Exclusive Extracellular Bioreduction of Methyl Orange by Azo

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Exclusive Extracellular Bioreduction of Methyl Orange by Azo Reductase-Free Geobacter sulf urreducens Yi-Nan Liu,† Feng Zhang,† Jie Li, Dao-Bo Li, Dong-Feng Liu,* Wen-Wei Li, and Han-Qing Yu* CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, China S Supporting Information *

ABSTRACT: Azo dyes are a class of recalcitrant organic pollutants causing severe environmental pollution. For their biodecolorization, the azo reductase system was considered as the major molecular basis in bacteria. However, the intracellular localization of azo reductase limits their function for efficient azo dye decolorization. This limitation may be circumvented by electrochemically active bacteria (EAB) which is capable of extracellular respiration. To verify the essential role of extracellular respiration in azo dye decolorization, Geobacter sulf urreducens PCA, a model EAB, was used for the bioreduction of methyl orange (MO), a typical azo dye. G. sulf urreducens PCA efficiently reduced MO into amines. Kinetic results showed that G. sulf urreducens PCA had the highest decolorization efficiency among the currently known MO reducing bacteria. Electrons from acetate oxidization by this strain were transferred by the respiratory chain to MO. The mass and electron balances, fluorescent probing and proteinase K treatment experimental results indicate that the biodecolorization of MO by G. sulf urreducens PCA is an exclusive extracellular process. OmcB, OmcC and OmcE were identified as the key outer-membrane proteins for the extracellular MO reduction. This work deepens our understanding of EAB physiology and is useful for the decontamination of environments polluted with azo dyes. The contribution of extracellular respiration to pollutants reduction will broaden the environmental applications of EAB.

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INTRODUCTION Because of their facile synthesis and favorable stability, azo dyes are currently the most widely used dyes in the textile industry. However, some unfixed azo dyes will inevitably be drained in wastewater.1−4 The complex, aromatic molecular structure and electron-deficient azo linkage make azo dyes stable and resistant to biodegradation.1,5 With the production of large amount of wastewater from the textile industry, the discharge of azo dyes into environment will cause serious pollution problems. Many physicochemical technologies are efficient for azo dye removal but suffer from high costs. Biodegradation, with its low cost, eco-friendliness, and good compatibility with current facilities, has attracted extensive interest.6,7 Due to their electrondeficient nature, azo dyes are difficult to be bio-oxidized directly. Instead, they are usually first bioreduced into amines under anaerobic conditions and then oxidized and degraded under aerobic conditions.4 Thus, bioreduction (also called biodecolorization) is the basic prerequisite for the biodegradation of azo dyes. Bacteria are a primary class of microbes with azo dye biodecolorization abilities. The intracellular azo reductase system was widely recognized as the major molecular basis for the biodecolorization of azo dyes by bacteria.8,9 However, the presence of polar groups, such as sulfonate acid groups, in most azo dyes prevent them from crossing the cell membrane. © XXXX American Chemical Society

This mass transfer limitation severely constrains the function of azo reductase in azo dye decolorization. Some outer-membrane proteins have also been found to play a role in the biodecolorization of azo dyes.4,10−12 In light of the polar structure of azo dyes and the barrier of the cell membrane, the key contribution of the outer-membrane proteins, as an alternative pathway for azo dye reduction, needs to be reevaluated.4,10 The extracellular reduction pathway can avoid steric blocking of azo dyes and might achieve a higher decolorization efficiency than azo reductase. Thus, bacterial strains with transmembrane electron transfer channels should be a promising candidate for the biodecolorization of azo dyes. Electrochemically active bacteria (EAB) can respire on extracellular electron acceptors, such as electrodes and minerals, through extracellular electron transfer (EET).13,14 Their EET ability perfectly fits the demands of the extracellular reduction pathway of azo dyes.15 Thus, EAB with a strong EET ability should also be efficient reducers of azo dyes. Geobacter sulf urreducens is a model EAB and exists predominately in sedimentary and subsurface environments.16,17 Among EAB, a Received: Revised: Accepted: Published: A

April 24, 2017 June 29, 2017 July 3, 2017 July 3, 2017 DOI: 10.1021/acs.est.7b02122 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology potent EET ability in Geobacter sulf urreducens has been revealed.16 Furthermore, neither monomeric flavin-free azo reductase genes nor polymeric flavin-dependent azo reductase genes (azoA from Enterococcus faecalis,18 azoA from Pigmentiphaga kullae,19 and acpD from Escherichia coli20) were found in the genome of G. sulfurreducens PCA (http://www.genome.jp/ kegg/). All of these features make Geobacter sulf urreducens PCA an ideal candidate for elucidating the extracellular decolorization of azo dyes. Therefore, this study aims to investigate the possibility of exclusive extracellular biodecolorization of azo dyes by G. sulfurreducens PCA and explore the underlying mechanism. The biodecolorization of methyl orange (MO), as a model azo dye, was investigated. The decolorization products were identified and the kinetic parameters of MO decolorization by G. sulfurreducens PCA were calculated. Furthermore, respiration inhibitors, fluorescent probe NA-MR and proteinase K treatment experiments were conducted to identify the location of the responsible enzymes. Finally, the performance of MO bioreduction by G. sulf urreducens PCA and its mutant strains was compared. The role of extracellular c-type cytochromes in MO bioreduction was evaluated. These findings deepen our knowledge about EAB physiology and expand the application of EAB for the degradation of pollutants.

dicumarol and 0.2 mM antimycin A). All inhibitors were dissolved in ethanol for stock solutions, except for dicumarol which was dissolved in 0.05 mM NaOH.12,23 Bacteria Inactivation and Proteinase K Treatment. Bacteria were inactivated for 15 min in a high-pressure steam sterilization pot at 121 °C. The proteinase K treatment was similar to previous reports.24−26 During the process of proteinase K treatment, bacteria were concentrated to approximately OD600 = 5.5 (in 10 mM HEPES buffer solution with 500 μM MgCl2) and incubated with 1 U/ml proteinase K. After 1 h incubation, 5 mM proteinase inhibitor-phenylmethylsulfonyl fluoride (PMSF) was added. The treated bacteria were washed twice with 10 mM HEPES buffer solution containing PMSF to remove residual proteinase K activity. The microbial vitalities were analyzed by BacLight RedoxSensor Green Vitality Kit (Invitrogen Co.) before and after proteinase K treatment. Analysis. Decolorization products of MO were analyzed by high-performance liquid chromatography (1260 Infinity, Agilent Inc.).15 For the HPLC mobile phase, a solution of 50% ammonium acetate (10 mM, pH 4.0) and 50% methanol was used. A HC-C18 column with a UV detector (detection wavelength: 240 nm) was used to analyze 10 μL of sample. The MO concentration was measured using a UV−vis spectrophotometer (UV-2401PC, Shimadzu Co., Japan) by recording the characteristic absorbance at 465 nm. The acetate concentration was measured by gas chromatography (GC7890, column DBFFAP, Agilent Inc.) as previously described.27 The biomass of bacteria was determined by measuring the protein concentration with a BCA Protein Assay kit (Sangon Biotech, China). Fluorescent Method for Locating the Dye Decolorization Sites. The azo probe NA-MR, which could locate the intracellular azo reductase, was prepared as reported previously (Supporting Information (SI) Figure S1).28 G. sulf urreducens PCA cells in stationary phase were harvested by centrifugation (6000g, 5 min), washed with DSMZ basal medium and added into DMA without MO. The strain concentration in media was adjusted to OD600 = 0.4. NA-MR (50 μM) was also added into media as a substitute to MO. The strains sampled at different incubation times were observed with an epifluorescent microscope (BX51, Olympus Co., Japan). G. sulf urreducens PCA cells were also stained with 4,6-diamidino-2-phenylindole (DAPI, a nucleic acid sensor). The microbial cultivation solution was centrifuged (6000g, 5 min) to remove cells and obtain supernatant. The emission spectra of supernatant under an excitation wavelength of 450 nm were recorded by a fluorescence spectrometer (AQUALOG, HORIBA Jobin Yvon Co., Japan). The emission intensities at 520 nm were used to quantify the reduction extent of NA-MR.

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MATERIALS AND METHODS Bacteria Strains and Growth Conditions. Wild-type G. sulfurreducens PCA and its mutant strains, ΔomcB, ΔomcC, ΔomcS, ΔomcE, ΔomcZ, ΔpilA, were gifted by Prof. Liang Shi from the China University of Geosciences (China). All strains were cultured in modified DSMZ medium with 50 mM fumarate and 20 mM acetate as the electron acceptor and donor, respectively.21 The strains were cultured at 30 °C until the stationary phase was reached (ca. 5 days). Shewanella oneidensis MR-1 and Aeromonas hydrophila ATCC7966 were cultured in Luria−Bertani broth (LB) until late stationary phase at 30 °C. Biodecolorization Experiments. Two types of decolorization media were used for decolorization experiments with G. sulfurreducens PCA. Decolorization medium A (DMA) was used to confirm the decolorization of MO and identify the reduction products. The DMA, with 72 μM acetate as the electron donor and 40 mg/L MO as the electron acceptor, had a similar composition to the modified DSMZ medium. Decolorization medium B (DMB) was used to study the MO decolorization kinetics. Thus, the concentration balance between the electron donor and acceptor was maintained. The DMB formulation was similar to that of DMA, except for the concentrations of acetate (20 mM) and MO (ranging from 8 mg/L to 200 mg/L). With the excessive amount of acetate, the acetate concentration would not be limiting in the decolorization process. Strains in stationary phase were harvested by centrifugation (6000g, 5 min), washed with DSMZ basal medium and added into DMA or DMB. For S. oneidensis and A. hydrophila, their anaerobic media without electron acceptors were used as the decolorization media.15,22 The concentration of bacteria in all decolorization media was adjusted to OD600 = 0.1. The medium in each serum vial was sparged with N2:CO2 (80:20) mixed gas to ensure an anaerobic atmosphere. During the decolorization experiments, samples were collected in an anaerobic glovebox. Respiratory chain inhibitors were added to the medium as required (final concentration: 0.1 mM capsaicin, 0.1 mM rotenone, 0.2 mM

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RESULTS Confirmation of MO Decolorization by G. sulf urreducens PCA. MO can absorb visible light intensively. Once G. sulf urreducens PCA was added to DMA, the decolorization of MO could be seen by naked eye. The orange color of MO disappeared gradually, which was consistent with the change in the MO concentration. The pH of DMA was approximately 7 and kept stable during the decolorization process (change within one unit). The change in MO concentration was measured by the characteristic absorbance at 465 nm under neutral pH conditions. As shown in Figure 1, 40 mg/L MO was completely decolored within approximately 5 h. In contrast, the control with heat-inactivated bacteria showed no obvious B

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Figure 1. Decolorization of MO by wild-type G. sulfurreducens. The insert image shows UV−vis spectra at different incubation times (0 and 5 h).

decolorization. Thus, the contribution of the abiotic process, such as chemical degradation and adsorption, for MO removal can be excluded (SI Figure S2). The decolorization of MO in our system was ascribed solely to biochemical process. The decolorization ability of G. sulf urreducens PCA was not limited to MO, and other azo dyes such as amaranth and m-methyl red could also be decolorized (SI Figure S3). The UV−vis spectra of the culture supernatant during the decolorization process were also collected (Figure 1, insert graph). It shows a decreased peak intensity over time in the broad maximum peak centered at 465 nm, a characteristic absorption peak representing energy absorption of the azo linkage electron. Simultaneously, a new adsorption peak centered at 247 nm appeared and amplified gradually. These results were consistent with previous reports on MO bioreduction.15 The new peak could be attributed to the decolorization products aromatic amines. Calculation of Kinetic Parameters. To compare the decolorization efficiency of G. sulf urreducens PCA to other EAB, the decolorization abilities of two other strains, including Shewanella oneidensis MR-1 and Aeromonas hydrophila ATCC7966, at identical bacteria concentrations were also tested (OD600 = 0.1, Figure 2A). The first-order kinetic constant of MO decolorization (40 mg/L) by G. sulfurreducens PCA (0.632 h−1) was 3.36 times greater than that of Shewanella oneidensis (0.188 h−1), and was hundred-fold higher than that of Aeromonas hydrophila (0.007 h−1). This result suggests that G. sulf urreducens PCA is superior to other EAB in MO decolorization. The Michaelis−Menten equation has been widely used to describe enzymatic reactions, and has the same grade as firstorder kinetics.29,30 The reaction rates obtained from the firstorder fitting curve of different MO concentrations (Figure 2B) were fitted by the Michaelis−Menten equation (Adj. R2=0.966). The Vmax of the reaction was 49.05 (mg L−1)/h (40.00 μM/h·mg protein normalized by protein concentration), and the Km value was 187.30 mg/L (Figure 2C). Mass and Electron Balances. The mass and electron balance of decolorization were confirmed by measuring the concentrations of the reactants and products. MO (tR = 10.8 min) and its potential products were identified by HPLC analysis. The main decolorization products were 4-aminobenzenesulfonic acid (4-ABA, tR = 2.5 min) and N,Ndimethylbenzene-1,4-diamine (DPD, tR = 2.9 min) (SI Figure S4A). Figure 3 shows the concentration change of the products and reactants during the decolorization process. The concentrations of acetate and MO decreased, whereas those of 4-ABA and

Figure 2. Kinetics of MO decolorization. Comparison of MO decolorization rates by G. sulfurreducens, Shewanella oneidensis and Aeromonas hydrophila (A). Decolorization results at different MO concentrations (8, 24, 40, 80, 120, 240 mg/L) and their fitting curves (B). Michaelis−Menten equation fitting results (C) of decolorization rates by G. sulfurreducens PCA.

Figure 3. Mass and electron balance during the decolorization process. The amount of electrons transferred from acetate (HAc) to MO, was calculated from the change in concentration of products (4aminobenzenesulfonic acid and N,N-dimethylbenzene-1,4-diamine) and reactants (acetate and MO) during the process of MO decolorization.

DPD proportionally increased. After 9-h decolorization, the MO concentration dropped to zero, whereas the 4-ABA and DPD increased to 18.0 mg/L and 15.2 mg/L, respectively. The molar ratio of the concentration increase for the two products (4-ABA and DPD) approached 1.00 (0.985). Likewise, the ratio between the increased concentration of the products (4-ABA) and decreased concentration of the reactants (MO) was also C

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Environmental Science & Technology close to 1 (0.857). These stoichiometric ratios illustrate that one mole of MO was transformed into one mole of 4-ABA and DPD. The decolorization process of MO could be interpreted as a break in the azo linkage and a production of aromatic amines. The relationship between the acetic acid and MO degradation was also examined. The reduction of one mole of MO into aromatic amines requires four moles of electrons, while oxidization of one mole of acetate releases eight moles of electrons. The concentration decrease (3.9 mg/L, 0.065 mM) of acetic acid was about half the value (0.516) of MO. This ratio is very close to the theoretical, stoichiometric ratio between MO reduction and the total oxidization of acetate. Moreover, the protein concentration changed slightly from 3.75 to 3.82 μg/mL during the decolorization process, suggesting that the released electrons from acetate oxidation were not used for bacterial growth. The results demonstrated that MO reduction consumed nearly all the electrons released from acetate respiration and was directly coupled to the respiration of G. sulf urreducens PCA. Since the respiration of G. sulfurreducens PCA usually involves an extracellular process, such a consistency suggests that the MO decolorization by G. sulfurreducens PCA might be an extracellular process. The stoichiometric equation of the MO decolorization reaction by G. sulf urreducens PCA is shown in SI Figure S4B. Effect of Respiratory Chain Inhibitors on MO Decolorization. After confirming the decolorization of MO by G. sulf urreducens PCA, the decolorization mechanism was subsequently studied. As a highly polar molecule, MO cannot easily pass the cell membrane, thus the MO bioreduction should be mainly through extracellular process. The extracellular reduction of MO by G. sulf urreducens PCA needs sufficient amount of electrons to be generated by acetate oxidization and transferred to the exterior of the cell through respiratory chain proteins. To test the potential role of respiratory chain proteins on MO decolorization, different respiration inhibitors were added to the decolorization system (Figure 4A). The inhibition ratios were calculated from the relative decrease in the first-order kinetic constant. Among these inhibitors, rotenone and capsaicin (inhibitors of complex I) have the strongest inhibitory effect. The apparent inhibition ratio was as high as 89.9%. Dicumarol (the inhibitor of coenzyme Q) also had a potent inhibitory effect on decolorization, and its apparent inhibition ratio was 49.74%. However, antimycin A (the inhibitor of complex III) did not affect the decolorization efficiency of MO. These results indicate that some components of the respiratory chain, such as Complex I and coenzyme Q, are involved in the electron transfer process for MO reduction and Complex III is not involved in this process. A similar result has also been reported for the Fe (III) reduction by G. sulf urreducens.31 Therefore, the extracellular respiration of G. sulfurreducens might also conduct through a similar inner membrane electron transfer process. Extracellular Decolorization of Azo dyes by G. sulfurreducens PCA. As a model EAB, the entire genome of G. sulf urreducens PCA has been sequenced.32 However, neither monomeric flavin-free azo reductase genes nor polymeric flavindependent azo reductase genes were annotated in the genome of G. sulf urreducens. Several known azo reductases genes (azoA from Enterococcus faecalis, azoA from Pigmentiphaga kullae, and acpD from Escherichia coli)18−20 were also queried against the genome of G. sulfurreducens PCA. No significantly similar

Figure 4. Decolorization of MO by treated wild-type (A, B) and mutant strains (C) of G. sulf urreducens PCA. Different respiration inhibitors (A) and proteinase K (B) were added, respectively. The insert image of B shows the cell activity before and after proteinase K treatment.

sequences to known azo reductases were found. Thus, the decolorization of MO by G. sulfurreducens PCA may proceed by a pathway independent of azo reductase. In other words, the biodecolorization of azo dyes might not be an intracellular process. To reconfirm the above hypothesis, an azo probe NA-MR was synthesized to clarify the decolorization site of azo dyes by G. sulf urreducens PCA, i.e., outer-membrane proteins or intracellular enzymes. This probe can readily penetrate through the cell membrane. In addition, it can serve as a nonfluorescent probe with azo linkage, but exhibit strong fluorescent after azo linkage breaks. This azo probe had been used to verify the intracellular decolorization ability.28 The as-synthesized endproduct was characterized by 1H NMR and was identified as NA-MR (Figure S5). The NA-MR images of G. sulf urreducens PCA are shown in Figure 5. Unlike the intracellular azo dyes decolorization by Shewanella strains reported previously, in our study no obvious fluorescence in G. sulfurreducens PCA was observed after 6-h incubation (Figure 5A−C). Although the fluorescence intensity of the supernatant increased (Figure 5D), this result should be attributed to the extracellular reduction rather than an intracellular process. The intracellular fluorescence signal, as an indicator of intracellular reduction of azo probe, suggests that the intracellular components of G. sulf urreducens PCA could not reduce azo dyes effectively. Thus, the intracellular components in G. sulf urreducens PCA had no contribution to the biodecolorization of azo dyes. D

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The overall mechanism of MO decolorization by G. sulf urreducens PCA is illustrated in Figure 6. Acetate is utilized

Figure 5. Fluorescent imaging of azo probe (NA-MR). The imaging of samples at 0 h (A), 6 h (B) and DAPI staining control at 6 h (C); the fluorescence intensity of supernatant at 0 and 6 h (D).

Figure 6. Schematic of the proposed decolorization mechanism of MO by G. sulf urreducens PCA. The solid lines depict the decolorization reaction pathway; the dashed lines show the electron flow from the interior to the exterior of the cell.

Predominant Role of Outer-Membrane Proteins in MO Decolorization by G. sulfurreducens PCA. Numerous studies have shown that the outer-membrane proteins are involved in the decolorization of MO.1,12,15 To explore the role of the outer-membrane proteins in the MO decolorization, proteinase K was used to digest the outer-membrane proteins of G. sulf urreducens PCA. By controlling the exposure dose, proteinase K could digest the outer-membrane proteins without disturbing the periplasmic and intracellular proteins.25 After proteinase K digestion, the treated cells lost the ability to decolor MO (Figure 4B). Meanwhile, the cell activity of the treated cells only decreased slightly compared to control cells (Figure 4B, insert graph), demonstrating that the bacteria vitality was not acutely affected by proteinase K treatment. In combination with the NA-MR imaging results, these results clearly show that the outer-membrane proteins play predominant roles in the MO decolorization MO by G. sulfurreducens PCA. As G. sulfurreducens PCA does not express known azo reductases, extracellular electron transfer is an alternative way to decolor MO. Outer-membrane cytochromes have been reported to play a prominent role in electron acceptor reduction in G. sulf urreducens PCA.33−38 Some outermembrane proteins, for example, OmcB, OmcC, OmcZ, OmcS, and OmcE, and PilA, have been identified as the key proteins in extracellular reduction of EAB. The performance of MO decolorization by G. sulfurreducens PCA and its mutant strains was compared (Figure 4C). Deletion of omcB, omcC, and omcE modestly inhibited the decolorization inhibitory ratios of ΔomcB, ΔomcC, and ΔomcE were 84.0%, 57.7% and 64.9%, respectively. These negative effects were limited and far from complete inhibition. Deletion of omcS, omcZ, and pilA had no obvious negative impact on the MO decolorization (SI Figure S6). Surprisingly, ΔomcS and ΔomcZ even showed a slightly improved decolorization than wild-type bacteria. These results suggest that OmcB, OmcC, and OmcE are involved in the decolorization process and may act as extracellular, terminal azo reductases. Notably, the remaining decolorization ability of the mutant strains indicates that there are still some unknown azo reductases or alternative pathways in G. sulf urreducens PCA for MO decolorization.

by G. sulfurreducens PCA as an electron donor to generate acetyl-CoA, which is further transformed in the TCA Cycle. With the reducing force formed in the TCA Cycle, electrons are transferred via the respiratory chain to the outer-membrane proteins, where some key enzymes such as OmcB, OmcC and OmcE reduce MO into colorless products (4-ABA and DPD).

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DISCUSSION In this work, the robust decolorization ability of G. sulf urreducens PCA was demonstrated. This bacterium showed much higher decolorization ability than other reported strains. The kinetic parameters of the decolorization process were calculated and the decolorization mechanism was elucidated. Unlike the previous studies with S. oneidensis15 or Geobacter metallireducens,39 this is the first report about the exclusive extracellular decolorization of azo dyes by azo reductase-free bacteria. Biodecolorization of MO by various microbial strains has been widely investigated. G. sulfurreducens PCA, the newly confirmed azo biodecolorization strain, is demonstrated to have a potent ability to decolor MO. Compared to other decolorization strains listed in Table 1, G. sulfurreducens PCA has a faster decolorization rate at a lower biomass concentration. In addition, the G. sulfurreducens PCA showed a much higher Vmax value than other strains.8,40 From a kinetic perspective, G. sulf urreducens PCA should be the best strain for MO decolorization reported so far. The azo probe experimental results demonstrate that no intracellular reduction of azo dye occurred in G. sulf urreducens PCA. Since MO molecules cannot readily enter the cells due to their highly polarity, EET is a suitable and logical way to decolorize MO. Compared with the intracellular decolorization, the extracellular MO decolorization can be completed catalyzed by the outer-membrane proteins directly, which avoids the diffusion process of azo dyes across the cell membrane. Furthermore, the extracellular decolorization can also keep azo dyes and its decolorization products out of cells, which will protect bacteria from the toxic effects of azo dyes and their products, and sustain microbial metabolic activity. These E

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Environmental Science & Technology Table 1. Comparison of MO Decolorization by G. sulf urreducens PCA with Other Strains strain

electron donor

C0(MO) (mg/L)

initial biomass

efficiency

ref

Lactobacillus casei Caldicellulosiruptor saccharolyticus Shewanella oneidensis Candida zeylanoides Aeromonas hydrophila Shewanella oneidensis Geobacter sulf urreducens

sucrose glucose lactate yeast extract glycerol lactate acetate

75.3 100 100 65.5 40 40 40

10 (OD600) 0.2 (OD620) 4−6 × 106 CFU ml−1 1.3 (OD640) 0.1 (OD600) 0.1 (OD600) 0.1 (OD600)

100% (2 h) 100% (6 h) ca. 95% (10h) 100% (22 h) 5% (7h) 64% (7h) 100% (5 h)

43 44 15 45 this study this study this study

Consequently, the powerful decolorization of MO may be attributed to the potent extracellular electron transfer ability of G. sulfurreducens PCA. Similarly, G. sulf urreducens PCA is also one of the most productive strains in terms of power and current density when used in bioelectrochemical systems. MO reduction and electricity production by G. sulf urreducens PCA are related to some extent. Both functions depend on a strong EET ability. However, these two functions were based on different extracellular reduction processes. The contrast between MO and an electrode in physical and chemical properties results in completely distinct, interfacial reactions. For example, OmcZ, an essential cytochrome C for electricity production, did not play an important role in MO decolorization (Table 2). Hence, the decolorization performance of a strain cannot simply be derived from its electricity producing ability. Based on the resemblance in chemical properties, the degradation of other polar organic pollutants, such as carbon tetrachloride and pentachlorophenol, by G. sulf urreducens may also share similar reduction mechanisms to MO decolorization. Significance of This Work. The potential applications of these findings are as follows. First, due to the strong EET ability, G. sulf urreducens PCA can decolorize MO much faster than other strains. The contribution of EET to pollutant degradation would broaden the environmental application range of G. sulfurreducens PCA. Second, azo dyes decolorization could be a new function of EAB. MO may be an ideal candidate for understanding the specialization of outer membrane cytochromes in the presence of different electron acceptors (e.g., soluble or insoluble electron acceptors). Third, due to extensively distribution of EAB in sedimentary environments, they might play an essential role in the transformation of pollutants.

advantages endow the extracellular decolorization a significant advantage in microbial transformation of pollutants over the intracellular decolorization, resulting in the superior MO decolorization efficiency of G. sulf urreducens PCA. We further confirmed that extracellular cytochromes were responsible for the MO decolorization. The genome of G. sulfurreducens PCA contains 111 putative genes for cytochrome C, 32 and at least 30 of those are outer membrane cytochromes.41 The specialized functions of those proteins are complicated and not fully understood. It is difficult to elucidate the roles of all of the cytochrome Cs in the decolorization of MO. Therefore, we tested several representative cytochrome Cs that have been reported to play important roles in extracellular reduction. Among these proteins, OmcB, OmcC, and OmcE were found to be involved in MO decolorization. Notably, deletion of omcB, omcC, and omcE only modestly inhibited the MO decolorization, suggesting that some other outer-membrane proteins also contributed to the MO decolorization. These proteins might complement each other. When one of those responsible proteins was knocked out, the rest could compensate to decolor MO. A similar phenomenon was also found in the reduction of soluble electron acceptors (such as anthraquinone-2,6-disulfonate, humic acid and ferric citrate) by G. sulfurreducens PCA.35 However, when the electron acceptors were insoluble (such as iron oxide and an electrode), one or two types of extracellular cytochrome Cs played pivotal roles in electron acceptor reduction (Table 2). Thermodynamically, MO had a relatively high redox potential (ca. + 0.2 V).42 All cytochrome Cs tested were able to reduce MO. Therefore, the results show that the specialization of extracellular cytochrome Cs did not depend on thermodynamics alone. The specialization of extracellular cytochrome Cs may be associated with the type of electron acceptor.

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Table 2. Electron Acceptor Utilization Phenotypes of Different Outer Membrane Protein Mutants of G. sulf urreducens PCA16a insoluble electron acceptor

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org/. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b02122. Additional figures on the synthesis route of NA-MR (Figure S1), MO concentration profiles when inactivated bacteria were added (Figure S2), decolorization of other azo dye by G. sulfurreducens PCA (Figure S3), putative reaction equation and chromatogram of MO and its decolorization products (Figure S4), 1H NMR spectrum of the synthesized probe NA-MR (Figure S5), decolorization of MO by other G. sulf urreducens PCA mutant strains (Figure S6) (PDF)

soluble electron acceptor

mutant strain

electrode

Fe (III) oxide

ferric citrate

humic substances

MO

ΔomcB ΔomcS ΔomcE ΔomcZ ΔomcC ΔpilA

− + ND * − +

* * + − ND +

+ − − + − −

− + + − ND −

+ − + − + −

a Phenotypes of electron acceptor utilization in different mutants are classed into nearly no reduction (*), partially impaired reduction (+), and unaffected reduction (−). ND: not determined.

F

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AUTHOR INFORMATION

Corresponding Authors

*(D-.F.L.) Fax: +86 551 63606698; e-mail: dfl@mail.ustc.edu. cn. *(H.-Q.Y.) Fax: +86 551 63601592; e-mail: [email protected]. ORCID

Feng Zhang: 0000-0002-8809-6910 Dong-Feng Liu: 0000-0003-4782-0033 Wen-Wei Li: 0000-0001-9280-0045 Han-Qing Yu: 0000-0001-5247-6244 Author Contributions †

Y.-N.L. and F.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (21477120, 21590812, 51538012, and 21607146), and the Collaborative Innovation Center of Suzhou Nano Science and Technology of the Ministry of Education of China for the support.

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DOI: 10.1021/acs.est.7b02122 Environ. Sci. Technol. XXXX, XXX, XXX−XXX