Combination of microbial oxidation and biogenic schwertmannite immobilization: A potential remediation for highly arsenic-contaminated soil

Chemosphere 181 (2017) 1e8

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Combination of microbial oxidation and biogenic schwertmannite immobilization: A potential remediation for highly arseniccontaminated soil Zhihui Yang a, b, Zijian Wu a, Yingping Liao c, Qi Liao a, b, Weichun Yang a, b, *, Liyuan Chai a, b a

Department of Environmental Engineering, School of Metallurgy and Environment, Central South University, Lushan South Road 932, Changsha, Hunan, 410083, PR China Chinese National Engineering Research Center for Control & Treatment of Heavy Metal Pollution, Changsha, 410083, PR China c Administration of Quality and Technology Supervision of Hunan Province, Changsha, Hunan, 410083, PR China b

h i g h l i g h t s  The strain YZ-1 was able to oxidize As(III) to As(V) efficiently in the soil.  Biogenic schwertmannite (Bio-SCH) has the advantage of immobilizing As(V).  Microbial oxidation and Bio-SCH immobilization were combined for As-contaminated soil.  The combination is superior to individual methods for treating As-contaminated soil.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 January 2017 Received in revised form 27 March 2017 Accepted 10 April 2017 Available online 12 April 2017

Here, a novel strategy that combines microbial oxidation by As(III)-oxidizing bacterium and biogenic schwertmannite (Bio-SCH) immobilization was first proposed and applied for treating the highly arseniccontaminated soil. Brevibacterium sp. YZ-1 isolated from a highly As-contaminated soil was used to oxidize As(III) in contaminated soils. Under optimum culture condition for microbial oxidation, 92.3% of water-soluble As(III) and 84.4% of NaHCO3-extractable As(III) in soils were removed. Bio-SCH synthesized through the oxidation of ferrous sulfate by Acidithiobacillus ferrooxidans immobilize As(V) in the contaminated soil effectively. Consequently, the combination of microbial oxidation and Bio-SCH immobilization performed better in treating the highly As-contaminated soil with immobilization efficiencies of 99.3% and 82.6% for water-soluble and NaHCO3-extractable total As, respectively. Thus, the combination can be considered as a green remediation strategy for developing a novel and valuable solution for As-contaminated soils. © 2017 Published by Elsevier Ltd.

Handling Editor: T. Cutright Keywords: Microbial oxidation Biogenic schwertmannite Immobilization Arsenite Arsenate

1. Introduction Arsenic (As) is a ubiquitous and carcinogenic metalloid element in the environment (Kim et al., 2012; Liang et al., 2016). The widespread contamination of As in soil has become an increasing rek et al., 2013; Lee environmental and toxicological concern (Koma

* Corresponding author. Department of Environmental Engineering, School of Metallurgy and Environment, Central South University, Lushan South Road 932, Changsha, Hunan, 410083, PR China. E-mail address: [email protected] (W. Yang). http://dx.doi.org/10.1016/j.chemosphere.2017.04.041 0045-6535/© 2017 Published by Elsevier Ltd.

et al., 2011b). Mining and smelting operations on As-bearing minerals are among the most serious sources of As contamination where the total As concentration in the soil can be three or four magnitude higher than that in non-contaminated soils (usually below 10 mg kg 1) (Adriano, 2001; Mikutta et al., 2014; Otones et al., 2011; Tang et al., 2016; Yang et al., 2014). Highly Ascontaminated soils allow As mobilization and subsequent leaching into ground or surface water, and can pose a sever risk to drinking water and food safety, and eventually human health. Significant health problems caused from As poisoning has arisen in some mine and their vicinity areas (Li and Ben, 2014; Pearce et al.,

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2012). Therefore, active remediation of As-contaminated soil is of great importance. Among soil remediation techniques, immobilization is a potentially reliable and relatively inexpensive alternative to remediation of the soils that have been highly polluted by metal(loid)s (Mallampati et al., 2012; Sun et al., 2016; Yang et al., 2015). Such technology is typically performed by mixing the contaminated soil with amendments that can immobilize contaminants (e.g., metal(loid)) through adsorption and/or precipitation reactions (Lei et al., 2017; Makris et al., 2009). However, remediation of the As-contaminated soils by immobilization is challenging as As exists mainly in two different oxidation states, arsenate (As(V)) and arsenite (As(III)) (Hyun et al., 2011), and As(III) is more mobile in the environment and more toxic to organisms than As(V) (Bertin and Lett, 2009; Burton et al., 2014). Indeed, due to its higher mobility, As(III) is more bioavailable than As(V) in soils (Bolan et al., 2015). Since As(V) can be more strongly retained with metal (hydr) oxides (e.g. Fe hydroxide/oxyhydroxide) as compared to As(III) (Yang et al., 2002; Yolcubal and Akyol, 2008; Zhang et al., 2015b), oxidation of As(III) to As(V) is proposed to facilitate As retention by metal (hydr)oxides, and is often used as an approach to remediate the As-contaminated waters and soils (Corsini et al., 2015; Khuntia et al., 2014). For this reason, As-remediation technologies should rely on a two-step approach, involving an initial oxidation of As(III) to As(V) followed by sequestration of As(V) (Bertin and Lett, 2009; Pous et al., 2015). Traditionally, chemical techniques using various chemical oxidants (such as permanganate, chlorine, chloramines and ozone) to oxidize As(III) to As(V) (Dodd and Vu, 2006; Lee et al., 2011a), suffer from high costs and generation of harmful byproducts (Bahar et al., 2012). Microbial transformation of As(III) to As(V) could be an eco-friendly and cost-effective alternative to chemical techniques (Bachate et al., 2012; Corsini et al., 2014), and it has been applied in bioremediation of the As-contaminated water (Bachate et al., 2012; Corsini et al., 2015; Ike et al., 2008; Kao et al., 2013; Michel et al., 2007). Many bacteria were reported to oxidize As(III) (Bahar et al., 2012; Ma, 2012; Majumder et al., 2013; Zhang et al., 2015a). However, the bacteria with strong As(III) oxidation ability and high As resistance were rare, and the remediation of the As-contaminated soil, especially for the highly As-contaminated soil by such As(III)-oxidizing bacterium is scanty. After the microbial As(III) oxidation, the subsequent remediation step can be carried out using iron-containing materials for As immobilization in the contaminated soil. Although the combined use of As(III) oxidizing bacteria and iron oxide as adsorbent for removing As from water has been reported (Corsini et al., 2014), no information is available regarding such combination for remediation of the As-contaminated soil. In the present study, the combination of microbial As(III) oxidation and biogenic schwertmannites (Bio-SCH) immobilization was applied to the highly As-contaminated soils. Bio-SCH was selected for the subsequent As immobilization, considering its high efficiency of As sequestration and slightly effect on soil pH (Liao et al., 2011; Chai et al., 2016). Moreover, Bio-SCH can be naturally formed during Fe(II) oxidation by microorganisms (e.g. Acidithiobacillus ferrooxidans), and is widespread in acidic iron and sulfate-rich environments as a common abundant iron mineral (Burton et al., 2007; Guo et al., 2015). Therefore, the application of Bio-SCH might be a promising technique for in-situ immobilization of As in soils from some As-contaminated mine areas. This combination could be regarded as “green remediation”, because the application of microorganisms and Bio-SCH is eco-friendly and hence is an ideal option to lower the hazardous effects of heavy metals on living beings without destroying soil properties. For microbial As(III) oxidation, we used a novel As(III)-oxidizing

bacterial strain Brevibacterium sp. YZ-1 (strain YZ-1), which was previously isolated from the highly As-contaminated soil in an abandoned realgar mine area (Yang et al., 2017). The main objectives of this study were to (1) examine the ability of strain YZ-1 to oxidize As(III) in soils; (2) investigate the potential application of combination of microbial oxidation and Bio-SCH immobilization for remediation of As-contaminated soils. 2. Materials and methods 2.1. Soil sampling The As-contaminated soil samples (Soil A and Soil B) were collected from the surface layer (0e20 cm) of an abandoned realgar mine area located in Shimen County, Hunan Province, central south of China. The sampling locations for Soil A and Soil B belong to mining site and smelting and processing plants, respectively. The soils contained extremely high As concentration, which can reached up to 5240.8 mg kg 1. Both As(III) and As(V) could be detected in the soils. According to FAO/UNESCO soil classification system, the soils were classified as calcaric cambisol. Prior to physicochemical analyses, the soil samples were air-dried at room temperature, ground, and passed through a150-mm nylon sieve (100 mesh). The soil pH, cation exchange capacity and organic matter were analyzed as described previously, and the results were shown in Table S1 in Supplementary Material. 2.2. As(III) oxidizing bacterium The As(III)-oxidizing bacterium strain YZ-1(CGMCC No.8329) was used in this work. This strain is alkaligenous, and exhibits high resistance to As(III) (1500 mg L 1 As(III) in solution) and strong As(III) oxidation ability, which oxidized 74.6% of 100 mg L 1 As(III) into As(V) within 72 h (data not shown). Prior to use, the strain cells were grown to early exponential phase in LB medium. 2.3. Microbial As(III) oxidation by stain YZ-1 A batch test was conducted to investigate the capability of As(III) oxidation of strain YZ-1 in the As-contaminated soils. 20 g of Soil A (or Soil B) was placed in a 150-ml conical flask, and mixed with 100 mL nutrient medium containing 5  107 cfu mL 1 of the stain YZ-1. Then, the mixture was incubated in a shaker at 30  C. During the incubation, the moisture content of soil was adjusted every 2 d by adding sterile deionized water equivalent to the loss of water. Soil samples were withdrawn at 0, 1, 3, 5, 7 and 10 d, air dried at room temperature, and then passed through a 100-mesh sieve. The sieved soil samples were used for determination of As content. Meanwhile, the control experiment was performed with the same procedure but without addition of strain YZ-1. All the experiments were carried out in triplicate. Various culture factors affecting As(III) oxidation of strain YZ-1 in contaminated soil were examined. The experiments were performed as described above. The following conditions were evaluated: (i) Carbon source: 5 g L 1 of glucose or sucrose, and different concentrations of glucose (3e12 g L 1); (ii) Nitrogen source: 5 g L 1 of yeast extract or ammonium nitrate, and different concentrations of yeast extract (0e12 g L 1); (iii) pH: varied from 6 to 10. 2.4. Bio-SCH immobilization Bio-SCH was synthesized through the oxidation of ferrous sulfate by Acidithiobacillus ferrooxidans (CGMCC No.1.6369) and characterized as described by Chai et al. (2016). In the test of As immobilization in contaminated soil by Bio-SCH, 20 g of Soil B was

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placed in a 150-ml conical flask, and mixed with Bio-SCH dosage of 5 wt%. Deionized water was added to the mixture with soil moisture content at 100% of water-holding capacity followed by immobilization for 35 d at room temperature. Then, the mixtures were withdrawn, air dried at room temperature, and then passed through a 100 mesh sieve. The sieved soil samples were used for determining As content and pH. 2.5. Combined remediation by stain YZ-1 and Bio-SCH The test combining microbial As(III) oxidation and Bio-SCH immobilization was carried out with Bio-SCH dosage of 5 wt% before the pretreatment by stain YZ-1 as above described. The nutrient medium for stain YZ-1 contained 5 g L 1 of glucose, 7 g L 1 of yeast extract and 2 g L 1 of NaCl with pH adjusted to 8.0. After 10 d of incubation, the soil sample was mixed with Bio-SCH for 35 d. Following the experiment, the soil sample was performed as described above for further analysis. All experiments were conducted in triplicate. Results are presented as the mean and the standard error. 2.6. Determination of As content in soils

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in As(III) oxidation because they could be used by the microorganisms as electron source to achieve As(III) oxidation and hence producing an improvement in As(III) removal efficiency in the contaminated soil. Considerable reduction in water-soluble As(III) concentration by strain YZ-1 with glucose and sucrose as carbon sources occurred with removal efficiency of 74.4% and 68.4% after 10 d, respectively (Fig. 1a). In contrast, after 10 d incubation litter water-soluble As(III) oxidation was observed in the control experiment without carbon source. Based on the higher water-soluble As(III) removal achieved with glucose addition, different dosage of this carbon source was examined. As shown in Fig. 1b, when the dosage of glucose exceeded more than 5 g L 1, the tends of watersoluble As(III) concentration along with the incubation time was similar (around 10 mg kg 1 after 10 d). Therefore, the dosage of 5 g L 1 of glucose was selected for further studies.

3.1.2. Nitrogen source Effect of nitrogen source on As(III) oxidation was also examined (Fig. 2). The concentration of water-soluble As(III) in the contaminated soil declined from initial concentration of 41.38 to 10.20 and 20.6 mg kg 1 for yeast extract and ammonium nitrate as nitrogen source, respectively (Fig. 2a). Clearly, yeast extract was better than

For water-soluble and NaHCO3-extractable As determination, 5 g of soil samples were mixed with 50 mL of deionized water and 0.5 M sodium bicarbonate solution, respectively, and vigorously shaken for 2 h and centrifuged at 4000 rpm for 5 min, the supernatants were filtered and analyzed for As concentration. To determine total As content in the soil, 0.2 g soil was digested with 10 mL of aqua regia (5 mL concentrated hydrochloric acid and 5 mL concentrated nitric acid) in boiling water bath for 2 h. Meanwhile, a certified reference material (yellow-red soil, GBW-07405) was used for quality assurance. The digest solution was diluted and determined for As concentration. The chemical forms of As fraction in the soil were determined using a five-step sequential extraction procedure presented by Wenzel et al. (2001). Briefly, five different extracting solutions were used sequentially to obtain five chemical forms including: nonspecifically adsorbed As (F1), specifically adsorbed As (F2), As associated with the amorphous and poorly-crystalline hydrous iron and aluminum oxides (F3), As bound to the well-crystalline hydrous iron and aluminum oxides (F4) and As associated with residual phases (F5). The detailed conditions are summarized in Table S2 (in Supplementary Material). After each extraction step, the samples were centrifuged at 8000 rpm for 15 min, filtered, and analyzed for As concentration. Total As and As(III) concentrations in solution were measured by Hydride Generation-Atomic Fluorescence Spectrometer (AFS-8X, Beijing Titan Instruments Co., Ltd). To determine As(III) selectively, samples were adjusted to pH 5.5 by acetate buffer. Determination of total As in solution was performed by treating the sample (1 mL) with reducing agents (3 mL concentrated hydrochloric acid, 5 mL 5% thiourea solution and 5 mL 5% ascorbic acid) for 30 min. Prior to the analysis, the treated sample was diluted to 50 mL. The As(V) concentration in solution was calculated as the difference between the total As concentration and As(III) concentration (Lee et al., 2011a). All analyses were performed in triplicate with data reported as mean values. 3. Results 3.1. Factors affecting As(III) oxidation in contaminated soil 3.1.1. Carbon source The addition of different carbon sources is of great importance

Fig. 1. Effect of different carbon sources addition on As(III) oxidation in the soil (a) and effect of glucose concentration on As(III) oxidation in the soil (b).

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Fig. 3. Effect of initial medium pH on As(III) oxidation by strain YZ-1 in the soil.

As(III) removal is shown in Fig. 3. The highest removal efficiency (82.6%) was achieved in the medium with pH 8.0 which hence was selected for further studies. At initial medium pH6.0, the lowest removal efficiency of water-soluble As(III) was observed. This could be explained that strain YZ-1 was susceptible in this acidic conditions, in which As(III)-oxidizing enzyme system inside the bacterial cells was less active (Anderson et al., 1992). The increase in soil pH from the initial value of 6.40e6.63 for initial medium pH6 further proved strain YZ-1 was alkaligenous.

3.2. As remediation by microbial As(III) oxidation

Fig. 2. Effect of different nitrogen sources on As(III) oxidation in the soil (a) and effect of yeast extract concentration on As(III) oxidation in the soil (b).

ammonium nitrate for removing water-soluble As(III). This is likely due to the superior bioavailability of organic nitrogen source for the strain cells than inorganic nitrogen source. However, the concentration of water-soluble As(III) decrease a little in the control without nitrogen source, and only 18.8% of the removal efficiency was achieved after 10 d. It is clearly indicated that the addition of nitrogen source can promote As(III) oxidation by strain YZ-1 in the contaminated soil. Besides, the effect of different dosage of yeast extract on water-soluble As(III) removal by strain YZ-1 was examined (Fig. 2b). The removal efficiency of water-soluble As(III) with 3 g kg 1 yeast extract was significantly lower (34.5% after 10 d) compared with those of other dosages yeast extract. When 7e12 g kg 1 of yeast extract added, no significant difference was observed in removal efficiency of water-soluble As(III). Therefore, the addition of carbon and nitrogen source allows a significant increase in water-soluble As(III) oxidation, which is potentially a simple and economic bioremediation strategy for the Ascontaminated soil. 3.1.3. pH effect The pH is an important factor on physiology and As(III) oxidation of bacteria. The effect of initial medium pH on water-soluble

The remediation effectiveness of the As-contaminated soil by the strain YZ-1 was evaluated under optimum culture condition. Fig. 4a shows the trends in water-soluble concentrations of total As, As(III) and As(V) in the contaminated soil with the bioremediation by the strain YZ-1. Water-soluble As(III) concentration in the soil was appreciably reduced with the increasing time. The removal efficiency of water-soluble As(III) reached 87.7% after 7 d and 92.3% after 10 d. While water-soluble As(V) concentration only slightly increased. The water-soluble total As concentration declined from the initial concentration of 106.86 to 73.68 mg kg 1 after 10 d. NaHCO3-extractable As is considered as labile fraction and is available for plant uptake (Ghosh et al., 2004). Thus the change in NaHCO3-extractable As was also conducted to evaluate remediation of the As-contaminated soil by the strain YZ-1. The remediation effectiveness of NaHCO3-extractable total As, As(III) and As(V) by the strain YZ-1 in the soil was shown in Fig. 4b. The concentrations of NaHCO3-extractable total As and As(III) decreased obviously with prolonging incubation time while NaHCO3-extractable As(V) concentration changed very little. After 10 d, the NaHCO3-extractable As(III) concentration declined from 60.96 mg kg 1 to 9.51 mg kg 1, and the NaHCO3-extractable total As concentration decreased by 30.97%, suggesting that As became less liable in the soil with As(III) oxidation by strain YZ-1. In the control experiment without the addition of strain YZ1 cells, no significant decrease in the concentrations of watersoluble and NaHCO3-extractable As(III) was observed (Fig. S1, in Supplementary Material). It is demonstrated that As remediation in the soil was contributed to As(III) oxidation by the stain YZ-1. The As(III) in the soil was oxidized to less toxic and mobile As(V) by microbial oxidation, proving the potential application of the As(III)oxidizing bacterial strain in remediation of As-contaminated soil.

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Fig. 4. Changes in water-soluble As(a) and NaHCO3-extractable As (b) concentration with microbial oxidation.

3.3. Comparison of three remediation methods Although As(III) oxidation by strain YZ-1 promoted As remediation in the soil, the immobilization efficiency of As was inadequate. The combination of microbial As(III) oxidation by the strain YZ-1 and immobilization by a biogenic schwertmannite (Bio-SCH) with chemical formula of Fe8O8(OH)5.19(SO4)1.41 was tested in order to improve As remediation in the soil, meanwhile individual methods were also included for comparison. Fig. 5 shows changes of the water-soluble As and NaHCO3-extractable As concentration before and after three remediation methods. The final removal efficiencies of the water-soluble total As were 99.25%, 85.59% and 31.04% for combined process, single Bio-SCH immobilization and microbial oxidation, respectively. Compared with the single remediation process, the combined process showed significantly enhanced removal efficiency of water-soluble total As. The removal efficiencies of water-soluble As(V) reached 87.63% and 98.78% for Bio-SCH immobilization and combined process, respectively. However, the concentration of water-soluble As(V) slightly increased after microbial oxidation. Despite the significant decrease of water-soluble total As in Bio-SCH immobilization process, the removal efficiency of water-soluble As(III) was inadequate (82.3%), whereas the combined process and single microbial oxidation process enhanced the water-soluble As(III) removal

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Fig. 5. Changes of water-soluble As(a) and NaHCO3-extractable As (b) concentration before and after remediation.

efficiencies to 100% and 92.3%, respectively. Therefore, Bio-SCH immobilization is inclined to immobilize water-soluble As(V) effectively, while the microbial oxidation has been shown favorable water-soluble As(III) removal. Benefitting from the advantages of the two process, the combination of microbial oxidation and the followed Bio-SCH immobilization has significant superiority in both water-soluble As(III) and As(V) removal of the As-contaminated soil. The combined process enhanced the removal efficiencies of NaHCO3-extractable total As (82.63%), compared with single microbial oxidation (30.97%) and Bio-SCH immobilization (67.79%) (Fig. 5b). The removal efficiencies of the NaHCO3-extractable As(III) were in the following order: combined process (100%) > microbial oxidation (84.45%) > Bio-SCH immobilization (64.24%). The removal efficiency of combined process was 35.76% higher than single Bio-SCH immobilization. Moreover, microbial oxidation displays insignificant effect on NaHCO3-extractable As(V) removal and its concentration only decreased by 8.5%. Unlikely the concentration of NaHCO3-extractable As(V) decreased significantly by BioSCH immobilization and combined process, and the removal efficiencies could be up to 69.28% and 75.32%, respectively. Such results reflected that the combined process is superior to single process. Sequential extraction is also commonly used to evaluate the

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mobility and bioavailability of As in soil (Yoon et al., 2016). In general, the binding strength of As to soil increased positively with the increasing dissolution strength of sequential extraction solutions, which means that the As extracted in the earlier fractions is more mobile and bioavailable than that in the later fractions (Kim et al., 2014). Fig. 6 shows the distribution of As fractions in soil by different remediation methods. As fractions in the soil for the control were present in the following order: F3 (61.4%) > F4 (18.51%) > F5 (13.2%) > F2 (3.96%) > F1 (2.93%). The distribution patterns of As fraction with microbial oxidation treatment was not significantly different from that of the control. However, notable changes have taken place in the distribution patterns of As fraction after combined process and Bio-SCH immobilization. For example, the proportions of F1 and F2 after combined process were obviously reduced, and accounted for only 0.2% and 1.54% of the total As, respectively. This indicates that the unstable As fractions were converted to more stable fraction, lowering the mobility and bioavailability of As in soil. Similar results were also observed after Bio-SCH immobilization.

4. Discussion In the present study, the ability of the stain YZ-1 to oxidize As(III) in the contaminated soil was tested. Direct evidence of oxidation of As(III) to As(V) by the strain YZ-1 in the soil was clearly demonstrated as shown in Fig. 4. The strain YZ-1 was capable to alter the As oxidation by markedly decreasing the proportion of As(III) in the water-soluble and NaHCO3-extractable fractions. During incubation of biotic treatments, the injected carbon source glucose can act as electron acceptor, and aldehyde group in the glucose can be reduced to primary alcohol, along with alkali generation. This can be confirmed by the increase of As(III)-containing (100 mg L 1) culture medium pH from 7.1 to 9.0 in 72 h (Fig. S2, in Supplementary Material). Bio-SCH used for As immobilization in this study is capable of sequestering As in the contaminated soil effectively. The critical process for As adsorption by Bio-SCH is the ligand exchange reaction of sulfate and active hydroxyl groups with anionic As (Wang et al., 2015), which is likely to favor the adsorption of As(V). Compared to As(V), As(III) is readily desorbed iron oxide ore such as goethite and hematite, and is more weakly adsorbed at low loadings to schwertmannite (Johnston et al., 2016). It is also observed that Bio-SCH has the advantage in immobilizing As(V) as shown in Fig. 5. Schwertmannite is known to be metastable with respect to goethite, transforming over time via dissolution of schwertmannite

and simultaneous precipitation of goethite. This transformation is likely to affect the behavior of arsenic immobilized on schwertmannite. Recently, it has been found that schwertmannite transformation is slow under oxic condition, and can be significantly inhibited after adsorbing As (Liao et al., 2011). Moreover the formation of goethite via partial transformation of As adsorbed schwertmannite significantly decreased As mobilization (Burton et al., 2010). Thus, the Bio-SCH after adsorbing As is stable, and meanwhile the As adsorbed on Bio-SCH is difficult to release during the transformation of schwertmannite to goethite. It is clearly indicated that combination of microbial oxidation and Bio-SCH immobilization is superior to the individual methods for remediation of the high As-contaminated soil (Fig. 5). Oxidation of As(III) to As(V) may also promote As immobilization since As(V) is more easily immobilized in solid phase (Yamamura and Amachi, 2014). In the subsequent Bio-SCH immobilization, the immobilization of As is markedly improved. Moreover, this study demonstrates that the As immobilization performance is significantly dependent on As species in the soil. In common environments As exists in different species, affecting its mobility and bioavailability significantly (Wu et al., 2017; Yamamura and Amachi, 2014). Mining and smelting processes are the main sources for As contamination in China, and soils in these areas have extra high As concentration (Tang et al., 2016; Yang et al., 2014). Single technique is difficult to achieve effective remediation of the highly As-contaminated soils. Karn and Pan (2016) found the application of microbial As(III) oxidation by Acinetobacter sp. XS21 can remove As(III) from soluble-exchangeable fraction and removed 70% of the As(III) in the soil. However, the concentration of the soluble-exchangeable As is still up to 52 mg kg-1. Combined technique of microbial As(III) oxidation and Bio-SCH immobilization developed in the present study is obviously superior to single technique, and can be considered as the more promising technique in As-contaminated soil remediation. In the microbial As(III) oxidation, the use of high As(III)-tolerant and strong As(III)oxidizing microorganism, with inexpensive carbon and nitrogen sources was more eco-friendly and cost-effective compared with chemical oxidation. The desirable features of microbial As(III) oxidation by the stain YZ-1 could facilitate the remediation of the As(III)-contaminated soil and decrease bioavailability and toxicity of As(III). Additionally, the used Bio-SCH in As immobilization is prepared by an autotrophic bacteria of Acidithiobacillus ferrooxidans which can use ferrous sulfate as energy resource and does not require exotic carbon source under normal temperature. More importantly, Bio-SCH can be in-situ formed in some mine areas where soil is rich in Fe(II) and sulfate, and Fe(II)-oxidizing bacteria (e.g. Acidithiobacillus ferrooxidans) can be widely found in the environment (Burgos et al., 2012). Thus, the application of Bio-SCH may be developed as an in-situ and low-cost remediation for the As-contaminated soils. Taking advantages of the microbial oxidation and Bio-SCH immobilization, the combination is emerging as a green remediation strategy for developing novel and valuable solution for the highly-As contaminated soils. 5. Conclusions

Fig. 6. The distribution of As fractions in soil before and after remediation.

This study provides evidence that the remediation process combining microbial oxidation and Bio-SCH immobilization is superior to the individual methods for treating the highly Ascontaminated soil. In microbial oxidation, As(III) was oxidized to less toxic and less mobile As(V) by the strain YZ-1, resulting in significant decreases in the water-soluble As(III) (92.3%) and NaHCO3-extractable As(III) (84.4%). However, the concentrations of water-soluble and NaHCO3-extractable As(V) slightly increased after microbial As(III) oxidation process. Bio-SCH immobilization has

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the advantage of immobilizing As(V) in terms of the remarkable decreases in the concentrations of water-soluble and NaHCO3extractable As(V). Compared with single microbial oxidation and Bio-SCH immobilization, the combination exhibited high performance in As immobilization with removal efficiencies of 99.3% and 82.6% for the water-soluble and NaHCO3-extractable total As, respectively. The present study suggest that the combination of microbial oxidation and Bio-SCH immobilization has potential in remediation of the highly As-contaminated soils. Certainly, field studies are needed to closely evaluate its suitability for the Ascontaminated soils. Acknowledgment This study was financially supported by National Natural Science Foundation of China (Grant Number: 51304252), and Special Program on Environmental Protection for Public Welfare (Grant Number: 201509050), for financial support. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2017.04.041. References Adriano, D., 2001. Trace Elements in Terrestrial Environments. Biogeochemistry Bioavailability and Risk of Metals. Springer, New York. Anderson, G.L., Williams, J., Hille, R., 1992. The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase. J. Biol. Chem. 267, 23674e23682. Bachate, S.P., Khapare, R.M., Kodam, K.M., 2012. Oxidation of arsenite by two bproteobacteria isolated from soil. Appl. Microbiol. Biotechnol. 93, 2135e2145. http://dx.doi.org/10.1007/s00253-011-3606-7. Bahar, M.M., Megharaj, M., Naidu, R., 2012. Arsenic bioremediation potential of a new arsenite-oxidizing bacterium Stenotrophomonas sp. MM-7 isolated from soil. Biodegradation 1, 803e812. http://dx.doi.org/10.1007/s10532-012-9567-4. Bertin, P.N., Lett, M., 2009. Biochimie Arsenic in contaminated waters: biogeochemical cycle, microbial metabolism and biotreatment processes. Biochimie 91, 1229e1237. http://dx.doi.org/10.1016/j.biochi.2009.06.016. Bolan, N., Mahimairaja, S., Kunhikrishnan, A., 2015. Bioavailability and ecotoxicity of arsenic species in solution culture and soil system: implications to remediation. Env. Sci. Pollut. Res. 22, 8866e8875. http://dx.doi.org/10.1007/s11356-0131827-2. Burgos, W.D., Borch, T., Troyer, L.D., Luan, F., Larson, L.N., Brown, J.F., Lambson, J., Shimizu, M., 2012. Schwertmannite and Fe oxides formed by biological low-pH Fe(II) oxidation versus abiotic neutralization: impact on trace metal sequestration. Geochim. Cosmochim. Acta 76, 29e44. http://dx.doi.org/10.1016/ j.gca.2011.10.015. Burton, E.D., Bush, R., Sullivan, L.A., Mitchell, D.R., 2007. Reductive transformation of iron and sulfur in schwertmannite-rich accumulations associated with acidified coastal lowlands. Geochim. Cosmochim. Acta 71, 4456e4473. Burton, E.D., Johnston, S.G., Kocar, B.D., 2014. Arsenic mobility during flooding of contaminated soil: the effect of microbial sulfate reduction. Env. Sci. Technol. 48, 13660e13667. Burton, E.D., Johnston, S.G., Watling, K., Bush, R.T., Keene, A.F., Sullivan, L.a., 2010. Arsenic effects and behavior in association with the fe(II)-catalyzed transformation of schwertmannite. Environ. Sci. Technol. 44, 2016e2021. http:// dx.doi.org/10.1021/es903424h. Chai, L., Tang, J., Liao, Y., Yang, Z., Liang, L., Li, Q., Wang, H., Yang, W., 2016. Biosynthesis of schwertmannite by Acidithiobacillus ferrooxidans and its application in arsenic immobilization in the contaminated soil. J. Soils Sediments 16, 2430e2438. http://dx.doi.org/10.1007/s11368-016-1449-7. Corsini, A., Colombo, M., Muyzer, G., Cavalca, L., 2015. Characterization of the arsenite oxidizer Aliihoeflea sp. strain 2WW and its potential application in the removal of arsenic from groundwater in combination with Pf -ferritin. Ant. Van Leeuwenhoek 108, 673e684. http://dx.doi.org/10.1007/s10482-015-0523-2. Corsini, A., Zaccheo, P., Muyzer, G., Andreoni, V., Cavalca, L., 2014. Arsenic transforming abilities of groundwater bacteria and the combined use of Aliihoeflea sp. strain 2WW and goethite in metalloid removal. J. Hazard. Mater. 269, 89e97. http://dx.doi.org/10.1016/j.jhazmat.2013.12.037. Dodd, M.C., Vu, N.D.U.Y., 2006. Kinetics and mechanistic aspects of As (III) oxidation by aqueous chlorine, chloramines, and ozone: relevance to drinking water treatment. Env. Sci. Technol. 40, 3285e3292. Ghosh, a. K., Bhattacharyya, P., Pal, R., 2004. Effect of arsenic contamination on microbial biomass and its activities in arsenic contaminated soils of Gangetic West Bengal, India. Environ. Int. 30, 491e499. http://dx.doi.org/10.1016/

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