Anaerobic Arsenite Oxidation by an Autotrophic Arsenite-Oxidizing

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Anaerobic arsenite oxidation by an autotrophic arseniteoxidizing bacterium from an arsenic-contaminated paddy soil Jun Zhang, Wuxian Zhou, Bingbing Liu, Jian He, Qirong Shen, and Fang-Jie Zhao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es506097c • Publication Date (Web): 23 Apr 2015 Downloaded from http://pubs.acs.org on April 28, 2015

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Environmental Science & Technology

1

Anaerobic arsenite oxidation by an autotrophic arsenite-oxidizing bacterium

2

from an arsenic-contaminated paddy soil

3 4

Jun Zhang1, Wuxian Zhou1, Bingbing Liu1, Jian He2, Qirong Shen1, Fang-Jie

5

Zhao1, 3*

6 7

1

8

Innovation Center for Solid Organic Waste Resource Utilization, College of

9

Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing

Jiangsu Key Laboratory for Organic Waste Utilization, Jiangsu Collaborative

10

210095, China

11

2

College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China

12

3

Sustainable Soils and Grassland Systems Department, Rothamsted Research,

13

Harpenden, Hertfordshire AL5 2JQ, U.K.

14 15

* Author for correspondence

16

Email: [email protected]

17

Telephone: +86 25 84396509

18

Fax: +86 25 84399551

1

ACS Paragon Plus Environment


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19

ABSTRACT:

20

Microbe-mediated arsenic (As) redox reactions play an important role in the

21

biogeochemical cycling of As. Reduction of arsenate [As(V)] generally leads to As

22

mobilization in paddy soils and increased As availability to rice plants, whereas

23

oxidation of arsenite [As(III)] results in As immobilization. A novel chemoautotrophic

24

As(III)-oxidizing

25

As-contaminated paddy soil. The isolate was able to derive energy from the oxidation

26

of As(III) to As(V) under both aerobic and anaerobic conditions using O2 or NO3- as

27

the respective electron acceptor. Inoculation of the washed SY cells into a flooded soil

28

greatly enhanced As(III) oxidation to As(V) both in the solution and adsorbed phases

29

of the soil. Strain SY is phylogenetically closely related to Paracoccus niistensis with

30

a 16S rRNA gene similarity of 96.79 %. The isolate contains both the denitrification

31

and ribulose 1,5-bisphosphate carboxylase/oxygenase gene clusters, underscoring its

32

ability to denitrify and to fix CO2 while coupled to As(III) oxidation. Deletion of the

33

aioA gene encoding the As(III) oxidase subunit A abolished the As(III) oxidation

34

ability of strain SY and led to increased sensitivity to As(III), suggesting that As(III)

35

oxidation is a detoxification mechanism in this bacterium under aerobic and

36

heterotrophic growth conditions. Analysis of the aioA gene clone library revealed that

37

the majority of the As(III)-oxidizing bacteria in the soil were closely related to the

38

genera Paracoccus of α-Proteobacteria. Our results provide direct evidence for As(III)

39

oxidation by Paracoccus species and suggest that these species may play an important

40

role in As(III) oxidation in paddy soils under both aerobic and denitrifying conditions.

bacterium,

designated

strain

SY,

41

2


was isolated

from an

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42

TOC Art

43

-

As(V)

NO3 Aio

As(III)

Nar, Nir, Nor, Nos

500 nm

N2

Paracoccus strain SY: A chemoautotrophic As(III)-oxidizer

3



44

INTRODUCTION

45

Arsenic (As) is a toxic metalloid that is widely distributed in the environment.

46

Inorganic As is a carcinogen;1 chronic exposure to As results in a wide range of

47

adverse health effects.2 Humans are exposed to As mainly through drinking water and

48

diet.3, 4 It has been recognized that rice is an important dietary source of inorganic As,

49

particularly for the population consuming rice as the staple food.5-7 Rice crops grown

50

under anaerobic paddy environment tends to accumulate more As than other cereal

51

crops.8 This is because anaerobic conditions in paddy soil lead to the mobilization of

52

As(III), which is taken up inadvertently and efficiently by the silicic acid transport

53

pathway in rice roots.9 In some areas in south and southeast Asia, paddy soils have

54

been contaminated with As due to irrigation of As-laden groundwater or mining and

55

smelting activities,10, 11 resulting in further elevation in the transfer of As from soil to

56

the food chain.

57

The environmental behavior, fate and toxicity of As to organisms are strongly

58

influenced by its chemical speciation. As(III) is generally thought to be more mobile

59

in the environment and more toxic to organisms than As(V).12 As(V) is the

60

predominant form of As in soil under aerobic conditions. The strong adsorption by

61

minerals such as iron oxides/hydroxides renders the bioavailability of As(V) relatively

62

low in soil.12-14 In contrast, As(III) is less strongly adsorbed than As(V).15 When soil

63

is flooded to grow paddy rice, reductive dissolution of iron oxides/hydroxides and

64

reduction of As(V) facilitate the release of As, predominantly in the As(III) form, into

65

the soil solution,16,

66

Growing rice under aerobic or alternate wet-dry conditions has been shown to

67

decrease As accumulation in rice markedly.17, 19

17

resulting in increased bioavailability of As to rice plants.18

68

Soil microorganisms play an important role in both As(V) reduction and As(III)

69

oxidation. Bacteria capable of either oxidizing As(III) or reducing As(V) coexist and

70

are ubiquitous in soils.20-22 Microbial As(V) reduction can be carried out via the

71

arsenate respiratory reductase (Arr) which uses As(V) as the terminal electron

72

acceptor during anaerobic respiration, or via the arsenate reductase (ArsC) as a part of

73

the As detoxification mechanism.23, 24 Conversely, As(III) can be oxidized to As(V) by 4


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74

both heterotrophic and chemoautotrophic oxidizing bacteria, which have been

75

identified from environments such as gold mine wastewater, arsenic contaminated

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soils, lake water, sediment, and geothermal environments.25-30 The As(III)-oxidizing

77

chemoautotrophic microorganisms use the energy and reducing power from As(III)

78

oxidation for CO2 fixation and cell growth under both aerobic27, 29, 31 and anaerobic

79

nitrate-reducing28,

80

(Aio), a heterodimeric periplasmic enzyme containing molydopterin.32 The gene

81

(aioA) encoding the subunit A of Aio was successfully amplified from the anaerobic

82

denitrifying As(III)-oxidizer Sinorhizobium sp. DAO10,33 suggesting a role for Aio in

83

anaerobic As(III) oxidation in anoxic environments. Recently, a new group of arsenite

84

oxidases, ArxA, was identified in the chemoautotrophic arsenite oxidizer

85

Alkalilimnicola ehrlichii strain MLHE-1, which catalyzes As(III) oxidation coupled to

86

nitrate reduction.34 Degenerate primers have been designed to examine As(III)

87

oxidase–like genes in environmental samples,35 providing evidence that a variety of

88

aerobic As(III) oxidizers are widespread in As-contaminated environments.

30

conditions. As(III) oxidation is mediated by arsenite oxidase

By transforming As(III) to As(V), As(III) oxidizers attenuate As(III)

89 90

bioavailability and toxicity in the environment.36

91

was found to account for significant proportions (10-30 %) of the As in the pore water

92

37, 38

93

predict the presence of negligible As(V).The presence of As(V) could be due to the

94

activities of anaerobic As(III) oxidizers. A number of anaerobic As(III) oxidizers have

95

been isolated from lake water and sediments.28, 30 However, little is known about

96

microbes capable of oxidizing As(III) under anaerobic conditions in paddy soils. Such

97

microorganisms could play an important role in the As redox cycle and thus affect As

98

mobility in paddy soils. In the present study, we isolated a novel autotrophic

99

microorganism Paracoccus sp. strain SY from an As-contaminated paddy soil and

100

demonstrated its ability to oxidize As(III) under both aerobic and anaerobic conditions.

101

When added to a paddy soil, Paracoccus sp. strain SY also markedly altered As

102

speciation and extractability.

In anaerobic paddy soils As(V)

, even though thermodynamic calculations based on the redox potential would

103 5



104

MATERIALS AND METHODS

105

Enrichment, isolation and routine

106

Growth Conditions and Medium Composition.

107

cultivation of the pure cultures were performed using a mineral salts medium (MSM)

108

under anaerobic conditions with pH adjusted to 7.2. MSM contained the following (g

109

l-1): 7.9 g Na2HPO4·7H2O, 1.5 g KH2PO4, 0.3 g NH4Cl, 0.1 g MgSO4·7H2O, 10 ml l−1

110

vitamin solution,30 and 5 ml l−1 trace elements solution (SL-10, DSMZ GmbH, 2010).

111

The medium was amended with 10 mM HCO3- (NaHCO3), 1 mM As(III) (NaAsO2)

112

and 5 mM NO3– (KNO3). The isolated As(III)-oxidizer was cultured in chemically

113

defined medium (CDM, Supporting Information Table S1)39 with or without As(III) to

114

test its As tolerance. Lactate was included in CDM as an alternate electron donor. All

115

incubations were carried out in the dark at 30 °C. The following antibiotics were used

116

at the indicated concentrations: ampicillin (Ap), 100 µg ml-1; spectinomycin (Sm),

117

100 µg ml-1 and gentamicin (Gm), 30 µg ml-1.

118 119

Enrichment and Isolation Procedures. An As-contaminated soil was collected from

120

the surface layer (0-15 cm) of a paddy field in Shangyu, Zhejiang Province, China.

121

The soil contained 340 mg As kg-1 due to contamination from nearby mining activities.

122

Enrichment cultures were established under anaerobic conditions by adding the

123

As-contaminated soil to the MSM medium (5% wt/vol) containing 1 mM As(III), 10

124

mM HCO3- and 5 mM NO3–. Aliquots of 60 ml were anaerobically dispensed into 100

125

ml serum bottles with N2/CO2 (80:20, v/v) in the headspace. The bottles were sealed

126

with rubber stoppers and aluminum crimp seals, and incubated statically at 30 °C in

127

the dark. Once oxidation of the added As(III) had occurred and NO3– was reduced, 3

128

ml of the enrichment was transferred to 57 ml of fresh liquid medium and incubated at

129

30 °C. After several rounds of subculturing, the enrichment was serially diluted and

130

spread onto plates prepared with the defined medium containing 0.5‰ (wt/vol) yeast,

131

1 mM As(III), 10 mM HCO3- and 5 mM NO3-. After incubation anaerobically, a

132

number of single colonies were picked and tested for their ability to oxidize As(III)

133

under nitrate reducing conditions using a qualitative KMnO4 screening method.40 Pure 6


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cultures with an As(III) oxidation activity were preserved in a 50% glycerol-medium

135

solution at −80 °C.

136

An isolate with a strong As(III)

137

Strain Identification and Characterization.

138

oxidation ability, designated strain SY, was morphologically and physiologically

139

characterized, and identified according to the diagnostic tables of bacteria proposed

140

by Cowan and Steel.41 Partial 16S rRNA genes were amplified by PCR using primers

141

27F (5′-AGAGTTTGATCCTGGCTCAG-3′, Escherichia coli positions 8−27) and

142

1492R (5′-TACCTTGTTACGACTT-3′, Escherichia coli positions 1507−1492) and

143

sequenced. The obtained 16S rRNA gene sequences were subjected to BLAST search

144

(http://www.ncbi.nlm.nih.gov/BLAST/) to determine the sequence identity. Based on

145

the BLASTN search results for the 1400 base pair segment of the 16S rRNA gene

146

from SY, sequence alignments were performed with additional 16S rRNA sequences

147

from other As(III)-oxidizers and environmental isolates obtained from GenBank. A

148

neighbor-joining phylogenetic tree, based on the sequence of the 16S rRNA gene, was

149

generated using the MEGA 5.0 software.42 To confirm the autotrophic nature of strain

150

SY coupled to As(III) oxidation and the reduction of NO3–, the gene clusters for

151

denitrification and CO2 fixation were manually obtained from the draft genome of

152

strain SY. Sequences of related taxa were obtained from the GenBank database.

153

Strain SY was tested for the ability to

154

Metabolic Profile and Chemolithotrophy.

155

utilize various organic carbon sources as the carbon and electron donors and to respire

156

inorganic electron acceptors under anoxic conditions. Electron donors tested for

157

growth under aerobic or denitrifying (anoxic with 5 mM NO3– added as the electron

158

acceptor) conditions included As(III) (5 mM), formate (10 mM), acetate (10 mM),

159

propionate (10 mM), succinate (10 mM), glucose (10 mM), lactate (10 mM), citrate

160

(10 mM), SO32- (5 mM), S0 (0.125 mM), and S2O32- (5 mM). Where organic C was

161

not included, 10 mM HCO3– was supplied as the C source for chemoautotrophic

162

growth. Electron acceptors tested for autotrophic growth coupled to As(III) oxidation

163

included O2 (air in the headspace), nitrate, nitrite, sulfate, thiosulfate, fumarate, 7



164

tungstate, selenate, selenite, Fe(III)-nitrilotriacetic acid and As(V) (all at 5 mM except

165

O2). Flasks with active culture and cell-free background control were established in

166

triplicate. After incubation for 2 weeks, results were considered positive if the

167

supplied electron donors supported significant cell growth.

168

Strain SY was tested for its

169

Arsenite Tolerance and Oxidation by Strain SY.

170

tolerance to As(III) in CDM medium. Overnight cultures were diluted 1:100 in CDM

171

medium supplemented with increasing concentrations of As(III) and incubated at

172

30 °C for 36 h before the absorbance at OD600 nm was measured. The As(III)-oxidizing

173

ability of strain SY was tested under both aerobic and denitrifying conditions. Aerobic

174

autotrophic growth of strain SY was evaluated in MSM medium amended with 10

175

mM HCO3– and 1.0 mM As(III). Aerobic tubes were placed on an orbital shaker (180

176

rpm) to ensure aerobiosis. The active tubes, sterile controls (with autoclaved cells),

177

and cell-free background controls were set up in triplicate. The active tubes were

178

inoculated with 1 ml of active culture (OD < 0.1), which had been previously grown

179

on 10 mM HCO3- and 1 mM As(III), and incubated at 30 °C. Microbial growth was

180

measured spectrophotometerically at 600 nm. Arsenic species in the solution were

181

determined using high-performance liquid chromatography and inductively coupled

182

plasma mass spectrometry (HPLC-ICP-MS). The rate of anaerobic As(III) oxidation

183

coupled with nitrate reduction was determined in SY cultures amended with 5 mM

184

nitrate as the electron acceptor and 1.0 mM As(III) as the electron donor. The active

185

tubes were inoculated with 1 ml of active culture (OD < 0.1). Growth was tested using

186

sealed Hungate tubes with N2/CO2 (80:20, v/v) headspace for anaerobic conditions.

187

Active tubes and cell-free background controls were established in triplicate.

188

Microbial growth and As species were measured as described above.

189

To investigate the capability of As(III)

190

Arsenite Oxidization in Soil by Strain SY.

191

oxidation by strain SY in an As-contaminated soil, washed cells of strain SY were

192

inoculated into a soil slurry. The soil used in the experiment was collected from a

193

mining-impacted paddy field near Shantou, Guangdong province, containing 554 mg 8


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kg-1 of total arsenic. This soil was chosen for its high As mobility based on

195

preliminary tests. Soil slurry (a mixture of 50 g and 50 ml of distilled water) was

196

dispensed into 100 ml serum bottles, and the bottles were flushed with a N2/CO2

197

(80:20, v/v) gas stream and sealed with butyl rubber stoppers and aluminum caps.

198

Strain SY was pregrown in MSM medium with 5 mM nitrate and acetate under

199

anaerobic conditions to reach 0.3 OD600nm. The culture (5 ml) was centrifuged (10,000

200

g, 5 min), and the cells were washed 3 times with sterile 0.85% NaCl and resuspended

201

in 1 ml of 0.85% NaCl. The cell suspension (1 ml) was inoculated into the soil slurry.

202

A control treatment (no inoculation of SY cells) was also prepared. Each treatment

203

was replicated 4 times. After incubation for 14 days, the slurries were centrifuged

204

(5000 g, 10 min), and the supernatant (soil pore water) was filtered through a 0.22-µm

205

filter. To prevent precipitation of Fe hydroxide and changes in As species, 1 ml of 1.5

206

M HNO3 was added to 9 ml of the filtrate immediately after filtration.45 Adsorbed As

207

in the remaining soil paste was extracted with 0.6 M ortho-phosphoric acid and 0.1 M

208

ascorbic acid as described by Giral et al.46 Arsenic species in the supernatant and

209

phosphoric acid extracts were determined by HPLC-ICP-MS.

210

Strain SY contains a putative aioA gene

211

Disruption of aioA Gene in Strain SY.

212

that possibly encodes the As(III)-oxidase subunit A. To investigate its function, aioA

213

gene in strain SY was disrupted through a single-crossover event. A 550-bp DNA

214

fragment in the middle of the gene was generated by PCR using the genomic DNA of

215

strain

216

(5′-ATTGAATTCGAGGGACGGAAGTAACCTTCCTGGTG-3′)

217

underlined) and (5′-ACTGTCGACGAACTGAACTACACCTATG-3′) (XhoI site

218

underlined). The resulting product was then cloned between EcoRI and XhoI sites of

219

the suicide plasmid pEX18 to give pEXDA. pEXDA was delivered into strain SY

220

from E. coli SM10λpir via conjugal transfer, and the transconjugants were selected on

221

LB plates supplemented with antibiotics Sm and Gm. The aioA-disrupted mutant,

222

designated SY∆aioA, was confirmed by PCR. The ability of SY∆aioA to oxidize

223

As(III) under both aerobic and denitrifying conditions were tested using a cell

SY

as

the

template

and

9


the (EcoRI

primers site


224

suspension assay as described above. Strains SY and SY∆aioA were assayed for

225

As(III) tolerance in CDM medium. Overnight cultures were inoculated to an initial

226

turbidity of 0.05 OD600nm in a fresh CDM medium supplemented with increasing

227

concentrations of As(III) and incubated at 30 °C for 12 h before measurement of the

228

absorbance at 600 nm.

229 230

Construction of aioA Gene Clone Library and Phylogenetic Analysis. A clone

231

library of putative aioA genes from the As-contaminated paddy soil (Shangyu) was

232

constructed to assess the diversity of aioA. 40 g of the soil were mixed with 40 g of

233

water in 100 ml glass tubes, to which 5 mmol nitrate kg-1 soil was added. The tubes

234

were incubated anaerobically under As(III)-oxidizing chemoautotrophic conditions

235

for 60 days. DNA was then extracted from the soil slurry using a FastDNA Spin kit

236

(MP Biomedicals). Putative aioA genes were amplified according to the PCR

237

protocols described by Quéméneur et al.35 The amplified products (approximately

238

1100 bp) were confirmed on 1% agarose gels by electrophoresis and purified with a

239

QIAquick PCR Purification kit (Qiagen). The PCR products were ligated into the

240

pEASY-T3 vector (Transgene, Beijing, China), and transformed into Escherichia coli

241

Top10 cells according to the manufacturer’s instructions. Seventy clones were

242

randomly selected for sequencing. For aioA gene phylogeny, the obtained nucleotide

243

sequences were compared with As(III) oxidase sequences from the GenBank database

244

using BlastX, excluding those with a low similarity to the existing genes in NCBI.

245

Phylogenetic analysis was carried out as described above.42 Representative sequences

246

were selected from each operating taxonomic units (OTU) determined with a cutoff

247

value of 97% using the method described by Mothur.47

248

Samples (1 ml) were taken from sealed anaerobic tubes by

249

Analytical Methods.

250

piercing through the stoppers using sterile 1.0 ml syringes with 16-gauge needles. All

251

liquid samples were filtered through a 0.2 µm membrane filter immediately after

252

sampling. Arsenic speciation was determined by HPLC-ICP-MS (Perkin Elmer

253

NexIon 300X) as described previously.11 Arsenic species were separated using an 10


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254

anion exchange column (Hamilton PRP X-100, 250 mm diameter) and NH4H2PO4 (6

255

mM, pH 6.0) as the mobile phase. The signals of

256

were measured by ICP-MS set up in the He gas collision mode. The peaks were

257

quantified by comparison with external standards using integrated calibration curves.

258

Total As concentrations of soils were determined following aqua-regia digestion and

259

quantification of As by ICP-MS. A certified reference soil material (obtained from the

260

Institute of Geophysical and Geochemical Exploration, China) was included in the

261

analysis, which gave a recovery of 108% compared with the certified value. The

262

concentrations of nitrate and nitrite were determined using SEAL Analytical

263

segmented continuous-flow Auto Analyzer 3.

75

As and

115

In (internal standard)

264 265

Nucleotide Sequence Accession Numbers. The sequences determined in this study

266

have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases

267

under accession numbers KP881610 (nar gene cluster), KP881607 (nir-nor gene

268

cluster), KP881608 (nos gene cluster), KP881609 (rbc gene cluster), KP881606 (aio

269

gene cluster) and KC894855 (16S rRNA gene).

270 271 272

RESULTS

273 274

Isolation of an As(III)-oxidizing Bacterium. An autotrophic As(III)-oxidizing

275

bacterium was isolated from an As-contaminated paddy soil in Shangyu, Zhejiang

276

province, China, and designated strain SY. The colonies of strain SY grown on the

277

CDM plate were circular, convex, and pink. Cells of the strain were non-spore

278

forming, Gram negative, non-motile, and coccoids to short rods (Supporting

279

information Figure S1). The strain showed positivity for oxidase and catalase, nitrate

280

reduction, urease, but was negative for the indole reaction, hydrolysis of gelatin. The

281

morphological and biochemical characteristics of strain SY fitted with the description

282

of the genus Paracoccus. Growth (OD600nm) of strain SY was not inhibited at the

283

As(III) concentrations of 10, 50, 100 µM (Supporting Information Figure S2). At 1 11



284

mM As(III) growth was inhibited by 9%. The lag phase of growth increased from 6 h

285

in the control to 30 h at 10 mM As(III) (Supporting information Figure S2). These

286

data suggest that strain SY is highly tolerant to As(III).

287

Strain SY was able to derive energy from the oxidation of As(III) to As(V) under

288

both aerobic and denitrifying conditions. When SY was grown autotrophically, NO3–,

289

NO2– or O2 could be used as an electron acceptor coupled to As(III) oxidation. Other

290

electron acceptors tested could not support autotrophic growth and As(III) oxidation

291

(Supporting information Table S2). When strain SY was grown in a MSM medium

292

with CO2-HCO3- as the carbon source under aerobic conditions (aerobic autotrophic),

293

thiosulfate, sulfide and elemental sulfur as well as As(III) could support growth,

294

suggesting that strain SY has broad versatility in the use of electron donors to support

295

autotrophic growth. In contrast, under anaerobic denitrifying conditions, strain SY

296

could only grow autotrophically when As(III) was supplied as the electron donor

297

(Supporting information Table S2).

298

In addition to chemolithoautotrophic growth on As(III) and HCO3- under aerobic

299

and denitrifying conditions, the strain was tested for heterotrophic growth

300

qualitatively on organic compounds. Within 24 h of inoculation, strain SY also grew

301

in MSM medium containing either formate, acetate, propionate or lactate as the

302

carbon and energy source.

303

The partial 16S rRNA gene sequence

304

Phylogenetic Characterization of Strain SY.

305

of SY (1409 nt; GenBank accession no. KC894855) was determined. BLASTN search

306

analysis revealed that the 16S rRNA gene of the isolate SY showed 96.79 % similarity

307

to Paracoccus niistensis KCTC 22789 (FJ842690), a strict aerobic bacterial strain

308

isolated from a forest soil.48 The similarity to other Paracoccus species was
0.05, Student’s t test).

340

Control experiments confirmed that no As(III) was oxidized and no NO3– was reduced

341

in the medium when no inoculum or autoclaved cells were added (data not shown).

342

Autotrophic growth of SY was thus dependent on As(III) oxidation coupled to NO3–

343

reduction. This suggests that the chemoautotrophic bacterium SY fixes CO2 and 13



344

couples nitrate reduction with As(III) oxidation.

345 346

Strain SY Mediated As(III) Oxidation in Paddy Soil. A batch experiment was

347

conducted to determine the effect of strain SY on As speciation in a soil slurry using

348

an As-contaminated paddy soil. The soil slurry was incubated under flooded

349

conditions for 2 weeks with the additions of NO3- and the washed cells of strain SY.

350

In the control treatment without the addition of SY cells, As(III) was the dominant As

351

species, accounting for 76% and 94% of the total As in the pore water and the

352

phosphoric acid extractable fraction, respectively (Figure 3). In contrast, in the

353

treatment with the addition of SY cells, As(V) became the dominant As species,

354

accounting for 78% and 89% of the total As in the pore water and the phosphoric acid

355

extractable fraction, respectively (Figure 3). Total As concentration in the pore water

356

decreased by 18% in the SY inoculated soil compared with the control, whilst the

357

phosphoric acid extractable As decreased by 51%, suggesting that more As became

358

unextractable by phosphoric acid in the soil slurry inoculated with SY cells.

359 360

The Role of AioA in As(III) Oxidation and Detoxification. To investigate the role

361

of AioA in As(III) oxidation and detoxification, a deletion mutation in the aioA gene

362

was created. The SY∆aioA mutant lost the ability to oxidize As(III) at all As(III)

363

concentrations tested under both aerobic (Figure 4A) and denitrifying conditions

364

(Figure 4B). Moreover, mutant cells grew significantly poorer than the wild-type

365

strain at 1 – 10 mM As(III) under heterotrophic and aerobic conditions (Figure 4C).

366

Based on the 24 h growth data, the EC50 of As(III) was calculated as 2.9 and 0.8 mM

367

for WT and ∆aioA mutant, respectively, indicating that the mutant lost 72% of the

368

As(III) tolerance compared with WT.

369

To investigate the diversity of aioA genes in

370

Diversity of Arsenite Oxidase Genes.

371

the As-contaminated paddy soil (Shangyu), the aoxBM1-2F/3-2R primers designed

372

for aioA35 were used to amplify partial As(III) oxidase large subunit genes (aioA) for

373

the construction of an aioA gene clone library. When the soil was incubated under the 14


Page 14 of 28

Page 15 of 28


374

chemoautotrophic denitrifying conditions, OTUs related to the genome sequences of

375

the As(III)-oxidizers Rhodobacter capsulatus (60%) and Ensifer (29%) genera of

376

α-Proteobacteria dominated the library. Almost all of the 63 aioA sequences obtained

377

in this study were assigned to two major groups within Proteobacteria sequences

378

(Supporting information Figure S3). Thirty eight out of the 63 aioA-like sequences

379

showed 88.5−100% similarities with the putative AioA of strain SY, indicating the

380

abundance of the SY-like aioA gene in the soil, whereas the AioA-SY-34 had only 67%

381

identity to Burkholderia multivorans in the family of Burkholderiaceae (Supporting

382

information Figure S3). To date, the majority of known As(III)-oxidizing strains

383

belonging to α-Proteobacteria are autotrophs, suggesting a predominance of

384

autotrophic metabolism among them.33, 35 There were no γ-Proteobacteria sequences

385

detected under As(III)-oxidizing chemoautotrophic conditions.

386 387

DISCUSSION

388 389

Microorganisms capable of linking anoxic As(III) oxidation to denitrification have

390

been identified in anaerobic sediments and sludges.28, 50, 51 The use of nitrate as an

391

electron acceptor may be an important link in the biogeochemical cycling of the two

392

arsenic species, As(III) and As(V), under anaerobic conditions. Despite the

393

importance of As redox reactions for As bioavailability and uptake by rice crops, the

394

coupling between As(III) oxidation and denitrification in paddy soils has not been

395

investigated. A pot study52 showed that the addition of nitrate resulted in decreased As

396

accumulation by rice, suggesting a possible link between denitrification and As(III)

397

oxidation in paddy soil.

398

In the present study, we isolated and functionally characterized a novel As(III)

399

oxidizer from an As-contaminated paddy soil. Strain SY was able to utilize NO3- as an

400

electron acceptor for the oxidation of As(III) under anaerobic conditions. On the basis

401

of the 16S rRNA gene sequence, strain SY was closely related to Paracoccus

402

niistensis and other Parcoccus species. Members of the genus Paracoccus are aerobic

403

or denitrifying microorganisms, and many species are able to derive energy for 15



404

growth from the reduction of NO3-. To the best of our knowledge, strain SY is the first

405

Paracoccus species identified as being capable of growth using As(III) as the electron

406

donor. Autotrophic As(III)-oxidizers are able to obtain reducing power for CO2

407

fixation and energy from the oxidation of As(III).30 Based on the stoichiometry of

408

As(III) oxidation coupled to nitrate reduction by strain SY (Figure 2B, C), the reaction

409

can be expressed as follows: 5H3AsO3+2NO3–→5HAsO42–+N2+8H++H2O. This is

410

consistent with the stoichiometry of anaerobic As(III) oxidation reported for other

411

bacteria.30 Denitrification has been observed in the free-living form of many species

412

of Paracoccus and the denitrification gene clusters have been detected in other strains.

413

The presence of aioA, denitrification (nar, nir, nor, and nos) and CO2 fixation (rbc)

414

gene clusters in strain SY further underscores its ability for As(III) oxidation coupled

415

to denitrification to support autotrophic growth.

416

Strain SY, like other Paracoccus isolates, was able to utilize formate, acetate,

417

propionate, and lactate as electron donors and a source of carbon for heterotrophic

418

growth under both aerobic and denitrifying conditions. The ability of strain SY to

419

grow using organic substrates as its electron donor with oxygen or NO3- as its electron

420

acceptor means that it is both a facultative chemoautotroph and a facultative anaerobe.

421

These results suggest that strain SY has diverse metabolic abilities and support the

422

phylogenetic evidence that strain SY belongs to a novel species in the genus

423

Paracoccus.

424

Previous studies have demonstrated that AioA is the large subunit of As(III)

425

oxidases located in the periplasm involved in aerobic As(III) oxidation.32 However,

426

Rhine et al. amplified the aioA gene from the anaerobic As(III)-oxidizer

427

Sinorhizobium sp. strain DAO10,33 suggesting that AioA may also be responsible for

428

in anaerobic As(III) oxidization. Deletion of the aioA gene in strain SY abolished its

429

As(III) oxidation ability under both aerobic and denitrification conditions, indicating

430

that AioA is involved in both aerobic and anaerobic As(III) oxidation. Deletion of the

431

aioA gene in strain SY sensitized the bacterium toward As(III) under aerobic and

432

heterotrophic growth conditions, supporting the notion that As(III) oxidation is a

433

detoxification mechanism under these conditions. In contrast, oxidation of As(III) 16


Page 16 of 28

Page 17 of 28


434

provides energy for its chemoautotrophic growth. Furthermore, phylogenetic analysis

435

of the aioA clone library indicated that aioA sequences closely related to that of strain

436

SY were present abundantly in the As-contaminated paddy soil under the

437

As(III)-oxidizing chemoautotrophic condition.

438

When added to a flooded soil slurry, strain SY was capable of altering the

439

oxidation status of As by markedly increasing the proportion of As(V) in both the soil

440

pore water and in the phosphoric acid-extractable fraction, and decreasing the

441

concentrations of As in both fractions. This oxidation requires the presence of nitrate,

442

which would be lost via denitrification. However, the amount of nitrate required

443

would not be large considering the stoichiometry of As(III) oxidation coupled to

444

denitrification and the fact that nitrogen is a major element whilst As is a trace

445

element in soils. The SY aioA gene was found to be abundant in the paddy soil tested,

446

suggesting that Paracoccus sp. plays an important role in As redox cycling in

447

anaerobic paddy soils. Enhancing anaerobic As(III) oxidation by Paracoccus sp. may

448

provide an effective way to decrease As bioavailability in paddy soil and As

449

accumulation by rice. Furthermore, our study also suggests that the process of

450

anaerobic As(III) oxidation coupled to denitrification could be widespread in the

451

paddy environment, potentially playing a significant role in the biogeochemical

452

cycling of As.

453 454

ACKNOWLEDGEMENTS

455

The study was supported by the Natural Science Foundation of China (grant Nos.

456

41330853 and 31200087), China Postdoctoral Science Special Foundation

457

(2013T60546), the Innovative Research Team Development Plan of the Ministry of

458

Education of China (grant no. IRT1256), the Priority Academic Program

459

Development of Jiangsu Higher Education Institutions (PAPD) and the 111 project

460

(B12009). 17



461 462

Supporting Information Available:

463

Fig S1, transmission electron micrograph of a strain SY cell; Fig. S2, effects of

464

arsenite on the growth strain SY; Fig. S3. neighbor-joining phylogenetic tree of AioA

465

amino acid sequences from the enrichment of As-contaminated paddy soil under

466

As(III)-oxidizing chemoautotrophic conditions; Table S1, the composition of CDM

467

medium; Table S2, electron donors tested for growth by isolate SY with either 5 mM

468

NO3- or O2 as the terminal electron acceptor. This information is available free of

469

charge via the Internet at http://pubs.acs.org/.

470 471 472

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473

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47. Schloss, P. D.; Westcott, S. L.; Ryabin, T.; Hall, J. R.; Hartmann, M.; Hollister, E.

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microbe-mediated arsenic redox changes in the rhizosphere. Environ. Sci. Technol.

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Maier, R. M.; Field, J. A. Molecular characterization and in situ quantification of

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620

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621

2008, 27 (4), 881-887.

622

23



623

List of Figures:

624 625

Figure 1. Phylogenetic dendrogram of strain SY, a new Paracoccus species, and other

626

closely related As(III)-oxidizers, based on comparisons of 1400 base segments of 16S

627

rRNA genes. Bootstrap values (expressed as percentages of 1000 replicates) are

628

shown at the branch points, and the bar equals 5 % difference.

629 630

Figure 2. Strain SY mediated As(III) oxidation to As(V) under aerobic condition with

631

10 mM HCO3– as the sole C-source without NO3– (A) or denitrifying condition with

632

10 mM HCO3– as the sole C-source and 5 mM NO3– as the electron acceptor (B).

633

Nitrate removal under denitrifying condition (C). The data are means ± SD (n= 3).

634 635

Figure 3. Effect of strain SY inoculation on As speciation in the soil solution (A) and

636

the phosphoric acid-extractable fraction (B) in a flooded soil slurry. CK: control

637

without SY inoculation; SY: with SY inoculum. The data are means ± SD (n= 3).

638 639

Figure 4. As(III) oxidation by the wild-type strain SY and the SY∆aioA mutant under

640

aerobic (A) and denitrifying conditions (B), and the effect of aioA deletion (SY∆aioA)

641

on the growth of SY strain under different As(III) concentrations (C). The data are

642

means ± SD (n= 3).

24


Page 24 of 28

Page 25 of 28


Figure 1. Sinorhizobium sp. DAO10(DQ336178)

86 100 77

Agrobacterium albertimagni AOL15(AF316615)

100 arsenite-oxidising bacterium NT-26(AF159453)

99

arsenite-oxidising bacterium NT-25(AF159452) Ochrobactrum tritici(AJ242584) 100

α-Proteobacteria

Ancylobacter sp. OL1(DQ986318) Thiobacillus sp. S1(DQ986319)

100

Bosea sp. WAO(DQ986321)

98

Nitrobacter winogradskyi (AY055796) Paracoccus sp. SY (KC894855) Paracoccus niistensis KCTC 22789 (FJ842690)

100 100

Nitrococcus mobilis ATCC 25380 (L35510)

100

γ-Proteobacteria

Alkalilimnicola ehrlichii MLHE-1 (AF406554) 96

Acidithiobacillus ferrooxidans (AJ879997) Hydrogenophaga sp. CL3 (DQ986320)

100

96

arsenite-oxidizing bacterium NT-14 (AY027497)

91

Thiomonas sp. B3 (AJ549220)

100

β-Proteobacteria

Azoarcus sp. DAO1(DQ336177) 100

arsenite-oxidizing bacterium NT-10 (AY027500) Alcaligenes fecalis HLE (AY027506) Nitrosomonas eutropha Nm57(AY123795)

61

Ralstonia sp. 22 (EU304284) Sulfolobus acidocaldarius (U05018)

Archrea

Hydrogenobaculum acidophilum (AY268103) Aquificaceae

0.05

25



Figure 2. A

C

B

26


Page 26 of 28

Page 27 of 28


Figure 3. A

B

27



Figure 4. A

B

C

28


Page 28 of 28

Anaerobic Arsenite Oxidation by an Autotrophic Arsenite-Oxidizing

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