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consumption, was responsible for the promotion effect of BP-4 on QJX-1 Mn(II). 16 oxidation. Our findings highlight the dual effects that MPs can have...
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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.

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TOC ART

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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and promotion.

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INTRODUCTION

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Anthropogenic organic compounds,1,

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

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

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

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

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

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

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

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

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of aquatic ecosystems.

2

such as pharmaceuticals and personal care

Bacteria can affect MP fate directly (biodegradation15,

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or co-metabolism of

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

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21

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

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

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

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

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

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remains unknown whether there are exceptions to this rule.

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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,

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

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

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

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

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

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

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

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

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might affect bacterial growth and function.

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MATERIALS AND METHODS

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Bacterial Strain and Culture Preparation. The manganese-oxidizing bacterium

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

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

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

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

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

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Culture Center (CGMCC; accession number 6630).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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(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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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concentrations (SI Text S4).

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

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

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

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

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

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

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

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

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

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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

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

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

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

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cDNA library construction. Details on RNA sequencing and transcriptomic analysis

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were listed in Text S5.

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

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database (Bioproject: PRJNA376037).

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

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

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

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

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

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supernatant was subjected to total protein concentration measurement and protein

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analysis. This experiment was run in parallel.

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Total protein concentrations were measured using a BCA Protein Assay Kit

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

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

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

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

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protein analysis (SI Text S6).

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

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

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

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

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Proteome Discoverer software according to the manufacturer’s instructions on

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

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

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

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without BP-4 addition).

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

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superoxide

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chemiluminescence

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

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

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

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

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

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

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

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

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

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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)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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removal of BP-4.

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

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

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

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

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

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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

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

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

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Mn-oxidizing capacity of the co-culture.

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Unless noted, all experiments were run in triplicate.

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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earlier onset on Mn(II) oxidation.

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

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

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

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

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

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

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

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

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

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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

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

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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

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

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

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

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

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

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

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morphology (Figure S4).

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

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

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

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

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

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

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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).

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Through this pathway, more phosphoenolpyruvate can be produced, with

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

274

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

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

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

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

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

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

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

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

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reaction42,

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

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two pathways. We further confirmed that superoxide production was mainly

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

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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

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

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

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

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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

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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

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

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(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

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

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

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

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

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

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

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

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

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

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

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

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response and promotion of superoxide generation.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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was not universal for all bacteria.

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ENVIRONMENTAL IMPLICATIONS

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Micropollutants widely exist in natural and engineered waters and are well

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

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

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

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

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

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

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

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

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

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

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

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

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ASSOCIATED CONTENT

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Supporting Information

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

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

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

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

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figures and tables.

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AUTHOR INFORMATION

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*Corresponding Author

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

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS

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

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

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

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

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FIGURE CAPTIONS

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

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

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

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

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

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

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

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

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

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

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

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overlap and are shown as only pink.

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

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

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

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

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

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

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

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process related to Mn(II) oxidation.

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

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

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

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

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

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

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

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

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± 0.2°C.

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

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

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

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

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

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

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= 30 ± 0.2°C, t = 72 h.

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