Effective stabilization of arsenic in contaminated soils with biogenic manganese oxide (BMO) materials

Journal Pre-proof Effective stabilization of arsenic in contaminated soils with biogenic manganese oxide (BMO) materials Ya-nan Wang, Yi Song, Huawei Wang, Yingjie Sun, Yiu Fai Tsang, Xiangliang Pan PII:

S0269-7491(19)33965-X

DOI:

https://doi.org/10.1016/j.envpol.2019.113481

Reference:

ENPO 113481

To appear in:

Environmental Pollution

Received Date: 20 July 2019 Revised Date:

27 September 2019

Accepted Date: 22 October 2019

Please cite this article as: Wang, Y.-n., Song, Y., Wang, H., Sun, Y., Tsang, Y.F., Pan, X., Effective stabilization of arsenic in contaminated soils with biogenic manganese oxide (BMO) materials, Environmental Pollution (2019), doi: https://doi.org/10.1016/j.envpol.2019.113481. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Effective stabilization of arsenic in contaminated soils

2

with biogenic manganese oxide (BMO) materials

3

Ya-nan Wang1, Yi Song1, Huawei Wang1,3*, Yingjie Sun1, Yiu Fai Tsang2,

4

Xiangliang Pan3

5

1

6

Qingdao University of Technology, School of Environmental and Municipal

7

Engineering, Qingdao 266033, China;

8

2

9

Hong Kong, Tai Po, New Territories, Hong Kong, China;

Qingdao Solid Waste Pollution Control and Resource Engineering Research Center,

Department of Science and Environmental Studies, The Education University of

10

3

11

Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011,

12

China.

13

*Corresponding author:

14

Dr. Huawei Wang: [email protected]; [email protected]

15

Abstract

Xinjiang Key Laboratory of Environmental Pollution and Bioremediation, Xinjiang

16

The role of biogenic manganese oxide (BMO) materials on the stabilization of

17

arsenic (As) in contaminated soil was investigated. Experimental results indicated that

18

the addition of BMO proved to be highly effective to stabilize As in soils. Soluble

19

content of As decreased 49.5-67.4% with a dosage of 0.013-0.063% BMO. X-ray

20

absorption near edge structure results confirmed that BMO is mainly responsible for

21

oxidizing As(III) to As(V) through a two-step pathway. Five-step sequential extraction

22

results indicated that such extractable fractions of As decreased, and consequently 1

23

residual fractions increased, which can decrease the risk of As in contaminated soils.

24

BMO had a higher efficiency in stabilizing As than two types of abiotic Mn oxides.

25

High throughput sequencing analysis indicated that the bacterial community and

26

diversity significantly changed after BMO treatment. High amounts of BMO (with a

27

dosage of 0.063%) significantly increased the abundance of Proteobacteria phylum,

28

including Massilia, Phenylobacterium and Sphingomonas genera. These findings

29

suggested that BMO can be considered a high effectiveness, low cost, and

30

environmental friendliness material for the remediation of As contaminated soils.

31

Keywords: Biogenic Mn oxide (BMO); arsenic contaminated soil; bacterial

32

community; TCLP; soil remediation.

33

1. Introduction

34

Arsenic (As) is a toxic metalloid in group VA of the periodic table. Despite its

35

low abundance in the Earth’s crust (0.0001%), As is widely distributed in nature

36

(Oremland and Stolz, 2003). Anthropogenic sources, including mining and smelting

37

of slag, fossil fuel combustion and excessive use of As-based pesticides, contribute to

38

As found in the environment (Shi et al., 2019). As contamination in soils is a global

39

concern because consumption of As-containing rice is a potential pathogenic threat to

40

human health (Rai et al., 2019; Zhou et al., 2018). Therefore, assessing remediation

41

for sustainable management of arsenic in soils is urgently needed.

42

Immobilization of As in soils is mainly associated with iron and aluminum

43

oxides (Lin et al., 2019; Mikkonen et al., 2019). Iron (oxyhydr)oxides, especially

44

poorly crystalline Fe(III) minerals (ferrihydrite), provide the main reactive sites for 2

45

As(III) and As(V) adsorption in soils (Li et al., 2018; Komárek et al., 2013). The main

46

issue in the application of ferrihydrite to stabilize As in contaminated soil is

47

ferrihydrite could be reduced to dissolved Fe2+ ions or crystal structure shift from

48

poorly crystallinity to secondary minerals with high crystallinity by Fe(III) or sulfate

49

reducing bacteria such as Geobacteraceae and Shewanella, leading to the decrease of

50

As binding points and the increase of As release (Fan et al., 2018; Poggenburg et al.,

51

2016). Moreover, the As(V) adsorbed to ferrihydrite could be reduced to As(III) by

52

arsenate reducing bacteria, such as, resulting in an increase release of As from soils to

53

aqueous phase due to the decrease of As adsorption properties and chemical stability

54

(Qiao et al., 2017).

55

Oxidation of As(III) to As(V) by Mn oxides is considered to be a promising

56

remediation because this process can decrease the release of As(III) by changing its

57

valence state and increasing its absorption ability on mineral surfaces (Chen et al.,

58

2018; Ociński

59

Mn oxides have been used for the remediation of As contaminated soils. Xu et al.

60

(2017) indicated that indigenous Mn oxides retard As migration into porewater due to

61

change in redox potential; their studies also indicated that the addition of synthetic Mn

62

oxides to soils decreases As migration. In addition, a recent study reported by Li et al.

63

(2019) also confirmed that the addition of synthetic nanostructured Mn oxides

64

(α-MnO2) decreases the effective As content in paddy soils.

et al., 2016; Wang et al., 2016). For this purpose, various synthetic

65

Among Mn oxides, biogenic Mn oxide (BMO) is one of the most reactive

66

minerals in terrestrial and aquatic environments (Spiro et al., 2009). Widespread 3

67

Mn(II)-oxidizing bacteria Pseudomonas putida (Villalobos et al., 2003) and Bacillus

68

(Web et al., 2005) and funguses Ascomycete (Miyata et al., 2006) and Acremonium

69

(Watanabe et al., 2013) contribute to the formation of BMO. BMOs are mainly

70

amorphous or poorly crystalline minerals, similar to δ-MnO2, birnessite and vernadite

71

(Bargar et al., 2009). Recently, interest in the application of BMOs for metal

72

decontamination has increased due to their promising adsorption abilities and

73

oxidative capacities (Tian et al., 2018; Wang et al., 2019a). Compared to chemically

74

synthesized Mn oxides, BMO is an environmentally friendly stabilizing and oxidizing

75

agent that it can be produced under environmentally benign conditions without

76

requiring excessive energy use (Gautam et al., 2019), therefore BMO has a low

77

environmental toxicity risk and is a cost-effective biogenic material. Although several

78

studies (Bai et al., 2016; He et al., 2019a; Watanabe et al., 2013) have been performed

79

on the oxidation and sequestration of As(III) by BMO, most of these studies have

80

focused on the reaction mechanisms of As in aqueous solution. Regarding the role of

81

BMO on the stabilization of As in contaminated soils is still poorly understood.

82

The aim of this study is to investigate the role of BMO for the stabilization of As

83

in polluted soils. In this work, the BMO were collected and their ability to stabilize As

84

in soils was performed by leaching tests and bacterial community analysis. The

85

stabilization efficiency of As was also compared with abiotic BMO. The results of this

86

study could facilitate the potential application of BMO for As remediation in

87

contaminated soils.

88

2. Materials and Methods 4

89

2.1 Soil samples

90

Surface soil samples (0–20 cm depth) were collected from contaminated

91

agricultural soil in Shandong Province, China. The soil samples were air–dried,

92

disaggregated and sieved through a 60 mesh sieve (＜0.3 mm). The chemical

93

composition of the soil samples was determined by X-ray fluorescence spectroscopy

94

(XRF). Soil pH and electrical conductivity (EC) were monitored after extracting of

95

the soil with deionized water (DW) at a ratio of 1:5 (w/v). The content of organic

96

matter (OM) was determined after ignition of the soil at 600oC for 2 h. The total As

97

and water soluble As contents were extracted by aqua regia digestion and DW,

98

respectively. The main physicochemical properties of the soil samples are shown in

99

Table 1.

100

Table 1 Physicochemical properties and chemical composition of As contaminated soil sample. OM (%)

EC (ds/m)

pH

2.96 ± 0.12

0.057 ± 0.004

4.93 ± 0.05

Total As (mg/kg)

Soluble As (mg/kg)

24.98 ± 0.59

2.19 ± 0.02

Chemical composition (mass %) SiO2

Al2O3

Fe2O3

K2O

CaO

Others

66.43

14.51

8.98

4.34

1.94

3.80

101 102

2.2 BMO preparation

103

In this work, P. putida strain MnB1 (ATCC 23483), a typical Mn(II) oxidizing

104

bacterium, was selected because it is widely used for toxic metal decontamination

105

(Liu et al., 2018; Wang et al., 2019a). The medium ATCC #279, which composed of

106

0.15 g/L ammonium iron sulfate, 0.15 g/L sodium citrate, 0.15 g/L, 0.075 g/L yeast

107

extract 0.05 g/L sodium pyrophosphate, and a initial pH of 6.8, was used to produce

5

108

BMO. Mn(II) carbonate with a concentration of 1 g/L was added to the culture

109

medium to provide a source of Mn(II). A final cell density of approximately 7.5×108

110

cells/mL was inoculated with #279 medium. According to our previous study (Wang

111

et al., 2015), two steps are involved in bacterial oxidation of Mn(II) carbonate. The

112

first step is the dissolution of Mn(II) carbonate and the release of dissolved Mn(II)

113

ions, and bacterial exopolymers (EPS) catalyze the dissolution of Mn(II) carbonate

114

(Eq. 1); the second step is the bacterial oxidation of Mn(II) ions to Mn(IV) (Eq. 2).

115

Dissolution reaction: MnCO3( s ) + H → Mn

116

Oxidation reaction: Mn

+

2+

2+

+ HCO3−

bacteria + 12 O 2 +H 2 O → MnO2( s ) + 2H +

(1) (2)

117

After two days of rotation incubation (25oC, 30 rpm), BMO was harvested with a

118

concentration of 0.63 ± 0.034 g/L. These newly formed BMOs were kept at 4oC in the

119

dark for further use. Some BMO samples were lyophilized at -80oC for 24 h in a

120

vacuum freeze dryer. The freeze-dried samples were analyzed by scanning electron

121

microscopy (SEM) coupled with energy dispersive spectroscopy (EDS) and

122

high–resolution transmission electron microscopy (HRTEM) (Wang et al., 2019a).

123

2.3 Stabilization experiments

124

Stabilization experiments were conducted by adding increasing amounts of BMO

125

to fixed amounts of soils (50 g) with a dosage of 0.013%, 0.025%, 0.038%, 0.050%

126

and 0.063%. The five treatments are referred to as B1, B2, B3, B4 and B5. B0 denotes

127

untreated soil. After adding 100 ml DW, the BMO and soil samples were

128

homogenized by stirring for 10 min at 150 rpm. The slurry samples were incubated at

129

30oC using an artificial climate box. At different reaction times (15, 30, 45 and 60 6

130

days), the BMO-treated soil samples were air-dried and sieved through a 60 mesh

131

sieve. These samples were further used to investigate stabilization efficiency of As

132

through leaching tests. In addition, the immobilization of BMO was compared to that

133

of abiotic Mn oxides. Two types of abiotic Mn oxides, namely, micro–MnO2 and

134

nano–MnO2, were purchased from Sangon Biotech (Shanghai) Co., Ltd., China and

135

Beijing DK Nano Technology Co., Ltd., China, respectively. Typical dosages of 1%,

136

5% and 10% were added according to a previous study (Álvarez-Ayuso et al., 2013).

137

The soil samples were incubated at 30oC for 30 days in the dark, and then used for the

138

leaching tests.

139

2.4 Leaching tests

140

Two leaching methods were used to evaluate the stabilization efficiency of As in

141

soils by BMO. First, the BMO–treated soils were extracted by DW to simulate the

142

water leaching conditions. The soil samples were leached with DW at a liquid to solid

143

ratio of 20 mL/g and shaken on a reciprocating shaker at 200 rpm for 60 min at 25oC.

144

Second, the toxicity characteristic leaching procedure (TCLP) was used to estimate

145

the process of leaching harmful components under mild acidic conditions (Wang et al.,

146

2019b). The soil samples were leached with a 0.1 M acetic acid buffer solution (initial

147

pH of 2.64 ± 0.05) at a liquid to solid ratio of 20 mL/g and rotated for 18 h at 30 rpm.

148

All experiments were performed in triplicate. After the leaching tests, the solutions

149

with suspended particulates were centrifuged at 4000 rpm for 10 min and filtered

150

through a 0.22 µm hydrophilic polyestersulfone membrane (Durapore PVDF,

151

Millipore), and then the speciation and concentration of As in the solution were 7

152

determined. Before the instrumental analysis, the solution samples were kept at 4oC in

153

the dark.

154

2.5 As species and sequential extraction procedure

155

The valence shift of As in the soil samples before and after BMO treatment were

156

determined by X-ray adsorption near-edge structure (XANES) at the Beijing

157

Synchrotron Radiation Facility (BSRF), China. In addition, a sequential extraction

158

procedure reported by Tessiser et al. (1979) is used to evaluate the As fraction in soils.

159

The changes of As fractions are helpful for further understanding the potential

160

leaching risk of As in soils.The procedure involved five extraction steps: 1 M MgCl2

161

at pH 7.0, 1 M acetic acid/sodium acetate at pH 5.0, 0.04 M NH2OH·HCl at 96oC,

162

0.02 M HNO3 at 85oC, and HNO3-H2O2. The fractions of As were accordingly

163

referred to as: (1) exchangeable (T1), (2) carbonate (T2), (3) Fe/Mn oxide (T3), (4)

164

organic matter (T4), and (5) residual phase (T5).

165

2.6 DNA extraction and high-throughput sequencing

166

After 30 days of reaction, the untreated and BMO–treated soil samples were

167

collected for the analysis of the bacterial community compositions. Total DNA was

168

extracted using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad,

169

California, USA) following the manufacturer’s instructions. The extracted DNA

170

samples were used for further sequencing with the Illumina MiSeq platform at Sangon

171

Biotech Co., Ltd. (Shanghai, China). The primers used for sequencing were 341F

172

(5’-CCTACGGGNGGCWGCAG-3’)

173

(5’-GACTACHVGGGTATCTAATCC-3’) primers.

and

8

805R

174

2.7 Instrumental analysis

175

The speciation and concentration of As in solution were determined using

176

AFS-810 hydride generation atomic florescence spectroscopy (Beijing Jitian, China)

177

coupled with a Shimazduo liquid chromatograph. The total As in the soil samples

178

(0.1000 g) was digested by a mixture of HNO3 and HCl at a ratio of 2:1 using a

179

WX-6000 microwave digestion system. The solution pH was detected using a pH

180

electrode (Metrohm 702, Switzerland).

181

3. Results and discussion

182

3.1 Characterization of contaminated soil sample and BMO materials

183

As shown in Table 1, the soil was weakly acidic with a pH value of 4.93 and

184

contained a medium OM content (2.96%). The XRF results indicated that the main

185

components of the soil are SiO2 (66.43%), Al2O3 (14.51%), Fe2O3 (8.98%), K2O

186

(4.34%), and CaO (1.94%), which are indicative of a typical Si-Al clay mineral. The

187

total As content was 24.98 mg/kg in soil, and the soluble As content was 2.19 mg/kg,

188

accounting for 8.8% of the total As content. The valence states of As in raw soil

189

sample were determined based on XANES analysis. As shown in Fig. 1, a maximum

190

absorbance at 11862 eV was observed, indicating that As is primarily in the +III state

191

in untreated soil sample (Zhang et al., 2014).

192

9

3.5

11862 eV

As in raw soil As in BMO-treated soil

Normalized absorbance

3.0 2.5

11871 eV 2.0 1.5 1.0 0.5 11850

193 194 195

11860

11870

11880

11890

Energy (eV)

Fig. 1 Normalized XANES spectra of the soil samples before and after BMO treatment.

(b)

(a) For EDS

wt. % C 4.79 O 43.66 P 0.64 Mn 49.82 Fe 1.10

2 µm

196 (c)

(d)

197 198 199

Fig. 2 SEM and HRTEM images of BMO, (a) SEM of BMO; (b) EDS of BMO for selected area; (c, d) HRTEM of BMO.

200 201

The SEM image (Fig. 2a) showed that aggregated particles of BMO with little

202

geometric regularity adhered with cells to the surface of undecomposed Mn(II)

203

carbonate. The EDS results (Fig. 2b) for the selected area indicated that the two main 10

204

elements in BMO were Mn and O, accounting for 93.47% of the total elemental mass.

205

The HRTEM results ((Fig. 2c, d)) also indicated that the form of BMO was mainly

206

layer phyllomanganate, which was consistent with previous studies (Learman et al.,

207

2011; Villalobos et al., 2003).

208

3.2 Changes in As species and soil pH after BMO treatment

209

As K-edge XANES spectra were used to determine the state shift before and

210

after BMO treatment. Fig. 1 shows that the As XANES spectra in BMO-treated

211

sample are identical to that of Na2HAsO4, indicating that As(III) in soil sample was

212

completely converted to As(V) on BMO surface. Bai et al. (2016) had observed that

213

As(III) could be completely oxidized and adsorbed to As(V) by in situ formed BMO

214

in aquatic ecosystems. In addition, He et al. (2019a) also confirmed that As(III) can be

215

oxidized and removed by in situ formed BMO from a manganese-oxidizing aerobic

216

granular sludge in wastewater.

217

The pH variation in soil samples after BMO treatment is presented in Fig. S1.

218

The pH values ranged from 5.09 to 6.34 after BMO treatment, which were higher than

219

the pH of the untreated soil (4.93). The increase in the soil pH values may be mainly

220

ascribed to the consumption of H+ ions during the oxidation of As(III) by BMO in soil

221

(Eqs. 3 and 4).

222

MnO2 + H2AsO3- + H+ →Mn2+ + HAsO42- + H2O

(3)

223

MnO2 + HAsO32- + H+ →Mn2+ + AsO43- + H2O

(4)

224

3.3 Changes in As fractions after BMO treatment

225

The As fractions before and after BMO treatment are shown in Fig. 3. In the 11

226

untreated soil, the dominant fraction of As was T5, accounting for 69.7% of the total

227

As, followed by T3 (15.5%), T2 (8.1%), T1 (4.4%) and T4 (2.2%). After BMO

228

treatment, the T1 fraction percentage decreased, while the T5 fraction increased. After

229

15 days of BMO treatment, the percentage of the T1 fraction significantly decreased

230

as BMO amount increased and was 2.7%, 1.7%, 1.7%, 1.1% and 0.9% for B1, B2, B3,

231

B4 and B5, respectively. The percentage of the T5 fraction was 73.7%, 76.8%, 77.3%,

232

77.6% and 77.8% for B1, B2, B3, B4 and B5, respectively. Then, the As fractions

233

increased slightly or remained stable for the remainder of the reaction stage

234

(15–45days). The first three fractions of As (T1, T2 and T3) are generally considered

235

unstable and to be closely related to the mobility and bioavailability of As in soils.

236

After BMO treatment, the first three fractions of As decreased by 5.1-9.5%,

237

7.0-10.4% and 9.3-11.6%, respectively, while the residual fraction increased by

238

4.1-8.2%, 7.7-10.8% and 9.3-11.7% after 15, 30 and 45 days of reaction, respectively,

239

compared to the untreated soil. These results indicated that BMO treatment can

240

effectively decrease the mobility and bioavailability of As in soils. Similar results

241

were also reported by Li et al. (2019) and Xu et al. (2017) who observed an increase

242

in the residual fraction after the application of synthetic Mn oxides for As

243

mobilization in paddy soils.

244 (a)

(b) 100

90

90

90

80

80

80

30

30

30

20

20

20

10

10

Fraction /%

100

0

245

(c)

100

10

0 B0

B1

B2

B3

B4

B5

T5 T4 T3 T2 T1

0 B0

B1

B2

B3

12

B4

B5

B0

B1

B2

B3

B4

B5

246 247 248

Fig. 3 Changes of As fraction in untreated and BMO–treated soil samples: (a) 15 days; (b) 30 days; (c) 45 days. (T1, Exchangeable; T2, Carbonate; T3, Fe/Mn oxide; T4, Organic matter; T5, Residual).

249

3.4 Leaching tests

250

The solubility of As in soils treated with BMO was further evaluated by leaching

251

tests. Fig. 4a shows that the soluble As content in untreated soil was 2.19 mg/kg,

252

indicating the high leachability and mobility of As. However, a significant decrease in

253

As leaching with increasing BMO was observed in the BMO–treated soil samples.

254

The soluble As content decreased 49.5%, 60.2%, 60.4%, 63.9% and 67.4% for B1, B2,

255

B3, B4 and B5, respectively, after 15 days of reaction. The soluble As content further

256

slightly decreased when the reaction time was extended to 30, 45 and 60 days, which

257

indicated the BMO treatment reached equilibrium in terms of As leaching after 15

258

days of reaction. These results showed that BMO treatments could effectively reduce

259

As leaching from soils, which was consistent with the results from the As speciation

260

and fraction analysis.

261

2.0 Soluble As (mg/kg)

3.5

B0 B1 B2 B3 B4 B5

(a)

1.5

(b)

B0 B1 B2 B3 B4 B5

3.0 TCLP leaching As (mg/kg)

2.5

1.0

0.5

2.5 2.0 1.5 1.0 0.5

0.0

0.0 15

262 263 264

30

45

60

15

30

45

60

Time (d)

Time (d)

Fig. 4 Effects of reaction time on As leaching content by DW (a) and TCLP (b) tests for untreated and BMO–treated soil samples.

265 266

Similar trends were observed for the TCLP leaching tests (Fig. 4b). The TCLP As 13

267

leaching content significantly deceased in the BMO–treated samples. After 15 days of

268

reaction, the As content decreased from 2.93 mg/kg for the untreated sample to 2.1

269

mg/kg, 1.9 mg/kg, 1.7 mg/kg, 1.7 mg/kg and 1.7 mg/kg with stabilization efficiencies

270

of 27.7%, 33.1%, 39.9%, 40.3% and 41.1% for B1, B2, B3, B4 and B5, respectively.

271

The TCLP As content gradually decreased with increasing reaction time. For the B1

272

treatment, the soluble As content decreased by 11.0%, 18.7% and 23.8% after 30, 45

273

and 60 days, respectively, relative to 15 days of reaction.

274

The results obtained from the two leaching tests suggested that the application of

275

BMO effectively stabilized As in soils. This phenomenon can be mainly explained as

276

follows. BMO can rapid oxidize As(III) to As(V), and simultaneous BMO can

277

effectively stabilize As(V) through the formation of bidentate inner-sphere complexes

278

(Bai et al., 2016) (Eqs. 5 and 6). The shift of As fraction in soil samples from the

279

unstable fractions to the residual fraction can prevent As release. These results further

280

indicated that BMO could act an oxidation agent and As adsorbent.

281

≡MnOH + H2AsO3- →≡Mn-HAsO3- +H2O

(5)

282

≡MnOH + HAsO32- →≡Mn-AsO32- +H2O

(6)

283

3.5 Changes in bacterial diversity and community structure

284

As summarized in Table 2, high–quality sequence reads ranging from 35535 to

285

57676 were achieved for untreated and BMO-treated soil samples. The operational

286

taxonomic units (OTUs) of the samples were calculated at the 3% cut-off level.

287

Good’s coverage (0.99) indicated that sufficient sequencing depths were obtained. The

288

bacterial community diversity in untreated and BMO–treated soil samples is reflected 14

289

by Shannon and Simpson indices. The Simpson index for BMO–treated samples

290

decreased with increasing BMO. The Simpson indices were 0.50, 0.48, 0.35, 0.25 and

291

0.12 for B1, B2, B3, B4 and B5, respectively, which were significantly lower than

292

0.52 for the untreated soil sample. On the other hand, the Shannon indices had the

293

opposite tendency and increased with increasing BMO. Changed in both indices

294

indicated that the bacterial community diversity remarkably improved after BMO

295

treatment compared to that of the untreated sample. These results suggested that

296

bacterial succession occurred in the soil after BMO treatment.

297 298

Table 2 Diversity indices of bacterial community in the untreated and BMO–treated soil samples Items

Sequences

OTUs

B0 B1 B2 B3 B4 B5

48711 57676 52258 47484 35535 55025

939 1183 1183 932 745 1056

Microbial community diversity Simpson

Shannon

Goods coverage

0.52 0.50 0.48 0.35 0.25 0.12

1.78 1.81 1.85 2.11 2.36 3.53

0.99 0.99 0.99 0.99 0.99 0.99

299 (a) Others Gemmatimonadetes Verrucomicrobia unclassified Bacteroidetes Planctomycetes Actinobacteria Acidobacteria Chloroflexi Proteobacteria Firmicutes

80

60

40

100

20

301 302

Others Sphingobacterium Paludibaculum Azospira Cellulomonas Ralstonia Sphingomonas Geodermatophilus Acidobacteria Gp3 Ktedonobacter Phenylobacterium Massilia Pseudomonas Acidobacteria Gp1 Alicyclobacillus Symbiobacterium Paenibacillus Tumebacillus Oxalophagus Pullulanibacillus unclassified Bacillus

60

40

20

0

300

(b)

80 Relative abundance /%

Relative abundance /%

100

0

B0

B1

B2

B3

B4

B5

B0

B1

B2

B3

B4

B5

Fig. 5 Relative abundances of different bacterial community (a: phylum level; b: genus level) before and after 30 days of BMO reaction.

303 304

Taxonomic classification was determined to reveal the structure of the bacterial

15

305

community and the abundance of functional microbes in the soil samples. At the

306

phylum level (Fig. 5a), the most abundant phyla in the untreated sample were

307

Firmicutes, accounting for 93.7% of the total microbial sequences. The dominance of

308

Firmicutes has been reported for highly arsenic–contaminated sites (Chen et al., 2017;

309

Das et al., 2016). Although the dominant phylum for the BMO–treated samples was

310

still Firmicutes, the proportion of Firmicutes decreased significantly with increasing

311

BMO, while the proportion of other phyla, such as Proteobacteria, Chloroflexi,

312

Acidobacteria and Actinobacteria, significantly increased. A similar study by (Shao et

313

al., 2016) reported an increase in the relative abundance of Acidobacteria after the

314

addition of nano–MnO2 amendment. In addition, for the B5 treatment, the proportion

315

of Firmicutes decreased from 93.7 to 66.7%, while that of Proteobacteria increased

316

from 1.8 to 21.8%.

317

Fig. 5b further reveals the genus level distributions of the bacterial populations

318

after treatment with different dosages of BMO. The top three genera in untreated soil

319

were Bacillus (75.9%), unclassified (7.2%) and Pullulanibacillus (4.9%), accounting

320

for 88.1% of the total bacterial sequences. The structure of the microbial community

321

was altered when BMO was added to the soil system. The relative abundances of

322

Bacillus, Pullulanibacillus and Tumebacillus decreased, while the percentage of

323

unclassified, Oxalophagus, and Paenibacillus increased. Members of the genera

324

Symbiobacterium (8.2%), affiliated with the Firmicutes group, Massilia (2.0%) and

325

Phenylobacterium (2.4%) and Sphingomonas (1.5%), belonging to the Proteobacteria

326

group was present in higher amounts in the B5 treatment sample than other treatment 16

327

samples. These results showed that high amounts of BMO significantly improved the

328

bacterial relative abundance and community diversity.

329

In addition, BMO was produced by P. putida strain MnB1; however, the

330

proportion of Pseudomonas genera in all untreated and BMO–treated soil samples was

331

fairly low (˂1.5%), indicating that P. putida strain MnB1 was not the predominant

332

bacterial group after BMO treatment. These results suggested that the poorly

333

crystalline Mn oxide minerals in BMO play a dominant role in As stabilization and

334

changes of bacterial community. Results from a recent study by Wang et al. (2019c)

335

are consistent with those of this current study; i.e., poorly crystalline iron minerals in

336

biogenic flocs resulted in the removal of majority As(III). In addition, Fig. 2 shows

337

that BMO covering the surface of a cell may inhibit cell involvement in geochemical

338

reactions. Moreover, our recent study (Wang et al., 2019a) indicated that the bacterial

339

activity in BMO did not significantly affect Sb(III) removal, but adsorption and

340

re-oxidation of Mn(II) by BMO may provide new reactive sites for the detainment of

341

Sb(III).

342

3.7 Comparison of As stabilization by BMO and abiotic Mn oxides

343

The stabilization of As varied with type of soil, crystal structure of Mn oxides

344

and soil properties (Cui et al., 2018; He et al., 2019b; Yuan et al., 2019); thus, it is

345

difficult to evaluate the stabilization of As with other studies. In this work, the

346

stabilization of As after BMO treatment was compared to that with two types of

347

abiotic Mn oxides, micro–MnO2 and nano–MnO2. The As stabilization was calculated

348

per unit mass of amendment, and the results are shown in Fig. 6. The BMO stabilized 17

349

As at levels of 49.6-201.6 mg/kg·g-1 amendment in DW leaching tests and 41.8-165.8

350

mg/kg·g-1 amendments for TCLP leaching tests, which was significantly higher than

351

the values seen with micro–MnO2 (0.4-2.8 mg/kg·g-1 amendment for DW leaching

352

tests and 0.6-4.1 mg/kg·g-1 amendment for TCLP leaching tests) and nano–MnO2

353

(0.4-3.2 mg/kg·g-1 amendment for DW leaching tests and 0.5-4.4 mg/kg·g-1

354

amendment for TCLP leaching tests). These results further confirmed that BMO is

355

favorable for the stabilization of As in soils. 200

Stabilization efficiency of As (mg/kg g-1 amendments)

150

DW tests TCLP tests

100 50 6 4 2 0 1% 5% 10% 1% 5% 10% B1 MMO

NMO

B2 B3 B4

B5

BMO

356 357 358 359

Fig. 6 Effect of Mn oxides types on the stabilization of As. MMO and NMO represent micro–MnO2 and nano–MnO2, respectively.

360

4. Conclusions

361

This study demonstrated that the role of BMO for stabilization of As in the

362

contaminated soils. The experimental indicated that BMO can effectively stabilize As

363

in soils. XANES analysis indicated that As(III) in soils was completely oxidized by

364

BMO to form As(V). The addition of BMO led to a decrease in the exchangeable

365

fraction of As in soils and a remarkable increase in the residual phase. Stabilization

366

efficiency levels of 49.5-67.4% and 27.7-41.1% were found in the DW and TCLP

367

tests, respectively, after 15 days of reaction. The 16S RNA high-throughput

368

sequencing analysis suggested that the bacterial community diversity significantly 18

369

improved after BMO treatment. BMO significantly increased the abundance of the

370

Proteobacteria phylum, including Massilia, Phenylobacterium and Sphingomonas

371

genera. In comparison to abiotic Mn oxides, BMO treatment exhibits a higher As

372

stabilization efficiency of As. Overall, BMO can be used as a promising material for

373

the remediation of As in contaminated soils, however, further work is still needed to

374

verify its efficiency and the effect of environmental factors on field scales.

375

Acknowledgements

376

This work was supported by the Key Research and Development Plan of

377

Shandong Province (grant numbers 2018GSF117030), the Shandong Province Natural

378

Science Foundation (grant numbers ZR2018BEE037), the China CPSF-CAS Joint

379

Foundation for Excellent Postdoctoral Fellows (grant numbers 2016LH0048), the

380

China Postdoctoral Science Foundation (grant numbers 2016M600829) and Croucher

381

Chinese Visitorships 2018-2019.

382

Appendix A: Supplementary materials.

383

Supplementary data related to this article can be found.

384

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Highlights BMO was successfully used for the stabilization of As in soil. BMO effectively stabilize As in contaminated soil. Bacterial community was remarkably changed after BMO treatment. BMO had a higher efficiency in stabilizing As than abiotic MnO2.

Declaration of Competing Interest There are no conflicts to declare.