Structures of nitroaromatic compounds induce Shewanella oneidensis MR-1 to adopt different electron transport pathways to reduce the contaminants

Journal Pre-proof Structures of Nitroaromatic Compounds Induce Shewanella oneidensis MR-1 to Adopt Different Electron Transport Pathways to Reduce the Contaminants Hefei Wang, He-Ping Zhao, Lizhong Zhu

PII:

S0304-3894(19)31449-9

DOI:

https://doi.org/10.1016/j.jhazmat.2019.121495

Reference:

HAZMAT 121495

To appear in:

Journal of Hazardous Materials

Received Date:

23 February 2019

Revised Date:

15 October 2019

Accepted Date:

17 October 2019

Please cite this article as: Wang H, Zhao H-Ping, Zhu L, Structures of Nitroaromatic Compounds Induce Shewanella oneidensis MR-1 to Adopt Different Electron Transport Pathways to Reduce the Contaminants, Journal of Hazardous Materials (2019), doi: https://doi.org/10.1016/j.jhazmat.2019.121495

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Structures of Nitroaromatic Compounds Induce Shewanella oneidensis MR-1 to Adopt Different Electron Transport Pathways to Reduce the Contaminants Hefei Wang1, 2, He-Ping Zhao1, Lizhong Zhu1, 2, *

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1. College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China;

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2. Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control,

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Hangzhou 310058, China.

*Corresponding Author: Prof. Lizhong Zhu, 1. College of Environmental and

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Resource Sciences, Zhejiang University, Hangzhou 310058, China; 2. Key Laboratory of Organic Pollution Process and Control, Zhejiang province, Zhejiang

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University, Hangzhou 310058, China; E-mail: [email protected]

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Graphical abstract

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Highlights

• Shewanella oneidensis MR-1 (MR-1) effectively reduced nitroaromatic compounds

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(NACs).

• Through constructing mutant stains, we found ten NACs with single benzene ring

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and short alkyl chain were reduced via both Mtr respiratory pathway and NfnB

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related pathway.

• Three NACs, 2,5-ditertbutyl-nitrobenzene, 2-nitrobiphenyl and 2,2'-dinitrobiphenyl were reduced only via Mtr respiratory pathway.

• van der Waals volume is the possibly primary leading factor for the different electron pathways employed by MR-1 to reduce NACs.

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Abstract Nitroaromatic compounds (NACs) are one class of typical refractory biodegradable organic pollutants detected in various environmental media. The reductive transformation of NACs by the electrochemically active bacterium Shewanella oneidensis MR-1 is a possible and cost-effective option for the removal of NACs. However, little information on the respiratory pathway employed by S. oneidensis

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MR-1 to reduce NACs is available. In the present study, we investigated the reduction

of NACs with different nitro or alkyl moieties by S. oneidensis MR-1 and eight

constructed mutants with the deletion of the mtrA, mtrB, mtrC/omcA, cymA, napA,

4-nitrotoluene,

1,2-dinitrobenzene,

4-ethylnitrobenzene,

1-tert-butyl-4-nitrobenzene,

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nitrobenzene,

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napB, nrfA, and nfnB genes to explore these key functional enzymes. The reduction of

1,3-dinitrobenzene,

1,4-dinitrobenzene,

2,4-dinitrotoluene,

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2,6-dinitrotoluene and 2,4,6-trinitrotoluene occurs via both the Mtr respiratory

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pathway and NfnB related pathway. However, 2,5-ditertbutyl-nitrobenzene, 2-nitrobiphenyl and 2,2'-dinitrobiphenyl are reduced only via the Mtr respiratory

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pathway. The van der Waals volume of NACs was the key factor in determining the reduction by S. oneidensis MR-1 according the correlation analysis. Our study

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provides new insights into the environmental adaption of S. oneidensis MR-1 and facilitates the bioremediation of NAC-related contamination.

Key word: Nitroaromatic pollutants; Shewanella oneidensis MR-1; Reduction mechanism; Molecular structures 3

1. Introduction Nitroaromatic compounds (NACs) are widely used as nitroaromatic explosives and raw materials for industrial manufacturing and are one kind of refractory biodegradable organic pollutants detected in soil and groundwater [1-3]. Due to the electron-deficient property of aromatic nitro groups, removal of NACs by oxidation

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reactions is typically difficult or poses a threat to living organisms, as reaction products which are more toxic than the parent compound can be formed under certain conditions [2, 4]. Reductive transformation, especially bio-reduction, has been

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considered a reliable and cost-effective alternative for the removal of NACs with high activation energy [5]. A number of recent studies have therefore focused on the

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reduction of the nitro group, the isolation of effective degrading strains, and the

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characterization of nitroreductases [3, 5, 6]; however, research on biodegradation mechanisms at gene level is limited.

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The bacterium Shewanella can reduce diverse electron acceptors including NACs owing to its incredible respiratory versatility [7, 8] and has great

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bioremediation potential for contaminated soil and water [9-11]. Recently, it has been

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found to respire on various NACs [9]. In fact, Shewanella has as many as 42 c-type cytochromes, which constitute a complicated respiratory network [12-15]. These cytochromes can be found in pathways such as the Mtr respiratory pathway and the periplasmic nitrate reduction system (NAP). The Mtr respiratory pathway includes cytochrome CymA attached to the cytoplasmic membrane [12, 16], a multiheme complex MtrCAB and dozens of periplasmic terminal reductases [13, 14]. The 4

respiratory chain extends electrons transferred from the oxidation of a carbon source to the cell surface or periplasm where the contaminants (electron acceptors) are reduced [15]. The NAP system encoded by operon napDAGHB and the nrfA gene was considered to be responsible for nitrate reduction according to the latest annotation [16]. Recently, napA, napB and the nrfA gene were reported to be essential for the reduction of nitrate and nitrite [17]. Considering NACs share a nitro group with

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nitrate, the NAP system may also be responsible for the reduction of NACs. However,

whether NACs are reduced by Shewanella strains via the Mtr respiratory pathway or

via the NAP system is still unknown. Other electron transport pathways and key

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functional proteins in Shewanella cells also need to be investigated.

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The reductive degradation of oxidized contaminants by Shewanella may proceed through different pathways depending on the properties of the contaminant,

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including molecular weight, hydrophobicity, and polarity [18-21]. Researchers have

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reported that some sparingly soluble or macromolecular organic environmental pollutants are unable to pass through the outer membrane, so their anaerobic reduction

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by Shewanella proceeds mainly via the Mtr respiratory pathway [19, 22, 23]. In contrast, freely diffusible gases or soluble species such as fumarate, nitrite, nitrate,

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trimethylamine N-oxide and thiosulfate are usually metabolized in the periplasm or cytoplasm [18, 24, 25]. What is perhaps surprising, however, is that a number of substrates that might typically be classified as ‘insoluble’ like nitrobenzene (NB) and 2,6-dinitrotoluene can also be partly reduced in the periplasm or cytoplasm [18, 19]. The reasons why this happens are still unknown. In addition to solubility, the impact 5

of the molecular structure, hydrophobicity, and polarity on the reductive degradation of NACs by Shewanella also remains unclear. In the present study, we investigated the transformation of 13 NACs by Shewanella oneidensis MR-1. By constructing mutant strains with the deletion of mtrA, mtrB, mtrC/omcA, cymA, napA, napB, nrfA, and nfnB genes, we compared the bio-reduction of NACs by S. oneidensis MR-1 and its mutant strains, and evaluated

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the roles of the Mtr respiratory pathway, NapAB, NrfA and NfnB in NAC

bio-reduction. Understanding how the NACs’ properties affect the removal

performance by Shewanella will help us to further understand the respiratory

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mechanism of NACs and provide more practical guidance for NAC remediation.

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2. Materials and methods 2.1 Bacterial strains

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S. oneidensis MR-1 and Escherichia coli WM3064 were grown aerobically in

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Lysogeny broth (LB) media at 30 °C and 37 °C, respectively. When required, the media were solidified by using 1.5% (wt/vol) agar. Diaminopimelic acid (DAP) was

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added with a final concentration of 100 μg∙mL-1 to allow the growth of the conjugation strain E. coli WM3064. Chloramphenicol at 34 μg∙mL-1 and 10 μg∙mL-1

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was added to the media for the cultivation of E. coli and Shewanella, respectively. 2.2 Strain constructions Plasmid and chromosomal DNAs were isolated using the SanPrep Column Plasmid Mini-Preps Kit (Sangon Biotech Co., Shanghai, China) and the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech Co.), respectively. A PCR 6

Amplification Kit (Takara Co., Dalian, China) was used to amplify the desired gene fragments. More information on primers and plasmids is listed in Table S1. Double Restrictive Digest enzymes were purchased from New England Biolabs Ltd (Beijing, China), and ligases were purchased from Vazyme Biotech Co., Ltd (Nanjing, China). Mutant strains with engineered defects in genes ∆mtrA, ∆mtrB, ∆mtrC/omcA, ∆cymA, ∆napA, ∆napB, ∆nrfA and ∆nfnB were constructed as described in Text S1 [26].

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2.3 Contaminants

Thirteen NACs were selected as model compounds for this study: NB (98-95-3), 1,2-dinitrobenzene (1,2-DNB, 528-29-0), 1,3-dinitrobenzene (1,3-DNB, 99-65-0), (1,4-DNB,

100-25-4),

4-nitrotoluene

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1,4-dinitrobenzene

(4-NT,

99-99-0),

118-96-7),

1-tert-butyl-4-nitrobenzene

4-ethylnitrobenzene (4-TNB,

3282-56-2),

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(2,4,6-TNT,

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2,4-dinitrotoluene (2,4-DNT, 121-14-2), 2,6-DNT (606-20-2), 2,4,6-trinitrotoluene (4-ENT,

100-12-9),

2,5-ditertbutyl-nitrobenzene

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(2,5-DTNB, 3463-35-2), 2-nitrobiphenyl (2-NPL, 86-00-0) and 2,2'-dinitrobiphenyl (2,2’-DNPL, 2436-96-6). Detailed information on the 13 exposure chemicals is shown

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in the Supporting Information (Table S2). 2.4 Reduction experiments

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Every spike in the reduction experiment was performed in triplicate. A synthetic

medium was used for anaerobic NACs bio-reduction tests, which contained (per litre): 3.74 g DL-sodium lactate, 11.91 g HEPES, 0.3 g NaOH, 1.5 g NH4Cl, 0.1 g KCl, 0.67 g NaH2PO4∙2H2O, 5.85 g NaCl. Fifty millilitres of the defined medium was added into each serum vial, followed by N2 aeration for 20 min. The serum vials were then 7

sealed with butyl rubber stoppers and autoclaved. Vitamins, amino acids, and trace mineral stock solutions were used to supplement the medium. NACs were subsequently added to the medium, and the initial concentration of NAC in each serum vial was set at 20 mg∙L-1. All S. oneidensis MR-1 and mutant strains were cultured in LB media at 30 °C, up to an OD600 of 2.3–2.6. Then, cells from these precultures were pelleted by centrifugation at 4000 g for 10 min and washed twice,

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and finally were resuspended in the defined medium. The initial OD600 value of MR-1 and mutant cells in the serum vial was 0.1. Triplicate bottles amended with

equal volumes of sterile medium, NACs, and an equal amount of heat-killed strains

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were set as negative controls. At each time interval, 1 mL sample was withdrawn and

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immediately centrifuged at 4000 g for 5 min. Concentrations of contaminants were measured in the supernatant. The collected cells were used for the gene expression

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2.5 Sample analysis

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analysis after the beginning of reduction.

The concentration of 2,5-DTNB was measured on a gas chromatography-mass

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spectrometer (GC/MS) instrument (7890B/5977A, Agilent Technologies, Santa Clara, USA) with an electron ionization (EI) ion source. The compound was separated on a

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DB-5 MS capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness) with helium as the carrier gas at a constant flow rate of 1 mL∙min-1. The oven temperature was initially 40 °C and then increased at a rate of 10 °C∙min-1 to 280 °C. The quantitative measurement was set at selected ion monitoring (SIM) mode. The monitored ion was m/z 190.0 and the retention time was 19.21 min. The concentrations of other NACs 8

were determined using an Agilent 1200 high performance liquid chromatograph (HPLC) equipped with a C18 reversed-phase column (5 μm, 4.6 mm ×150 mm) and an ultraviolet (UV) detector. To determine the concentration of NB, 1,2-DNB, 1,3-DNB, 1,4-DNB, 2,4-DNT, 2,6-DNT, 4-NT, 4-ENT, 4-TNB and 2,4,6-TNT, a gradient elution method was used with methanol-water as the mobile phase. The gradient elution procedure was performed as follows: 45% (v/v) methanol at 0 min,

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90% (v/v) methanol from 1.0 to 3.0 min, and 45% (v/v) methanol from 3.1 to 8.0 min.

The UV detector was set at 254 nm and the flow rate was kept as 0.3 mL∙min-1. The monitored peaks appeared at 4.58 (NB), 3.8 (1,2-DNB), 5.45 (1,3-DNB), 5.51

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(1,4-DNB), 6.13 (2,4-DNT), 6.16 (2,6-DNT), 6.71 (4-NT), 5.02 (7-ENT), 7.35

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(4-TNB) and 3.96 min (2,4,6-TNT). For the determination of 2-NPL and 2,2'-DNPL, the mobile phase was methanol/water (70/30, v/v) with a flow rate of 1.0 mL∙min-1.

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The UV detector was set at 230 nm and the retention times are 2.96 and 3.21 min,

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respectively.

2.6 Quantitative reverse transcription PCR

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RNA was extracted from 1.0 ml of cell suspension after 4 h incubation with NACs (OD600, 01-0.2) and frozen at -80 °C using an RNAprep pure kit (Tiangen

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Biotech Co., Beijing, China), according to manufacturer’s instructions and including the optional DNase treatment step. Then 2 μg RNA were reverse transcribed to cDNA using the FastKing gDNA Dispelling RT SuperMix (Tiangen Biotech Co.). Finally, 2 μL cDNA were analyzed using qPCR targeting the enzyme in the Mtr respiratory pathway and the intracellular reductase NfnB using Bestar MasterMix (SYBR Green) 9

(DBI, Ludwigshafen, Germany). The analysis was performed on the StepOne Real-Time PCR System (Applied Biosystems, USA). The primers used in the RT-PCR and standard curves are shown in Table S3. PCR parameters consisted of 10 min of Taq activation at 95 °C, followed by 40 cycles of PCR at 95 °C × 15 s, 55 °C × 30 s, 72 °C × 1.5 min and a final melt curve stage conducted from 60 to 95 °C. 2.7 Statistical Analysis

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The results are presented as mean ± standard error (n = 3). To understand the

interaction network between NfnB and other proteins in S. oneidensis MR-1, we did protein-protein interaction (PPI) analysis using STRING software [27]. Similarly,

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associations between CymA and other proteins were also analyzed (not shown). The

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reduction rates (r) of NACs by MR-1 wild type and mutants can be described by the following equation:

𝑑(𝐶𝑁𝐴𝐶𝑠 )

= 𝑘 ∙ (𝐶𝑁𝐴𝐶𝑠 )1

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𝑟=

𝑑𝑡

(1)

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where 𝐶𝑁𝐴𝐶𝑠 is the NAC concentration at different times, k is the rate constant and t is the reaction time. After integration, equation (1) can be transformed to equation (2) (2)

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− 𝑙𝑛 𝐶𝑁𝐴𝐶𝑠 = 𝑘 ∙ 𝑡

The value of k was determined by plotting − 𝑙𝑛 𝐶𝑁𝐴𝐶𝑠 against t.

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For expression of cymA and nfnB genes in S. oneidensis MR-1, a one-way

analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) test was performed using SPSS Statistics 20.0. The molecular descriptors including physical properties, chemical properties, and some molecular configuration indexes were chosen based on the previous studies, and their values were obtained from online 10

molecular descriptors data base MOLE db (http://michem.disat.unimib.it/mole_db/) [28]. Significant differences were based on p-values < 0.05. Correlation of molecular descriptors of NACs with the reduction was analyzed by linear regression using SPSS Statistics 20.0.

3. Results 3.1 Screening of genes responsible for the reduction of NACs

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To identify genes responsible for the reduction of NACs, we analysed the S. oneidensis MR-1 genome for proteins probably involved in this process. Three kinds of enzymes or protein complexes may be directly responsible for reduction of NACs:

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the Mtr respiratory pathway, the terminal reductase NapAB, NrfA in periplasm and

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NfnB enzyme in cytoplasm. Mutants with different phenotypes deleted in mtrA, mtrB, mtrC/omcA, cymA, napA, napB, nrfA and nfnB were constructed and used for

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reduction performance tests to screen for genes responsible for the reduction of NACs.

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Figure 1 shows the NB reduction performance of S. oneidensis MR-1 and its mtr-deleted mutant strains. NB was reduced effectively in 78 hours by the wild type

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culture with a reduction rate constant of 0.042 h-1. The reduction rate constant for NB by ∆napA, ∆napB, and ∆nrfA were 0.038, 0.039 and 0.048 h-1, respectively, which

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were comparable to that of the wild type strain. This results show that the S. oneidensis MR-1 strains defective in napA, napB, nrfA displayed no defects in comparison of wild MR-1 strain (Figure 1), suggesting that the napA, napB, and nrfA genes were not responsible for NB reduction. In contrast, mtrA, mtrB, or omcA/mtrC deletion mutant strains retained most of their degradation capabilities for NB but with 11

a lower reduction rate (the reduction rate constants are shown in Table 1). The slower reduction rates indicate that the Mtr respiratory pathway is involved in the reduction of NB, but is probably not the only electron transport pathway. The deletion of cymA and nfnB resulted in a significant drop in NB bio-reduction rate by 64.3% and 61.9%. The significant inhibition in NB reduction by ∆cymA and ∆nfnB implies that they play an important role in reducing this electron acceptor. The reduction mechanisms of

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other contaminants were same as NB reduction by MR-1 (data not shown).

Because the results indicated that the cymA and nfnB play important roles in the reduction of NB, PPI analysis was used to further understand the interaction network

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between NfnB and other proteins in S. oneidensis MR-1. NfnB is annotated as an

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oxygen-insensitive NAD(P)H nitroreductase, which is localized to cytoplasm. Ten proteins including Sye1, Sye2, Sye3, FadH, CobS, CobO, CobT, GltB, PhhA,

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SO_1667 participated in the interactions with NfnB (Figure S1). Interestingly, there

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was no interaction between NfnB and the Mtr respiratory pathway, meaning that these two electron transfer pathways are distinct and independent. NB is thus reduced by

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two non-overlapping electron transport pathways in S. oneidensis MR-1. 3.2 Reduction of different NACs by MR-1 and mutants

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Thirteen NACs were chosen for testing whether S. oneidensis MR-1 adopts

different electron delivery pathways to reduce NACs with different branched substituent groups. To investigate the impacts of nitro group number on electron transport pathway, 4-NT, 2,4-DNT and 2,4,6-TNT were selected as parallel electron acceptors. Figure 2 shows that the NACs with more nitro groups were removed much 12

faster. The reduction rate constants by wild type cultures were 0.017 for 4-NT, 0.317 for 2,4-DNT, and 1.183 h-1 for 2,4,6-TNT (data shown in Table 1, the reduction process followed first-order kinetics model). Compared with the wild type strain, reductions of 4-NT, 2,4-DNT and 2,4,6-TNT by the ∆cymA were greatly suppressed, with a decrease of 59.2%, 78.3% and 63.8% in the reduction rate, respectively. The reduction rate constants also decreased (by -29.4% to 48.5% compared with the wild

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type) for mutants ∆mtrA, ∆mtrB and ∆mtrC/omcA, demonstrating that the Mtr respiratory pathway was needed for the complete reduction of NACs. Knockout of nfnB also resulted in a significant drop in the bio-reduction of the three NACs. The

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reduction rate constants decreased by over 50% compared with the wild type strain.

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These results indicated both the Mtr respiratory pathway and NfnB protein are involved in 4-NT, 2,4-DNT and 2,4,6-TNT reduction.

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To study the effect of position of the nitro groups, 1,2-DNB, 1,3-DNB and

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1,4-DNB were selected as parallel electron acceptors. Little difference was found among their reduction profiles (Figure S2), suggesting that the position of nitro

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substitutes did not have a significant effect on reduction by S. oneidensis MR-1. Figure 3 depicts the reduction of NACs with different alkyl groups (4-NT,

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4-ENB, 4-TNB and 2,5-DTNB) by S. oneidensis MR-1 and its mutants. As shown in Table 1, the wild type culture was able to reduce 2,5-DTNB (rate constant = 0.003 h-1), 4-TNB (0.010 h-1) and NB (0.042 h-1). Compared with the wild type strain, dramatic changes were observed in the reduction of the four NACs by the mutant defective in cymA. Changes, albeit less dramatic, were also observed in the mtrA, 13

mtrB, and mtrC/omcA deletion strains, demonstrating that the Mtr respiratory pathway was needed for the complete reduction of 4-NT, 4-ENB, 4-TNB and 2,5-DTNB. The reduction rate constants of 4-NT, 4-ENB and 4-TNB by ∆nfnB decreased by more than 50% compared with the wild type, which indicated the crucial contribution of NfnB in reducing these three NACs. However, no decrease was observed in the reduction of 2,5-DTNB by nfnB deletion strains, with the reduction constant of 0.0024

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h-1 compared with 0.0022 h-1. Therefore, while the NfnB enzyme is clearly necessary for the reduction of 4-NT, 4-ENB and 4-TNB, it might not be the key enzyme for the reduction of 2,5-DTNB.

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To test the removal kinetics of NACs containing diphenyl rings by S. oneidensis

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MR-1 strains, 2-NPL and 2,2’-DNPL were selected as parallel electron acceptors. As shown in Table 1, the reduction rate constants for 2-NPL and 2,2’-DNPL by MR-1

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wild type were 0.012 and 0.028 h-1, respectively. The cymA, mtrA, mtrB and

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mtrC/omcA deletion mutants showed severe defects in their ability to reduce 2-NPL compared with the wild type strain. The ∆cymA mutant was only able to reduce 55%

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of 2-NPL, in contrast with the 90% reduced by the wild type strain. And the reduction profiles of 2-NPL by ∆mtrA, ∆mtrB, or ∆mtrC/omcA perfectly conforms to that of

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mutants defective in cymA (Figure 4a). However, ∆nfnB displayed no defect in reducing 2-NPL compared with the wild type strain. The Mtr respiratory pathway is therefore the key pathway involved in the reduction of 2-NPL, and cytoplasmic enzyme NfnB is not involved in this process.

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As shown in Figure 4b, though the reduction of 2,2'-DNPL had much faster rates compared with 2-NPL, their reduction patterns were similar: the Mtr respiratory pathway was the key pathway involved in the reduction of nitro-biphenyl and cytoplasmic enzyme NfnB was not involved. 3.3 The effect of NACs on the expression of nfnB and omcA-mtrABC in MR-1 To test whether the nfnB gene, cymA gene and omcA-mtrABC gene cluster

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abundances were differentially expressed when S. oneidensis MR-1 was respiring on

different NACs, we then carried out reverse-transcription PCR analysis (Figure 5 and

Figure S3). The cymA abundances ranged from (7.00.39)×104 to 1.09 0.10)×105 and

the

mtrB

abundances

ranged

from

(4.120.59)×105

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copies∙mL-1

to

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(9.840.46)×105 copies∙mL-1. It indicates that the expression level of each gene in cymA and omcA-mtrABC was quantitatively compared after cultivation of MR-1 cells

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for 4 hours in the presence of different NACs. The abundances of nfnB gene (for

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intracellular reduction of NACs) in MR-1 inoculated in NB, 2,4-DNT and 4-ENB were greatly induced in comparison with a slight induction for that in the cells

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inoculated in 2 5-DTNB and 2,2’-DNPL (5- to 24-fold) (Figure 5b). These results confirm that the nfnB gene is not involved in the anaerobic reduction of 2,5-DTNB,

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2-NPL and 2,2’-DNPL.

4. Discussion 4.1 The possible electron transport pathway In the present study, we have screened eight mutant strains of S. oneidensis MR-1 with deletions in functional genes and investigated the reduction mechanisms 15

of 13 NACs by wild type and mutant strains. The kinetic studies indicated that reduction of NACs with fewer alkyl groups and more nitro groups proceeded at a more rapid rate. This observation is likely because more nitro groups and fewer alkyl groups will lead to stronger electronegativity (evidenced by sum of Kier-Hall electrotopological states shown in Table 2) of the nitrogen atoms in NACs. The stronger the electronegativity of the nitrogen atom, the easier the reaction will be. The

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position of alkyl and nitro groups had little impact on the reduction rates (Table 1 and

Figure S2). Accordingly, the reduction rates of NACs are ranked as follows:

4-ENB > 4-NT > 4-TNB > 2-NPL > 2,5-DTNB.

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1,4-DNB > 1,2-DNB > 1,3-DNB > 2,4-DNT > 2,6-DNT > NB > 2,2’-DNPL >

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Mutants with different phenotypes corresponding to deletions in mtrA, mtrB, mtrC/omcA, cymA, napA, napB, nrfA and nfnB were constructed and their reduction

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of NACs was observed. The reduction of NACs by S.oneidensis MR-1 suggested that

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only the Mtr respiratory pathway was involved in the anaerobic reduction of 2,5-DTNB, 2-NPL and 2,2’-DNPL. Both the intracellular enzyme NfnB and the Mtr

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respiratory pathway located at the outer membrane in S. oneidensis MR-1 were involved in the reduction for the other NACs. PPI network analysis indicated no

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interaction between NfnB and CymA, MtrA, MtrB or MtrC (members of the Mtr respiratory pathway), which implies that NfnB is involved in a separate electron transfer pathway from the Mtr respiratory chain. This result is also consistent with previous work [18]. The differences in reduction mechanism were further verified by examining gene expression. All of the genes in the Mtr respiratory pathway (cymA, 16

mtrA, mtrB and mtrC) were expressed after inoculation of MR-1 cells with different NACs (Figure 5, Figure S3). However, the expression level of nfnB gene in MR-1 cells inoculated in 2,5-DTNB, 2-NPL and 2,2’-DNPL was only induced slightly in comparison with the significant induction in the cells inoculated with the other NACs. This result further corroborates the hypothesis that nfnB gene is not involved in the reduction of 2,5-DTNB, 2-NPL or 2,2’-DNPL. The deletion of cymA dramatically

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reduced the capability of the mutant to reduce NACs, indicating that CymA is a key protein involved in the bio-reduction of NACs. Previous researchers have demonstrated that CymA is a member of the NapC/NirT family of menaquinol

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dehydrogenases and is essential for the respiratory reduction of nitrate, nitrite,

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fumarate, DMSO, soluble complex forms of Fe(III) and extracellular particulate substrates [12].

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Based on our observations, we propose a possible model for the electron delivery

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path. For the reduction of 2,5-DTNB, 2-NPL and 2,2’-DNPL, electron flow from the menaquinone pool (MQ) to the membrane-bound c-type cytochrome CymA continues

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through the Mtr respiratory enzyme complexes composed of MtrA, MtrB, MtrC and OmcA. MtrC and OmcA are located on the extracellular face of the outer membrane,

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where they directly transfer electron to these pollutants. In addition to this pathway, the other 10 NACs may also partly enter into the cells and then are reduced by the nitroreductase NfnB in cytoplasm. It is still unclear why the reduction mechanisms for 2,5-DTNB, 2-NPL and 2,2’-DNPL are different from the other NACs. One possible explanation is that diphenyl rings and alkyl groups with long carbon chains can 17

change the properties of NACs (such as increase the molecular dimension and hydrophobicity), which may further induce the MR-1 cells to reduce them extracellularly [18]. 4.2 Key properties of NACs influencing the electron transport pathway Correlations between reduction of NACs by mutants defective in nfnB and their properties were analyzed. The reduction efficiencies of NACs by ∆nfnB (Table 2)

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were recorded when the removal rate by the wild type strains reached 50%. This

reduction efficiency would be used for estimating the impacts of nfnB deletion on

reduction of NACs. The three maximum values of removal efficiency were 42%, 44%

-p

and 52% observed in the reduction of 2,5-DTNB, 2-NPL and 2,2'-DNPL, respectively.

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The removal rates of the other 10 NACs by ∆nfnB were below 33%, indicating that the reduction of 2,5-DTNB, 2-NPL and 2,2'-DNPL was only affected slightly after the

lP

deletion of nfnB gene. This is in agreement with the main conclusion that the nfnB

na

gene is not responsible for the reduction of 2,5-DTNB, 2-NPL and 2,2'-DNPL. The correlations between reduction efficiency and 10 molecular descriptors of

ur

NACs were analysed (Table 2, Figure 6). The van der Waals volume (p < 0.01), total atom number, polarizability and molecular weight of NACs were positively correlated

Jo

with the reduction efficiencies of them by nfnB deletion strains (p < 0.05). The van der Waals volume was considered as the primary factor for the reduction mechanism transformation of NACs because of the strongest correlation. The reduction of NACs with larger size and higher polarizability should thus be relatively less affected after the deletion of nfnB gene, which is in accordance with our observations. One possible 18

reason for this phenomenon could be that smaller molecules like NB, 2,4,6-TNT, and 4-TNB could partly enter into the cells and be reduced in the cytoplasm with the involvement of NfnB. The deletion of nfnB can thus have great impact on the reduction efficiency. However, 2,5-DTNB, 2-NPL and 2,2'-DNPL are too large to penetrate the outer membrane of MR-1 and are thus reduced entirely outside the cells. The Log Kow and asphericity coefficient (Figure 6) were slightly correlated with the

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involvement of NfnB, suggesting that factors related to hydrophobicity and shape of

NACs partly contributed to the choice of reduction mechanism. This is markedly different from previous observations that sparingly soluble electron acceptors were

-p

mainly reduced extracellularly by MR-1 [19, 23]. A possible reason for the difference

re

is that previous studies were all conducted with different electron acceptors. The vastly different natures of varied electron acceptors can all have great impacts on the

lP

reduction mechanism. It is hard to identify the dominant factor in acceptor reduction

na

without considering the impacts of other properties. The specific reduction mechanism used for any given electron acceptor is likely a comprehensive result of

ur

many influencing factors, like van der Waals volume and polarizability. How to quantitatively determine the contribution of these numerous factors needs further

Jo

study. The sum of Kier-Hall electrotopological states, which represents the electronegativity of NACs, was a key factor influencing the reduction rate and had no correlation to the involvement of NfnB. The differences in reduction mechanisms provide guidance for the bioremediation of NAC contamination. Larger NACs are mainly reduced by MR-1 via the Mtr 19

respiratory pathway. We can enhance the reduction process by accelerating the electron transfer between the Mtr respiratory pathway and extracellular pollutants. However, for the reduction of smaller NACs, both methods to enhance activities of the Mtr respiratory pathway and catalysis of NfnB can be used. Some preliminary experiments were conducted to find proper enhancement methods. Different dosages of riboflavin, which is widely used as an electron mediator, were added to the wild

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type cultures with 2,4-DNT and 2,2’-DNPL as electron acceptors (Figure S4). After riboflavin was exogenously added, an obvious increase of the reduction rate of 2,4-DNT and 2,2’-DNPL by MR-1 was observed, and more riboflavin

-p

supplementation led to a slight increase in the reduction rate of NACs. This result is

re

consistent with previous reports that riboflavin enhanced the reduction of methyl orange and nitrobenzene [10, 18]. Other materials, such as biochar and humic acid can

lP

also be used to promote the extracellular electron transport process [29, 30].

na

Intracellular reduction processes relying on the NfnB can be enhanced by promoting the transmembrane transport of NACs and enzymatic activity, and the specific

ur

enhancement methods warrant further research. 5. Conclusion

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In this work, we demonstrate that different mechanisms were employed by S.

oneidensis MR-1 to reduce NACs with different structures. van der Waals volume of NACs is the primary factor for the transformation of reduction mechanism. For the reduction of molecules with small sizes, like nitrobenzene, 2,4,6-trinitrotoluene, and 1-tert-butyl-4-nitrobenzene, both the nitroreductase NfnB in cytoplasm and the Mtr 20

respiratory pathway on the outer membrane of MR-1 cells are involved. However, 2,5-ditertbutyl-nitrobenzene, 2-nitrobiphenyl and 2,2'-dinitrobiphenyl were reduced only via the Mtr respiratory pathway. Polarizability and molecular weight also partly contribute to the choice in reduction mechanism. Insight into the reduction mechanism of NACs with different structures is valuable to understand environmental adaption of Shewanella strains and other bacteria. Considering that the reduction of

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NACs can take different pathways dependent on different molecular properties, we can accordingly choose suitable enhancement methods for better removal efficiency

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for bio-remediation.

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Acknowledgements

This project was supported by the National Key Research and Development Program

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of China (2017TFA0207002), the Science and Technology Research Program of

na

Zhejiang (2015C03022), the National Natural Science Foundations of China (21621005), and the Fundamental Research Funds for the Central Universities

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(2019FZA6005). We thank Professor Hanqing Yu from University of Science and Technology of China for his kind provision of the strain and the assistance of the

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experiments. We are also grateful to Professor Shulin Zhuang from Zhejiang University for modifying the manuscript and Dr Yaqi Sheng for her assistance on the operation of GC-MS.

Notes The authors declare no competing financial interest. 21

Reference [1] Yuan, Y., Xi, B., He, X., Tan, W., Gao, R., Zhang, H., Chao Y., Zhao Y., Huang C., Li, D., Compost-derived humic acids as regulators for reductive degradation of nitrobenzene, J. Hazard. Mater. 339 (2017) 378-384. [2] Wang, J., Lu, H., Zhou, Y., Song, Y., Liu, G., Feng, Y., Enhanced

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mediator-functionalized polyurethane foam, J. Hazard. Mater. 252 (2013) 227-232.

(p-NP)

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macroalga Portieria hornemannii, Biotechnol. Bioeng. 93 (2006) 401-412. [5] Wang L., Liu Y. L., Ma J., Zhao F., Rapid degradation of sulphamethoxazole and

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[6] Chunli Z, Jiti Z, Jing W, et al. Isolation and characterization of a nitrobenzene degrading yeast strain from activated sludge, J. Hazard. Mater. 160 (2008) 194-199. 22

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[10] Cai P. J., Xiao X., He Y. R., Li W. W., Chu J., Wu C., He M. X., Zhang Z.,

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transformation via direct or indirect electron transfer by a sulfate reducing enrichment culture, Environ. Pollut. 242 (2018) 738-748.

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[12] Myers J. M., Myers C. R., Role of the tetraheme cytochrome CymA in anaerobic electron transport in cells of Shewanella putrefaciens MR-1 with normal levels of

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outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1, Mol. Microbiol. 393 (2001) 722-730. [15] Sturm G., Richter K., Doetsch A., Heide H., Louro R. O., Gescher J., A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime, ISME J. 9 (2015) 1802. [16] Romine M F, Carlson T S, Norbeck A D, et al. Identification of mobile elements

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[17] Gao H., Yang Z. K., Barua S., Reed S. B., Romine M. F., Nealson K. H.,

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Fredrickson J. K., Tiedje J. M., Zhou J., Reduction of nitrate in Shewanella

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oneidensis depends on atypical NAP and NRF systems with NapB as a preferred electron transport protein from CymA to NapA, ISME J. 3 (2009) 966-976.

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[18] Liu D. F., Min D., Cheng L., Zhang F., Li D. B., Xiao X., Sheng G. P., Yu H. Q.,

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Anaerobic reduction of 2, 6-dinitrotoluene by Shewanella oneidensis MR-1 Roles of Mtr respiratory pathway and NfnB, Biotechnol. Bioeng. 114 (2017) 761-768.

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[19] Gralnick J. A., Newman D. K., Extracellular respiration, Mol. Microbiol. 65 (2007) 1-11.

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[20] Ayyasamy, P. M., Chun, S., Lee, S., Desorption and dissolution of heavy metals from contaminated soil using Shewanella sp.(HN-41) amended with various carbon sources and synthetic soil organic matters, J. Hazard. Mater. 161 (2009) 1095-1102. [21] Chen X. J., Xu M. Y., Wei J., Sun G. P., Two different electron transfer pathways 24

may involve in azoreduction in Shewanella decolorationis S12, Appl. Microbiol. Biot. 86 (2010) 743-751. [22] Logan B. E., Exoelectrogenic bacteria that power microbial fuel cells, Nat. Rev. Microbiol. 7 (2009) 375. [23] Liu F., Xu M. Y., Chen X. J., Yang Y. G., Wang H. J., Sun G. P., Novel strategy

cells, Environ. Sci. Technol. 49 (2015) 11356-11362.

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for tracking the microbial degradation of azo dyes with different polarity in living

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USA. 103 (2006) 4669-4674.

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[25] Shirodkar S., Reed S., Romine M., Saffarini D., The octahaem SirA catalyses dissimilatory sulfite reduction in Shewanella oneidensis MR-1, Environ.

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Microbiol. 13 (2011) 108-115.

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[26] Wang F., Xiao X., Ou H. Y., Gai Y. B., Wang F. P., Role and regulation of fatty acid biosynthesis in the response of Shewanella piezotolerans WP3 to different

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temperatures and pressures, J. Bacteriol. 191 (2009) 2574-2584. [27] Szklarczyk D., Franceschini A., Wyder S., Forslund K., Heller D., Huerta-Cepas

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J., Simonovic M., Roth A., Santos A., Tsafou K. P., Kuhn M., Bork P., Jensen L. J., Von Mering C., STRING v10: protein–protein interaction networks, integrated over the tree of life, Nucleic acids Res. 43 (2014) 447-452.

[28] Ballabio, D., Manganaro, A., Consonni, V., Mauri, A., & Todeschini, R., Introduction

to

MOLE

DB-on-line 25

Molecular

Descriptors

Database,

MATCH-Commun. Math. Co. 62 (2009) 199-207 [29] Xu, S., Adhikari, D., Huang, R., Zhang, H., Tang, Y., Roden, E., Yang, Y., Biochar-facilitated microbial reduction of hematite, Environ. Sci. Technol. 50 (2016) 2389-2395. [30] Nevin, K. P., Lovley, D. R., Potential for nonenzymatic reduction of Fe(III) via electron shuttling in subsurface sediments, Environ. Sci. Technol. 34 (2000)

Jo

ur

na

lP

re

-p

ro of

2472-2478.

26

Figures and tables in the manuscript Table 1. Reduction rate constants (h-1) of NACs by MR-1 strains and mutants. Electron acceptors

kMR-1

k∆mtrA

k∆mtrB

k∆mtrC/omcA k∆cymA

k∆nfnB

-0.0421 -0.0350 -0.0258 -0.0277

-0.0152 -0.0160

1,2-dinitrobenzene

-0.4562 -0.3164 -0.2262 -0.2471

-0.0729 -0.1231

1,3-dinitrobenzene

-0.4169 -0.2830 -0.1936 -0.2420

-0.0724 -0.1241

1,4-dinitrobenzene

-0.5020 -0.3512 -0.3038 -0.2673

-0.0850 -0.1296

2,4-Dinitrotolunte

-0.3711 -0.1347 -0.1921 -0.1909

-0.0816 -0.1042

2,6- Dinitrotolunte

-0.3503 -0.1782 -0.2036 -0.2074

-0.0848 -0.1166

2,4,6-Trinitolunte

-1.1829 -1.0119 -1.2788 -1.3055

-0.4279 -0.5757

2-Nitrobiphenyl

-0.0122 -0.0034 -0.0031 -0.0032

-0.0031 -0.0083

2,2’-Dinitrobiphenyl

-0.0277 -0.0155 -0.0157 -0.0176

-0.0104 -0.0384

4-Nitrotoluene

-0.0173 -0.0217 -0.0102 -0.0154

-0.0069 -0.0076

4-Ethylnitrobenzene

-0.0252 -0.0256 -0.0125 -0.0138

-0.0074 -0.0077

1-Tertbutyl-4-nitrobenzene

-0.0101 -0.0083 -0.0073 -0.0082

-0.0063 -0.0050

2,5-Ditertbutyl-nitrobenzene -0.0022 -0.0017 -0.0018 -0.0016

-0.0008 -0.0024

Jo

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na

lP

re

-p

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Nitrobenzene

27

f oo

Table 2. Relative reduction rate constants and some molecular descriptors of the electron acceptors employed in this research. NB

of NACs by ∆nfnB (%)

33.7

18.6

17.7

Mw

123.11

168.11

182.13

Wv

9.21

10.63

12.23

tAN

14.00

16.00

19.00

lg Kow

1.86

1.68

2.18

Polarizability

9.00

11.00

electron

ASC

0.47

0.42

acceptor

tPSA (NO) Kier-Hall

RG

4-ENT

4-TNB 2,5-DTNB

2-NPL

2,2'-DNPL

19.9

29.3

42.3

44.0

52.4

227.13

137.14

151.16

179.22

235.32

199.21

244.20

13.65

10.81

12.41

15.60

22.00

16.41

17.83

21.00

17.00

20.00

26.00

38.00

24.00

26.00

1.99

2.37

2.85

3.72

5.63

3.57

3.39

12.00

14.00

11.00

13.00

16.00

24.00

17.00

18.00

0.38

0.22

0.47

0.65

0.61

0.36

0.29

0.13

e-

27.8

45.80

91.64

91.64

137.46

45.82

45.82

45.82

45.82

45.82

91.64

43.30

43.30

45.00

60.67

29.30

30.83

34.58

41.50

39.00

54.67

0.07

0.13

0.11

0.14

0.06

0.10

0.08

0.08

0.08

0.11

2.50

2.59

2.82

2.04

2.56

2.78

3.16

2.65

2.65

Jo ur

FRB

4-NT

28.6

Pr

of

na l

Properties

1,3-DNB 2,4-DNT 2,4,6-TNT

pr

Reduction efficiencies

2.04

Mw, molecular weight; Wv, van der Waals volume; tPSA (NO), total polar surface area calculated using N,O polar coefficients; Kier-Hall, sum of Kier-Hall electrotopological states; ASC, asphericity; FRB, fraction of rotatable single bonds; tAN, total atom number; RG, radius of gyration. The parameters were obtained from online molecular descriptors data base MOLE db (http://michem.disat.unimib.it/mole_db/)

28

Figure Legends Figure 1. Anaerobic reduction of NB at 20 mg∙L-1 by S. oneidensis MR-1 wild-type and derivative strains (∆mtrA, ∆mtrB, ∆mtrC/omcA, ∆cymA, ∆napA, ∆napB, ∆nrfA, ∆nfnB,). Figure 2. Reduction kinetics of NACs with different number of nitro groups by MR-1 and some mutants. (a) 4-NT, (b) 2, 4-DNT and (c) 2, 4, 6-TNT.

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Figure 3. Reduction performance of NACs with alkyl groups differed in number of carbon atoms MR-1 and mutants. (a) 4-NT, (b) 4-ENB, (c) 4-TNB and (d) 2, 5-DTNB.

re

and mutants: (a) 2-NPL and (b) 2, 2’-DNPL.

-p

Figure 4. Reduction performance of 2-nitrobiphenyl and 2, 2’-nitrobiphenyl by MR-1

Figure 5. Expression of (a) cymA, (b) nfnB genes in S.oneidensis MR-1 after

lP

cultivated for 4 h in the substrates containing NB, 2,4-DNT, 4-ENB, 2,5-DTNB and

na

2,2’-DNPL.Asterisks indicate a statistically significant difference (p < 0.05). Figure 6. Correlation of physical and chemical properties of NACs with the reduction

ur

efficiency of them by ∆nfnB: (a) van der Waals volume; (b) tAN, total atom number; (c) molecular weight; (d) Log Kow; (e) polarizability; (f) asphericity; (g) sum of

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Kier-Hall electrotopological states; (h) FRB, fraction of rotatable single bonds; (i) Radius of gyration. (j) tPSA (NO), total polar surface area calculated using N,O polar coefficients.

29

Concentration (mg·L

-1

)

20 Control mtrA mtrC/omcA napA nrfA

16 12

MR-1 mtrB cymA napB nfnB

8 4

0

20

40

60

80

Jo

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na

lP

re

-p

Time (h)

ro of

0

Figure 1. 30

(a) -1

Concentration (mg·L )

20 16 12 Control MR-1 mtrA mtrB mtrC/omcA cymA

8 4

nfnB

0 0

20

40

60

80

ro of

Time （ h）

(b) -1

Concentration (mg·L )

20

-p

16 12

4

re

Control MR-1 mtrA mtrB mtrC/omcA cymA

8

0

lP

nfnB

0

2

4

6

8

10

12

na

Time (h)

(c)

-1

Concentration (mg·L )

20 16

Jo

ur

Control MR-1 mtrA mtrB mtrC/omcA cymA

12

8

nfnB

4 0 0

2

4

6 Time (h)

Figure 2. 31

8

10

12

-1

Concentration (mg·L )

20

20

16

16

12

12 Control MR-1 mtrA mtrB mtrC/omcA cymA nfnB

8 4 0

Control MR-1 mtrA mtrB mtrC/omcA cymA nfnB

8 4

(a) 0

0

20

40

60

80

0

(b)

20

40

ro of

20 16 12

16

0

20

(c)

re

0

12

Control MR-1 mtrA mtrB mtrC/omcA cymA nfnB

-p

Control MR-1 mtrA mtrB mtrC/omcA cymA nfnB

(d)

0

40

80

Jo

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na

Time (h)

60

lP

-1

Concentration (mg·L )

20

4

80

Time (h)

Time (h)

8

60

Figure 3.

32

0

40

80 Time (h)

120

(a)

16 12 Control MR-1 mtrA mtrB mtrC/omcA cymA nfnB

8 4 0 0

40

80

120

160

Time (h)

(b)

-p

20 16

re

12 Control MR-1 mtrA mtrB mtrC/omcA cymA nfnB

8 4 0

10

20

30

Time (h)

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na

0

lP

-1 Concentration (mg·L )

200

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-1 Concentration (mg·L )

20

Figure 4.

33

40

50

60

-1

cymA abundance (copies·mL )

(a) 1.2x10

a

5

a

b 9.0x10

c

4

d

6.0x10

4

3.0x10

4

d

6

7x10

6

6x10

6

5x10

6

4x10

6

3x10

6

2x10

6

1x10

6

a

(b) b

lP

re

b

-p

8x10

2,4-DNT 4-ENB 2,5-DTNB 2-NPL 2,2'-DNPL

na

-1

nfnB abundance (copies·mL )

NB

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0.0

c

d

d

0

2,4-DNT 4-ENB 2,5-DTNB 2-NPL 2,2'-DNPL

Jo

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NB

Figure 5.

34

(a) 60

(d)

2

R = 0.519 p < 0.05

p < 0.05

(e)

2

2

R = 0.580 p < 0.05

R = 0.416 p < 0.1

40

20

15

20

20

30

van der Waals volume

(f)

tAN

(g)

2

R = 0.03

200

2

250

Molecular weight

(h)

2

4

LogKow

(i)

R =0.075

6 10

15

20

2

Polarizability

(j)

2

R = 0.019

R =0.131

re

60

2

R = 0.394 p < 0.1

40 150

lP

40

na

20

30

45 60 Kier-Hall

0.08 0.12 FRBs

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0.2 0.4 0.6 Asphericity

Figure 6. 35

2.0

25

ro of

10

Reduction efficiency (%)

(c)

2

R = 0.438

-p

Reduction efficiency (%)

(b)

2

R = 0.634 p < 0.01

2.5 3.0 RG

40

80 120 tPSA