The effect of an antimony resistant bacterium on the iron plaque fraction and antimony uptake by rice seedlings

Environmental Pollution xxx (xxxx) xxx

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The effect of an antimony resistant bacterium on the iron plaque fraction and antimony uptake by rice seedlings* Jiumei Long a, Dongsheng Zhou a, Bingyu Li b, c, Yimin Zhou b, c, Yongjie Li b, c, Ming Lei b, c, * a b c

College of Life Sciences & Environment, Hengyang Normal University, Hengyang, 421008, PR China College of Resource & Environment, Hunan Agricultural University, Changsha, 410128, PR China Hunan Engineering Research Center for Safe and High-Efficient Utilization of Heavy Metal Pollution Farmland, Changsha, 410128, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 September 2019 Received in revised form 20 November 2019 Accepted 22 November 2019 Available online xxx

Iron plaque (IP) is crucial in mitigating antimony (Sb) uptake and accumulation in rice plants, while, few studies focused on the effect of the iron plaque-associated Sb resistant bacteria on IP and Sb uptake into rice plants. Here, the effect of a Sb resistant bacterium (GenBank accession No. MH345840, with potential of conversion soluble Sb(III) into insoluble Sb2O3) on IP and Sb(III)/Sb(V) uptake under hydroponic condition was investigated. The results showed that in the presence of Sb(III), a large quantity of bacterial cells consorted with IP on rice roots, the bacterial inoculum altered the IP fraction distribution without enhancing its amount. However, it reduced Sb(III) uptake into rice roots. On contrary, seldom bacterial cells associated with the IP on rice roots in the presence of the Sb(V), the bacterial inoculum increased the IP amount slightly, and did not decline the Sb(V) uptake into rice roots. It also showed that the bacterial inoculum decreased Sb concentrations in rice shoots greatly in both Sb(III) and Sb(V) supplied treatments. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Antimony resistant bacteria Sb(III) and Sb(V) Rice plants Iron plaque fraction Sb uptake

1. Introduction Antimony (Sb) is a chalcophilic metalloid belonging to group 15 of the periodic table, which is similar to arsenic (As). Although Sb has not yet be identified as human carcinogen, the Council of the European Communities and the U.S. Environmental Protection Agency listed it as a priority pollutant due to its toxicity (Council of the European Communities, 1976; U. S. Environmental Protection Agency, 1979a, b). It was regarded that elemental Sb is more toxic than its salts and inorganic species of Sb are more toxic than the organic ones. The studied showed that Sb(III) compounds are about 10 times more toxic than Sb(V) species (Smichowski, 2008; He et al., 2012). Both natural and anthropogenic sources are responsible for the Sb contamination in the environment. It reported that about 5 tonnes year
1 of Sb are released into the atmosphere by volcanoes (Hinkley et al., 1999), resulting in Sb contamination in the atmosphere. Besides, rock weathering and soil runoff are also

* €rg Rinklebe. This paper has been recommended for acceptance by Dr. Jo * Corresponding author. College of Resource & Environment, Hunan Agricultural University, Changsha, 410128, PR China. E-mail address: [email protected] (M. Lei).

important natural sources contributed to Sb contamination. Among the anthropogenic sources, Sb related mining and/or smelting activities were considered as the greatest contributors to Sb contamination in the environment (He, 2007; Liu et al., 2007; Zhang et al., 2009; He et al., 2012). Moreover, with extensive application of Sb in industry and for items, Sb contamination become more and more ubiquitous throughout the environment (Nriagu, 2005; Filella et al., 2009). Sb in contaminated soil is “phyto-available” for plants (Ainsworth et al., 1990; Hammel et al., 2000; He, 2007), thereby it might enter the food chain and threat human health. Rice is a staple food for more than three billion people worldwide. A number of previous studies reported that rice plant could take up and accumulate both Sb(III) and Sb(V) (He and Yang, 1999; Wu et al., 2011; Huang et al., 2012; Ren et al., 2014; Cui et al., 2015; Ding et al., 2015; Cai et al., 2016). These studies proposed that the rice root is the major uptake and accumulation tissue for Sb. A variety of factors were assumed to influence the Sb uptake into rice plant, such as rice cultivars, rice growth stage, co-exist ions with Sb and Sb speciation. For example, Zhang et al. (2017) reported that hybrid genotypes of rice plant could take up significantly more Sb than indica genotype ones. Our earlier study showed that there were

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significantly differences of Sb uptake into rice among different growth stages, tillering and jointing stages were identified as the most efficient growth stages for rice to take up Sb (Long et al., 2019). Besides, as a kind of typical wetland plant, rice plant induces a large number of iron plaque (IP) on rice roots under natural condition, through oxidation of soluble reductive Fe2þ in the rice rhizosphere (Chen et al., 1980; Fu et al., 2014). It proposed that the fractions of IP on rice root were ferrihydrite, siderite and a small amount of goethite (Hansel et al., 2002; Liu et al., 2005). Extensive studies have proved that IP is a critical “barrier” for rice plant to take up Sb in the rhizosphere (Huang et al., 2012; Ren et al., 2014; Cui et al., 2015), thereby, IP is important to abate the Sb accumulation in rice grains. Since metal resistant and plant growth-promoting bacteria with the potential of decreasing heavy metal accumulation in rice plants, these metal resistant bacteria attracted wide attention recent years (Rajkumar et al., 2012). Li et al. (2017) reported that an inoculum of cadmium (Cd) immobilized bacterium immobilized 79～96% of soluble Cd in the rice rhizosphere, and thus decreased 45～72% of Cd in the rice grains. Mitra et al. (2018) also found that a Cd immobilized bacterium decreased 73～97% of Cd in the rice rhizosphere and reduced the Cd in rice plant greatly. Lin et al. (2010) demonstrated that sulfur reducing bacteria (SRB) in the rhizosphere of rice plant changed the pH and Eh, which in turn immobilized the lead (Pb) and reduced the Pb uptake into rice plant. It was worth noting that there are substantial iron plaqueassociated bacteria in rice rhizosphere (Weiss et al., 2003), which may impact the IP formation and mediate the “barrier” function of IP on the heavy metals. Some scientists have proved that iron oxidizing bacteria (IOB) could promote the formation of IP and reduce the heavy metal uptake by rice plant (Neubauer et al., 2007). Lakshmanan et al. (2015) reported that an As immobilized bacterium increased the oxidation capacity of the rice rhizosphere and the IP formation, thus it ameliorated the As uptake by rice roots. Dong et al. (2016) also demonstrated that an As/Cd immobilized bacterium decreased the As/Cd accumulation in rice plants through promoting the IP formation. However, there is little information about the effect of bacteria on IP fractions, which was relevant to the “barrier” capacity of the IP (Fu et al., 2016). Moreover, the application of Sb resistant bacteria to reduce Sb uptake into rice plants has not yet been reported. The overall goal of this study was to examine the IP fractions, Sb species in rice roots and shoots when rice seedling exposed to Sb(III) or Sb(V) in the presence of Sb resistant bacterium inoculum. The specific aims of the present study were to: 1) investigate whether the Sb resistant bacterium inoculum affects IP amounts and fractions on rice roots exposed to Sb(III) or Sb(V); 2) evaluate the effect of Sb resistant bacterium inoculum on Sb(III)/Sb(V) uptake and translocation in rice seedlings. 2. Materials and methods All the chemicals and reagents used in the present study were of analytical or guaranteed grade. Deionized water (18.2 MU cm) used in this study was sterilized. All plastic and glass containers were acid washed, and glass containers were further sterilized before use. 2.1. The isolation and preparation of the Sb resistant bacterium The Sb resistant bacterium used in the present study was isolated from Sb contaminated soils of Xikuangshan (27440 4600 N, 111270 3900 E) area in Hunan province of China, the world’s largest Sb mine. The isolation, screening and subsequent identification of

the bacterial strain was performed according to the method of Jiang et al. (2008). The sequence of 16S rDNA of the strain was deposited in the GenBank (Accession number is MH345840). The strain is rod shaped and belongs to Ochrobactrum. sp, and it showed extremely high Sb resistance (MIC of Sb(III) > 20 mM). To prepare the bacterial inoculum to rice rhizosphere, the strain was initially grown in Luria-Bertani’s (LB) medium at 28  C for 10 h, harvested by centrifugation at 4000g, and washed twice with sterile distilled water. The cell pellets were then re-suspended in sterile distilled water to obtain a 108 CFU mL
1 suspension, which was used as the bacterial inoculum. 2.2. The effect of the bacterial strain on soluble Sb(III) With the incubation of the isolated strain in liquid medium containing soluble Sb(III), precipitation was noticed at the bottom of the culture. To study the effect of the isolated strain on soluble Sb(III), the strain was cultured in 200 mL liquid LB medium containing 20 mM Sb(III) at 28  C with 150 r min
1, shaking for 7 d. Every 24 h, 2 mL of aliquots from culture was sampled and filtered through 0.45 mm nylon filter membrane for chemical analysis. Sb(III) and Sb(V) in solutions were diluted and determined by liquid chromatography coupled with atomic fluorescence spectroscopy (LC/AFS 6000, Haiguang, China). The control treatment (CK) without bacterial strain inoculation was also conducted as described above. At the end of the incubation, the precipitation was scraped and lyophilized in a vacuum freeze drier (LC-10N-50A, LICHEN Co. Ltd.). Observations and micrographs of the precipitation was then performed using scanning electron microscopy-Energy Dispersive Spectroscopy (SEM-EDS, SEM: QUANTA Q400, Thermo Fisher Scientific, USA; EDS: GENESIS, EDAX Inc, USA) with an accelerating voltage of 12 kV. Meanwhile, X-ray diffraction (XRD) measurement for the precipitation powder was carried out with a Philips 1830/40 X-ray powder diffractometer using Cu Kᾳ radiation (40 kV and 30 mA). The precipitation powder was also placed on the Al platform for X-ray photoelectronic energy spectrum (XPS) measurement (ESCALAB 250Xi, Thermo Fisher Scientific, USA) using the Al Ka X-ray line of 1486.6 eV excitation energy at 150 W. 2.3. Nutrient solution experiment with rice seedlings The germination and following cultivation of rice seedlings in nutrient solution was performed based on the method of Long et al. (2019). The composition of the nutrient solution were: 28.7 mg L
1 NH4H2PO4, 0.71 mg L
1H3BO3, 164.10 mg L
1 Ca(NO3)2, 0.02 mg L
1 CuSO4, 2.66 mg L
1ferric tartrate, 60.19 mg L
1 MgSO4, 0.45 mg L
1 MnCl2, 151.65 mg L
1 KNO3 and 0.055 mg L
1 ZnSO4 (Liu et al., 2005). After four weeks of cultivation, each six uniform size of rice seedlings were transplanted to a plastic bottle (100 mm  97 mm) with 500 mL of nutrient solution. The different treatments in the present study were performed by adding Sb(III), Sb(V) and/or Fe2þ into the nutrient solution directly. The bacterial inoculum was injected to the rice rhizosphere. According to the previous studies (Huang et al., 2012; Cui et al., 2015), 20 mM of soluble Sb(III) or Sb(V) had no obvious adverse effect on the growth of rice seedlings, so the concentration of Sb(III)/Sb(V) in the nutrient solution for rice seedling was set as 20 mM. To simulate the ferrous ion level in soils under waterlogged condition, the concentration of Fe2þ in the nutrient solution was set as 1 mM. As soon as the rice seedlings transplanted into the plastic bottle with nutrient solution, 50 mL of bacterial inoculum was injected to the rice rhizosphere. In the present study seven treatments were designed and the specific were shown in Table 1. All the treatments were prepared in triplicates. Rice seedlings in all the treatments were cultivated for the following 72 h.

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Table 1 The specific of the treatments performed in the present study. Treatment

Sb(III) (mM)a

Sb(V)(mM)b

Fe2þ(mM)

Bacterial inoculum (mL)

CK SbIII SbIII þ Fe SbIII þ Fe þ B SbV SbV þ Fe SbV þ Fe þ B

0 20 20 20 0 0 0

0 0 0 0 20 20 20

0 0 1 1 0 1 1

0 0 0 50 0 0 50

a b

Sb(III) solution was prepared with K(SbO)C4H4O6. Sb(V) solution was prepared with H6KO6Sb.

2.4. Analysis of IP on rice root 2.4.1. Observation of IP on rice root Rice seedlings were harvested and washed gently with sterile distilled water three times. Then the rice roots were cut into segments and immersed in glutaraldehyde in phosphate buffer (pH 7.2) at 4  C for 2 h, and washed three times with phosphatebuffered saline (PBS, pH 7.2). The rice roots were subsequently dehydrated in an ethanol solution series (30e95%) and lyophilized in a vacuum freeze drier (LC-10N-50A, LICHEN Co. Ltd.), then similar sized roots in different treatments were sputter-coated with gold-palladium. Observations and micrographs were performed using scanning electron microscopy coupled with an energy dispersive X-ray spectrometry (SEM-EDS, SEM: QUANTA Q400, Thermo Fisher Scientific, USA; EDS: GENESIS, EDAX, USA.) with an accelerating voltage of 12 kV. 2.4.2. IP fraction and amount analysis After 72 h of cultivation, rice seedlings were harvested from nutrient solution in different treatments. Rice roots were washed with distilled water several times, separated from rice plants, dried with filter paper gently and weighted precisely. Then a modified sequential extraction procedure was conducted to analyze the various fractions of IP on the rice roots. The modified sequential extraction procedure was based on the method of Poulton & Canfield (2005), which was developed to analyze the iron fractions in modern or ancient sediment. In the present study, we employed the step 1 and 2 of Poulton & Canfield (2005) to extract carbonate-associated Fe (including siderite and ankerite) and easily reducible Fe oxides (including ferrihydrite and lepidocrocite), respectively. Since previous studies demonstrated the fractions of IP on rice roots were ferrihydrite (81e100%), siderite (30e50%) and a minor of goethite (Hansel et al., 2002; Liu et al., 2006), it was not applicable to perform the further extraction steps due to the minor amounts of the other IP fractions on rice roots. Given the common extractant for IP on rice roots is DCB (dithionite-citrate-bicarbonate), we used DCB to extract all the other minor IP fractions after step 1 and 2. The details of the modified sequential extraction procedure used in the present study was shown in Table 2. 2.5. Sb species determination in rice roots and shoots Rice seedlings were harvested from the nutrient solution,

washed with distilled water several times and dried with filter paper. Rice roots and shoots were separated and weighted precisely. Then rice shoots were frozen in freezing chamber of refrigerator (
18  C), and rice roots were transferred into cold DCB extractant (Long et al., 2019) to remove the IP at 4  C before frozen in refrigerator. All the frozen rice roots and shoots were cut into fragments, ground in mortar with liquid nitrogen, and transferred into 50 mL polypropylene centrifuge tubes with addition of 10 mL 0.1 M citric acid. The mixtures were shaken (200 rpm) at 25  C for 4 h, sonicated for 1 h, and centrifuged (4000 rpm) for 15 min. The supernatant was collected in another 50 mL centrifuge tube. The above steps were repeated twice for the residue in centrifuge tubes with 5 mL 0.1 M citric acid, and the supernatant was transferred into the same centrifuge tube. The final extracted solution was made up to 50 mL with deionized water, passed through 0.45 mm filter membrane and stored in refrigerator at 4  C immediately (Okkenhaug et al., 2012; Ren et al., 2014). The Sb species in extracted solution was determined within 24 h using liquid chromatography coupled with atomic fluorescence spectroscopy (LC/AFS 6000, Haiguang, China). A Hamilton PRP-X100 10 mm anion-exchange column was used to separate Sb species. The mobile phase consisted of 10 mM EDTA and 1.5 mM potassium hydrogen phthalate (Ren et al., 2014). 2.6. Data analysis All experiments were carried out in three replicates. All results were presented as means ± standard deviations. The significance of the IP amounts, IP adsorbed Sb, Sb concentrations in rice roots and shoots between different treatments was analyzed by Tukey’s test (p < 0.05), and the variance analysis on different fractions of IP was carried out with one way ANOVA using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Effect of the bacterial strain on soluble Sb(III) According to the dynamic of Sb(III)/Sb(V) concentration in liquid LB medium containing 20 mM Sb(III) (Fig. 1), Sb(III) in liquid LB medium with bacterial inoculum decreased steeply in the first 3 days (from 20.00 to 12. 34 mM), and continuously slightly decreased in the following days, with the Sb(III) concentration

Table 2 The sequential extraction of IP on rice roots. Extraction step

Extraction methods

The target IP fractions

Terminology

Step 1 Step 2 Step 3

Na Acetate, pH 4.5, 24 h Hydroxylamine-HCl, 48 h DCB extractanta

Siderite and ankerite Ferrihydrite and lepidocrocite Others

Fe1 Fe2 Fe3

a

DCB extractant includes 0.03M sodium citrate, 0.125M sodium bicarbonate, and 0.6 g sodium dithionite in 100 mL of extractant.

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Fig. 1. Sb concentration changes in liquid medium containing 20 mM Sb(III). A is the treatment inoculated with the bacterium, and B is the CK without bacterial inoculation, respectively.

(Fig. 1-B). Moreover, there was no precipitation presented in the culture of the CK treatment. From Fig. 2, it was found that the precipitation formed in liquid medium mentioned above is crystalline, cubic like particle aggregation without bacterial cell associated, indicating it was formed in extracellular liquid medium. It also showed that these particles aggregated compactly, and the particle size was relatively large, which was up to ～1 mm. According to the typical EDX spectrum of the precipitation (Fig. 2-D), the content of element Sb was the highest, and those of Ca and O were also relatively high. The X-ray diffraction (XRD) patterns of the precipitation showed the typical crystal structure of the particles (Fig. 3-A). Referring to the Joint Committee on Powder Diffraction Standard (JCPDS) number 431071, the precipitation was identified as cubic crystalline Sb2O3. Further, the XPS analysis result (Fig. 3-B) showed that the binding energy cores of Sb3d5/2 and Sb3d3/2 were both detected, the peak area of Sb 3d5/2 is about 1.5 times of that of Sb 3d3/2, which was consistent with previous study (Huang and Ruiz, 2006). After analyzed the peak cores of Sb 3d with XPSPEAK41 software, we found that there was only Sb(III) in the precipitation, and this was consistent with the XRD analysis result. 3.2. SEM-EDS analysis of the overall IP on rice roots

being 2.91 mM at the end of the incubation. Moreover, a small quantity of Sb(V) was measured in the liquid medium after 7 days incubation, which was 0.63 mM (Fig. 1-A). After 2 days incubation, precipitation was observed on the bottom of the culture. By contrast, in CK treatment, Sb(III) slightly decreased through the whole incubation period, and low concentration of Sb(V) was also determined, which was 0.49 mM at the end of the incubation

The present study used SEM imaging to visualize the overall IP formation on rice roots of different treatments (Fig. 4). It showed that IP displayed as rough and polymer particulate deposition on the surface of rice roots. The distribution of these deposition was irregular and uneven on the root surface in all the treatments. From the imagines at 2000 times magnification, we observed that the polymer deposition amounts in SbIII, SbIII þ Fe, SbIII þ Fe þ B and

Fig. 2. The SEM imagines (A, B and C are the imagines at 2000  , 20000  and 80000  , respectively) and typical EDX spectrum (D) of the precipitation appeared at the bottom of the culture.

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Fig. 3. The optical analysis of the precipitation formed in the liquid medium. A is the XRD patterns and B is the XPS analysis of the precipitation, respectively.

SbV þ Fe þ B treatments were relatively more than those in others (CK, SbV and SbV þ Fe treatments). From the imagines at 10000 times magnification, it showed that the particulate polymer spots in SbIII þ Fe, SbIII þ Fe þ B treatments were apparently much larger than those in other treatments, which was consistent with the EDS analysis of Fe element (Fig. 5). Interestingly, a large amount of bacterial cells coupling with particulate polymer was observed in SbIII þ Fe þ B treatment. In comparison, seldom bacterial cell was observed in SbV þ Fe þ B and other treatments. 3.3. The IP fraction and the adsorbed Sb on rice roots As shown in Fig. 6-A, the total amounts of IP (FeT) in treatments with Fe2þ supply (SbIII þ Fe, SbIII þ Fe þ B, Sbv þ Fe and Sbv þ Fe þ B treatments) were much higher than those without Fe2þ supply (CK, SbIII and SbV treatments). In specific, the FeT in SbIII þ Fe treatment was the highest (668.38 mg kg
1), and that in SbIII þ Fe þ B was also relatively high (639.62 mg kg
1). The FeT in Sbv þ Fe þ B and Sbv þ Fe treatments were much lower, which were 410.86 and 358.81 mg kg
1 respectively. Among the treatments without Fe2þ supply, the FeT in SbIII treatment was relatively high (94.03 mg kg
1), and that in CK and SbV treatments were the lowest (71.63 and 71.33 mg kg
1respectively, p > 0.05). With respect to the distribution of IP fractions, there were two patterns in treatments with or without Fe2þ supply. Specifically, in treatments with Fe2þ supply (except for SbIII þ Fe þ B treatment), the IP fraction distribution was as follows: Fe2 > Fe1 [ Fe3, while in SbIII þ Fe þ B and treatments without Fe2þ supply, it was as: Fe1> Fe2  Fe3 (others). Compared with the other treatments with Fe2þ supply, the fraction

distribution in SbIII þ Fe þ B treatment implied that the bacterial inoculum probably enhanced the formation of Fe1 when rice seedlings were exposed to Sb(III). In most of the treatments, the total amount of adsorbed Sb (SbT) was generally consistent with the FeT, it turned out that more IP adsorbed more Sb. Similarly, the adsorbed Sb on IP fractions were also consistent with the IP fraction amounts (Fig. 6-B). It was worth noting that although the FeT in SbIII þ Fe þ B treatment was significantly lower than that in SbIII þ Fe treatment (p < 0.05), the SbT in SbIII þ Fe þ B treatment was significantly higher than that in SbIII þ Fe treatment (p < 0.05), which were 1854.41 and 1656.97 mg kg
1 respectively. This might indicate that the bacterial inoculum in SbIII þ Fe þ B treatment increased the Sb quantity adsorbed on IP. However, there was no similar phenomenon in Sbv þ Fe or Sbv þ Fe þ B treatments. 3.4. Sb species in rice roots and shoots As shown in Fig. 7-A, the total Sb concentration (SbT) in rice root of SbIII treatment was 41.12 mg kg
1, which was the highest among all the treatments. With the supply of Fe2þ and bacterial inoculum, the SbT in rice roots reduced to 33.92 and 28.59 mg kg
1 in SbIII þ Fe and SbIII þ Fe þ B treatments respectively. The SbT in rice root of SbV treatment was the lowest (6.57 mg kg
1). Compared with the SbV treatment, the SbT significantly increased in SbV þ Fe and SbV þ Fe þ B treatments, which were 15.85 and 15.82 mg kg
1 respectively. In the case of Sb species in rice roots, both Sb(III) and Sb(V) were detected in the SbIII, SbIII þ Fe, SbIII þ Fe þ B and SbV þ Fe þ B treatments. Sb(III) in SbIII and SbIII þ Fe treatments

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Fig. 4. SEM imagines of IP on rice roots of different treatments. A1, B1, C1, D1, E1, F1, and G1 are the SEM imagines at 2000  in CK, SbIII, SbIII þ Fe, SbIII þ Fe þ B, SbV, SbV þ Fe, and SbV þ Fe þ B, respectively. A2, B2, C2, D2, E2, F2, and G2 are the SEM imagines at 10 000  in CK, SbIII, SbIII þ Fe, SbIII þ Fe þ B, SbV, SbV þ Fe and SbV þ Fe þ B, respectively.

accounted for 35.50 and 35.37% of the total Sb respectively, significantly lower than Sb(V) (p < 0.01). By contrast, Sb(III) accounted for 54.49 and 58.72% of total Sb in SbIII þ Fe þ B and

SbV þ Fe þ B treatments. Furthermore, there was only Sb(V) found in the rice roots of SbV and SbV þ Fe treatments. In rice shoots, the SbT in SbIII treatment was the highest among

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Fig. 4. (continued).

all the treatment (14.65 mg kg
1), and that in SbIII þ Fe treatment was comparable (p > 0.05). The SbT in SbIII þ Fe þ B was significantly lower than those two treatments mentioned above (6.53 mg kg
1). The lowest SbT was found in SbV and SbV þ Fe þ B treatments, which was 3.14 and 2.77 mg kg
1 respectively (p > 0.05). With respect to Sb species, both Sb(III) and Sb(V) can be found in SbIII and SbIII þ Fe treatments, and they accounted for 44.57 and 23.33% of the SbT respectively. However, There was only Sb(V) detected in all the other treatments. 4. Discussion 4.1. Immobilization of soluble Sb(III) by the bacterial strain Given the substantial decrease of Sb(III), presence of a small amount of Sb(V) as well as the formation of precipitation in the

liquid medium through the incubation experiment, it was assumed that a large quantity of soluble Sb(III) was immobilized as insoluble Sb2O3, and the Sb resistant bacterium showed potential of immobilization of soluble Sb(III). Bio-mineralization by microbe was an important element immobilization mechanism in the environment, in which soluble elements were subjected to co-preciptation with carbonates (Kranz et al., 2010; Li et al., 2013; Perri et al., 2018). However, no XRD patterns of carbonates of the precipitation were found in the present study. Therefore, the precipitation process mediated with the Sb resistant bacterium was not the same as that of bio-mineralization, and the mechanism involved remains unclear for date. Considering the Sb2O3 was identified as nontoxic substance in the environment (Oorts et al., 2008), the formation of the precipitation might be related to the Sb(III) detoxification mechanism by the Sb resistant bacterium.

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Fig. 5. EDS spectra collected onto the rectangular zones of Fig. 4. A3, B3, C3,D3, E3, F3, and G3 are the EDS spectra collected in CK, SbIII, SbIII þ Fe, SbIII þ Fe þ B, SbV, SbV þ Fe and SbV þ Fe þ B, respectively.

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Fig. 6. Fractions of IP and adsorbed Sb in different treatments. A is the fractions of IP, B is the IP fraction adsorbed Sb; The results were based on fresh weight of rice roots; FeT¼Fe1þFe2þFe3, SbT¼Sb1þSb2þSb3, respectively.

Fig. 7. Sb in rice roots and shoots in different treatments. A is about Sb in rice roots, and B is about Sb in rice shoots. All the Sb concentrations were based on the fresh weight of rice seedlings; SbT¼Sb(III)þSb(V).

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4.2. The effect of Sb resistant bacterial on IP Considering the remarkably more bacterial cell consorted with IP in SbIII þ Fe þ B than that in SbV þ Fe þ B treatment, it probably because the stress of more toxic Sb(III) caused greater increase of rice root exudate (Luo et al., 2017; Fu et al., 2017). Since studies demonstrated that root exudates are an important source of nutrients for microorganisms in the rhizosphere (Macario et al., 2003; Yuan et al., 2016), bacterial cells in SbIII þ Fe þ B treatment thereby thrived using the root exudate as nutrients and subsequently colonized on the rice rhizosphere. The IP amount analysis showed that the supply of Fe2þ drastically enhanced the IP formation in the presence of both Sb(III) and Sb(V), which was similar to the previous studies (Huang et al., 2012; Cui et al., 2015; Wang et al., 2019). The IP sequential extraction results showed that small amounts of IPs on rice roots in treatments without Fe2þ supply were also formed. This demonstrated that Fe(III) as tartrate ferric (in the nutrient solution) could induce a limited quantity of IP on rice root, which was consistent with the study of Fu et al. (2011). Interestingly, the IP amounts were much higher in the presence of Sb(III) than those of Sb(V). It might be due to the tolerance strategy of rice to form more IP to cope with the more toxic Sb(III), which was similar to the previous study (Wu et al., 2013). Some studies have verified that IP is dominated by ferrihydrite (Liu et al., 2004; Hansel et al., 2002; Liu et al., 2006; Seyfferth & Angelia, 2015). The highest fraction of easily reducible Fe oxides in SbIII þ Fe, SbV þ Fe, and SbV þ Fe þ B treatments was consistent with these previous studies. Considering the significant higher amount of carbonate-associated Fe in SbIII þ Fe þ B treatment, it was likely attributed to the fact that substantial amounts of bacterial cells consorted with the IP on rice root according to the SEM imagines. Thus, the carbonate-associated Fe was induced greatly with the mediation of the bacterial respiration, which have been documented earlier (Zhang et al., 1999; Roh et al., 2003). Compared with SbIII þ Fe þ B treatment, the bacterial cells on rice root was less pronounced in SbV þ Fe þ B treatment, thus, the distribution of IP fraction was not different from the “normal” situation. It was worthnoting that when rice seeding exposed to Sb(III), the bacteria inoculum did not enhance the total IP amount. However, the sequestration of Sb on IP increased. Considering much higher carbonate-associated Fe of the IP in SbIII þ Fe þ B treatment, it might indicate that the adsorption capacity of Sb by carbonateassociated Fe was relatively higher than by other fractions. Meanwhile, since the bacterium with potential of conversion soluble Sb(III) into insoluble Sb2O3, it was also hypothesized that a proportion of Sb(III) in the rhizosphere was converted into insoluble Sb2O3 and co-occurred with IP. Consequently, compared with SbIII þ Fe, more Sb immobilized on IP in treatment SbIII þ Fe þ B. This indicated that the Sb resistant bacterium could help to immobilize the soluble Sb(III) on IP in rice rhizosphere, thus decrease the Sb(III) uptake by rice roots. 4.3. The effect of the bacterial strain on Sb accumulation in rice seedlings Much higher Sb concentrations in rice roots of treatments added with Sb(III) than those with Sb(V) indicated that rice plants preferentially take up Sb(III), which have been strongly evidenced earlier (Ren et al., 2014; Cui et al., 2015). Higher concentrations of Sb in rice shoots were also found in the treatments added with Sb(III) than those with Sb(V). This implied that uptake and accumulation of Sb in rice root was critical for Sb to possess human health risk through rice grain consumption, and efficient strategies should be taken into consideration to control the Sb uptake in rice

root. Compared with the SbIII treatment, greater amounts of IPs with lower concentrations of Sb in rice roots in SbIII þ Fe and SbIII þ Fe þ B treatments were observed, this firmly evidenced IP could efficiently reduce the Sb(III) uptake by rice roots (Ren et al., 2014). However, it was opposite about the IP on Sb(V) uptake by rice root, since compared with SbV treatment, higher amounts of IPs with more Sb in the rice roots of SbV þ Fe and SbV þ Fe þ B treatments were found. Cui et al. (2015) found that IP barrier for Sb(V) uptake by rice was less pronounced than that for Sb(III), and Huang et al. (2012) noticed that Sb concentrations in roots increased significantly with the increment of IP formation for some rice cultivars in Sb(V) treatment. Thus, this was not the first time that scientists noticed the IP is likely to stimulate the uptake of Sb(V) by rice. Whether the IP reduces or enhances metal(oid) uptake depends on several factors, such as the element, element chemical speciation and the amount of IP formed around the roots (Batty et al., 2000; Cui et al., 2015). For example, previous studies demonstrated that the uptake of As(III) into rice was enhanced by IP, while that of As(V) was decreased (Chen et al., 2005; Liu et al., 2005; Seyfferth et al., 2010). We hypothesized that the effect of the IP on Sb(III) and Sb(V) uptake was different. Both Sb(III) and Sb(V) could be easily adsorbed on IP of rice root (Ren et al., 2014; Cui et al., 2015; Cai et al., 2016), the hypothesis is that adsorbed Sb(III) might be hard to be taken up by rice root due to its much stronger affinity to IP (Leuz et al., 2006; Guo et al., 2014; Sazakli et al., 2015), therefore, IP served as barrier when rice root takes up Sb(III). To the opposite, due to the weak affinity of Sb(V) to IP, adsorbed Sb(V) could be taken up by rice roots easily, and IP might act as reservoir to facilitate the uptake of Sb(V) by rice root. The dominant species of Sb in rice roots was Sb(V) (except for SbIII þ Fe þ B and SbV þ Fe þ B), this was consistent with previous studies (Okkenhaug et al., 2012; Ren et al., 2014). However, the Sb(III) in rice roots accounted for 54.49 and 58.72% of the total Sb in SbIII þ Fe þ B and SbV þ Fe þ B treatments, indicating that bacterial activities increased the proportional of Sb(III) in rice roots. Interestingly, Sb in rice shoots of SbIII þ Fe þ B were much lower than that of SbIII and SbIII þ Fe treatments, similarly, Sb in rice shoot of SbV þ Fe þ B was also lower than that of SbV þ Fe treatment. Considering the relative higher Sb(III) proportion in rice roots of SbIII þ Fe þ B and SbV þ Fe þ B treatments, it might imply that the rice plant is relatively hard to translocate Sb(III) into aboveground parts, which was also implied by Okkenhaug et al. (2012). It might also indicate that in both Sb(III) and Sb(V) supplied treatments, bacterial activities reduced the translocation of Sb from root to shoot through increasing the proportion of Sb(III) in rice roots. It is widely accepted that when As, the analog of Sb, absorbed by rice plants, the As(V) reduction into As(III) will be catalyzed by As(V) reductase, As(III) is then complexed with thiols, such as glutathione and phytochelatins, and sequestered in the vacuoles (Xu and Zhao, 2007; Liu et al., 2010; Xu et al., 2017). Consequently, a large proportion of As was confined in cells of rice root as complexed As. The reduction of As(V) and complexation of As(III) by thiol peptides are considered to be the main mechanism of As detoxification in plants (Xu and Zhao, 2007). Although the mechanism of Sb detoxification in rice plant is not yet clear, it was hypothesized that the bacterial activities might involve in Sb detoxification process, enhancing reduction of Sb(V) into Sb(III) and limiting the translocation of Sb from rice root to shoot. 5. Conclusion IP formation was largely promoted by the supply of Fe2þ in the rice rhizosphere, and more IP formed on the rice roots exposed to Sb(III) than that exposed to Sb(V). The IP served as an obvious

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barrier for rice plant to take up Sb(III), but it was less pronounced for rice plant to take up Sb(V). The IP fraction distribution on rice roots was as: Easily reducible Fe oxides > Carbonate-associated Fe > Others. However the bacterial inoculum altered the fraction distribution when rice plants exposed to Sb(III), which was as: Carbonate-associated Fe > Easily reducible Fe oxides > Others. The Sb resistant bacterium enhanced the sequestration of Sb(III) on IP and decreased the Sb uptake by rice roots. The bacterial inoculum increased the proportion of Sb(III) in the rice roots when rice plants exposed to both Sb(III) and Sb(V), and greatly decreased the Sb concentrations in rice shoots. Declaration of competing interest We declared that we have no conflicts of interest to this work. We have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript. CRediT authorship contribution statement Jiumei Long: Conceptualization, Methodology, Writing - review & editing. Dongsheng Zhou: Writing - original draft, Project administration. Bingyu Li: Writing - original draft, Investigation. Yimin Zhou: Software, Validation. Yongjie Li: Software, Validation. Ming Lei: Supervision. Acknowledgement The authors are grateful for financial support from the National Science Foundation of China (41671475), and the Ministry of Science and Technology of China (2018YFD0800700). The authors are also extremely thankful to the anonymous reviewers that work in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113670. References Ainsworth, N., Cooke, J.A., Johnson, M.S., 1990. Distribution of antimony in contaminated grassland: 2-small mammals and invertebrates. Environ. Pollut. 65 (1), 79e87. Batty, L., Baker, A., Wheeler, B., Curtis, C., 2000. The effect of pH and plaque on the uptake of Cu and Mn in Phragmites australis (Cav.) Trin ex. Steudel. Ann. Bot. 86, 647e653. Cai, F., Ren, J., Tao, S., Wang, X., 2016. Uptake, translocation and transformation of antimony in rice (Oryza sativa L.) seedlings. Environ. Pollut. 209, 169e176. Chen, C., Dixon, J., Turner, F., 1980. Iron coatings on rice roots: morphology and models of development. Soil Sci. Soc. Am. J. 44 (5), 1113e1119. Chen, Z., Zhu, Y.G., Liu, W.J., Meharg, A.A., 2005. Direct evidence showing the effect of root surface iron plaque on arsenite and arsenate uptake into rice (Oryza sativa) roots. New Phytol. 165 (1), 91e97. Council of the European Communities, 1976. Council directive 76/substances discharged into aquatic environment of the community. Official J. L 23e29. Cui, X.D., Wang, Y.J., Hockmann, K., Zhou, D.M., 2015. Effect of iron plaque on antimony uptake by rice (Oryza sativa L.). Environ. Pollut. 2015 (204), 133e140. Ding, Y., Wang, R., Guo, J., Wu, F., Xu, Y., Feng, R., 2015. The effect of selenium on the subcellular distribution of antimony to regulate the toxicity of antimony in paddy rice. Environ. Sci. Pollut. Res. 22 (7), 5111e5123. Dong, M.F., Feng, R.W., Wang, R.G., Sun, Y., Ding, Y.Z., Xu, Y.M., et al., 2016. Inoculation of Fe/Mn-oxidizing bacteria enhances Fe/Mn plaque formation and reduces Cd and as accumulation in rice plant tissues. Plant Soil 404 (1e2), 75e83. Filella, M., Williams, P.A., Belzile, N., 2009. Antimony in the environment: knowns and unknowns. Environ. Chem. 6, 95e105. Fu, Y.Q., Yang, X.J., Ye, Z.H., Shen, H., 2016. Identification, separation and component analysis of reddish brown and non-reddish brown iron plaque on rice (Oryza

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