Benzophenone-4 promotes the growth of a Pseudomonas sp. and

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Benzophenone-4 promotes the growth of a Pseudomonas sp. and biogenic oxidation of Mn(II) Yangyang Chang, Yaohui Bai, Yang Huo, and Jiuhui Qu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05014 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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Environmental Science & Technology

Benzophenone-4 promotes the growth of a Pseudomonas sp. and biogenic oxidation of Mn(II)

Yangyang Chang†$#, Yaohui Bai†*, Yang Huo†#, Jiuhui Qu† †Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. $

School of Environmental Science and Technology, Key Laboratory of Industrial

Ecology and Environmental Engineering (Ministry of Education), Dalian University of Technology, Dalian 116024, China. #

University of Chinese Academy of Sciences, Beijing 100049, China.

ACS Paragon Plus Environment


TOC ART


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ABSTRACT Interactions between microbes and micropollutants (MPs) play a crucial

2

role in water purification or treatment. Current studies have generally focused on the

3

direct degradation or co-metabolism of MPs. Considering the increasing interest in

4

and importance of the roles of MPs in microbial metabolism, we adopted a

5

Mn(II)-oxidizing Pseudomonas sp. QJX-1 using tyrosine (Tyr) as the sole carbon and

6

nitrogen source to investigate the effects of seven MPs on its growth and function. Six

7

MPs exhibited an inhibition effect on bacterial growth and Mn(II) oxidation. Only

8

benzophenone-4 (BP-4) promoted the growth of QJX-1 and biogenic oxidation Mn(II),

9

but its concentration was not directly coupled to growth, which was unexpected.

10

RNA-seq data suggested that the addition of BP-4 did not significantly change the

11

basic metabolic function of QJX-1, but stimulated the upregulation of the pyruvate

12

and gluconeogenesis metabolic pathways of Tyr for QJX-1 growth. Furthermore,

13

protein identification and extracellular superoxide detection indicated that Mn(II)

14

oxidation was largely driven by the formation of superoxide in response to Tyr

15

starvation; the acceleration of superoxide production, due to BP-4 accelerating Tyr

16

consumption, was responsible for the promotion effect of BP-4 on QJX-1 Mn(II)

17

oxidation. Our findings highlight the dual effects that MPs can have on the growth

18

and function of a single strain in aquatic ecosystem, i.e. the co-existence of inhibition

19

and promotion.



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INTRODUCTION

21

Anthropogenic organic compounds,1,

22

products (PPCPs), endocrine disrupting chemicals (EDCs), and pesticides, are widely

23

found in natural3 (e.g., surface4 and ground5 water) and engineered water

24

environments (e.g., wastewater treatment plant effluent6). Generally, they are referred

25

to as micropollutants (MPs) due to their detectable concentrations at ng–µg·L-1 levels.

26

Many of these pollutants have negative effects on aquatic life, including bacteria,

27

invertebrates (Daphnia magna), algae, and fish.7-14 Compared with all other

28

organisms, bacteria are the most ubiquitous and abundant. Interactions between MPs

29

and bacteria occur in a broad range and are essential to maintain the self-purification

30

of aquatic ecosystems.

2

such as pharmaceuticals and personal care

Bacteria can affect MP fate directly (biodegradation15,

31

16

or co-metabolism of

32

MPs17-19) or indirectly (formation of biogenic metal oxides and then MP oxidation,20,

33

21

34

or positively23, 24 (MPs are used as growth substrates) affect bacterial growth and

35

function, and impact the organic matter flux controlled by bacteria25. Until now, our

36

knowledge on the interaction between bacteria and MPs in aquatic ecosystems is still

37

limited. For instance, previous findings showed that MPs increased bacterial growth

38

and function only when they were used as energy and nutrient sources.24 However, it

39

remains unknown whether there are exceptions to this rule.

40

e.g., biogenic manganese oxide (BMO)). The presence of MPs can also negatively22

In natural and engineered waters, bacteria primarily grow using natural organic


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matters (NOMs) as energy sources to remove MPs directly or indirectly. Accordingly,

42

we selected a Mn(II)-oxidizing bacterium Pseudomonas sp. QJX-1 (maybe affecting

43

the MP fate directly and indirectly) as the model strain and tyrosine (Tyr, a NOM) as

44

its energy source to evaluate the impacts of seven MPs on bacterial growth and

45

function. Surprisingly, we found that benzophenone-4 (BP-4) promoted QJX-1

46

growth and biogenic oxidation of Mn(II) but not via its metabolism. This challenges

47

our understanding about the roles of MPs in bacteria. So we focused on how BP-4

48

promoted the bacterial growth and biogenic oxidation of Mn(II) using molecular

49

approach (RNA and protein assays). Our study provides new evidence on how MPs

50

might affect bacterial growth and function.

51

MATERIALS AND METHODS

52

Bacterial Strain and Culture Preparation. The manganese-oxidizing bacterium

53

Pseudomonas sp. QJX-1 (GenBank accession number KM242057) was isolated from

54

wetlands in Zhejiang Province, China. Details on its physiological and Mn-oxidation

55

characteristics are as described previously.26 Briefly, QJX-1 is a rod-shaped bacterium

56

that can catalyze the transformation of Mn(II) to BMO, with a maximum oxidation

57

rate at 30°C and pH 7.5. The strain is deposited in the China General Microorganism

58

Culture Center (CGMCC; accession number 6630).

59

Modified peptone-yeast extract glucose (PYG) medium buffered with 10 mM

60

N-2-hydroxyethylpiperazine-Nʹ-2-ethanesulfonic acid (HEPES) (pH 7.2)27 was used

61

for bacterial enrichment, and consisted of 500 mg·L-1 of MgSO4·7H2O, 60 mg·L-1 of



62

CaCl2·2H2O, 250 mg·L-1 of peptone, 250 mg·L-1 of yeast extract, and 250 mg·L-1 of

63

glucose. For each test, a single isolated colony of QJX-1 on a solid medium plate was

64

first transferred to liquid PYG medium and incubated at 30°C in the dark at 170 rpm.

65

Bacterial cells were then collected at the exponential growth phase (~48 h) and

66

washed three times using sterilized solution A (500 mg·L-1 of MgSO4·7H2O, 60

67

mg·L-1 of CaCl2·2H2O, 7 mg·L-1 of K3PO4, 10 mM HEPES, pH 7.2) by centrifugation

68

at 5000 g for 5 min at room temperature. Finally, the bacterial precipitate was

69

resuspended in sterilized solution A and used as the inoculum for the following study.

70

Selection of Organic Matter for QJX-1 Growth. We used amino acids as

71

representatives of the NOMs used by bacteria for growth in aquatic ecosystems.

72

Amino acids are easily utilized by bacteria without the need of an additional nitrogen

73

source due to their simple structures and -NH2 groups28. Ten amino acids at a final

74

carbon concentration of 100 mg·L-1 (in solution A) were tested as energy substrates

75

for QJX-1 growth, respectively. They included lysine (203 mg·L-1), threonine (248

76

mg·L-1), glutamic acid (245 mg·L-1), histidine (215 mg·L-1), isoleucine (182 mg·L-1),

77

valine (195 mg·L-1), DL methionine (248 mg·L-1), tryptophan (155 mg·L-1),

78

phenylalanine (153 mg·L-1), and tyrosine (Tyr, 176 mg·L-1). Prior to use, these amino

79

acids were filter-sterilized through a 0.22-µm membrane. To determine which amino

80

acid was optimal for growth yield, the QJX-1 cells were inoculated in 100 ml of each

81

amino acid solution for 72 h of culture, respectively. The optical density of each

82

culture at 600 nm (OD600) was measured using a U-3010 UV-vis spectrophotometer

83

(Hitachi, Japan) equipped with 10 mm quartz cuvettes. To obtain enough biomass for


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the following RNA-seq and protein analysis, the amino acid with the maximum

85

bacterial growth yield was selected as the substrate to explore the impacts of MPs.

86

Effect of Different Micropollutants on QJX-1 Growth and Mn(II) Oxidation. To

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determine the effects of MPs on QJX-1 growth and Mn(II) oxidation, QJX-1 strains

88

were incubated with and without different MPs at a specific concentration (50 µg·L-1)

89

in Tyr medium (selected according to the above criteria) containing 5.5 mg·L-1 of

90

MnCl2. For MP selection, we thought they better belong to different categories of

91

MPs and be frequently detected in aquatic environment. Hence, seven MPs, including

92

two UV filters (benzophenone-3 (BP-3) and BP-4)29, two pesticides (glyphosate30 and

93

2,4-dichlorophenoxyacetic acid (2,4-D)31), an additive (bisphenol A (BPA))32, an

94

antibiotic (tetracycline)33, and a bactericide (triclosan)34 were selected and tested. At

95

different incubation times, the OD600 and residual Mn(II) concentrations of the

96

cultures with MP addition were determined and compared with those of the control

97

(without MP addition). The decrease in residual Mn(II) concentration reflected the

98

production of BMO. Concentrations of Mn(II) were measured using inductively

99

coupled plasma/optical emission spectrometry (ICP-OES, Agilent, USA).

100

Growth and Mn(II) Oxidation of QJX-1 with BP-4 Addition. To investigate the

101

effect of BP-4 on QJX-1 growth and Mn(II) oxidation, QJX-1 strains were cultured in

102

Tyr media containing different concentrations of BP-4 (0, 5, 50, 500, and 5000 µg·L-1).

103

Under different incubation times, the OD600 and the residual concentrations of Mn(II)

104

and Tyr were measured, respectively. The Tyr concentration was analyzed using an



105

Agilent 1290 ultra-performance liquid chromatography (UPLC) system equipped with

106

a Waters ACQUITY UPLC ® HSS T3 column (2.1 mm × 100 mm × 1.8 µm), and

107

identified by an Agilent 6460 triple quadrupole mass spectrometry (USA) (details in

108

Supporting Information (SI), Text S1). BP-4 was determined by a Waters high

109

performance liquid chromatography system equipped with a Waters C18 column (2.1

110

mm × 100 mm × 1.8 µm), and identified by tandem mass spectrometry (Waters, USA)

111

(HPLC-MS) (SI Text S2)35. To further clarify the effect of BP-4 on the QJX-1 growth,

112

the living and dead cells of/from each experiment were quantified by flow cytometry

113

(BD influx, USA) (SI Text S3). Additionally, the scanning electron microscope (SEM)

114

was used to reveal the physiological change in the cells at different BP-4

115

concentrations (SI Text S4).

116

To determine whether BP-4 could be utilized by QJX-1 via metabolic or

117

co-metabolic pathways, a BP-4 (183 mg·L-1) and NH4Cl (19 mg·L-1) mixture and a

118

BP-4 (183 mg·L-1) and Tyr (176 mg·L-1) mixture were tested as energy sources for

119

QJX-1 growth, respectively. The OD600 and residual concentrations of Mn(II), Tyr,

120

and BP-4 were measured after 72 h of cultivation.

121

Role of BP-4 in the Growth and Mn(II) Oxidation of QJX-1.

122

RNA Extraction, Sequencing, and Transcriptomic Analysis. QJX-1 cells with and

123

without BP-4 (50 µg·L-1) were incubated in Tyr medium containing 5.5 mg·L-1 of

124

MnCl2. The cells were harvested after 18 h (before Mn(II) oxidation) and 36 h (during

125

Mn(II) oxidation) by centrifugation at 12000 g for 5 min at 4°C. The cell pellets were


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then subjected to RNA extraction using TRNzol reagent (Tiangen, China) according

127

to the manufacturer’s instructions. RNA integrity was evaluated by an Agilent 2100

128

Bioanalyzer (USA). Only samples with an RNA integrity number above 7.0 were

129

chosen for the next step. RNA was stored at -80°C immediately after extraction until

130

cDNA library construction. Details on RNA sequencing and transcriptomic analysis

131

were listed in Text S5.

132

All original RNA-seq datasets were archived in the NCBI Sequence Read Archive

133

database (Bioproject: PRJNA376037).

134

Protein Analysis. Cell pellets from QJX-1 with and without BP-4 addition were

135

harvested at 18 h as described in the above section. The obtained precipitates were

136

washed three times using phosphate buffer solution and re-dissolved in 1 mL of lysis

137

buffer (8 M urea, 1 mM phenylmethanesulfonyl fluoride, 1 mM cocktail, pH = 8).

138

After centrifugation of the above lysis buffer for 30 min (12000 g) at 4°C, the

139

supernatant was subjected to total protein concentration measurement and protein

140

analysis. This experiment was run in parallel.

141

Total protein concentrations were measured using a BCA Protein Assay Kit

142

(Tiangen, China) according to the manufacturer’s instructions. Based on this, equal

143

amounts of proteins from the two groups (QJX-1 with or without BP-4 addition) were

144

subjected to proteolytic cleavage into peptides and then labeled with a proper dose of

145

TMT (six-plex) labeling reagent. The TMT-labeled peptides were separated for

146

protein analysis (SI Text S6).



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The MS/MS spectra from each LC-MS/MS run were searched against the

148

selected Pseudomonas sp. KT2440 fasta from UniProt using in-house Proteome

149

Discoverer software (Version 1.4; Thermo-Fisher, USA). The search criteria are listed

150

in Text S6 (SI). Relative protein quantification was also carried out using the

151

Proteome Discoverer software according to the manufacturer’s instructions on

152

reporter ion intensities per peptide. Differentially expressed proteins in two groups

153

were identified based on a fold change in relative expression level of > 1.5

154

(upregulation) or < 0.67 (downregulation) (QJX-1 with BP-4 addition vs QJX-1

155

without BP-4 addition).

156

Extracellular Superoxide Detection. The presence of Tyr can interfere with the

157

superoxide

158

chemiluminescence

159

2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3(7H)-one

160

(MCLA)36. This might be because oxidation of the amino acids by peroxy radicals

161

results in the emission of visible CL.37 To eliminate this interference, PYG medium,

162

instead of Tyr medium, was used for incubation of QJX-1 cells. Extracellular

163

superoxide formed from the QJX-1 culture without Mn(II) was detected using an

164

infinite SPARK 10 M microplate reader (Tecan, Swiss).36 At a pre-designed

165

incubation time, 280 µL of bacterial suspension was added into each well of an

166

acid-washed white 96-well plate, and CL emissions were read. Then, 2 µL of MCLA

167

was added to the wells containing QJX-1 culture at room temperature. The CL

168

emissions from the plate were read for 15 min using an acquisition time of 1 s·well-1.

detection

based

on

the

interaction

between

superoxide

(CL)


and probe

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Finally, 5 µL of superoxide dismutase (SOD, 50 KU·L-1) was added to the above

170

culture to prevent reaction between superoxide and MCLA. The CL signal for each

171

well was read for an additional 3 min. The last 10 CL signal values (in stable stage) in

172

each well were averaged before and after SOD addition. The CL intensity of each

173

sample was calculated by subtracting the average CL signal value before MCLA

174

addition from that after MCLA addition. To investigate the relationship between

175

superoxide production and Mn(II) oxidation, QJX-1 cells with Mn(II) addition were

176

also cultivated. The residual soluble Mn(II) concentrations were determined at the

177

time of extracellular superoxide detection. Additionally, to further clarify the

178

relationship between Mn(II) oxidation and Tyr starvation, the residual concentrations

179

of Tyr and Mn(II) were determined when QJX-1 were incubated with gradient of Tyr

180

concentrations (132 mg·L-1, 176 mg·L-1, 220 mg·L-1 and 264 mg·L-1).

181

Removal of BP-4 by BMO and Bacteria. We have investigated and demonstrated

182

that BP-4 cannot be removed through bacterial biodegradation based on the above

183

method, which determined whether BP-4 could be utilized by QJX-1 via metabolic or

184

co-metabolic pathways. The detail was listed in the section “Growth and Mn(II)

185

Oxidation of QJX-1 with BP-4 Addition” of MATERIALS AND METHODS.

186

Therefore, BP-4 may be removed via two remaining ways only, that is, adsorption by

187

BMO and bacteria and/or reaction with BMO. In the incubation experiment, the BMO

188

production was too small to detect any effect on BP-4 fate. So we enriched a large

189

number of BMO by centrifuging 72 h-incubated culture (5 L). To avoid the oxidation

190

possibility of BP-4 by superoxide produced from living bacteria, the collected



191

precipitates were further dried in a freeze drier for the oxidation and adsorption

192

experiments with BMO. The freeze-dried BMO was incubated with 1 mg·L-1 or 5

193

mg·L-1 of BP-4 at 30°C with shaking (170 rpm), respectively. After 72 h of incubation,

194

the BP-4 concentrations were determined using HPLC-MS. To clarify the roles of

195

adsorption and oxidation of BMO in BP-4 removal, BMO was dissolved using

196

ascorbic acid. The BP-4 concentrations were then measured again. The incubation

197

mixtures were also analyzed using an UPLC equipped with a quadrupole-time of

198

flight mass spectrometer (UPLC-QTOF-MS; Waters, USA) to identify the

199

transformation products of BP-4 (SI Text S7).38 In addition, QJX-1 cells were cultured

200

with and without 1 mg·L-1 or 5 mg·L-1 of BP-4 for 72 h at 30°C and 170 rpm,

201

respectively. The BP-4 concentrations were measured to determine the role of living

202

bacteria in the removal of BP-4. Concurrently, QJX-1 cells were collected and

203

freeze-dried. 1 mg·L-1 or 5 mg·L-1 of BP-4 were incubated with the freeze-dried

204

bacteria while shaking (170 rpm) at 30°C. After 72-h incubation, the BP-4

205

concentrations were measured to determine the role of bacterial absorption in the

206

removal of BP-4.

207

Effect of BP-4 on other bacterial growth and Mn(II) Oxidation. Arthrobacter sp.

208

QXT-31 and Sphingopyxis sp. QXT-31 were used to determine whether BP-4 could

209

promote the growth and/or Mn(II) oxidation of other bacteria. For these two strains,

210

Mn(II) oxidation did not occur in the monoculture of either strain, but occurred in the

211

co-culture.39,40 The monocultures and co-culture of these two strains with and without

212

BP-4 (50 µg·L-1) were incubated with 5.5 mg·L-1 of Mn(II). After different incubation


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times, OD600 was measured to investigate the effect of BP-4 on the growth of

214

Arthrobacter sp. and Sphingopyxis sp. We also determined the residual Mn(II)

215

concentration in co-culture of the two strains to evaluate the effect of BP-4 on the

216

Mn-oxidizing capacity of the co-culture.

217

Unless noted, all experiments were run in triplicate.

218

RESULTS AND DISCUSSION

219

Utilization of Natural Organic Matter for QJX-1 Growth. To simplify the

220

incubation system and elucidate the impacts of MPs on the growth and function of

221

QJX-1, we investigated ten amino acids as energy substrates for QJX-1 cultivation

222

(Figure S1). No QJX-1 growth was observed after the addition of lysine, threonine,

223

histidine, phenylalanine, or DL methionine in the media. Based on consumption rates,

224

amino acids supporting growth were, from high to low, tyrosine > glutamic acid >

225

valine ≈ tryptophan > isoleucine. Accordingly, we used Tyr as the sole carbon and

226

nitrogen source for the growth of QJX-1 in the following studies.

227

Effect of Different MPs on QJX-1 Growth and Mn(II) Oxidation. Seven selected

228

MPs (50 µg·L-1) were tested to investigate their impact on QJX-1 pure culture (Figure

229

1). Six MPs exhibited inhibition effects on the growth and Mn(II) oxidation of QJX-1,

230

consistent with previous research.22 Only BP-4 significantly promoted the growth of

231

QJX-1 and its Mn(II) oxidation capacity. To further verify this, we incubated QJX-1

232

with different concentrations of BP-4. We observed that the OD600 significantly



233

increased with increasing BP-4 concentration (Figure S2). Flow cytometry was used

234

to count living and dead cells. As expected, living cells increased with increasing

235

BP-4 concentration, with few dead cells observed (Figure 2A), demonstrating that

236

BP-4 promoted QJX-1 growth. Concurrently, Mn(II) concentration decreased with

237

increasing BP-4 concentration, indicating that the addition of BP-4 also promoted the

238

transformation of Mn(II) to BMO (Figure 2B). Combined the growth and Mn(II)

239

oxidation data, it was deduced that the increased cell growth could account for the

240

earlier onset on Mn(II) oxidation.

241

Utilization of BP-4 by QJX-1. Given the biodegradability of organic compounds, we

242

determined whether QJX-1 could utilize BP-4 as a carbon source to accelerate growth

243

(Figure S3). However, no bacterial growth was observed in media containing BP-4

244

and NH4Cl, suggesting that BP-4 could not serve as a direct carbon source for QJX-1.

245

When the same concentration of BP-4 was mixed with Tyr as a medium, QJX-1 grew

246

and oxidized Mn(II) to BMO. Although some BP-4 removal was observed in the

247

Tyr-containing incubations, taken all together the results suggest that QJX-1 uses Tyr

248

rather than BP-4 as the carbon source for its growth. We concluded that the promotion

249

effect of BP-4 on QJX-1 growth was most likely neither via direct microbial

250

degradation nor via co-metabolism of BP-4. This challenges our understanding about

251

the roles of MPs in bacteria. Thus, it is essential to reveal how BP-4 promotes the

252

growth of QJX-1 and biogenic oxidation of Mn(II).

253

Effect of BP-4 on Metabolic Pathway of QJX-1. We compared the expressed gene


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abundances of QJX-1 with and without BP-4 addition at two incubation times (before

255

and during Mn(II) oxidation). The PCoA of metatranscriptomic data (Figure 3A)

256

indicated that the gene expressions of QJX-1 at the two time points differed obviously;

257

however, the differences between BP-4 addition and no BP-4 addition were not

258

obvious, and did not exceed the differences between replicates. This suggests that

259

BP-4 does not significantly change the basic metabolic function of QJX-1.41

260

Additionally, SEM images showed the addition of BP-4 also did not change the cell

261

morphology (Figure S4).

262

To explore how BP-4 promoted QJX-1 growth, we focused on the differentially

263

expressed genes between QJX-1 with and without BP-4 addition before Mn(II)

264

oxidation (18 h). Based on an FDR < 0.05, 136 transcripts were found to be

265

upregulated and 51 transcripts were found to be downregulated. To reveal the possible

266

metabolic pathway related to QJX-1 growth affected by BP-4, GO pathway

267

enrichment analysis of the upregulated genes was performed. Results showed that the

268

top significantly enriched pathway was the pyruvate metabolic process (FDR = 9.28 ×

269

10-8) (Figure 3B), in which the activities of pyruvate, water dikinase (FDR = 9.59 ×

270

10-6) and pyruvate, phosphate dikinase (FDR = 1.42 × 10-3) were significantly

271

upregulated. These two enzymes are involved in pyruvate carboxylation (Figure S5).

272

Through this pathway, more phosphoenolpyruvate can be produced, with

273

phosphoenolpyruvate gluconeogenesis (FDR = 1.85 × 10-3) then enhanced for glucose

274

production (Figure S5). Accordingly, it appears that BP-4 mainly stimulates pyruvate

275

metabolism and gluconeogenesis of Tyr to accelerate the utilization of Tyr by QJX-1,



276

thus promoting bacterial growth. This conclusion was supported by our findings that

277

QJX-1 consumed Tyr faster after the addition of BP-4 (Figure S6).

278

To explore how BP-4 promoted Mn(II) oxidation of QJX-1, we further searched

279

RNA-seq data and found SOD transcripts and peroxidase transcripts in all samples.

280

This suggests the generation of superoxide and hydrogen peroxide. It has been

281

previously proved that manganese oxide can be generated through a direct peroxidase

282

reaction42,

283

(Table S2). Thus, it satisfies the requirement of Mn(II) oxidation through the above

284

two pathways. We further confirmed that superoxide production was mainly

285

responsible for Mn(II) oxidation based on the significant decrease in Mn(II) oxidizing

286

capacity of QJX-1 when incubated with SOD (it has the capacity of a scavenging

287

superoxide) (Figure S7). To understand the role of BP-4 in the Mn(II) oxidation of

288

QJX-1, the abundances of SOD transcripts and peroxidase transcripts were compared

289

in the QJX-1 cultures with and without BP-4. Results showed that peroxidase activity

290

was significantly upregulated with BP-4 addition (FDR = 4.68 × 10-2) at mRNA level

291

before Mn(II) oxidation (18 h) (Figure 3B, black arrow). Based on this evidence, we

292

performed protein analysis, which revealed that both SOD and peroxidase were

293

upregulated (Figure 4A). Thus, the presence of BP-4 probably accelerated the

294

production of superoxide and hydrogen peroxide, and therefore promoted Mn(II)

295

oxidation of QJX-1. To directly demonstrate this, we compared the production of

296

superoxide and Mn(II) oxidation in QJX-1 cells with and without BP-4 addition

297

(Figure 4B and 4C). Results showed the production of superoxide (reflected in the CL

43

or/and by producing superoxide and degrading hydrogen peroxide44


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signal) was in line with the Mn(II) oxidation. The time-points of CL signal increasing

299

and peaking in the culture with BP-4 were both obviously earlier than that without

300

BP-4 (Figure 5B). Thus, we concluded that the superoxide was generated more

301

rapidly with BP-4 addition. According to previous research, bacterial superoxide can

302

be produced as a stress response to starvation,45 namely superoxide production and its

303

mediated Mn(II) oxidation occur under a substrate-deficient status. To verify this in

304

our culture systems, we investigated the Mn(II) oxidation capacity of QJX-1 at

305

different Tyr concentrations. It was observed that less tyrosine led to the faster

306

formation of manganese oxide (Figure S8).The result directly provides evidence to

307

back up that the Mn(II) oxidation is largely driven by the formation of superoxide in

308

response to starvation. Our previous results also demonstrated this and showed that

309

BP-4 could accelerate the consumption of Tyr (Figure S6), leading to a starvation

310

response and promotion of superoxide generation.

311

Adsorption and Oxidation of BP-4 by BMO. We observed that BP-4 concentration

312

decreased during QJX-1 incubation (Figure S3). Because BMO has high adsorption

313

and oxidation properties for organic matters, 20, 21 we further investigated its role in

314

BP-4 removal. To amplify the effect, we freeze-dried large volumes of in situ-formed

315

BMO by QJX-1 and then mixed the freeze-dried BMO and BP-4 in deionized water

316

for a batch adsorption/oxidation experiment. Figure 5A showed that BP-4 could be

317

significantly removed when in contact with BMO; dissolving BMO by adding

318

ascorbic acid resulted in a significantly decrease in BP-4 removal, due to the release

319

of BP-4 adsorbed by BMO. Additionally, bacterial adsorption was very small. These



320

results reveal that BP-4 removal was mainly due to the adsorption to BMO. During

321

the reaction between freeze-dried BMO and BP-4, an transformation product was

322

observed (Figure 5B) and its structure was proposed (Figure 5C) based on its

323

molecular ion peaks and a previous study on BP-4 oxidation by ozone.46 Above all,

324

we demonstrated that BP-4 could be adsorbed and oxidized by in situ-formed BMO.

325

Effect of BP-4 on the Growth and Mn(II) Oxidization of Other Bacteria. We used

326

Arthrobacter sp. and Sphingopyxis sp. (Figure S9) to investigate whether the

327

promotion role of BP-4 also occurred in other bacteria. Based on previous studies,39,40

328

Mn(II) oxidation did not occur in the monoculture of either strain, but occurred in the

329

co-culture. Results showed that BP-4 inhibited the growth of the monocultures and

330

co-culture of these two strains (Figures S9a–c), and delayed Mn(II) oxidation of the

331

co-culture (Figure S9d). Thus, the promotion effect of BP-4 on growth and function

332

was not universal for all bacteria.

333

ENVIRONMENTAL IMPLICATIONS

334

Micropollutants widely exist in natural and engineered waters and are well

335

accepted as potential inhibitors of the growth and function of microbes. This study

336

showed that the presence of BP-4 (a UV filter) promoted the growth of Pseudomonas

337

sp. QJX-1 (a single bacterium) and biogenic oxidation of Mn(II) but not via its

338

metabolism, which is contrary to our previous understanding. Although this might be

339

a special phenomenon that does not occur frequently in aquatic ecosystems (the

340

promotion effect of BP-4 did not apply to other bacteria (Figure S9)), it still provides


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341

new insight into the interactions between microbes and MPs. That is, our findings

342

highlight the need to consider the combined effects of MPs when studying their

343

impacts on aquatic ecosystems, particularly the co-existence of inhibition and

344

promotion (via metabolism and non-metabolism) on the growth and function of

345

microbes. In addition, it is not yet clear why or how BP-4 interacts with a seemingly

346

unrelated metabolic system, and this knowledge gap will motivate future research.

347

ASSOCIATED CONTENT

348

Supporting Information

349

The Supporting Information is available free of charge on the ACS Publications

350

website. Details include determination of Tyr and BP-4 concentrations, quantification

351

of living/dead QJX-1 cells by flow cytometry, proteomic analysis, analysis of the

352

transformation products of BP-4, and effect of BP-4 on other bacteria, and additional

353

figures and tables.

354

AUTHOR INFORMATION

355

*Corresponding Author

356

* Phone: +86-10-62849160; fax: +86-10-62849160; e-mail: [email protected].

357

Notes

358

The authors declare no competing financial interest.



359

ACKNOWLEDGEMENTS

360

This work is financially supported by the National Natural Science Foundation of

361

China (No. 51420105012, 51578537, and 51290282) and the Chinese Academy of

362

sciences (ZDRW-ZS-2016-5-6). The authors thank the Protein Chemistry Facility at

363

the Center for Biomedical Analysis of Tsinghua University for sample analysis.


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364

FIGURE CAPTIONS

365 366

Figure 1. Effects of different MPs on the growth (t = 18 h, before Mn(II) oxidation,

367

lower subfigure) and Mn(II) oxidation (t = 36 h, during Mn(II) oxidation, upper

368

subfigure) of a Pseudomonas sp. QJX-1. The medium contained 176 mg·L-1 of Tyr (C

369

and N source), 5.5 mg·L-1 of Mn(II), and mineral salts. Each added MP was 50 µg·L-1.

370

The control represents no MP addition. Black and blue dashed lines represent average

371

OD600 and average Mn(II) removal efficiency of the control, respectively. Data are

372

means ± standard deviations for triplicate assays. pH = 7.2, T = 30 ± 0.2°C.



373 374

Figure 2. Effect of different concentrations of BP-4 on the (A) growth and (B) Mn(II)

375

oxidation of a Pseudomonas sp. QJX-1. Data are means ± standard deviations for

376

triplicate assays. pH = 7.2, T = 30 ± 0.2°C. Note: in Figure A, the dead QJX-1 cells

377

with different concentrations of BP-4 added were similar, and therefore the five lines

378

overlap and are shown as only pink.


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379 380

Figure 3. Effect of BP-4 on gene expression of QJX-1. (A) Principal coordinate

381

analysis (PCoA) of metatranscriptomic data retrieved from QJX-1 cells with and

382

without BP-4 after 18 h (before Mn(II) oxidation) and 36 h (during Mn(II) oxidation)

383

of incubation; (B) Significant enrichment analysis of GO terms for genes upregulated

384

in QJX-1 with BP-4 addition compared with those in QJX-1 without BP-4 addition

385

after 18 h of incubation (before Mn(II) oxidation). Gray arrows denote the metabolic

386

pathway related to QJX-1 growth and black arrow denotes the possible metabolic

387

process related to Mn(II) oxidation.



388 389

Figure 4. Effect of BP-4 on superoxide production and Mn(II) oxidation. (A) Fold

390

change of superoxide dismutase (SOD) and peroxidase in BP-4-induced QJX-1 cells

391

compared to that without BP-4 addition. Fold change above the dashed line indicates

392

that the protein is upregulated (> 1.5). Error bars represent average deviation of two

393

biological replicates. (B) Extracellular superoxide detection in QJX-1 cultures

394

incubated with or without BP-4 in PYG medium (without MnCl2); (C) Residual

395

concentration of Mn(II) in QJX-1 cultures incubated with or without BP-4 in PYG

396

medium. Data are means ± standard deviations for triplicate assays. pH = 7.2, T = 30

397

± 0.2°C.


Page 24 of 30

Page 25 of 30


398 399

Figure 5. Adsorption and oxidation of BP-4 by formed biogenic manganese oxide

400

(BMO). (A) Adsorption capacity of dried BMO, dissolved dried BMO (dissolved

401

using ascorbic acid), and living and dried bacteria for BP-4; (B) Total ion

402

chromatogram (full scan mode) of BP-4 (1 mg·L-1), dried BMO, and their mixture in

403

water using UPLC-QTOF-MS. (C) Mass spectra of BP-4 transformation product and

404

BP-4 in dried BMO plus BP-4 reaction. Inserts in (C) are their structures. pH = 7.2, T

405

= 30 ± 0.2°C, t = 72 h.



406

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Benzophenone-4 promotes the growth of a Pseudomonas sp. and

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