. Fei, Q., et al., 2004. Effects of low-molecular-weight organic acids and residence time on desorption of Cu, Cd, and Pb from soils. Chemosphere 57, 253e263.
8
H. Ma et al. / Chemosphere 239 (2020) 124706
Hees, P.A.W.V., et al., 2003. Low molecular weight organic acid adsorption in forest soils: effects on soil solution concentrations and biodegradation rates. Soil Biol. Biochem. 35, 1015e1026. Jing, Y.D., et al., 2007. Effects of pH, organic acids, and competitive cations on mercury desorption in soils. Chemosphere 69, 1662e1669. Jones, D.L., 1998. Organic acids in the rhizosphere e a critical review. Plant Soil 205, 25e44. Jones, D.L., Darrah, P.R., 1995. Influx and efflux of organic-acids across the soil-root interface of Zea-Mays L and its implications in rhizosphere C flow. Plant Soil 173, 103e109. Jones, D.L., et al., 2003. Organic acid behavior in soils e misconceptions and knowledge gaps. Plant Soil 248, 31e41. Khademi, Z., et al., 2009. Organic acid mediated nutrient extraction efficiency in three calcareous soils. Aust. J. Soil Res. 47, 213e220. Lebrun, J.D., et al., 2012. Assessing impacts of copper on soil enzyme activities in regard to their natural spatiotemporal variation under long-term different land uses. Soil Biol. Biochem. 49, 150e156. Li, X., et al., 2019. Mechanisms into the removal and translocation of cadmium by Oudemansiella radicata in soil. Environ. Sci. Pollut. Control Ser. 1e11. Liu, H., et al., 2015. Mitigation mechanism of Cd contaminated soils by different levels of exogenous low-molecular-weight organic acids with Phytolacca americana. RSC Adv. 5, 47e49. Liu, H., et al., 2018. Effect of modified coconut shell biochar on availability of heavy metals and biochemical characteristics of soil in multiple heavy metals contaminated soil. Sci. Total Environ. 645, 702e709. Liu, M.Y., et al., 2012. Research on Differences of Component and Quantity of Organic Acids in the Root Exudates Among the Three Green Manures. World Automation Congress, pp. 1e4. Lu, H., et al., 2007. Low-molecular-weight organic acids exuded by Mangrove (Kandelia candel (L.) Druce) roots and their effect on cadmium species change in the rhizosphere. Environ. Exp. Bot. 61, 159e166. LV, K., 1995. Cellular mechanisms of aluminum toxicity and resistance in plants [review]. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 237e260. Mahmud, J.A., et al., 2018. Insights into citric acid-induced cadmium tolerance and phytoremediation in Brassica juncea L.: coordinated functions of metal chelation, antioxidant defense and glyoxalase systems. Ecotoxicol. Environ. Saf. 147, 990e1001. Marschner, H., 2012. Mineral nutrition of higher plants/Horst Marschner. J. Ecol. 76, 1250. Morrissey, E.M., et al., 2015. Dynamics of extracellular DNA decomposition and bacterial community composition in soil. Soil Biol. Biochem. 86, 42e49. Nemati, K., et al., 2011. Speciation of heavy metals by modified BCR sequential extraction procedure in different depths of sediments from Sungai Buloh, Selangor, Malaysia. J. Hazard Mater. 192, 402e410. Onireti, O.O., et al., 2017. Combined effects of low-molecular-weight organic acids on mobilization of arsenic and lead from multi-contaminated soils. Chemosphere 170, 161e168. Pan, F., et al., 2016. Endophytic bacterium Sphingomonas SaMR12 promotes cadmium accumulation by increasing glutathione biosynthesis in Sedum alfredii Hance. Chemosphere 154, 358e366. Pan, J., Yu, L., 2011. Effects of Cd or/and Pb on soil enzyme activities and microbial community structure. Ecol. Eng. 37, 1889e1894. Pascual, J.A., et al., 2000. Soil microbial activity as a biomarker of degradation and remediation processes. Soil Biol. Biochem. 32, 1877e1883. €ttger, M., 1991. Contribution of organic acids to the acidification of Petersen, W., Bo the rhizosphere of maize seedlings. Plant Soil 132, 159e163. Poli, A., et al., 2009. Heavy metal resistance of some thermophiles: potential use of a-amylase from Anoxybacillus amylolyticus as a microbial enzymatic bioassay. Res. Microbiol. 160, 99e106. Rajendran, M., et al., 2019. Effect of sulfur and sulfur-iron modified biochar on cadmium availability and transfer in the soil-rice system. Chemosphere 222,
314e322. Rasouli-Sadaghiani, M.H., 2014. Effects of low-molecular-weight organic acids on phosphorus sorption characteristics in some calcareous soils. Turk. J. Agric. For. 36, 459e468. Saha, S., et al., 2012. Vermicompost and Soil Quality. Springer, Netherlands. Sanadi, D.R., 1982. Mitochondrial coupling factor-B - properties and role in Atp synthesis. Biochim. Biophys. Acta 683, 39e56. Sebastian, A., Prasad, M.N.V., 2018. Exogenous citrate and malate alleviate cadmium stress in Oryza sativa L.: probing role of cadmium localization and iron nutrition. Ecotoxicol. Environ. Saf. 166, 215e222. Shrestha, B., et al., 2013. An evaluation of the impact of multiwalled carbon nanotubes on soil microbial community structure and functioning. J. Hazard Mater. 261, 188e197. Strobel, B.W., 2001. Influence of vegetation on low-molecular-weight carboxylic acids in soil solutionda review. Geoderma 99, 169e198. €m, L., et al., 2001. Organic acid behaviour in a calcareous soil: sorption reactions Stro and biodegradation rates. Soil Biol. Biochem. 33, 2125e2133. Tangaromsuk, J., et al., 2002. Cadmium biosorption by Sphingomonas paucimobilis biomass. Bioresour. Technol. 85, 103e105. Wang, C., et al., 2016a. Application of acclimated sewage sludge as a bioaugmentation/bio-stimulation strategy for remediating chlorpyrifos contamination in soil with/without cadmium. Sci. Total Environ. 579, 657e666. Wang, S., et al., 2017a. Differential effects of citric acid on cadmium uptake and accumulation between tall fescue and Kentucky bluegrass. Ecotoxicol. Environ. Saf. 145, 200e206. Wang, S., et al., 2016b. Effect of low-molecular-weight organic acids on nanohydroxyapatite adsorption of cadmium and lead. J. Biomater. Tissue Eng. 6, 433e439. Wang, T., et al., 2014. Immobilization of Cd in soil and changes of soil microbial community by bioaugmentation of UV-mutated Bacillus subtilis 38 assisted by biostimulation. Eur. J. Soil Biol. 65, 62e69. Wang, Y., et al., 2017b. Vermicompost and biochar as bio-conditioners to immobilize heavy metal and improve soil fertility on cadmium contaminated soil under acid rain stress. Sci. Total Environ. 621. Wu, B., et al., 2016. Mycoextraction by Clitocybe maxima combined with metal immobilization by biochar and activated carbon in an aged soil. Sci. Total Environ. 562, 732e739. Wu, B., et al., 2018. Response of soil micro-ecology to different levels of cadmium in alkaline soil. Ecotoxicol. Environ. Saf. 166, 116e122. Wu, B., et al., 2019a. The performance of biochar-microbe multiple biochemical material on bioremediation and soil micro-ecology in the cadmium aged soil. Sci. Total Environ. 686, 719e728. Wu, C., et al., 2019b. Effect of sulfur-iron modified biochar on the available cadmium and bacterial community structure in contaminated soils. Sci. Total Environ. 647, 1158e1168. Wu, L., et al., 2007. Manipulating soil metal availability using EDTA and lowmolecular-weight organic acids. Phytoremediation 23, 291e303. Wu, L.H., et al., 2003. Effects of EDTA and low molecular weight organic acids on soil solution properties of a heavy metal polluted soil. Chemosphere 50, 819e822. Xiao, K., et al., 2017. Remediation performance and mechanism of heavy metals by a bottom-up activation and extraction system using multiple biochemical materials. ACS Appl. Mater. Interfaces 9, 30448. Yang, H.L., Liao, B.H., 2010. Extraction condition for heavy metals from contaminated soils by using low molecular weight organic acids. J. Agro-Environ. Sci. 29, 2330e2337. Yin, D., et al., 2016. Varying effect of biochar on Cd, Pb and as mobility in a multimetal contaminated paddy soil. Chemosphere 152, 196e206. Zhan, X., et al., 2010. Interactive effect of dissolved organic matter and phenanthrene on soil enzymatic activities. J. Environ. Sci. 22, 607e614. Zhang, G.Z., et al., 2011. Effects of exogenous citric on nutrients of old manured loessal soil. J. Northwest For. Univ. 26, 47e51.