Novel Strategy for Tracking the Microbial Degradation of Azo Dyes

Subscriber access provided by UNIV LAVAL

Article

A Novel Strategy for Tracking the Microbial Degradation of Azo Dyes with Different Polarity in Living Cells Fei Liu, Meiying Xu, Xingjuan Chen, Yonggang Yang, Haiji Wang, and Guoping Sun Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02003 • Publication Date (Web): 10 Sep 2015 Downloaded from http://pubs.acs.org on September 18, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22

Environmental Science & Technology

1

A Novel Strategy for Tracking the Microbial

2

Degradation of Azo Dyes with Different Polarity in

3

Living Cells

4

Fei Liu, Meiying Xu*, Xingjuan Chen, Yonggang Yang, Haiji Wang, and Guoping Sun

5 6

State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of

7

Microbiology.100 Central Xianlie Road, Guangzhou 510070, P.R. China ， E-mail:

8

[email protected]

9

KEYWORDS: microbial degradation, azo dyes, FRET, fluorescence tracking, degradation

10

pathways

11

ABSTRACT: Direct visualization evidence is important for understanding the microbial

12

degradation mechanisms. To track the microbial degradation pathways of azo dyes with different

13

polar characterizations, sensors based on the FRET (fluorescence resonance energy transfer)

14

from 1, 8-naphthalimide to azo dyes were synthesized, in which the quenched fluorescence will

15

recover when the azo bond was cleaved. In living cells, the sensor tracking experiment showed

16

that the low polar and hydrophobic azo dye can be taken up into the cells and reduced inside the

17

cells, whereas the highly polar and hydrophilic azo dye can be reduced only outside the cells

18

because of the selective permeability of the cell membranes. These results indicated that there

ACS Paragon Plus Environment

1


Page 2 of 22

19

were two different bacterial degradation pathways available for different polarity azo dyes. To

20

our knowledge, no fluorescent sensor has yet been designed for illuminating the microbial

21

degradation mechanisms of organic pollutants with different characteristics.

22

INTRODUCTION

23

Microorganisms can detoxify or remove many environmental pollutants because of their

24

diverse metabolic capabilities, providing noninvasive and cost-effective methods to protect us

25

from pollution.1 The traditional strategies for understanding microbial degradation mechanisms

26

usually relied on genetic engineering and protein separation and purification technologies,

27

complicating the monitoring of the mode of action and the transfer process of pollutants in vivo.2-

28

4

29

remain in the conjectural stage, notably for those organic pollutants that are anthropogenic

30

sources and are only difficultly or partially degraded.5-8 Describing the metabolic reductive

31

processes will assist the understanding of microbial degradation mechanisms. Thus, substantial

32

attention has been focused on developing new methods to reveal the details of microbial

33

degradation, notably visualization methods that provide direct evidence.

Because of the lack of strong evidence, most studies of microbial degradation mechanisms

34

During the past decade, a substantial number of fluorescent chemosensors have been

35

published and have attracted attention because of their simplicity, high selectivity and sensitivity

36

in fluorescent assays. These sensors can be conveniently used as a tool to analyze guest species

37

and sense biologically important species in vitro and in vivo to clarify their function in living

38

systems.9-11 However, examples of fluorescent sensors applied in microbial systems to study

39

microbial degradation are rare. This study provides a fluorescence method to track the microbial

40

degradation processes of azo dyes, which are commonly used in a number of industries and have


2

Page 3 of 22


41

been identified as a dominant toxic organic pollutant around the world, to better understand

42

microbial degradation mechanisms.12

43

Azo dyes are compounds bearing a functional group with an azo bond (R–N=N–R). Over the

44

last few years, the carcinogenicity and other toxic effects of azo dyes have raised significant

45

concerns.13-16 A prolonged intake of azo dyes can result in the formation of tumors, allergies,

46

respiratory problems and birth defects.17-18 To date, many azo dye degrading bacteria have been

47

found, and select studies have implied that the ability of bacteria to reduce azo dyes is mostly

48

attributed to enzymes (azoreductases) that cleave the azo bond inside or outside the cells. These

49

enzymes might be related to the characteristics of the azo dyes.19-25 However, no direct

50

visualization evidence has been provided. To understand the microbial degradation mechanisms

51

of the azo dyes, we designed a new fluorescence method utilizing the different polarity to track

52

the mode of action and transfer processes of azo dyes during microbial degradation.

53

Materials and methods

54

1. Strains and growth conditions:

55

Shewanella decolorationis strain S12T (CCTCCM203093T = IAM 15094T) was isolated from

56

activated sludge from a textile-printing wastewater treatment plant and preserved in our

57

laboratory.26-27 Strain S12-22 was a mutant of strain S12 constructed by transposon insertion in

58

ccmA 170 bp downstream of the initiation codon, resulting in a deficiency in producing mature c-

59

type cytochromes (Fig S5, S6).28

60

Strain S12 and S12-22 were grown aerobically in standard luria-bertani medium (LB) or

61

anaerobically inlactate medium (LM) containing 2.0 g/l lactate, 2.0 g/l yeast extract, 12.8 g/l


3


Page 4 of 22

62

Na2HPO4·7H2O, 3 g/l KH2PO4, 0.5 g/l NaCl, and 1.0 g/l NH4Cl at 33°C (asdescribed

63

previously).28 The defined medium could be supplemented with lactate or 1 mM NADH.

64

2. Analytical procedures:

65

Decolorizing reactions were conducted in an anaerobic station at 33°C for each assay in

66

triplicate. An NADH or lactate and sensors were added to the defined medium, which was then

67

purged with pure nitrogen gas for more than 5 min. Intact cells of strain S12 and S12-22 reduced

68

0.05 mM NA-MR and NA-OG (Scheme 1) within 7 h when NADH or lactate were existed in

69

the defined medium.

70

3. HPLC conditions:

71

The degradation products of NA-MR and NA-OG were analyzed by HPLC. Ethanol was

72

added to the supernatants, and the suspensions were filtered through 0.22-µm pore-sized acetyl

73

cellulose membranes. The HPLC system employed an Athena C18-WP-100 V column (4.6 mm /

74

3150 mm); a detection wavelength of 280 nm; a flow rate of 3 ml/min; and water: methanol ratio

75

of 9:91(v/v) as the eluent.

76

4. Absorption and Fluorescence Measurements.

77

Absorption spectra were measured on a Thermo Scientific MULTISKAN GO. Fluorescence

78

spectra were obtained with a Perkin Elmer LS 45 Fluorescence Spectrometer. The spectra were

79

obtained every 2 h (0-6 h) in the recovered supernatants. Cells were removed by centrifugation,

80

and the supernatant was recovered by filtering through 0.22-µm pore-sized acetyl cellulose

81

membranes.

82

5. Fluorescence imaging:


4

Page 5 of 22


83

The fluorescence imaging of NA-MR and NA-OG in cells were obtained with a spectral

84

CLSM (Confocal Laser Scanning Microscopy, LSM 700, Zeiss). Before the CLSM analysis, the

85

cells were separated from the culture by centrifugation (6000 g) for 2 min and then washed twice

86

with a sterilized phosphate buffer solution (PBS) to remove residual nutrients. The probes (50

87

µM) were loaded into LM medium (1 mM NADH for NA-MR) with strain S12, S12-22 or

88

Pseudomonas stutzeri cells and inoculated for 2, 4 and 6 h, respectively.

89

6. Synthesis of azo dye Sulfo-red and sensor NA-MR, NA-OG:

90

Synthesis of Sulfo-red:

91

Diluted HCl (5 mL of conc. HCl, 58 mmol) was added slowly to p-aminobenzoic acid (1 g,

92

7.24 mmol) in water at 0oC and the mixture was stirred for 15 min. A solution of NaNO2 (0.75g,

93

11 mmol) in water is dropped into the slurry mixture. The resulting clear mixture was added

94

dropwise to a solution of N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (1.8 g, 6 mmol).

95

After stirring for 2 h at 0-5 °C, it was stirred at room temperature for 24 h. Heat the mixture to 70

96

°C for 3 minutes to dissolve most of the precipitate. When all the dye is dissolved, add 4 g of

97

sodium chloride, and cool the mixture in an ice bath. The precipitate was filtered and washed

98

with water to give compound Sulfo-red as an orange solid (1.2 g) in 46.8 % yield.

99

Synthesis of sensor NA-MR and NA-OG:

100

Compound b (shown in the Scheme S1.) (0.17 mL, 0.58 mmol) and (Benzotriazol-1-yl-

101

oxytripyrrolidinophosphonium hexafluorophosphate) PyBOP (0.56 g, 0.32 mmol) were added to

102

the solution of TEA (0.22 mL, 0.58 mmol) and Sulfo-red (0.50 g, 0.29 mmol) in an hydrous

103

dimethylformamide (5 mL). The mixture was stirred at room temperature for 2 days. The

104

mixture was poured into ice water and filtered off the precipitate. The precipitate was washed

105

with ethanol. The crude product was purified by silica gel column chromatography eluting with

106

dichloromethane/ethanol (6/4) to give NA-MR as a red solid (109 mg, 65%). With the same


5


107

experiment method, we got the probe NA-OG.

108

7. Test method of octanol-water partition coefficient (Log KOW)

Page 6 of 22

109

The octanol-water partitioning coefficients of the four azo dyes were measured at 25 ˚C

110

following the related literatures.29-30 10 mL of deionized water containing 100 µg of azo dyes

111

were mixed with 10 mL of n-octanol in ten glass bottles of 20 mL. The bottles were sealed with

112

rubber plug, and shaken for 5 min. After standing for 24 hours and phase separation, the

113

concentrations of azo dyes in two aqueous phases were measured by HPLC.

114

Results and discussion

115

Azo reduction and spectrum response.

116

Because azo dyes are non-fluorescent and quenching dyes, when a new fluorophore is

117

designed to connect with the azo dye by an alkyl chain, the fluorescence of the molecule will be

118

quenched. Nevertheless, the fluorescence will recover when the azo moiety is degraded by

119

microorganisms. This reduction is examined here using strain S12 with lactate as the electron

120

donor (in LM medium) and the sensors as the electron acceptor.31 To track the azo dye

121

degradation, new fluorescent sensors NA-MR and NA-OG (Scheme 1) were developed based on

122

the FRET mechanism by linking fluorescent moieties with low polar azo-moieties and highly

123

polar sulfonated azo-moieties, respectively.

124 125

Scheme 1. Chemical structures of the sensors NA-MR and NA-OG and microbial reduction.

126


6

Page 7 of 22


127

Decolorizing reactions were conducted in an anaerobic station for each assay. The bacterial

128

metabolism of azo dyes is initiated in most cases by a reductive cleavage of the azo bond,

129

resulting in the formation of colorless aromatic amines. The azo bond is an important targeted

130

position; under anaerobic conditions, the decolorizing reaction works in conjunction with a direct

131

enzymatic reaction involving the azoreductase. However, if the extracellular environment is

132

aerobic, then this reduction mechanism will be inhibited by oxygen because of the preferential

133

oxidation of the reduced redox mediator by oxygen rather than by the azo dye.32-33 The proposed

134

fluorescence sensing mode of NA-MR and NA-OG for the decolorizing tracking experiment is

135

displayed in Scheme 1. The generation of the fluorescence is mainly because of the azo bond

136

reduced by the microorganism. In solution, after reduction by the cells of strain S12, a

137

naphthalimide derivative was the main product and fluorescence appeared.

138

As illustrated in Figure. 1, NA-MR and NA-OG have an absorption peak at 420 nm in the

139

recovered supernatants. Intact cells of strain S12 has high azoreduction efficiency when lactate

140

or NADH is existed in the defined medium. In total, 0.05 mM of the probes NA-MR and NA-

141

OG are completely reduced within 7 h, and the absorptions band of NA-MR and NA-OG

142

gradually decrease with an apparent color change from dark yellow to bright yellow. The

143

fluorescence changed from none to yellow at 520 nm (Fig 1e). With the degradation of the

144

probes in the solution, the main fluorescence signal was produced by the generation of the

145

naphthalimide derivative, which is mainly produced in this degradation process. These results

146

indicated that the fluorescence is mainly because of the azo bond being reduced by the enzymes

147

in strain S12, showing a good selection to the azo bond and that no other chemical bond is

148

broken in the sensors.


7

a)

0.35

Absorption intensity


0.30 0.25 0.20 0.15 0.10 0.05 0.00 360

149

400

440

480

520

400

320

240

160

80

0 480

560

c)

0.5

Absorption intensity

0.4

520

560

600

640

680

640

680

Wavelength(nm)

Wavelength(nm)

Fluorescence Intensity

d) 300

0.3

0.2

0.1

0.0 360

150

Fluorescence intensity

b)

Page 8 of 22

420

480

Wavelength(nm)

540

600

250 200 150 100 50 0 480

520

560

600

Wavelength(nm)

151 152

Figure 1. Changes in the absorption spectra of NA-MR (a) and NA-OG (c) (50µM) with strain

153

S12 in the standard LM medium (1 mM NADH for NA-MR) and the fluorescence spectra of

154

NA-MR (b) and NA-OG (d). λex = 420 nm. e) Photographs of two probes in solution after 0, 4,

155

and 6 h. The visible absorption and fluorescence observed in recovered supernatants.

156 157

In order to further scrutinize the relationship between the molecular property and the tracking

158

degradation mechanisms, the n-octanol-water partition coefficient (log KOW) of four azo dyes,

159

sulfo-red, methyl-red (chemical structures shown in scheme S1), including NA-OG and NA-MR,

160

were measured (Table S1). The log KOW is a key parameter of hydrophobicity which is an

161

intrinsic property that relates to a chemical’s tendency to partition between a polar and a

162

nonpolar phase. Based on the results shown in Table S1, the azo dyes and their corresponding


8

Page 9 of 22


163

fluorescent sensors contained similar hydrophilic natures although the log KOW values of the

164

corresponding fluorescent sensors were higher than their parent azo dyes. The partition

165

coefficient Log KOW of sulfo-red and NA-OG were 0.25 and 1.16 at 25 °C, which explained the

166

predominantly hydrophilic nature of high polar azo dyes, and the Log KOW of methyl-red and

167

NA-MR were 4.62 and 6.05 which shown good hydrophobicity. Compared to the high polar azo

168

dyes, the log KOW of low polar azo dyes methyl-red and NA-MR is at least 4-5orders of

169

magnitude higher, suggesting that methyl-red and NA-MR were more powerful than sulfo-red

170

and NA-OG to penetrate through the live cell membranes barriers. We also found that the

171

degradation rates for strain S12 decreased when the azo dyes (methyl-red and sulfo-red) were

172

connected

173

molecular size and structure may influence on the affinities of the involved enzymes to the

174

targeted compounds and the degradation rates of the azo dyes will be reduced when their

175

molecular size increased.

the

fluorophore

(shown

in

Fig

S1),

which

indicated

that

the

176 177

Degradation products analysis

178

NA-MR and NA-OG degradation products were analyzed by HPLC (Fig. 2). The HPLC and

179

MS analysis of the test solutions confirmed that the reduction products of NA-MR and NA-OG

180

with strain S12 in an anaerobic station were NA-M and NA-O (chemical structures shown in

181

scheme 1). As shown in Figure 2a, we monitored the degradation of NA-MR in the supernatants

182

just after inoculation using HPLC, and a single peak was shown at 5.7 min corresponding to

183

NA-MR. After 3 h of culturing with NA-MR, a sharp peak at 2.2 min was apparent. Finally, the

184

probe NA-MR completely disappeared, and the NA-M was left as the only production in

185

solution after 6 h. To confirm the identity of the peaks, we used an ESI-TOF-MS analysis to


9


Page 10 of 22

186

detect the mass at m/z 443 and 574, which represent NA-MR and NA-M, respectively (Fig.

187

S2a). In Figure 2b, the degradation products were analyzed by HPLC for the probe NA-OG, and

188

a single peak was detected at 1.1 min. After 3 h of culturing, the peak with a retention time of

189

2.7 min appeared, but the peaks at 1.1 min remained. After 6 h, some NA-OG probes were still

190

present in the solution, and a slower degradation rate was noted for NA-OG than for NA-MR.

191

Two peaks were isolated and analyzed by ESI-TOF-MS, and these peaks were confirmed to be

192

NA-OG and NA-O (Fig. S2b). Compared with previous research,28 degradation product NA-M

193

and NA-O were fluorophore, which could track the microbial degradation processes of azo dye

194

using the fluorescence. In this process, we did not find another portion of the molecule;

195

therefore, we predicted that the remaining portions of the probe molecules were presumably

196

further biodegraded and used as carbon or energy sources by the bacteria.

197 198

Figure 2.HPLC chromatograms of the culture supernatant of probes NA-MR and NA-OG, a)

199

NA-MR (50 µM) was incubated with S12 cells for 0 h, 3 h and 6 h in the standard LM


10

Page 11 of 22


200

medium(1 mM NADH for NA-MR); b) NA-OG (50 µM) with S12 in the standard LM

201

medium(1 mM NADH for NA-MR) for 0 h, 3 h and 6 h.

202

Fluorescence tracking imaging

203 204

The potential utility of NA-MR for tracing azoreduction in living cells of strain S12 was

205

then investigated by confocal microscopy. Strain S12 was grown anaerobically in the standard

206

LM medium. We monitored the change of the fluorescence in the cells of strain S12 over 6

207

hours. Those cells are almost non-fluorescent at the beginning, but the fluorescence gradually

208

increased as time progressed. Two hours later, a fluorescence signal in the green channels could

209

be detected (Fig. S7a). After 6 hours, the confocal microscopic images exhibited intense

210

fluorescence in the green channel, corresponding to NA-M (Fig. 3a-d), and the fluorescence

211

image intensity did not disappear as the time progressed. We speculate that the NA-MR would

212

continuously enter the cells and that the NA-M diffuses away from the cells, which is congruent

213

with the fluorescence intensity increasing in the supernatant with a balanced concentration

214

occurring in the cells. Moreover, there are a lot of reductases in the endoplasm or cell membrane.

215

So we think, it is very likely the partitioning and accumulation of fluorescent degradation

216

moieties of low polar and hydrophobic azo dye will occur in the cell membrane and endoplasm.

217

This result demonstrated that NA-MR can be efficiently degraded to NA-M by strain S12 and

218

displays a good stain in the cell.


11


Page 12 of 22

219 220

Figure 3. Degradation in the living cells of strain S12 imaged with NA-MR (a-d) and NA-OG

221

(e-h). The two probes (50 µM) were loaded into LM medium (1 mM NADH for NA-MR) with

222

the cells of strain S12 for 6 h. The images of the NA-MR (a) and NA-OG (e) with the cells of

223

strain S12 after 6 h incubation; b) the green image merged with bright-field; c) enlarged image of

224

the cells in (b); d) bright-field image merged with (c); f) DAPI-staining is shown in blue; h) the

225

bright-field image merged with the green (e) and blue (f) channel.

226 227

In Fig 3 (e-h), the probe NA-OG was used to track the azoreduction in living S12 cells and

228

assessed using confocal microscopy. To investigate the reduction process and imaging of the

229

probe NA-OG, DAPI staining (a commercially available nucleic acid sensor) was performed.

230

DAPI was mainly used to determine the presence of the cells. Initially, we monitored the change

231

of the fluorescence in the S12 cells over the first 2 hours; however, almost no fluorescence was

232

generated (Fig S7d). Over the next 4 h, some fluorescence emerged, but the signal remained

233

weak. Simultaneously, the supernatants displayed a distinct color change related to the

234

degradation of azo dyes by strain S12 (Fig 1e). Therefore, the reducing activity for the probe is

235

not dependent on the intracellular uptake of the probe, but this activity occurs outside the cells.


12

Page 13 of 22


236

The sulphonate group is unlikely to pass through the cell membranes of strain S12, and the

237

electron transport components, such as OmpA and OmpB (outer membrane protein A and B),

238

must be localized in the outer membrane of the bacterial cells, in which these components can

239

directly transmit electrons to the probes at the cell surface.34-36 In this process, several

240

degradation products, such as NA-O, could diffuse into the cells and produce a low fluorescence

241

in the cells. This indicated that the NA-OG dye is likely not degraded in the cell because of its

242

highly polar and hydrophilic characteristics, and this dye cannot migrate inside the cells through

243

the cell membrane barriers. As shown in Fig 3e, over the 6 h, only little weak fluorescence

244

emerged. Simultaneously, the supernatants displayed a distinct color change related to the

245

degradation of azo dyes by strain S12. Therefore, the reducing activity for the probe is not

246

dependent on the intracellular uptake of the probe, but this activity occurs outside the cells. If the

247

degradation occurred in the cell membrane, degradation product NA-O may accumulate

248

in the cell membrane and will appear bright fluorescence as Fig 3c. This result provided direct

249

evidence that the chemical molecular polarity interaction with the lipid bacterial cell membrane

250

prevents azo dyes with high polarity from penetrating bacteria cell membranes; therefore, the

251

high polarity azo dyes are reduced outside the cell. By contrast, the low polarity azo dyes can

252

penetrate the cell membrane, and the azo bond reduction can occur inside the cell.


13


Page 14 of 22

253 254

Figure 4. Degradation in the living cells of strain S12-22 imaged with NA-MR (a-d) and NA-

255

OG (e-h). The two probes (50 µM) were loaded into LM medium (1 mM NADH for NA-MR)

256

with the cells of strain S12-22 for 6 h. The image of the S12-22 cells incubated with NA-MR (a)

257

and NA-OG (e) for 6 h; b) the green channel merged with bright-field image; c) enlarged image

258

of the cells; d) bright-field image merged with (c); f) fluorescence image of DAPI compared with

259

NA-OG; h) bright-field image (g) merged with(e) and (f).

260

To further discuss the characteristics of the two probes, we investigated the application of the

261

sensors in strain S12-22. Strain S12-22 is a mutant of strain S12 with a transposon insertion in

262

ccmA with a result in a deficiency in producing mature c-type cytochromes (Fig S5, S6). Mature

263

c-type cytochromes are reported to be essential electron mediators for the extracellular

264

azoreduction of intact cells and defects of ccmA restricted the azoreduction outside the cells after

265

employing genetic engineering or protein separation and purification technologies.28

266 267

In this study, strain S12-22 displays a good azoreduction for NA-MR. The absorption band of

268

NA-MR gradually decreases, and all cells exhibited fluorescence increases at 6 hours (Fig. S3a,

269

S8). Confocal microscopic images showed a strong and clear fluorescent staining, and this result


14

Page 15 of 22


is congruent with the in vitro tests (Figure. 5a). Meanwhile, strain S12-22 was not able to reduce

271

NA-OG in 6 hours, as seen in Figure 5b. Almost no fluorescence change in the spectrum was

272

noted. The wild-type strain S12 was able to reduce NA-OG in the defined medium supplemented

273

with lactate or NADH, but the mutant strain S12-22 did not reduce NA-OG well in the identical

274

conditions. When cultivating the probe anaerobically in the standard LM medium, no

275

fluorescence was emitted from the S12-22 cells (Figure 4e). These results indicated that the

276

highly polar sulfonated azo dye NA-OG could not penetrate the cell membrane barriers and be

277

reduced by the mutant strain S12-22. Therefore, no apparent affects were noted in the aqueous

278

solution and cell images. The lowly polar azo dye NA-MR, which could penetrate through the

279

cell membrane barriers into the cell, was not influenced by the deficiency in the mature c-type

280

cytochromes and was reduced inside the cells of the mutant strain S12-22 (Figure 4a). Another

281

bacterial strain (Pseudomonas stutzeri, which cannot use LM medium for azoreduction in vivo)

282

was used to further test the two sensors. When the probes of NA-OG and NA-MR were used to

283

study the azoreduction of Pseudomonas stutzeri (Figure 5 and S4), no color change in the

284

aqueous solution and cell images was noted over 6 hours’ incubation (Figure S9).


a)

500 control NA-MR

400

300

200

100

0 S12

S12-22

pseudomonas

b)

300


270

250

control NA-OG

200 150 100 50 0 S12

S12-22

pseudomonas

285 286

Figure 5. The recovered supernatants of the maximum-fluorescence changes in spectra of NA-

287

MR (a) and NA-OG (b) (50 µM) in the cultures with 3 different bacteria in the standard LM


15


288

Page 16 of 22

medium within 7 hours.

289

In summary, the oxidoreductase catalyzed the reductive degradation of azo dyes depend on the

290

structure of the compounds. The two sensors showed a greater sensitivity towards the bacterial

291

reduction, which breaks the N=N double bond structure, and displayed a dramatic fluorescence

292

enhancement as the degradation of the azo moiety in vitro. The chemical structures of the sensors

293

NA-OG and NA-MR are distinct because of the sulfonic acid substituents on the dyes. NA-MR

294

(with no sulfonic acid substituent) is minimally polar, highly permeable and able to penetrate the

295

cell membranes, enabling the reduction of this compound inside the cells of the ccmA mutant

296

S12-22. However, NA-OG is a highly polar sulfonated azo compound with a low permeability

297

through the cell membranes. After removing c-type cytochromes, the bacterial membranes

298

restrict the transfer of electrons from the cytoplasm to the sulphonated sensor NA-OG. These

299

results indicate that the design of NA-OG and NA-MR may be a useful in investigating the

300

reduction processes of azo dyes with different characterizations in different bacteria. This study

301

may establish a new research model for microbial degradation and transformation mechanisms

302

based on a chemical fluorescent labeling technology.

303 304

ASSOCIATED CONTENT

305

Supporting Information. Synthesis, experimental details, additional spectroscopic data and

306

images. This material is available free of charge via the Internet at http://pubs.acs.org.

307

AUTHOR INFORMATION

308

Corresponding Author

309

*Tel.: +86 20 87684471. Fax: +86 20 87684587. E-mail: [email protected].


16

Page 17 of 22


310

Author Contributions

311

F.L., M.X., and G.S. designed the experiments. F.L.,X. C., Y. Y., and H. W. performed the

312

experiments. F.L. and M.X. analyzed the data and wrote the manuscript.

313

Author Contributions

314

The manuscript was written through contributions by all authors. All authors have given

315

approval to the final version of the manuscript.

316

Notes

317

The authors declare no competing financial interest.

318

ACKNOWLEDGMENTS

319

This research was supported by the National Natural Science Foundation (21307016); the

320

Natural Science Foundation of Guangdong, China (2014A030308019; S2013040014438); the

321

National Science Foundation for Excellent Young Scholars of China (51422803); Special

322

Foundation for the Science and Technology Innovation Leaders of Guangdong Province

323

(2014TX01Z038); the Special Fund for Agro-scientific Research in the Public Interest

324

(201503108); and the Guangdong Provincial Innovative Development of Marine Economy

325

Regional Demonstration Projects (GD2012-D01-002)

326

REFERENCES

327

1) Liu, S.; Suflita, J. M. Ecology and evolution of microbial populations for bioremediation.

328

Trends. Biotechnol. 1993, 11, 344-352.

329

2) Kazuya, W.; Maki, T.; Shigeaki, H. Stable augmentation of activated sludge with foreign

330

catabolic genes harboured by an indigenous dominant bacterium. Environ. Microbiol. 2002,

331

4, 577-583.


17


Page 18 of 22

332

3) Willy, V. Microbial ecology and environmental biotechnology. ISME Journal, 2007, 1, 4-8.

333

4) Suar, M.; Hauser, A.; Poiger, T.; Buser, H.; Müller, M.; Dogra, C.; Raina, V.; Holliger, C.;

334

van der Meer, R.; Lal, R.; Kohler, H. Enantioselective transformation of α-

335

hexachlorocyclohexane by the dehydrochlorinasesLinA1 and LinA2 from the soil bacterium

336

Sphingomonaspaucimobilis B90A. Appl. Environ. Microbiol. 2005, 71, 8514-8518.

337 338

5) Akira, H. Biodiversity of dehalorespiringbacteria with special emphasis on polychlorinated Biphenyl/Dioxin dechlorinators. Microbes. Environ. 2008. 23, 1-12.

339

6) Margaret, E. B.; Mark, C. Wa.; Toshiki, G. N.; Anthony, T. I.; Michelle, C. Y. Discovery and

340

characterization of heme enzymes from unsequenced bacteria: Application to microbial

341

lignin degradation. J. Am. Chem. Soc. 2011, 133, 18006-18009.

342 343 344 345 346 347 348 349 350 351 352 353 354

7) Dariusz, D.; Marcin, W.; Benedykt, W.; Sonja, N.; Jussi, M. Microbial degradation of microcystins. Chem. Res. Toxicol. 2013, 26, 841-852. 8) Martin, O.; Alexander, S. Microbial degradation of poly(amino acid)s. Biomacromolecules. 2004, 5, 1166-1176. 9) Wright, A. T.; Anslyn, E. V. Differential receptor arrays and assays for solution-based molecular recognition. Chem. Soc. Rev.2006, 35, 14-28. 10) Amendola, V.; Fabbrizzi, L. Anion receptors that contain metals as structural units. Chem. Commun. 2009, 513-531. 11) Yoon, J.; Kim, S. K.; Singh, N. J.; Kim, K. S. Imidazolium receptors for the recognition of anions. Chem. Soc. Rev. 2006, 35, 355-360. 12) Stolz, A. Basic and applied aspects in the microbial degradation of azo dyes. Appl. Microbiol. Biot. 2001, 56, 69-80. 13) Golka, K.; Kopps, S.; Myslak, Z. W. Carcinogenicity of azo colorants: influence of


18

Page 19 of 22

355 356 357 358 359 360 361


solubility and bioavailability. Toxicol. Lett.2004, 151, 203-210. 14) Feng, J.; Cerniglia, C. E.; Chen, H. Toxicological significance of azo dye metabolism by human intestinal microbiota. Front. Biosci. (Elite edition). 2012, 4, 568-586. 15) Saratale, R. G.; Saratale, G. D.; Chang, J. S. Bacterial decolorization and degradation of azo dyes: A review. J. Taiwan Inst. Chem. E. 2011, 42, 138-157. 16) Yahagi, T.; Degawa, M.; Seino, Y. Mutagenicity of carcinogenic azo dyes and their derivatives. Cancer Lett. 1975, 1, 91-96.

362

17) Gupta, V. K.; Jain, R.; Mittal, A.; Saleh, T. A.; Nayak, A.; Agarwal, S.; Sikarwar, S. Photo-

363

catalytic degradation of toxic dye amaranth on TiO2/UV in aqueous suspensions. Mat. Sci.

364

Eng. C-Mater. 2012, 32, 12-17.

365

18) Alves de Lima, R. O.; Bazo, A. P.; Salvadori, D. M. F.; Rech, C. M.; Oliveira, D. P.;

366

Umbuzeiro, G. A. Mutagenic and carcinogenic potential of a textile azo dye processing plant

367

effluent that impacts a drinking water source. Mutat. Res. 2007, 626, 53-60.

368 369

19) Rafii, F.; Cerniglia, C.E. Comparison of the azoreductase andnitroreductase from Clostridium perfringens. Appl. Environ. Microbiol.1993, 59, 1731-1734.

370

20) Suzuki, Y.; Yoda, T.; Ruhul, A.; Sugiura, W. Molecular cloning and characterization of the

371

gene coding for azoreductase from Bacillussp. OY1–2 isolated from soil. J. Biol. Chem.

372

2001, 276, 9059-9065.

373

21) Moutaouakkil, A.; Zeroual, Y.; Zohra-Dzayri, F.; Talbi, M.; Lee, K.; Blaghen, M.

374

Purification and partial characterization of azoreductase from Enterobacter agglomerans,

375

Arch. Biochem. Biophys. 2003, 413, 139-146.

376

22) Maier, J.; Kandelbauer, A.; Erlacher, A.; Cavaco-Paulo, A.; Gubitz, G. A new alkali-


19


Page 20 of 22

377

thermostable azoreductase from Bacillus sp. strain SF. Appl. Environ. Microbiol. 2004, 70,

378

837-844.

379

23) Ramalho, P. A.; Paiva, S.; Cavaco-Paulo, A.; Casal, M.; Cardoso, M. H.; Ramalho, M. T.

380

Physiology and biotechnology azo reductase activity of intact Saccharomyces cerevisiae cells

381

is dependent on the fre1p component of plasma membrane ferric reductase. Appl.

382

Environ .Microbiol. 2005, 71, 3882-3888.

383

24) Rau, J.; Stolz, A. Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli

384

function under anaerobic conditions as lawsone-dependent azo reductases. Appl. Environ.

385

Microbiol. 2003, 69, 3448-3455.

386 387 388 389

25) Ryan, A.; Laurieri, N.; Westwood, I.; Wang, C. J.; Edward, Lowe, E.; Sim, E. A Novel mechanism for azoreduction. J. Mol. Biol.2010, 400, 24-37. 26) Xu, M. Y.; Guo, J.; Kong, X. Y.; Chen, X. J.; Sun, G. P. Fe(III)-enhanced azo reduction by Shewanella decolorationis S12. Appl. Microbial. Biotechnol. 2007, 74(6), 1342-1349.

390

27) Hong, Y. G.; Xu, M. Y.; Guo, J.; Xu, Z. C.; Chen, X. J.; Sun, G. P. Respiration and growth

391

of Shewanella decolorationis S12 with an azo compound as the sole electron acceptor. Appl.

392

Environ. Microbiol. 2007, 73 (1), 64-72.

393

28) Chen, X. J.; Xu, M.; Wei, J. B; Sun, G. P. Two different electron transfer pathways may

394

involve in azoreduction in Shewanella decolorationis S12. Appl. Microbiol. Biotechnol.

395

2010, 86, 743-751.

396

29) Im, J.; W. Prevatte, C.; Campagna, S. R.; Löffler, F. E. Identification of 4-hydroxycumyl

397

alcohol as the major MnO2-mediated bisphenol A transformation product and evaluation of

398

its environmental fate, Environ. Sci. Technol. 2015, 49, 6214-2621.

399

30) United States Environmental Protection Agency (USEPA). Product Properties Test


20

Page 21 of 22

400 401 402 403 404 405 406


Guidelines; OPPTS 830.7550 Partitioning Coefficient. 1998. 31) Xu, M. Y.; Guo, J.; Zeng, G. Q.; Zhong, X. T.; Sun, G. P. Decolorization of anthraquinone dye by Shewanella decolorationis S12. Appl. Microbiol. Biotechnol. 2006, 71, 246-251. 32) Yoo, E.S.; Libra, J.; Adrian, L. Mechanism of decolourization of azo dyes in anaerobic mixed culture. J. Envion. Eng. Sci. 2001, 127, 844-849. 33) Chacko, J. T.; Subramaniam, K. Enzymatic Degradation of Azo Dyes-A Review. Int. J. Electrochem. Sc. 2011, 1, 1250-1260.

407

34) Robinson, T.; McMullan, G.; Marchant, R.; Nigam P. Remediation of dyes in textile effluent:

408

a critical review on current treatment technologies with a proposed alternative. Bioresource

409

Technol. 2001, 77, 247-255.

410

35) Duran, N.; Esposito, E. Potential applications of oxidative enzymes and phenoloxidase-like

411

compounds in wastewater and soil treatment: a review. Appl. Catal. B-Environ. 2000, 28, 83-

412

99.

413

36) Park, J. S.; Lee, W. C.; Yeo, K. J., Ryu, K. S.; Kumarasiri, M.; Hesek, D.; Lee, M.;

414

Mobashery, S.; Song, J. H.; Kim, S.; Lee, J. C.; Cheong, C.; Jeon, Y. H.; Kim, H. Mechanism

415

of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial

416

outer membrane. FASEB J. 2012, 26, 1-10.

417 418 419 420


21


421

Page 22 of 22

Insert Table of Contents Graphic and Synopsis Here

422 423

The sensors NA-MR and NA-OG were synthesized based on the FRET (fluorescence

424

resonance energy transfer) from 1, 8-naphthalimide to the azo dye, in which the quenched

425

fluorescence will recover when the azo bond is cleaved by the bacteria. In living cells, the sensor

426

tracking experiment showed that the lowly polar and hydrophobic azo dye can be taken up into

427

the cells and be reduced inside the cells, whereas the highly polar and hydrophilic azo dye can be

428

reduced only outside the cells because of the permeability of the cell membranes.


22

Novel Strategy for Tracking the Microbial Degradation of Azo Dyes

Recommend Documents