Bromate Reduction by Rhodococcus

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Bromate Reduction by Rhodococcus sp. Br-6 in the Presence of Multiple Redox Mediators Naoko Tamai, Takahiro Ishii, Yusuke Sato, Hiroko Fujiya, Yasuyuki Muramatsu, Nobuaki Okabe, and Seigo Amachi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02261 • Publication Date (Web): 09 Sep 2016 Downloaded from http://pubs.acs.org on September 11, 2016

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Environmental Science & Technology

1

Revised (es-2016-02261r)

2

Bromate Reduction by Rhodococcus sp. Br-6 in the Presence of Multiple

3

Redox Mediators

4 †

†

†

5

Naoko Tamai,

6

Muramatsu,‡, § Nobuaki Okabe,‡ and Seigo Amachi*,

Takahiro Ishii,

Yusuke Sato,

†

Hiroko Fujiya,

Yasuyuki

†

7 8 9

†

Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo-city, Chiba

271-8510, Japan.

10

‡

11

171-8588, Japan

12

§

Department of Chemistry, Gakushuin University, Mejiro 1-5-1, Toshima-ku, Tokyo,

Deceased 2 July 2016.

13 14 15

AUTHOR INFORMATION

16

Corresponding Author

17

*Phone: +81 47 308 8867; fax: +81 47 308 8867; e-mail: [email protected].

18

1 ACS Paragon Plus Environment


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ABSTRACT: A bromate (BrO3–)-reducing bacterium, designated Rhodococcus sp.

21

strain Br-6, was isolated from soil. The strain reduced 250 µM bromate completely

22

within 4 days under growth conditions transitioning from aerobic to anaerobic

23

conditions, while no reduction was observed under aerobic and anaerobic growth

24

conditions. Bromate was reduced to bromide (Br–) stoichiometrically, and acetate

25

was required as an electron donor. Interestingly, bromate reduction by strain Br-6

26

was

27

2,6-dichloroindophenol (DCIP). Cell free extract of strain Br-6 showed a

28

dicumarol-sensitive diaphorase activity, which catalyzes the reduction of DCIP in the

29

presence of NADH. Following abiotic experiments showed that the reduced form of

30

DCIP was re-oxidized by ferric iron, and that the resulting ferrous iron reduced

31

bromate abiotically. Furthermore, activity staining of the cell free extract revealed

32

that one of diaphorase isoforms possessed a bromate-reducing activity. Our results

33

demonstrate that strain Br-6 utilizes multiple redox mediators, i.e., DCIP and ferric

34

iron, for bromate reduction. Since the apparent rate of bromate reduction by this

35

strain (60 µM day-1) was 3 orders of magnitude higher than that of known

36

bromate-reducing bacteria, it could be applicable to removal of this probable human

significantly

dependent

on

both

ferric

iron


and

a

redox

dye

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carcinogen from drinking water.

38 39

INTRODUCTION

40

Bromate (BrO3–) is a disinfection by-product of ozonation and chlorination of drinking

41

water and is formed from bromide ion (Br–).1 It is a genotoxic carcinogen and causes

42

renal cell tumors in rats.2 Thus, bromate is classified as a probable human carcinogen by

43

the US Environmental Protection Agency, with a maximum contaminant level (MCL) in

44

drinking water of 10 µg L-1 (78 nM).3 The World Health Organization also sets a

45

provisional guideline value of 25 µg L-1 (195 nM) for drinking water.4 Bromate formation

46

becomes a serious problem when bromide levels are above 100 µg L-1 because of salt

47

water intrusion, potassium mining, coal mining, and chemical production.1 Although

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bromate formation can be minimized, at most, 50% by water treatment options such as

49

ammonia addition5 and pH depression,6 complete elimination is difficult once bromate

50

forms.

51

Microbial reduction of bromate to innocuous bromide is a promising way to remove

52

bromate from drinking water. Hijnen et al.7 first found that mixed and pure cultures of

53

denitrifying bacteria supplemented with ethanol were capable of bromate reduction. van

54

Ginkel et al.8 enriched an acetate-oxidizing mixed microbial culture that utilized bromate

55

as a terminal electron acceptor for anaerobic growth. Bromate reduction in biological

56

activated carbon (BAC) filters,9 granular activated carbon,10 and a chlorate

57

(ClO3–)-reducing mixed microbial culture11 were also reported. Recently, two types of

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mixed microbial cultures, i.e., an elemental sulfur (S0)-oxidizing culture12 and a



59

sulfate-reducing culture,13 were found to reduce bromate. These studies demonstrate that

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bromate reduction can be catalyzed by a wide variety of bacteria, including denitrifiers,

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chlorate-reducers, sulfur-oxidizers, and sulfate-reducers. However, bacterial bromate

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reduction is still poorly understood, primarily because of the limited number of available

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isolates as well as the limited information on key enzymes that are involved in the

64

reaction.

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In this study, a bromate-reducing bacterium, Rhodococcus sp. strain Br-6, was newly

66

isolated from soil. The strain reduced bromate only under growth conditions transitioning

67

from aerobic to anaerobic conditions (transition conditions), and physiological analyses

68

revealed that the strain utilizes multiple redox mediators for bromate reduction. An

69

enzyme diaphorase that is involved in bromate reduction and possible application of

70

strain Br-6 for bromate removal from drinking water are also discussed.

71 72

MATERIALS AND METHODS

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Enrichment and isolation of bromate-reducing bacteria. Soil was collected from a

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residential area in Matsudo-city, Chiba, Japan. The enrichment culture was prepared by

75

inoculating 1 mL soil slurry (a mixture of 1 g wet weight of the soil and 50 mL distilled

76

water) into 19 mL minimal medium. The medium contained the following (per liter):

77

NH4Cl (0.535 g), KH2PO4 (0.136 g), MgCl2·6H2O (0.204 g), CaCl2·2H2O (0.147 g), trace

78

mineral element solution (1 mL) (Table S1), vitamin solution (1 mL) (Table S2), 1 g L–1

79

resazurin solution (1 mL), and NaHCO3 (2.52 g). In anaerobic incubation, minimal

80

medium (20 mL) was dispensed into a 60-mL serum bottle under an N2-CO2 (80:20) gas


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stream. The bottle was sealed with a thick butyl rubber stopper and aluminum cap. After

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autoclaving at 121°C for 20 min, sodium acetate and sodium bromate were added

83

separately from sterile anaerobic stock solutions to give final concentrations of 20 mM

84

and 250 µM, respectively. The final pH of the medium was 6.8 to 7.0. The bottle was

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incubated at 30°C. In some cases, 10 mM nitrate or 5 mM chlorate was also added.

86

Incubation under transition conditions was carried out similar to the anaerobic

87

conditions in the sealed serum bottle, but air substitution of the head space and the liquid

88

phase with N2-CO2 gas was omitted. In aerobic incubation, the medium (20 mL) was

89

dispensed into a 100-mL Erlenmeyer flask, and the flask was incubated on a rotary shaker

90

at 200 rpm. In both transition and aerobic incubations, 0.174 g L-1 of K2HPO4 was also

91

added to the minimal medium, but NaHCO3 was omitted. The pH of the medium for

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transition and aerobic incubation was adjusted to 7.0 with NaOH.

93

To isolate bromate-reducing bacteria, the slurry incubated with bromate for 5 days

94

under transition conditions was serially diluted and spread on minimal agar medium

95

containing 15 g L-1 of agar. After aerobic incubation, 8 bacterial colonies were removed

96

randomly, purified, and evaluated for their bromate-reducing capacities in the liquid

97

medium under transition growth conditions.

98 99

Sequencing and phylogenetic analysis of the 16S rRNA gene. Genomic DNA of

100

strain Br-6 was isolated as described previously.14 The 16S rRNA gene was amplified by

101

PCR using the bacterial consensus primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′)

102

and 1491R (5′-GGTTACCTTGTTACGACTT-3′). PCR products were purified using a



103

QIAquick PCR Purification kit (Qiagen, Hilden, Germany) and sequenced using a

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BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA)

105

and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems) using appropriate

106

sequencing primers.15 The obtained 16S rRNA gene sequences were subjected to a BLAST

107

search (http://www.ncbi.nlm.nih.gov/BLAST/) to determine sequence similarity. The

108

retrieved sequences were aligned using Clustal X version 2.0. The phylogenetic tree was

109

constructed using the neighbor-joining method.16

110 111

Bromate reduction in growing cultures. Strain Br-6 was grown in minimal medium

112

with 250 µM bromate under anaerobic, transition, and aerobic conditions. Acetate was

113

added as the electron donor and carbon source at final concentrations of 5 to 40 mM.

114

Potential redox mediators that were tested included resazurin sodium salt, resorufin

115

sodium salt, 2,6-dichloroindophenol sodium n-hydrate (DCIP), methylene blue trihydrate,

116

phenazine methosulfate (PMS), anthraquinone-1,5-disulfonic acid disodium salt

117

n-hydrate (AQDS), methyl viologen trihydrate (MV), and riboflavin. The apparent rate of

118

bromate reduction (µM h-1) was calculated by simply dividing the tested bromate

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concentration by the amount of time required for complete reduction of bromate.

120 121

Enzyme assays. Cells grown under transition conditions were disrupted by bead

122

beating (BioSpec Products, Bartlesville, OK, USA) with 0.1 mm zirconia-silica beads

123

(Waken B Tech, Kyoto, Japan) at 2,500 rpm for 5 min. After centrifugation at 18,000 × g

124

at 4°C, the supernatant was used as a crude enzyme solution. Diaphorase activities were


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assayed spectrophotometrically at 30°C by monitoring the oxidation of NADH (ε340 =

126

6.22 mM-1·cm-1) as an electron donor, or the reduction of substrates as the electron

127

acceptors.17–19 The molar extinction coefficients (mM-1·cm-1) for resazurin, resorufin, and

128

DCIP are 40.1 at 600 nm, 8.25 at 569 nm, and 16.1 at 600 nm, respectively. The reaction

129

mixture contained 20 mM Tris-HCl (pH 6.8), 200 µM NADH, 100 µM substrate, and an

130

appropriate amount of enzyme. In some cases, dicumarol, a specific inhibitor of

131

diaphorase,20,21 was also added. One unit (U) of diaphorase activity was defined as the

132

amount of enzyme protein required to oxidize 1 µmol of NADH or reduce 1 µmol of

133

substrate per min. Protein concentration was determined using the method described by

134

Bradford.22

135 136 137

Analytical techniques. In the presence of excess iodide under acidic conditions, bromate is reduced to bromide with the formation of triiodide (I3–).23

138

BrO3– + 9I– + 6H+ → 3I3– + Br– + 3H2O

139

Using this equation, bromate concentration was routinely and spectrophotometrically

140

determined. First, 1 g L-1 potassium iodide (700 µL) and 2N HCl (100 µL) were added to

141

700 µL of culture supernatant. The mixture was incubated for 15 min at room temperature

142

to yield triiodide, which was measured at 352 nm. In a preliminary experiment, it was

143

confirmed that the culture supernatant without bromate did not produce triiodide. The

144

detection limit of this method was approximately 5 µM. Bromide concentration was

145

determined by high-performance liquid chromatography (HPLC) with a size-exclusion

146

column (AsahiPak GS-220 7C) in combination with inductively coupled plasma mass



147

spectrometry (ICP/MS, Agilent7700). The detection limit of this method was

148

approximately 10 nM of bromide. Acetate was determined by HPLC (L-7000, Hitachi,

149

Tokyo, Japan) with an Aminex HPX-87H ion exclusion column (Bio-Rad Laboratories,

150

Hercules, CA, USA) using UV detection at 212 nm.

151 152

Bromate reduction by the reduced form of DCIP. To determine if bromate can be

153

reduced by the reduced form of DCIP (DCIPH2), DCIP (10 µM) was mixed with NADH

154

(150 µM) and the cell free extract in 20 mM Tris-HCl (pH 6.8). After the blue color of

155

DCIP disappeared, bromate (50 µM) was added. The mixture was prepared in a 10-mL

156

serum bottle under an N2 gas stream, and was incubated anaerobically at 30°C. In some

157

cases, 25 µM of ferric chloride hexahydrate (FeCl3·6H2O) and 335 µM of nitrilotriacetic

158

acid (NTA) were also added.

159 160

Abiotic experiments using ferric and ferrous iron. To determine if ferric iron can

161

re-oxidize DCIPH2, the following abiotic experiment was performed in 20 mM Tris-HCl

162

(pH 6.8). First, 50 µM DCIP was reduced to DCIPH2 with 50 µM ascorbic acid, and then

163

DCIPH2 was mixed with 40 to 120 µM of FeCl3·6H2O. NTA was also added at the same

164

ratio of that in the minimal medium (0.5 to 1.5 mM). Re-oxidation of DCIPH2 by ferric

165

iron was monitored spectrophotometrically at 600 nm. A further abiotic experiment was

166

also performed to determine if bromate can be reduced by ferrous iron. In this case, 50

167

µM of bromate was simply mixed with 100 to 300 µM of ferrous ethylenediamine sulfate

168

tetrahydrate in Tris-HCl (pH 6.8), and bromate reduction was monitored periodically.


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Bromate reduction by washed cell suspension. The cells grown for 4 days under

171

transition conditions were collected, washed twice with 20 mM Tris-HCl (pH 6.8), and

172

resuspended to the same buffer. In some cases, 335 µM NTA was added to the washing

173

buffer. The washed cell suspension was dispensed into a 60-mL serum bottle under an N2

174

gas stream. After the bottle was sealed, acetate, bromate, DCIP, FeCl3·6H2O, and NTA

175

were added to give final concentrations of 10 mM, 50 µM, 10 µM, 25 µM, and 335 µM,

176

respectively. The bottle was incubated at 30°C. In some cases, 50 to 200 µM of dicumarol

177

was also added.

178 179

Electrophoresis. The diaphorase activity and bromate-reducing activity in the cell free

180

extract were visualized and identified by sodium dodecyl sulfate-polyacrylamide gel

181

electrophoresis (SDS-PAGE). Electrophoresis was performed using 7% polyacrylamide

182

gel in 25 mM Tris-glycine buffer (pH 8.3) containing 0.1% SDS by the method described

183

by Laemmli.24 For the following activity staining, 2-mercaptoethanol was omitted from

184

the electrophoresis buffer. Proteins were visualized by staining with Coomassie brilliant

185

blue R-250 (CBB). Activity staining of diaphorase was performed by incubating the gel

186

with 20 mM Tris-HCl (pH7.0) containing NADH (0.4 mM) and DCIP (200 µM). Activity

187

of bromate reductase was visualized by incubating the gel anaerobically with 20 mM

188

Tris-HCl (pH7.0) containing NADH (0.3 mM), DCIP (20 µM), FeCl3·6H2O (50 µM),

189

NTA (300 µM), and bromate (100 µM). Anaerobic incubation of the gel was performed

190

with the Anaero Pack System (Mitsubishi Gas Chemical, Tokyo, Japan). After washing



191

the gel with distilled water twice, the gel was immersed in 0.1 M AgNO3 solution to form

192

a pale-yellow precipitate AgBr.

193

AgNO3 (aq) + Br- (aq) AgBr (s) + NO3- (aq)

194

Since AgBr is highly photosensitive, the gel was finally exposed to light to form black Ag.

195

For activity staining of ferric iron reductase, the gel was incubated anaerobically with 20

196

mM Tris-HCl (pH7.0) containing NADH (0.3 mM), FeCl3·6H2O (250 µM), NTA (250

197

µM), and ferrozine (1.25 mM). When ferrozine reacts with ferrous iron, it forms a

198

purplish red chelate compound.25

199 200

Nucleotide sequence accession number. The 16S rRNA gene sequence of strain Br-6

201

has been deposited in the DDBJ/EMBL/GenBank databases under an accession number

202

AB974352.

203 204

RESULTS

205

Enrichment of bromate-reducing bacteria. We first attempted to enrich dissimilatory

206

bromate-reducing bacteria, and incubated soil slurry with either 1 or 5 mM bromate under

207

anaerobic conditions. However, no significant reduction was observed, even after

208

incubation for 2 months (data not shown). This was probably due to a toxic effect of high

209

concentrations of bromate or possible intermediates of bromate reduction, such as bromite

210

(BrO2–) and hypobromite (BrO–).1 Thus, the slurry was then incubated with 250 µM

211

bromate under three types of incubation conditions, i.e., aerobic, transition, and anaerobic

212

conditions (Fig. S1).


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No reduction was observed under aerobic conditions, whereas only limited reduction

214

was observed under anaerobic conditions after cultivation for 45 days. When 10 mM

215

nitrate was added with bromate, complete reduction of bromate occurred under anaerobic

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conditions within 30 days, consisting with knowledge that certain denitrifiers are capable

217

of bromate reduction.7,26–28 In contrast, 5 mM chlorate did not stimulate bromate

218

reduction. Interestingly, 250 µM of bromate was reduced completely within 7 days under

219

transition conditions (Fig. S1), in which the slurry was incubated in sealed serum bottles

220

without air substitution. Under this condition, a shift from aerobic to anaerobic conditions

221

occurred gradually because of microbial O2 consumption, and the blue color of resazurin

222

in the medium changed to pink (the color of resorufin) and finally to clear (the color of

223

dihydroresorufin).

224 225

Isolation of bromate-reducing bacterium strain Br-6. Substantially faster reduction

226

of bromate under transition conditions implied that bromate-reducing bacteria are capable

227

of aerobic growth but reduce bromate only under anaerobic conditions. Thus, 8 aerobic

228

bacteria were randomly isolated from the slurry that was incubated under transition

229

conditions, and their bromate-reducing capacities were evaluated under transition growth

230

conditions. All of these isolates reduced 250 µM bromate completely within 7 days.

231

Among these, strain Br-6 was chosen for further analysis, since it demonstrated the fastest

232

rate of reduction. Analysis of an approximately 1,480-bp 16S rRNA gene sequence

233

revealed that the strain is most closely related to Rhodococcus equi, with a sequence

234

similarity of more than 99% (Figure S2).



235 236

Bromate reduction by growing cultures of strain Br-6. Strain Br-6 was grown under

237

aerobic, transition, and anaerobic conditions with 250 µM bromate and 20 mM acetate

238

(Figure 1). In the aerobic culture, the strain grew but did not reduce bromate significantly.

239

In the anaerobic culture, neither growth nor bromate reduction was observed. In the

240

transition culture, however, the strain reduced bromate completely within 4 days (Figure

241

1B). Acetate consumption was accompanied by bromate reduction and cell growth

242

(Figure 1C). Determination by HPLC-ICP/MS revealed that bromide was produced from

243

bromate stoichiometrically (Figure 1B). Bromate reduction was inhibited completely

244

when the transition culture was autoclaved at 121°C for 20 min (Figure 1B).

245

To understand whether acetate is required for bromate reduction, 5 to 40 mM of acetate

246

was added to the transition cultures (Figure 2). When 5 mM of acetate was added, acetate

247

was depleted within 2 days and bromate reduction ceased thereafter. However, if 5 mM

248

acetate was refed to the medium, bromate reduction proceeded again. Similarly, 250 µM

249

bromate was not reduced completely at an acetate concentration of 10 mM. Complete

250

reduction of bromate occurred at acetate concentrations of more than 20 mM. These

251

results indicate that acetate is required for bromate reduction as the electron donor under

252

our experimental condition. Since strain Br-6 was able to reduce bromate when it was

253

grown in a complex medium consisting of polypepton, yeast extract, and MgSO4 (data

254

not shown), its electron donor may not be restricted to acetate.

255 256

Bromate reduction is enhanced by redox mediators. Bromate reduction by strain


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Br-6 under transition conditions was enhanced by 4 µM resazurin (Figure 1B), which was

258

added to the medium as a redox indicator. The apparent rate of bromate reduction without

259

resazurin was 15.8 µM day-1, whereas that with resazurin was 63.0 µM day-1. The effect

260

of resazurin on bromate reduction could be substituted with 4 µM each of resorufin (a

261

reduced form of resazurin), DCIP, methylene blue, and PMS (Figure 3), but not for AQDS,

262

MV, and riboflavin (Figure S3). These results suggest that these compounds, all of which

263

are common artificial electron acceptors, function as redox mediators in bromate

264

reduction. Optimum concentrations of resazurin, resorufin, and DCIP were 20 µM, 20 to

265

40 µM, and 4 to 12 µM, respectively (Figure S4).

266 267

Diaphorase activity in cell free extract of strain Br-6. If redox mediators play a role

268

in bromate reduction, they should be oxidized by bromate and then reduced again

269

enzymatically. Diaphorase is known to catalyze NADH-dependent reduction of various

270

redox dyes such as DCIP, resazurin, and resorufin,17-19 and cell free extract of strain Br-6

271

actually showed diaphorase activity. In the presence of DCIP as the substrate,

272

DCIP-dependent NADH-oxidizing activity and NADH-dependent DCIP-reducing

273

activity were 147 and 132 mU mg protein-1, respectively. The activity was inhibited by

274

dicumarol, a specific inhibitor of diaphorase (Figure S5).20,21 Resazurin-dependent

275

NADH-oxidizing

276

NADH-oxidizing activity (15.2 mU mg protein-1) were also observed in the cell free

277

extract.

activity

(53.4

mU

mg

protein-1)

278


and

resorufin-dependent


279

Ferric iron is required for DCIP-dependent bromate reduction. In the following

280

experiments, we used only DCIP as the redox mediator, since its ability to enhance

281

bromate reduction was comparable to those of resazurin and resorufin (Figure 3). To

282

determine if DCIPH2 can directly reduce bromate, DCIP was first incubated with NADH

283

and the cell free extract in Tris-HCl buffer until the blue color of DCIP changed to clear.

284

Bromate was then added to the reaction mixture. As shown in Figure 4, bromate reduction

285

did not occur under this reaction condition. However, bromate reduction did occur when

286

Tris-HCl buffer was replaced with minimal medium (that did not contain resazurin),

287

indicating that certain ingredients in the minimal medium are necessary for bromate

288

reduction. Thus, one or several ingredients in the minimal medium were added to

289

Tris-HCl buffer until they compensated bromate reduction. As a consequence,

290

FeCl3·6H2O and NTA (a chelating agent), both of which are ingredients of trace mineral

291

element solution, were found to be required for DCIP-dependent bromate reduction

292

(Figure 4). No bromate reduction occurred in the absence of NTA or under aerobic

293

incubation conditions. These results demonstrate that not only DCIP but also ferric iron is

294

involved in bromate reduction.

295 296

Abiotic reactions with ferric and ferrous iron. From the experimental results

297

described above, we hypothesized that strain Br-6 utilizes multiple redox mediators for

298

bromate reduction, i.e., DCIP and ferric/ferrous iron. To test this hypothesis, two abiotic

299

experiments were carried out. First, DCIP was reduced to DCIPH2 by ascorbic acid and

300

mixed with FeCl3·6H2O and NTA to determine if ferric iron can re-oxidize DCIPH2. As


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shown in Figure S6A, ferric iron re-oxidized DCIPH2 in a dose-dependent manner. Both

302

ferric iron and NTA were indispensable for DCIPH2 re-oxidation, and bromate did not

303

directly re-oxidize DCIPH2. In the second experiment, ferrous iron was mixed with

304

bromate to determine if bromate can be reduced by ferrous iron. As shown in Figure S6B,

305

bromate was reduced by ferrous iron in a dose-dependent manner. After the reaction

306

proceeded for 24 h, bromate consumption in the presence of 100, 200, and 300 µM

307

ferrous iron was 20.7, 34.6, and 46.7 µM, respectively. This indicates that ferrous iron

308

reduces bromate with a Fe(II)/BrO3– molar ratio of 6.

309 310

Bromate reduction by washed cells of strain Br-6. Bromate reduction by washed

311

cells of strain Br-6 was determined under anaerobic incubation conditions. As shown in

312

Figure 5, the cells of strain Br-6 reduced bromate at a rate of 42.4 nmol h-1 mg dry cells-1

313

in the presence of DCIP, ferric iron, and NTA. When either DCIP or NTA was omitted,

314

the rates of bromate reduction decreased to 2.5 to 3.5 nmol h-1 mg dry cells-1. In the

315

absence of DCIP, ferric iron, and NTA, no significant reduction of bromate was observed

316

(less than 1.0 nmol h-1 mg dry cells-1). Bromate reduction rates in the presence of 50, 100,

317

200 µM dicumarol were 28.3, 22,5, and 17.9 nmol h-1 mg dry cells-1, respectively (data

318

not shown). A relatively high reduction rate of 34.1 nmol h-1 mg dry cells-1 was still

319

observed in the absence of ferric iron (Figure 5). This was probably due to the trace

320

amount of iron species remaining in the washed cells. To prove this, the cells were then

321

washed with 20 mM Tris-HCl containing 335 µM NTA to eliminate iron species from the

322

cells, and their bromate reduction was observed in the presence or absence of ferric iron.



323

As shown in Figure S7, the cells washed with NTA did not reduce bromate, while the

324

washed cells supplemented with ferric iron could reduce bromate at a reduction rate of

325

32.5 nmol h-1 mg dry cells-1. Under aerobic incubation condition, bromate reduction rate

326

of the washed cells decreased gradually, and 12 µM of bromate remained even after the

327

incubation for 24 h (Figure 5). At this point, however, we have no enough information to

328

explain this phenomenon, and more research is needed. Washed cells of strain Br-6

329

grown under aerobic condition reduced bromate with a same rate of the cells grown under

330

transition condition (data not shown), indicating that the enzyme involved in bromate

331

reduction are synthesized regardless of growth conditions.

332 333

Activity staining of diaphorase and bromate reductase. To obtain direct evidence

334

that diaphorase is involved in bromate reduction, SDS-PAGE of the cell free extract was

335

performed under non-denatured conditions, and the gels were stained either for

336

diaphorase or for bromate reductase activity (Figure 6). The staining of diaphorase was

337

performed based on DCIP-decolorizing activity in the presence of NADH. As shown in

338

Figure 6B, at least 6 bands were observed, of which 3 bands showed relatively high

339

intensity, suggesting that strain Br-6 has multiple isoforms of diaphorase. The staining of

340

bromate reductase was performed based on the reaction between bromide and AgNO3 to

341

form AgBr, and also on light sensitivity of AgBr to form a black Ag precipitate. As shown

342

in Figure 6D, only one band appeared, and its relative migration distance was same as one

343

of the diaphorase isoforms with high intensity. Finally, the gel was stained for ferric iron

344

reductase based on ferrozine method, since certain bacterial diaphorases possess


Page 16 of 37

Page 17 of 37


345

NADH-dependent ferric iron reductase activity.29 As shown in Figure 6C, the active

346

staining for ferric iron reductase produced two bands. Interestingly, both bands appeared

347

to correspond to diaphorase activity, and one of the two bands showed same relative

348

migration distance with bromate reductase (Figures 6C and 6D).

349 350

DISCUSSION

351

Certain denitrifiers7,26–28 and chlorate-reducers11 can reduce bromate in a co-metabolic

352

manner, since bromate can be recognized as a substrate by nitrate reductase30,31 and

353

chlorate reductase.32,33 On the other hand, the existence of specific bromate-reducing

354

bacteria has been proposed in several studies.8,34,35 For example, van Ginkel et al.8

355

enriched a bromate-reducing mixed microbial culture that utilized bromate as the sole

356

electron acceptor for growth. However, there are still many uncertainties regarding

357

bacterial bromate reduction, mainly because of the limited number of available isolates.

358

In this study, we isolated a bromate-reducing bacterium, Rhodococcus sp. strain Br-6, and

359

found that it utilized multiple redox mediators, i.e., DCIP and ferric/ferrous iron, to

360

accelerate bromate reduction.

361

As shown in Figure 1, growth and acetate consumption by strain Br-6 was much better

362

under transition than aerobic conditions. Although strain Br-6 was closely related to a

363

member of the strictly aerobic actinomycetes, R. equi (Figure S2), this strain may prefer

364

transition to aerobic environments at least under our experimental conditions. Although

365

acetate was required for bromate reduction as the electron donor (Figure 2), the ratio of

366

acetate consumed to bromate reduced (approximately 80 from Figure 2) was far greater



367

than that expected from the following equation, which expresses acetate oxidation

368

coupled to bromate reduction.

369

3C2H3O2− + 4BrO3− + 3H+ = 4Br− + 6CO2 + 6H2O

370

Thus, it is apparent that most of the acetate was oxidized irrespective of bromate

371

reduction in strain Br-6, and that its bromate reduction is not a respiratory process. Given

372

40 mL headspace (with 21% oxygen) and 20 mL liquid medium (with 7.56 mg L-1

373

oxygen), we can roughly estimate that about half of acetate was consumed with oxygen as

374

the electron acceptor, and that the remaining half of acetate was converted to biomass of

375

strain Br-6. Nevertheless, transition condition was required for bromate reduction by

376

strain Br-6. This was probably due to instability of the second mediator, ferrous iron (see

377

below), under oxic condition.

378

It was found that DCIP is an efficient redox mediator for bromate reduction by strain

379

Br-6 (Figures 3 and S3). In the presence of DCIP, the apparent rate of bromate reduction

380

by the transition culture (59.3 µM day-1) was 4 times higher than that in the absence of

381

DCIP (13.7 µM day-1) (Figure 3). In addition, bromate reduction by washed cells of strain

382

Br-6 was enhanced 17-fold in the presence of DCIP (Figure 5). However, the reduced

383

form of DCIP (DCIPH2) could not directly reduce bromate, and both ferric iron and NTA

384

were required for the DCIP-dependent bromate reduction (Figure 4). It is known that

385

bromate can be reduced by a redox reaction with ferrous iron.36

386

BrO3– + 6Fe2+ + 6H+ Br– + 6Fe3+ + 3H2O

387

Our abiotic experiments revealed that ferrous iron actually reduces bromate with a

388

Fe(II)/BrO3– molar ratio of 6 (Figure S6B), and that ferric iron can re-oxidize DCIPH2


Page 18 of 37

Page 19 of 37


389

(Figure S6A). These results strongly suggest that not only DCIP but also ferric/ferrous

390

iron is indispensable for efficient bromate reduction as a second redox mediator. NTA is

391

considered to be required for stabilization and solubilization of iron species as a chelating

392

agent. Xie et al.36 reported a similar bromate-reducing mechanism, in which ferric/ferrous

393

iron acts as a mediator for bromate reduction by humic acid. Such an electron-transferring

394

process may also play a significant role in bromate-reduction by zerovalent iron.37

395

Cell free extract of strain Br-6 showed the dicumarol-sensitive diaphorase activity.

396

Furthermore, activity staining of the cell free extract revealed that bromate reductase

397

activity actually correlated with one of diaphorase isoenzymes (Figure 6). These results

398

strongly suggest that diaphorase is involved in bromate reduction by strain Br-6. In Figure

399

7, we illustrate a proposed mechanism of bromate reduction by strain Br-6. First,

400

electrons derived from acetate are transferred from NADH to DCIP by diaphorase.

401

DCIPH2 then reduces ferric iron, and ferrous iron finally reduces bromate to bromide.

402

This model is energetically reasonable, since standard redox potentials (E0’) of

403

NAD+/NADH, DCIP/DCIPH2, Fe(III)-NTA/Fe(II)-NTA, and BrO3–/Br– couples are

404

–0.320, +0.217, +0.385, and +0.997 V, respectively. Bromate reduction was possible

405

when PMS (+0.080 V), methylene blue (+0.011 V), and resorufin (–0.051 V) were used

406

as the first redox mediators, but not with AQDS (–0.185 V), riboflavin (–0.208 V), and

407

methyl viologen (–0.446 V), suggesting that E0’ of the first redox mediators should be

408

between 0 to +0.2 V. In the absence of first redox mediator, diaphorase might reduce

409

ferric iron directly, since at least two diaphorase isoforms showed ferric iron reductase

410

activity (Figure 6C).



411

Strain Br-6 reduced bromate at an apparent rate of 60 µM day-1 in the transition culture

412

(Figure 3) or 42 nmol h-1 mg dry cells-1 in a washed cell suspension (Figure 5). Hijnen et

413

al.7 reported that the rate of bromate reduction by a denitrifying bacterium, Pseudomonas

414

fluorescens Br5, was 0.013 to 0.027 µM day-1. Davidson et al.35 isolated 15

415

bromate-reducing bacteria, including two Rhodococcus strains, but their bromate

416

reduction rates were less than 0.04 µM day-1. Bromate reduction rates by mixed microbial

417

cultures such as denitrifying bioreactors have been reported, and they are between 0.125

418

and 10.6 µM day-1.7,26,27,38 van Ginkel et al.8 reported an exception when they established

419

a continuously fed enrichment culture with acetate and bromate as the electron donor and

420

acceptor, respectively. The sludge originating from this enrichment culture reduced

421

bromate at an apparent reduction rate of 45 µM day-1 or 17 nmol h-1 mg dry weight of

422

sludge-1.8 Although most of above rate comparisons are overly simplified due to lack of

423

biomass information, the bromate reduction rate by strain Br-6 seems to be the highest

424

among the bromate-reducing bacteria isolated to date and is comparable to the highest

425

value of mixed microbial cultures. Therefore, it could be a promising microorganism that

426

is applicable to biological removal of bromate in drinking water. For example, treatment

427

of bromate-containing water with immobilized cells of strain Br-6 or with immobilized

428

diaphorase after ozonation might be a useful solution. To make it possible, further

429

screening of the first redox mediator, which is cheaper and more eco-friendly than DCIP,

430

is underway in our laboratory.

431 432

Supporting Information


Page 20 of 37

Page 21 of 37


433

Figure S1. Bromate reduction by soil slurry incubated under various conditions. Figure

434

S2. Phylogenetic tree showing the relationship between strain Br-6 and related

435

Rhodococcus species based on 16S rRNA gene sequences. Figure S3. Bromate reduction

436

by strain Br-6 in the presence of AQDS, MV, and riboflavin. Figure S4. Effect of various

437

concentrations of resazurin (A), resorfin (B), and DCIP (C) on bromate reduction by

438

strain Br-6. Figure S5. Inhibition of DCIP-dependent NADH-oxidizing activity by

439

dicumarol. Figure S6. Abiotic re-oxidation of DCIPH2 by ferric iron (A) and abiotic

440

reduction of bromate by ferrous iron (B). Figure S7. Bromate reduction by an

441

NTA-washed cell suspension of strain Br-6.

442



443

REFERENCES

444

(1) von Gunten, U.; Ozonation of drinking water: part II. disinfection and by-product

445

formation in presence of bromide, iodide or chlorine. Water Res. 2003, 37,

446

1469–1487.

447

(2) Kurokawa Y.; Maekawa A.; Takahashi M.; Hayashi Y. Toxicity and carcinogenicity of

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potassium bromate–a new renal carcinogen. Environ. Health Persp. 1990, 87,

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309–339.

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(3) United States Environmental Protection Agency (USEPA). National primary drinking water regulations: final rule. Federal Register 1989, 54, 27485–27541. (4) World Health Organization (WHO). Guidelines from drinking water quality, vol. 2, 2nd ed., Chemical Aspects, Geneva, 1996. (5) Song R.; Westerhoff P.; Minear RA.; Amy G. Bromate minimization during ozonation.

J. Am. Water Works Assoc. 1997, 89, 69–78. (6) Pinkernell U.; von Gunten U. Bromate minimization during ozonation: mechanistic considerations. Environ. Sci. Technol. 2001, 35, 2525–2531.

458

(7) Hijnen W. A. M.; Voogt R.; Veenendaal H. R.; van der Jagt H.; van der Kooij D.

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Bromate reduction by denitrifying bacteria. Appl. Environ. Microbiol. 1995, 61,

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239–244.

461

(8) van Ginkel C. G.; van Haperen A. M.; van der Togt B. Reduction of bromate to

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bromide coupled to acetate oxidation by anaerobic mixed microbial cultures. Water

463

Res. 2005, 39, 59–64.

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(9) Kirisits M. J.; Snoeyink V. L.; Inan H.; Chee-Sanford J. C.; Raskin L.; Brown J. C.


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Water quality factors affecting bromate reduction in biologically active carbon filters.

466

Water Res. 2001, 35, 891–900.

467 468 469

(10)

Kirisits M. J.; Snoeyink V. L.; Kruithof J. C. The reduction of bromate by

granular activated carbon. Water Res. 2000, 34, 4250–4260. (11)

van Ginkel C. G.; Middelhuis B. J.; Spijk F.; Abma W. R. Cometabolic

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reduction of bromate by a mixed culture of microorganisms using hydrogen gas in a

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gas-lift reactor. J. Ind. Microbiol. Biotechnol. 2005, 32, 1–6.

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(12)

Chairez M.; Luna-Velasco A.; Field J. A.; Ju X.; Sierra-Alvarez R. Reduction of

473

bromate by biogenic sulfide produced during microbial sulfur disproportionation.

474

Biodegradation 2010, 21, 235–244.

475

(13)

Assuncao A.; Martins M.; Silva G.; Lucas H.; Coelho M. R.; Costa M. C.

476

Bromate removal by anaerobic bacterial community: mechanism and phylogenetic

477

characterization. J. Hazard. Mater. 2011, 197, 237–243.

478

(14)

Hiraishi A. Direct automated sequencing of 16S rDNA amplified by

479

polymerase chain reaction from bacterial cultures without DNA purification. Lett.

480

Appl. Microbiol. 1992, 15, 210–213.

481 482 483 484 485 486

(15)

Weisburg W. G.; Barns S. M.; Pelletier D. A.; Lane D. J. 16S ribosomal DNA

amplification for phylogenetic study. J. Bacteriol. 1991, 173, 697–703. (16)

Saito N.; Nei M. The neighbor-joining method: a new method for

reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4, 406–425. (17)

Chakraborty S.; Sakka M.; Kimura T.; Sakka K. Cloning and expression of a

Clostridium kluyveri gene responsible for diaphorase activity. Biosci. Biotechnol.



487 488

Biochem. 2008, 72, 735–741. (18)

Chakraborty S.; Sakka M.; Kimura T.; Sakka K. Characterization of a

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dihydrolipoyl dehydrogenase having diaphorase activity of Clostridium kluyveri.

490

Biosci. Biotechnol. Biochem. 2008, 72, 982–988.

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(19)

Kurokawa J.; Asano M.; Nomoto S.; Makino Y.; Itoh N. Gene cloning and

492

characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum:

493

a useful enzymatic regeneration system of NAD+ from NADH. J. Biosci. Bioeng.

494

2010, 109, 218–223.

495 496 497 498 499

(20)

Bernofsky C.; Mills R. C. Diaphorases from Aerobacter aerogenes. J. Bacteriol.

1966, 92, 1404–1414. (21)

Tedeschi G.; Chen S.; Massey V. DT-diaphorase: Redox potential, steady-state,

and rapid reaction studies. J. Biol. Chem. 1995, 270, 1198–1204. (22)

Bradford M. M. A rapid and sensitive method for the quantification of

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microgram quantities of protein utilizing the principle of protein-dye binding. Anal.

501

Chem. 1976, 72, 248–254.

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Afkhami A.; Madrakian T.; Zarei A. R. Spectrophotometric determination of

503

periodate, iodate and bromate mixtures based on their reaction with iodide. Anal. Sci.

504

2001, 17, 1199–1202.

505 506 507 508

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Laemmli U. K. Cleavage of structural proteins during the assembly of the head

of bacteriophage T4. Nature 1970, 227, 680–685. (25)

Pal S. Identification of multiple soluble Fe(III) reductases in Gram-positive

thermophilic bacterium Thermoanaerobacter indiensis BSB-33. Int. J. Genom. 2014,


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2014, 850607. (26)

Hijnen W. A. M.; Jong R.; van der Kooij D. Bromate removal in a denitrifying

bioreactor used in water treatment. Water Res. 1999, 33, 1049–1053. (27)

Butler R.; Ehrenberg S.; Godley A. R.; Lake R.; Lytton L.; Cartmell E.

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Remediation of bromate-contaminated groundwater in an ex situ fixed-film bioreactor.

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Sci. Total Environ. 2006, 366, 12–20.

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Downing L. S.; Nerenberg R. Kinetics of microbial bromate reduction in a

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hydrogen-oxidizing, denitrifying biofilm reactor. Biotechnol. Bioeng. 2007, 98,

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Petrat F.; Paluch S.; Dogruöz E.; Dörfler P.; Kirsch M.; Korth H. -G.; Sustmann

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R.; de Groot H. Reduction of Fe(III) ions complexed to physiological ligands by

520

lipoyl dehydrogenase and other flavoenzymes in vitro. J. Biol. Chem. 2003, 278,

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46403–46413.

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Morpeth F. F.; Boxer D. H. Kinetic analysis of respiratory nitrate reductase

from Escherichia coli K12. Biochemistry 1985, 24, 40–46. (31)

Yamamoto I.; Shimizu H.; Tsuji T.; Ishimoto M. Purification and properties of

nitrate reductase from Mitsuokella multiacidus. J. Biochem. 1986, 99, 961–969. (32)

Kengen S. W.; Rikken G. B.; Hagen W. R.; van Ginkel C. G.; Stams A. J. M.

527

Purification and characterization of (per)chlorate reductase from the chlorate-respiring

528

strain GR-1. J. Bacteriol. 1999, 181, 6706–6711.

529 530

(33)

Wolterink A. F. W. M.; Schiltz E.; Hagedoorn P. –L.; Hagen W. R.; Kengen S.

W. M.; Stams A. J. M. Characterization of chlorate reductase from Pseudomonas



531 532

chloritidismutans. J. Bacteriol. 2003, 185, 3210–3213. (34)

Martin K. J.; Downing L. S.; Nerenberg R. Evidence of specialized

533

bromate-reducing bacteria in a hollow fiber membrane biofilm reactor. Water Sci.

534

Technol. 2009, 59, 1969–1974.

535

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Davidson A. N.; Chee-Sanford J.; Lai H. Y. M.; Ho C. –H.; Klenzendorf J. B.;

536

Kirisits MJ. Characterization of bromate-reducing bacterial isolates and their potential

537

for drinking water treatment. Water Res. 2011, 45, 6051–6062.

538 539 540 541 542 543

(36)

Xie L.; Shang C.; Zhou Q. Effect of Fe(III) on the bromate reduction by humic

substances in aqueous solution. J. Environ. Sci. 2008, 20, 257–261. (37)

Xie L.; Shang C. Role of humic acid and quinone model compounds in bromate

reduction by zerovalent iron. Environ. Sci. Technol. 2005, 39, 1092–1100. (38)

Kirisits M. J.; Snoeyink V. L. Reduction of bromate in a BAC filter. J. AWWA

1999, 91, 74–84.

544


Page 26 of 37

Page 27 of 37


545

FIGURE LEGENDS

546

Figure 1. Bromate reduction by strain Br-6 under aerobic, anaerobic, and transition

547

growth conditions. Growth (A), bromate and bromide concentrations (B), and acetate

548

consumption (C) are shown. Symbols represent the mean values obtained for triplicate

549

determinations, and bars indicate standard deviations. The arrow indicates the time at

550

which the transition culture was sterilized.

551 552

Figure 2. Acetate is required for bromate reduction by strain Br-6. Cells were grown

553

under transition growth conditions with 250 µM bromate and 5 to 40 mM of acetate.

554

Bromate reduction (A) and acetate consumption (B) are shown. Symbols represent the

555

mean values obtained for triplicate determinations, and bars indicate standard deviations.

556

Arrows indicate the time at which 5 mM acetate was refed to the culture.

557 558

Figure 3. Bromate reduction by strain Br-6 is enhanced by various redox mediators. Cells

559

were grown under transition conditions with 20 mM acetate, 250 µM bromate, and 4 µM

560

each of redox mediators. Symbols represent the mean values obtained for triplicate

561

determinations, and bars indicate standard deviations.

562 563

Figure 4. Ferric iron is required for DCIP-dependent bromate reduction. DCIP (10 µM)

564

was first enzymatically reduced to DCIPH2 using the cell free extract of strain Br-6 in the

565

presence of NADH. Bromate (50 µM) was then added to the reaction mixture, and the

566

mixture was incubated anaerobically. The results from when the reaction was performed



567

in Tris-HCl buffer (pH 6.8) or in minimal medium are shown. In some cases, 25 µM

568

FeCl3·6H2O and 335 µM nitrilotriacetic acid (NTA) were also added to Tris-HCl buffer.

569

The arrow indicates the time at which the reaction mixture was transferred from anaerobic

570

to aerobic conditions. Symbols represent the averages of duplicate experiments, and error

571

bars show the range of data. Ranges smaller than the symbols are not displayed.

572 573

Figure 5. Bromate reduction by a washed cell suspension of strain Br-6. Cells of strain

574

Br-6 were incubated anaerobically with acetate (10 mM), bromate (50 µM), DCIP (10

575

µM), FeCl3·6H2O (25 µM), and nitrilotriacetic acid (NTA, 335 µM). Approximately 5.4

576

mg dry cells were used in the experiment. Symbols represent the averages of duplicate

577

experiments, and error bars show the range of data. Ranges smaller than the symbols are

578

not displayed.

579 580

Figure 6. Activity staining of diaphorase, ferric iron reductase, and bromate reductase in

581

cell free extract of strain Br-6. The cell free extract was run on native PAGE gel, and the

582

gel was stained with Coomassie brilliant blue R-250 for total proteins (A). For diaphorase

583

activity assay, the gel was stained with NADH and DCIP for 10 to 60 min (B). For ferric

584

iron reductase activity assay, the gel was stained anaerobically with NADH, Fe(III)-NTA,

585

and ferrozine for 1 to 3 h (C). For bromate reductase activity assay, the gel was first

586

incubated anaerobically with NADH, DCIP, Fe(III)-NTA, and bromate (100 µM) for 16 h.

587

After washing, the gel was immersed in 0.1 M AgNO3 solution to form a pale-yellow

588

precipitate AgBr. Since AgBr is highly photosensitive, the gel was finally exposed to light


Page 28 of 37

Page 29 of 37


589

to form black Ag. The arrow indicates the band that showed diaphorase, ferric iron

590

reductase, and bromate reductase activities. For clarity, color tone and contrast of the gel

591

image was modified.

592 593

Figure 7. Proposed mechanism of bromate reduction by strain Br-6. Standard redox

594

potential (E0’) of each redox couple is also shown.

595



DCIP

y

NADH

Diaphorase

NAD+

DCIPH2

Fe(II)-‐NTA

Abio>c

Page 30 of 37

BrO3–

Abio>c

Fe(III)-‐NTA

Br–

TOC/Abstract Art ACS Paragon Plus Environment

Page 31 of 37


107

A

!

!

350

!

300

!

250

!

200

!

! 100! 150

50 106

! 0

!

2

!

4

!

6

!

days

!

8

!

B

Acetate (mM)

Cells mL-1

108

!

Bromate and bromide (µM)

109

!

0

!

0

!

2

!

4

!

6

!

!

8

!

days

25

!

20

!

15

!

10

!

5

!

0

!

C

Aerobic! Anaerobic! Transition with resazurin! Transition without resazurin! Transition sterilized at 2 d! Bromide (transition with resazurin)!

0

!

2

!

4

!

6

!

!

8

!

days

Fig. 1 ACS Paragon Plus Environment


A

300!

B

40! 5 mM! 10 mM! 20 mM! 40 mM! 5 mM refed at 4 d!

250!

Page 32 of 37

5 mM! 10 mM! 20 mM! 40 mM! 5 mM refed at 4 d!

30! Acetate (mM)!

Bromate (µM)!

200!

150!

20!

100! 10! 50!

0!

0! 0!

2!

4!

6!

8!

0!

days!

2!

4!

6!

8!

days!



Bromate (µM)!

Page 33 of 37

300

!

250

!

200

!

150

!

100

No mediator! Resazurin! Resorufin! DCIP! Methylene blue! PMS!

!

50

!

0

!0!

2

!

!

4 days

!

6

!

8

!



60

Bromate (µM)

!

50

40

Page 34 of 37

! ! !

! ! ! !!

30

20

Tris-HCl buffer! Minimal medium! Tris/Fe3+/NTA! Tris/Fe3+/NTA (aerobic)!

10

0

0

Tris/Fe3+ Tris/NTA!

10

!

20

!

! !

30

40

!

50

!

hours



60

!

50

!

40

!

30

!

20

!

10

!

Bromate (µM)

!

Page 35 of 37

0

!

No addition! DCIP/Fe3+/NTA! DCIP/Fe3+/NTA (aerobic)! Fe3+/NTA! DCIP/Fe3+! DCIP/NTA!

0

!

4

!

8

!

12

!

16

!

!

20

!

24

!

hours



A B C

Page 36 of 37

D


Page 37 of 37


NADH

Diaphorase!

NAD+ E0ʼ –0.320 V

Fe(II)! -NTA

DCIP!

Abiotic!

DCIPH2! +0.217 V

Fe(III)! -NTA +0.385 V

BrO3–! Abiotic!

Br–! +0.997 V


Bromate Reduction by Rhodococcus

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