Humic substances facilitate arsenic reduction and release in flooded

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Environmental Processes

Humic substances facilitate arsenic reduction and release in flooded paddy soil Jiangtao Qiao, Xiaomin Li, Fangbai Li, Tongxu Liu, Lily Y Young, Weilin Huang, Ke Sun, Hui Tong, and Min Hu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06333 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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Environmental Science & Technology

1

Humic substances facilitate arsenic reduction and release in flooded paddy soil

2 3 4 5

Jiang-tao Qiao1†, Xiao-min Li2†, Fang-bai Li1*, Tong-xu Liu1, Lily Y. Young3, Weilin Huang1, 3, Ke Sun4, Hui Tong1, and Min Hu1

6 7 8

1Guangdong Key Laboratory of Integrated Agro-Environmental Pollution Control and

9

Management, Guangdong Institute of Eco-Environmental Science & Technology, Guangzhou

10

510650, P.R. China

11

2SCNU Environmental Research Institute, Guangdong Provincial Key Laboratory of Chemical

12

Pollution and Environmental Safety & MOE Key Laboratory of Environmental Theoretical

13

Chemistry, South China Normal University, Guangzhou 510006, P.R. China

14

3Department of Environmental Sciences, Rutgers University, New Brunswick, NJ 08901, USA

15

4State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal

16

University, Beijing 100875, P.R. China

1 ACS Paragon Plus Environment


17

ABSTRACT

18

Organic matter is important for controlling arsenic reduction and release under anoxic

19

conditions. Humic substances (HS) represent an important fraction of natural organic

20

matter, yet the manner in which HS affect arsenic transformation in flooded paddy

21

soil has not been thoroughly elucidated. In this study, anaerobic microcosms were

22

established with arsenic-contaminated paddy soil and amended with three extracted

23

humic fractions (fulvic acid, FA; humic acid, HA; and humin, HM). The HS

24

substantially enhanced the extent of arsenic reduction and release in the order FA >

25

HA > HM. It was confirmed that microbially reduced HS acted as an electron shuttle

26

to promote arsenate reduction. HS, particularly FA, provided labile carbon to

27

stimulate microbial activity and increase the relative abundances of Azoarcus,

28

Anaeromyxobacter and Pseudomonas, all of which may be involved in the reduction

29

of HS, Fe(III) and arsenate. HS also increased the abundance of transcripts for an

30

arsenate-respiring gene (arrA) and over transcripts in arsenate-respiring Geobacter

31

spp. The increase in both abundances lagged behind the increases in dissolved

32

arsenate levels. These results help to elucidate the pathways of arsenic reduction and

33

release in the presence of HS in flooded paddy soil.

34

Keywords: Humic fractions; Reduced humic substances; Arsenate-respiring bacteria;

35

Gene transcription; Paddy soil


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INTRODUCTION

37

Arsenic (As) contamination in paddy fields has gained considerable attention because

38

rice is an important source of arsenic pollution in human diets.1-3 The biogeochemical

39

processes and toxicity of arsenic vary considerably with seasons in paddy fields.

40

During the flooding season, arsenic can be released into soil solution by reductive

41

dissolution of arsenic-bearing Fe(III) minerals and reduction of arsenate (As(V)) to

42

arsenite (As(III)), which increases the arsenic mobility and facilitates arsenic uptake

43

and accumulation in rice plants.4, 5

44

Microbes play a key role in arsenic mobilization in arsenic-contaminated

45

aquifers, sediments and soils,6-11 and organic matter is important in controlling the

46

rate and magnitude of microbially mediated arsenic release from these

47

arsenic-contaminated environments.10, 12 Several microcosm-based and in situ studies

48

used labile organic carbon (mainly acetate) to mimic the influx of easily degradable

49

organic matter and to promote the activity of indigenous microbial communities

50

potentially involved in Fe(III) reduction and arsenic release.10, 11, 13-15 Our recent study

51

demonstrated that the introduction of lactate into arsenic-contaminated paddy soil

52

stimulated microbial reduction of As(V), mainly by upregulating transcription of the

53

As(V) respiratory reductase gene arrA and overall transcription in As(V)-respiring

54

Geobacter spp.7

55

Humic substances (HS), which are operationally defined by their extraction from

56

soils using a traditional alkaline-acid extraction technique, constitute one of the

57

important fractions of soil organic matter (SOM).16 HS can influence the

58

transformation of arsenic in soils by both abiotic and biotic processess.17 First, arsenic

59

can interact with HS by adsorption and complexation due to the functional groups of

60

HS, such as carboxylic, amino, hydroxyl, phenolic and sulfhydryl groups.18 Second, 3 ACS Paragon Plus Environment


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owing to their redox properties, HS can act as an electron shuttle to accelerate the

62

electron transfer from humic-reducing bacteria to solid electron acceptors, such as

63

Fe(III) minerals.19,

64

reduced forms of quinone-like moieties in HS, which is accompanied by an increase

65

in arsenic release.12 The addition of anthraquinone-2,6-disulfonic acid, a proxy for the

66

quinone moieties in HS, increases both Fe(III) and As(V) reduction in arsenic-rich

67

sediments.10, 14 It is reasonable to consider, therefore, that HS could also function as an

68

electron shuttle between bacteria and As(V) to facilitate As(V) reduction. Third, HS

69

may increase the abundance of microbial species (e.g., humic-, Fe(III)- and

70

As(V)-reducing bacteria), as HS can either be utilized as a carbon source or enhance

71

interactions

72

bacteria-HS-organic matter complexes.14, 21 However, no study has extracted HS from

73

soil and investigated their effect on arsenic transformation in paddy soil.

20

between

Fe2+(aq) is substantially generated in an aquifer with more

bacteria

and

organic

matter

by

forming

ternary

74

HS are highly complex, and differences in chemical properties and

75

environmental effects between bulk HS and their fractions can be notable.22-26 The

76

objectives of our study were (i) to isolate and characterize different fractions of HS

77

(fulvic acid, FA; humic acid, HA; and humin, HM) to investigate their effect on

78

arsenic transformation in flooded paddy soil; (ii) to evaluate whether HS act as an

79

electron shuttle between microorganisms and As(V) or electron donors for microbial

80

arsenic reduction; and (iii) to profile the active microbial communities and functional

81

As(V)-reducing bacteria stimulated by different HS fractions. The results obtained

82

will provide a better understanding of the pathways of arsenic reduction and release

83

and their associated contributors in the presence of HS in flooded paddy soil.

84

MATERIALS AND METHODS 4 ACS Paragon Plus Environment

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Soil Sampling and Characterization. Soil was sampled from a drained paddy field

86

contaminated by arsenic-containing acid mine drainage in Shantou City, China. The

87

soil contains 0.3 g kg-1 total arsenic, 30.5 g kg-1 total iron, and 17.3 g kg-1

88

dithionite-citrate-bicarbonate (DCB)-extractable Fe, which represents crystalline and

89

amorphous Fe oxides.7

90

Extraction and Characterization of HS. The fractions of HS used in this study were

91

extracted from a subtropical peat soil (Zhongxiang International Ltd., Beijing) that

92

had a lower content of iron than the tested paddy soil, minimizing the interference of

93

iron in the electron-shuttling of HS.27 HA and FA were extracted following the

94

International Humic Substances Society (IHSS) method,28 and HM was prepared

95

based on previously described procedures.29 For purification, each fraction of HS was

96

concentrated in a rotating evaporator and purified using a dialysis membrane

97

(molecular weight cut-off: 10,000 Dalton, Sigma, USA) against deionized water

98

according to a procedure described previously.30 The water collected outside the

99

dialysis membrane was subjected to a Cl- test with AgNO3 (0.10 M) with a detection

100

limit of 0.81 μM. The fractions of HS were freeze-dried and then stored in a

101

desiccator at 25oC in the dark until use.

102

The physicochemical properties of HS were characterized by elemental

103

composition and solid-state cross-polarization magic-angle spinning

104

magnetic resonance (NMR) spectra. The electron transfer capacity (ETC) of HS,

105

including their electron-accepting capacity (EAC) and electron-donating capacity

106

(EDC), were evaluated by electrochemical reduction and oxidation methods25 using

107

an electrochemical workstation (CHI660D, Chenhua Co., Ltd., Shanghai, China). A

108

glassy carbon cylinder with a volume of 15 mL (Sigradur, HTW Germany) was used

109

as both the electrochemical reaction vessel and the working electrode, a saturated 5 ACS Paragon Plus Environment

13C

nuclear


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110

calomel electrode was used as the reference electrode, and a spiral Pt wire was used as

111

the counter electrode. For the EAC and EDC determination, amperometric

112

measurements were conducted at an applied potential of -0.49 V (vs. SHE) and +0.61

113

V

114

2,2’-azino-di-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as mediators, respectively.

115

Details of these analyses and the original current peaks (Figure S1) are provided in the

116

Supporting Information (SI).

117

Microcosm Experiments. Anaerobic microcosms were set up as previously

118

described;7 7 g of anoxic paddy soils (wet weight) were placed into sterile serum vials

119

containing 70 mL 30 mM bicarbonate buffer (pH 7.3), 1 mL L-1 trace element

120

solution and 1 mL L-1 vitamin solution,31 followed by the addition of 0.07 g individual

121

HS. After flushing with a mixture of N2:CO2 (80:20) for 40 min, the microcosms were

122

sealed with butyl rubber stoppers. Incubation and sampling were carried out at 30oC

123

in the dark without shaking in an anaerobic chamber (Coy Labs, Grass Lake, MI,

124

USA). All microcosms, including abiotic controls, were processed in triplicate (Table

125

S1). For the abiotic control, the fractions of sterile soil and HS were prepared by

126

irradiation with 50 kGy γ-rays from a 60Co radioactive source at a γ-irradiation facility

127

(Guangzhou Huada, China). The uncertainties in

128

within 2% by both alanine and dichromate dosimeters, which gives confidence in the

129

gamma dose applied. To evaluate the potential effect of microbially reduced HS on

130

arsenic reduction in paddy soil, additional experiments were conducted with

131

Shewanella oneidensis MR-132 and with incubation with sterile soil and HS in the

132

presence and absence of acetate. Details of the experiments are in the SI.

133

Arsenic and Iron Speciation. When sampling at intervals, three vials of each

134

treatment were centrifuged at 8,000 g for 10 min at room temperature. A total of 5 mL

(vs.

SHE)

at

the

working

electrode

60Co


using

diquat

and

γ-ray dosimetry agreed to

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of the supernatant then was then filtered through a sterile 0.22-μm filter for

136

determination of dissolved As(III) and As(V) concentrations using atomic

137

fluorescence spectroscopy (SA-20, Jitian, Beijing, China).8 Soil pellets were divided

138

into two subsamples. One subsample was immediately frozen in liquid nitrogen and

139

stored at -80oC until total RNA extraction; the other was used to sequentially

140

fractionate arsenic in soil using 1 M KH2PO4 + 0.1 M ascorbic acid (mainly targeting

141

As adsorbed on iron (oxyhydr)oxides,33 0.2 M NH4+-oxalate (predominantly targeting

142

As incorporated into iron (oxyhydr)oxides),34 and HNO3 + H2O2 (dissolving the

143

residual As remaining in the soil).33 Detailed descriptions of the PO4-As, oxalate-As,

144

and residual As extraction are in the SI. Dissolved Fe(II) and 0.5 M HCl-extractable

145

Fe(II), namely, HCl-Fe(II) were quantified by the methods previously described.35

146

16S rRNA High-Throughput Sequencing. Total community RNA was extracted

147

using the PowerSoil Total RNA Isolation Kit (Mio Bio Laboratories, USA) according

148

to

149

reverse transcription

150

with gDNA Eraser (Takara, Japan). The cDNA from 3 replicates of experiments at

151

each sampling time was amplified with primers 515F and 806R and then subjected to

152

Illumina sequencing.36 Details of the PCR amplification and sequence analysis are in

153

the SI. The sequence data have been submitted to the NCBI Short Read Archive in

154

Bioproject PRJNA471565 under the accession number SRP148109.

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Identification and Quantification of As(V)-respiring Gene and Bacteria.

156

Quantification of bacterial 16S rRNA and arrA gene transcripts was performed on the

157

CFX 384™ Real-Time PCR Detection System (Bio-Rad Laboratories, USA) with

158

SYBR Green I detection. The arrA gene was amplified by primers arrA-CVF1

159

(5’-CACAGCGCCATCTGCGCCGA-3’)

the

manufacturer's were

instructions.

Elimination

performed with

of

genomic

the PrimeScript

and 7

ACS Paragon Plus Environment

RT

DNA

and

reagent

kit

arrA-CVR1


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160

(5’-CCGACGAACTCCYTGYTCCA-3’).37 Details of the qPCR amplification, the

161

primers used for each reaction, and construction of the standard plasmid have been

162

previously described.7 The arrA gene transcript clone library was constructed using

163

RNAs extracted on day 12. Nested PCR amplification of the arrA gene (625 bp) was

164

achieved with primers AS1F/AS2R (the first PCR), and AS2F/AS1R (the nested

165

PCR).38 Details of PCR conditions, and the construction and further analysis of the

166

clone library are in the SI. The GenBank/EMBL/DDBJ accession numbers of the arrA

167

gene sequences are MH370058 to MH370104. Transcriptional patterns of active

168

Geobacter spp. were also quantified by qPCR using primers Geo494F and Geo825R39

169

as described above.

170

RESULTS AND DISCUSSION

171

Characteristics of FA, HA and HM. In comparison with HM, both FA and HA

172

showed higher C/H elemental ratios (Table 1), which indicates that they have higher

173

aromaticity and a higher degree of condensation.40 The

174

that whereas both FA and HA had higher percentages of carboxyl C than HM, FA

175

showed the highest percentage of carbonyl C and HA showed the highest percentage

176

of aromatic C (Table 1 and Figure S2). The EAC of different HS ranked as HA (0.62

177

± 0.04 mmole- g-1) > FA (0.48 ± 0.06 mmole- g-1) > HM (0.30 ± 0.07 mmole- g-1), and

178

the EDC ranked as FA (0.59 ± 0.08 mmole- g-1) > HA (0.54 ± 0.11 mmole- g-1) > HM

179

(0.26 ± 0.03 mmole- g-1) (Table 1). The iron contents were 0.3, 0.2, and 2.3 mg g-1 in

180

FA, HA, and HM, respectively. Since complexed iron in HS is also a redox-active

181

species,25,

182

0.7%, 0.9% and 13.2% for FA, HA and HM, respectively, if all Fe in the HS is

183

present as Fe(III) and each Fe(III) accepts one electron. Humic-bound iron can act as

41

13C

NMR spectra illustrated

the maximum possible contribution of the Fe to the measured EAC is


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an electron shuttle for microbial Fe(III) reduction,42 although the complexed iron

185

alone might not be sufficient to be responsible for the amount of electrons transferred

186

by the HS samples.43,

187

accept and donate electrons more efficiently than HM when acting as an electron

188

shuttle on a per gram basis.

189

44

Nevertheless, our results implied that HA and FA should

The EAC of HA was higher than that of FA, which is similar to earlier studies.25,

190

45

191

ratios and aromaticity data from

192

higher aromaticity (50.0%) than FA (43.5%), but FA had a higher C/H ratio and

193

contained higher percentages of carbon in carbonyl groups relative to HA (Table 1),

194

which can lead to high electron transfer capacity. HM usually has a high aromaticity,

195

but the aromaticities calculated from 13C NMR spectral data cover a wide range, from

196

35% to 92%.47 Jin et al. (2017) found that while the HM of a grassland soil had a

197

higher C/H than the corresponding HA, the HM of an agriculture soil showed a lower

198

C/H ratio than the corresponding HA.48 Hence, the C/H ratios and aromaticities of

199

HM could vary depending on HM origins. In this study, HM had the lowest C/H ratio

200

and aromaticity, which thus result in its low EAC and EDC (Table 1). The ratios of

201

total carbon to labile carbon were 11:1, 5:1, and 63:1 for HA, FA, and HM,

202

respectively (Table 1), suggesting that FA can provide a greater amount of

203

bioavailable carbon as an electron donor for microbial respiration than HA and HM.

204

Arsenic and Iron Transformation. Figure 1 illustrates the levels of soluble and

205

phosphate- and oxalate-extractable arsenic species over time as affected by the

206

different treatments. Dissolved As(III) increased over time and reached 4.7, 4.2 and

207

3.9 mg L-1 in the FA-, HA-, and HM-amended microcosms on day 30, respectively,

208

which was at least twice the concentration in the Soil control (1.7 mg L-1) (Figure 1a).

Generally, the EAC of HA and FA correlated positively with their C/H elemental 13C

NMR.25,

45, 46

In this study, HA also showed



209

Dissolved As(V) levels rapidly increased to 0.4-1.1 mg L-1 (FA > HA ≈ HM ≈ Soil) at

210

day 6, and then gradually declined to approximately 0.2 mg L-1 at day 30 (Figure 1b).

211

Compared with the control treatment, the addition of HS resulted in higher PO4- and

212

oxalate-As(III) and lower PO4- and oxalate-As(V) at day 30 (Figure 1c-1f), indicating

213

that HS enhanced the reduction of As(V) adsorbed on and incorporated into iron

214

(oxyhydr)oxides. As shown in Figure S3, the molar mass of different arsenic fractions

215

suggested that the dominant arsenic species was PO4-As(V) at the beginning and

216

dissolved As(III) at the end. In all biotic microcosms, the recovery rates of the sum of

217

the different arsenic species to the individual total arsenic content in the samples were

218

good, ranging from 90 ± 3% to 105 ± 2% (Table S2). The above results indicated that

219

the presence of HS enhanced As(V) reduction and As(III) release into the soil

220

solution.

221

The abiotic effect of HS on arsenic transformation was investigated using the

222

abiotic controls where the tested soil and HS were sterilized by γ-ray irradiation

223

(Figure S4). In the abiotic controls amended with HS, dissolved As(V) and As(III)

224

concentrations were 140-200 μg L-1 and 11-12 μg L-1 at day 30, respectively, which

225

were higher than the levels in samples without HS amendments (44 μg L-1 and 2 μg

226

L-1). HS can mobilize arsenic species adsorbed on iron (hydr)oxide surfaces by

227

competitive adsorption and redox reactions45 or by forming arsenic-organic-metal

228

complexes and mineral colloids.49 Among these abiotic effects, competitive

229

adsorption due to electrostatic interaction is considered to be the main mechanism,

230

and FA likely competes more strongly for adsorption with arsenic than HA because

231

FA has a smaller size and closer proximity to the iron (hydr)oxide surfaces, which

232

results in its stronger electrostatic interaction with arsenic.50 FA did not show a

233

stronger effect on arsenic release than HA and HM in the abiotic controls (Figure S4), 10 ACS Paragon Plus Environment

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probably because γ-ray irradiation can increase its average molecular weight due to

235

condensation of carboxylic groups and CO2 liberation.51 Nevertheless, FA without

236

any irradiation treatment likely contributes in part to the facilitation of arsenic release

237

in the biotic microcosms.

238

The formation of dissolved- and HCl-Fe(II) was higher in all biotic microcosms

239

than in the abiotic controls (Figure S5a). HS also had a stimulating effect on microbial

240

Fe(III) reduction, and samples with them released more Fe(II) than the soil control. In

241

the FA, HA, and HM amendments, dissolved Fe(II) levels increased over time and

242

reached 34, 15, and 7.6 mg L-1 at day 30, respectively, which was approximately 7-, 3-

243

and 1.5-fold that of the soil control (Figure S5a). The redox potentials of Fe(III)

244

(oxyhydr)oxides/Fe2+ couples ranged from -0.284 V to +0.15 V (pH=7),52 and those

245

of dissolved As(V)/As(III) couples ranged from +0.045 V to +0.054 V (pH=7).53 In

246

cultures with pure isolates, whether Fe(III) or As(V) was preferentially reduced

247

depended on whether the bacterium was an Fe(III)-reducer or As(V)-reducer,52,

248

while As(V) and Fe(III) can be simultaneously reduced in both pure and mixed

249

cultures.54 In our biotic microcosms, the initial increases in dissolved As(V) (Figure

250

1b) are predominantly due to microbial Fe(III) reduction, and As(V) and Fe(III)

251

reduction proceeded simultaneously (Figure 1 and S5). Regarding the substantial

252

increase in dissolved As(III) concentration, it is necessary to clarify how HS

253

facilitated As(V) reduction in flooded paddy soil.

254

Arsenic Reduction by Microbially Reduced HS. To generate microbially reduced

255

HS, another experiment with sterile HS and sterile soil was incubated with S.

256

oneidensis MR-1 and supplied with acetate. MR-1 is a humic- and Fe(III)-reducing

257

bacterium that cannot reduce As(V).55,

258

soil, both dissolved Fe(II) and dissolved As(V) levels increased after day 6 and

56

53

In the treatment of acetate+MR-1+sterile



259

remained at 28-36 mg L-1 and 3.1-4.6 mg L-1 during days 6-30, respectively (Figure

260

2d). Thus, As(V) release from the soil into solution is mainly due to the microbial

261

Fe(III) reduction by MR-1. Meanwhile, dissolved As(III) was barely detected in the

262

soil solution (Figure 2d), confirming that MR-1 cannot reduce As(V).

263

With the addition of sterile HS, dissolved As(III) increased over time, and

264

dissolved As(V) decreased after day 6. The dissolved As(III) and dissolved Fe(II)

265

concentrations were 3.0-3.4 mg L-1 and 21-30 mg L-1 on day 30, respectively (Figure

266

2a-c). It seems that HS facilitate As(V) reduction by functioning as an electron

267

shuttle, in which HS are reduced by MR-1 and the reduced HS transfer electrons to

268

As(V) for As(V) reduction. The decrease in dissolved As(V) being larger than the

269

increase in dissolved As(III) in Figure 2a-c and the slight decrease in dissolved As(V)

270

in Figure 2d are likely due to readsorption on and reincorporation into secondary

271

Fe(II)-Fe(III) minerals.57, 58

272

To confirm the electron shuttle effect of HS, two supplementary experiments

273

were performed. (i) MR-1 was incubated with sterile soil and sterile HS but without

274

acetate to verify whether the HS can serve as an electron donor for MR-1. The results

275

shown in Figure S6 demonstrate that the electron donor effect of HS on facilitating

276

anaerobic respiration of MR-1 for Fe(III) and HS reduction was small because HS

277

only slightly increased the dissolved Fe(II), As(III) and As(V) levels; these

278

concentrations were negligible relative to their higher production levels with acetate

279

first (Figure 2). (ii) FA and HA were incubated with MR-1, and the reduced FA and

280

HA (without any microorganisms) were then collected and mixed with dissolved

281

As(V) for reaction. It is confirmed that microbially reduced FA and HA can

282

abiotically reduce As(V) to As(III) (Figure S7).

283

In Figure 2, the sterile HS seems to facilitate only As(V) reduction, not Fe(III) 12 ACS Paragon Plus Environment

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reduction. In this experiment, acetate and MR-1 were supplied at concentrations as

285

high as 20 mM and 1010-1011 cells mL-1, respectively, both of which can provide a

286

sufficient number of electron donors and cells to maximize the microbial activity for

287

Fe(III) and HS reduction. Since MR-1 cannot reduce As(V), the reduced HS mainly

288

facilitated As(V) reduction when acting as an electron shuttle, given that microbially

289

available Fe(III) and HS could be fully reduced by MR-1. The extracellular electron

290

transfer performance of HS is positively correlated with their EAC and aromaticity.25,

291

26, 59

292

because reduced HS could be provided repeatedly by MR-1, maximizing the electron

293

shuttle effect on As(V) reduction. In the biotic microcosms with soil (Figure 1),

294

however, HS resulted in different levels of As(III) production, which should be

295

associated not only with their EAC and aromaticity (Table 1) but also with the

296

microorganisms stimulated by HS.

297

Bacterial Communities Stimulated by HS. An overall summary of high-throughput

298

sequencing libraries is provided in the SI. The profiling of bacterial 16S rRNA genes

299

identified 13 distinct phyla/classes (Figure S8) and 12 genera (Figure 3) at a relative

300

abundance > 1%. At the genus level, Variovorax (1-22%), Flavisolibacter (0.1-6%)

301

and Azoarcus (0.4-6%) represented the dominant genera in the soil control, and their

302

abundances greatly increased during days 0-6 (Figure 3d). With HA and HM, the

303

abundance of Variovorax was constant at 16-20% during days 2-30, which was

304

similar to its level in the soil control (Figure 3b-3d). Azoarcus was also one of the

305

dominant genera in samples with HA and HM, and its abundance (1-10%) was

306

slightly higher in these samples than in the soil control (Figure 3b-3d). In addition, a

307

gradual increase in Anaeromyxobacter (from 0.7% to 4%) was observed over time in

308

samples with HA (Figure 3b). Compared with amendments with the other HS, FA

Different HS with MR-1 led to similar production of dissolved As(III) (Figure 2)



Page 14 of 35

309

amendment resulted in a lower abundance of Variovorax (1.2-12%) during incubation

310

and a higher abundance of Azoarcus, particularly during days 2-6 (14-17%) (Figure.

311

3a). Meanwhile, FA amendment increased the abundance of Pseudomonas (0.5-8%),

312

Synechococcus (0-7%), and Anaeromyxobacter (1-4%).

313

The genus Variovorax contains a large number of heavy-metal-tolerant strains.62

314

The sequenced genome of V. paradoxus S110 provides some clues to the organism’s

315

arsenic tolerance by identifying the operon coding for an arsenic reductase complex.61

316

Similarly, whole-genome analyses find a putative arsenic reduction operon in the

317

genomes of Azoarcus members.62 Both Pseudomonas and Anaeromyxobacter have

318

been frequently detected in arsenic-contaminated sites,7,

319

respire humics, Fe(III) or As(V) under anoxic conditions.14,

320

Azoarcus might participate in As(V) reduction without HS, and HS, particularly FA,

321

might stimulate Azoarcus, Anaeromyxobacter, and Pseudomonas to reduce HS,

322

Fe(III) and/or As(V).

323

Identification and Quantification of As(V)-respiring Gene and Bacteria. This

324

study focused on transcriptional copy numbers of the As(V) respiration-related arrA

325

gene and associated bacteria since As(V)-respiring bacteria play a more important role

326

in

327

arsenic-contaminated environments.7,

328

transcription of bacterial 16S rRNA and arrA genes were stimulated overall (Figure

329

S9a and 9b).

arsenic

reduction

than

arsenic-resistant 65-67

14

and some of them can 63, 64

Variovorax and

microorganisms

in

anoxic

Following the addition of HS, the

330

The arrA gene transcripts clone library showed that more than 50% of the ArrA

331

sequences were related to putative ArrA sequences of Geobacter spp. and 46% of

332

them were related to ArrA sequences of uncultured bacteria (Figure S10 and Table

333

S3). Transcriptional patterns of Geobacter spp. revealed that FA stimulated the 14 ACS Paragon Plus Environment

Page 15 of 35


334

transcription of Geobacter spp. throughout the incubation the most (Figure S9c).

335

Geobacter was also detected in the bacterial communities of all microcosms and at

336

relatively high levels (0.5-1.3%) during days 6-30 (Figure 3). These results

337

emphasized the importance of Geobacter spp. in respiratory arsenic reduction in the

338

overall As(V) reduction in arsenic-contaminated paddy soil, which can be stimulated

339

by FA.

340

The increase in arrA gene and overall Geobacter spp. transcripts lagged behind

341

the increase in dissolved As(V) levels in all biotic microcosms after being normalized

342

against the copy numbers of 16S rRNA genes (Figure 4). This finding is different

343

from the results of our previous study, which showed a positive correlation between

344

the dissolved As(V) levels and the arrA gene/Geobacter spp. transcription after the

345

addition of lactate and biochar.7 The introduction of easily degradable organic carbon

346

(e.g., acetate and lactate) can promptly stimulate transcription of the arrA gene and

347

Geobacter species.7, 13 However, HS are a mixture of complex organic compounds,

348

and the majority of HS (i.e., HA and HM) are insoluble in water at circumneutral pH,

349

which leads to the low bioavailability of HS for microbes.16 This effect is supported

350

by the following results (i) HS had a limited electron donor effect on anaerobic

351

respiration of MR-1 in Fe(III) and HS reduction (Figure S6), and (ii) the transcription

352

levels of bacterial 16S rRNA gene and Geobacter spp. with HS were 10-100 times

353

lower than those with acetate and biochar in a previous study.7 As time elapses, it is

354

possible that HS are decomposed by soil microbes to more readily degradable

355

substrates with low molecular weight, stimulating respiratory As(V) reduction. With

356

FA, the substantial increase in the release of dissolved As(V) most likely stimulated

357

the expression of the arrA gene and the transcription in Geobacter species (Figure

358

4a). Such stimulation also likely contributed to the reduction of PO4- and 15 ACS Paragon Plus Environment


359

oxalate-As(V) (Figure 1c and 1d).

360

Environmental Implications. In summary, our results demonstrate that HS can

361

facilitate arsenic reduction and release via three main pathways: (1) functioning as an

362

electron shuttle, (2) providing labile carbon as an electron donor and increasing the

363

abundance of indigenous soil bacteria (i.e., Azoarcus, Anaeromyxobacter and

364

Pseudomonas), and (3) stimulating the transcriptions of the As(V) respiration-related

365

arrA gene and As(V)-respiring Geobacter species. Among the three fractions of HS,

366

FA is the most effective because it has a greater amount of labile carbon and higher

367

solubility and ETC. These characteristics not only favor the stimulation of microbial

368

activity but also accelerate electron transfer from microbes to both dissolved and

369

adsorbed As(V).19, 68, 69

370

Since SOM is thoroughly mixed with and often adheres to soil minerals, it has

371

traditionally been separated into several operationally defined fractions to better

372

understand its properties.16 Similarly, the fractions of HS in this study were subject to

373

traditional alkaline-acid extraction, but some conceptual problems have been raised

374

when defining “humic substances” by this extraction procedure.22, 23, 70 For example,

375

50–70% of the soil organic carbon is unextracted due to incomplete extraction,71 and

376

the alkaline solution at pH 13 can ionize compounds that would never dissociate

377

within the pH range of soil (3.5-8.5).16, 23 As such, the fractions of HS may not be the

378

best representatives of SOM.23

379

Recently, HA and FA have been found to share major chemical groups

380

irrespective of their sources. However, their molecular architectures may differ

381

substantially, which would influence their binding properties.73 In this study, the

382

ETCs of HS differed from each other, which was associated with the functional

383

groups involved in their ETC. Before an alternative approach of SOM isolation is 16 ACS Paragon Plus Environment

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Page 17 of 35


384

developed, the operational separation of HS into three fractions, in combination with

385

characterization of the individual fractions, could provide more information for a

386

better understanding of their environmental effects on arsenic transformation.

387

It is important to note that the relative contributions of FA, HA and HM to the

388

electron-shuttling effect on arsenic reduction and release may differ among soils. The

389

relative contributions of these fractions may also be different for the native FA, HA

390

and HM fractions in the studied paddy soil, as the observations in this study are based

391

on added fractions of HS that were isolated from a different soil system. Nevertheless,

392

paddy soil with a high content of HS, particularly with high FA levels, can be

393

predicted to highly elevate As(III) concentrations into soil solution during flooding

394

seasons. FA and HA are also commonly used as soil supplements in agriculture.74

395

Therefore, introducing HS into arsenic-contaminated paddy soil, particularly during

396

planting season, should be considered very carefully. However, an arsenic

397

remediation strategy using HS can still be developed and applied during the slack

398

season to respond to the demand for safe food and sustainable agriculture because this

399

environmentally

400

arsenic-contaminated paddy soil.

401

ASSOCIATED CONTENT

402

Supporting Information

403

Current responses of HS in EAC and EDC measurement (Figure S1),

404

spectra (Figure S2), molar mass of different As(III)/As(V) and total As (Figure S3),

405

different As(III)/As(V) species in abiotic controls (Figure S4), dissolved Fe(II) and

406

HCl-Fe(II) (Figure S5), dissolved Fe(II), As(III), and As(V) in MR-1+sterile

407

soil+sterile

HS

friendly

(Figure

substance

S6),

can

dissolved

effectively

As(III)


mobilize

in

arsenic

from

13C

NMR

microbially

reduced


408

FA/HA+dissolved As(V) (Figure S7), bacterial community at the phylum and class

409

levels (Figure S8), transcriptions of 16S rRNA, arrA gene and Geobacter spp. (Figure

410

S9), phylogenetic tree of putative ArrA sequences (Figures S10), experimental

411

treatments conducted (Table S1), molar mass of different As(III)/As(V), and total As

412

(Table S2), summary of arrA gene transcripts clone library (Table S3), additional

413

references.

414

AUTHOR INFORMATION

415

Corresponding Author

416

Phone: +86 20 37021396 ; Fax: +86 20 87024123; E-mail: [email protected].

417

Author Contributions

418

† These

419

Notes

420

The authors declare no competing financial interest.

421

ACKNOWLEDGMENTS

422

This work was supported by funding from National Natural Science Foundation of

423

China (41330857 and 41877043), Guangdong Natural Science Fund for Distinguished

424

Young Scholars (2017A030306010), Guangdong Academy of Sciences’ Projects

425

(2019GDASYL-0103050, 2018GDASCX-0501 and 2017GDASCX-0106), and

426

Guangdong Special Support Plan for High-Level Talents (Xiaomin Li).

427

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sediments of the Mekong Delta. Geobiology 2015, 13 (6), 581–587.

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(66) Lear, G.; Song, B.; Gault, A. G.; Polya, D. A.; Lloyd, J. R. Molecular analysis of

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arsenate-reducing bacteria within Cambodian sediments following amendment with 26 ACS Paragon Plus Environment

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Microbial mobilization of arsenic from sediments of the Aberjona Watershed.

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Environ. Sci. Technol. 1997, 31 (10), 2923–2930.

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freudenreichii and other fermenting bacteria. Appl. Environ. Microbiol. 1998, 64 (11),

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Field, J. A. Reduction of humic substances by halorespiring, sulphate-reducing and

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methanogenic microorganisms. Environ. Microbiol. 2002, 4 (1), 51–57.

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view. Environ. Sci. Technol. 2005, 39, 9009−9015.

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(71) Rice, J. A. Humin. Soil Sci. 2001, 166 (11), 848–857.

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organic matter decomposing at different depths in soil: an acid hydrolysis approach.

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653

FIGURES and TABLE

654

Table 1 Characteristics of the FA, HA and HM fractions.

655

Figure 1 Time-dependent concentrations of water-soluble (dissolved As(III)/As(V)),

656

phosphate-extractable

657

(oxalate-As(III)/As(V)) arsenic fractions in the microcosms set up with paddy soil and

658

amended with FA, HA and HM and the control (no amendment) over the course of

659

incubation (Soil+FA, Soil+HA, Soil+HM and Soil). Bars represent standard errors (n

660

= 3).

661

Figure 2 Time-dependent concentrations of dissolved As(III), As(V) and Fe(II) in

662

different anaerobic cultures set up with sterile paddy soil and Shewanella oneidensis

663

MR-1 with and without amendment of sterile FA, HA and HM. (a)

664

Acetate+MR-1+sterile soil+sterile FA; (b) Acetate+MR-1+sterile soil+sterile HA; (c)

665

Acetate+MR-1+sterile soil+sterile HM; (d) Acetate+MR-1+sterile soil. Bars represent

666

standard errors (n = 3).

667

Figure 3 Relative abundance of the potentially active bacterial community at the

668

genus level identified in the Soil+FA (a), Soil+HA (b), Soil+HM (c) and Soil (d)

669

microcosms during the incubation by 16S rRNA gene high-throughput sequencing.

670

Only the genera with relative abundance higher than 1% in at least two treatments

671

were selected for further analysis. The abundance is presented as the average

672

percentage of three replicates, classified by RDP classifier at a confidence threshold

673

of 97%.

674

Figure 4 Dissolved As(V) concentrations and transcriptional levels of the arrA gene

675

and Geobacter spp. normalized to the 16S rRNA gene in the Soil+FA (a), Soil+HA

676

(b), Soil+HM (c) and Soil (d) microcosms throughout the incubation. Bars represent

(PO4-As(III)/As(V)),


and

oxalate-extractable

Page 29 of 35

677


standard errors (n = 3).



678

Table 1 Characteristics of the FA, HA and HM fractions. Bulk elemental composition C (%)

679 680 681

Page 30 of 35

O (%)

N (%)

H (%)

C/H

13C-NMR

ETC

Aliphatic C

Aromatic C

Carboxyl C

Carbonyl C

(%)

(%)

(%)

(%)

0-93 ppm

93-165 ppm

165-190 ppm

190-220 ppm

Aromaticity

EACb EDCb

a

Total C:Labile Cc

(mmole- g-1)

FA

41.0

31.2

1.1

3.4

12.1

45.5

35.1

9.7

9.7

43.5

0.48

0.59

11:1

HA

40.2

30.0

0.9

3.6

11.2

42.2

42.2

10.4

5.2

50.0

0.62

0.54

5:1

HM

47.8

28.0

0.4

5.2

9.2

57.9

32.2

5.9

4.0

35.7

0.30

0.26

63:1

a Aromaticity

=100×aromatic C (93-165 ppm)/[aromatic C (93-165 ppm) + aliphatic C (0-93 ppm)]. The electron accepting and donating capacities (EAC and EDC) values were the average of replicates (n ≥ 3) of the data in Figure S1. c The labile carbon was extracted by 2.5 M H SO at 105oC for 30 min according to Rovira and Vallejo (2002).72 2 4 b


Page 31 of 35


TOC 45x19mm (600 x 600 DPI)



Figure 1 Time-dependent concentrations of water-soluble (dissolved As(III)/As(V)), phosphate-extractable (PO4-As(III)/As(V)), and oxalate-extractable (oxalate-As(III)/As(V)) arsenic fractions in the microcosms set up with paddy soil and amended with FA, HA and HM and the control (no amendment) over the course of incubation (Soil+FA, Soil+HA, Soil+HM and Soil). Bars represent standard errors (n = 3). 242x184mm (300 x 300 DPI)


Page 32 of 35

Page 33 of 35


Figure 2 Time-dependent concentrations of dissolved As(III), As(V) and Fe(II) in different anaerobic cultures set up with sterile paddy soil and Shewanella oneidensis MR-1 with and without amendment of sterile FA, HA and HM. (a) Acetate+MR-1+sterile soil+sterile FA; (b) Acetate+MR-1+sterile soil+sterile HA; (c) Acetate+MR-1+sterile soil+sterile HM; (d) Acetate+MR-1+sterile soil. Bars represent standard errors (n = 3). 204x126mm (300 x 300 DPI)



Figure 3 Relative abundance of the potentially active bacterial community at the genus level identified in the Soil+FA (a), Soil+HA (b), Soil+HM (c) and Soil (d) microcosms during the incubation by 16S rRNA gene high-throughput sequencing. Only the genera with relative abundance higher than 1% in at least two treatments were selected for further analysis. The abundance is presented as the average percentage of three replicates, classified by RDP classifier at a confidence threshold of 97%. 257x290mm (300 x 300 DPI)


Page 34 of 35

Page 35 of 35


Figure 4 Dissolved As(V) concentrations and transcriptional levels of the arrA gene and Geobacter spp. normalized to the 16S rRNA gene in the Soil+FA (a), Soil+HA (b), Soil+HM (c) and Soil (d) microcosms throughout the incubation. Bars represent standard errors (n = 3). 179x283mm (300 x 300 DPI)


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