In situ spectral kinetics of quinone reduction by c-type cytochromes in intact Shewanella oneidensis MR-1 cells

Colloids and Surfaces A: Physicochem. Eng. Aspects 520 (2017) 505–513

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In situ spectral kinetics of quinone reduction by c-type cytochromes in intact Shewanella oneidensis MR-1 cells Rui Han a,b,1 , Xiaomin Li a,1 , Yundang Wu a , Fangbai Li a , Tongxu Liu a,∗ a Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650, China b School of Environment and Energy, South China University of Technology, Guangzhou 510006, China

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• The in situ kinetics of quinone reduction by c-Cyts in intact cells was investigated. • The reaction between quinone and cCyts was influenced by the incubation conditions. • The reduction/oxidation of c-Cyts in living Shewanella cells was observed.

a r t i c l e

i n f o

Article history: Received 17 November 2016 Received in revised form 6 February 2017 Accepted 7 February 2017 Keywords: c-type cytochrome Quinone reduction Living cell suspension Shewanella oneidensis MR-1

a b s t r a c t Microbial quinone reduction is closely related to electron transport in the bacterial membrane. The outer membrane (OM) c-type cytochromes (c-Cyts) of microbes are involved in electron transport from the electron donor to quinone. However, the in situ redox status of OM c-Cyts during microbial quinone reduction processes remains poorly understood. In this study, diffuse-transmission UV/Vis spectroscopy was used to investigate the in situ spectral reaction of quinone analogue anthraquinone-2,6-disulfonate (AQDS) reduction by c-Cyts in intact Shewanella oneidensis MR-1 cells under different incubation conditions. The reduced c-Cyts decreased transiently at the beginning and then recovered gradually over time. Compared to the wt, cymA and mtrA exhibited distinctly low AQDS reduction rates and regeneration of reduced c-Cyt. The AQDS reduction rates decreased with increasing initial AQDS concentrations but increased with increasing cell density. The highest AQDS reduction rate and fastest c-Cyts recovery were obtained at 30 ◦ C and pH 7.0. The above-mentioned incubation conditions may affect AQDS reduction by influencing the metabolic rates of lactate and the thermodynamic properties of c-Cyts and AQDS. This study provides a case of directly examining the in vivo reaction properties of an outer-membrane enzyme during the microbial quinone reduction processes under non-invasive physiological conditions. © 2017 Elsevier B.V. All rights reserved.

∗ Corresponding author. E-mail address: [email protected] (T. Liu). 1 Rui Han and Xiaomin Li contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfa.2017.02.023 0927-7757/© 2017 Elsevier B.V. All rights reserved.

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1. Introduction Humic substances constitute a chemically heterogeneous and very abundant class of organic compounds that are widely distributed on the Earth’s surface [1,2]. As a main functional group of humic substances, quinines serve as electron-accepting moieties when microorganisms transfer electrons to extracellular humic substances [3,4], which is known as quinone respiration [2]. A variety of phylogenetic diversity microorganisms (e.g., Geobacter and Shewanella sp.) have been identified as humic-reducing bacteria that can use quinones as electron acceptors for anaerobic growth using a wide variety of organic substrates or H2 as electron donors [3–5]. In addition, quinone is widely used as an electron mediator for microbially catalysed metal reduction, as the reduced form of quinone can further donate electrons to other electron acceptors with lower redox potential [4,6]. Therefore, the redox cycle of quinone plays an important role in the natural biotic and abiotic processes of elemental cycles [7]. Due to the nature of quinones as electron mediators, the electron transfer between quinone and the outer membrane (OM) proteins is a key step in extracellular electron transfer (EET) [2,8]. The ctype cytochrome proteins (c-Cyts) such as OmcA and MtrC in the OM of S. oneidensis MR-1 have been identified to be involved in the EET to many quinones such as anthraquinone-2,6-disulfonate (AQDS) and anthraquinone-2-sulfonic acid (AQS) [8,9]. The roles of the OM c-Cyts (e.g., OmcA and MtrC) in EET were characterized by examining the performance of AQDS reduction by different Shewanella mutant deletions [8,9] or by using highly purified OM c-Cyts extracted from Shewanella species [10–12]. However, the purified proteins may behave differently from the protein complexes in live cells because the highly reactive enzymes may be easily altered during the purification treatments [13]. Hence, an in vivo study of the reaction between AQDS and c-Cyts in live cells will allow a more comprehensive understanding of microbial quinone reduction processes. Since the electron transfer centre of OM c-Cyts is a heme group with a large molar absorption coefficient, spectroscopic methods have been applied to monitor the OM c-Cyts in living cells under physiological conditions [13–15]. A diffuse-transmission (DT) mode in the UV/Vis spectroscopy can accurately measure the multi-heme c-Cyts in living whole cells [13], which can effectively subtract the strong spectral interference from light scattering on the cell surface. Recent studies have attempted to employ DTUV/Vis spectroscopy to monitor the heme groups in OM c-Cyts of different humic-reducing bacteria, such as S. putrefaciens 200 [16,17], S. decolorationis S12 [18], Aeromonas hydrophila HS01 [18], and Klebsiella pneumonia L17 [19,20], and have successfully analysed the in situ spectral kinetics of OM c-Cyts in different intact Shewanella species or Cr(VI) reduction [14,15]. In addition, AQDS can also be easily measured by the UV/Vis spectral system because the absorbance spectra of the oxidized and reduced forms of AQDS can be distinctly measured [21]. Therefore, DT-UV/Vis spectroscopy is a promising approach to simultaneously investigate the in situ spectral kinetics of AQDS and c-Cyts in a suspension of living humicreducing bacteria, such as S. oneidensis MR-1. The microbial AQDS reduction can be influenced by different environmental factors, such as the AQDS concentration, temperature, bacterial cell density and pH [2]. While previous studies primarily focused on the effects of incubation factors on the apparent AQDS reduction [2,7], it is still unknown how the in situ redox status of OM c-Cyts behaves over time during the microbial quinone reduction processes under various incubation conditions. In this study, AQDS reduction by a living cell suspension of S. oneidensis MR-1 was investigated by DT-UV/Vis spectroscopy with the following objectives: (1) directly examine in situ spectral kinetics of AQDS and c-Cyts in intact MR-1 cells of wild type and mutants;

(2) evaluate the effects of different incubation conditions, i.e., cell density, initial AQDS concentration, lactate concentrations, pH, and temperature, on the in vivo reaction between AQDS and c-Cyts; and (3) discuss the effects of various conditions on the electron transfer processes based on the experimental data and thermodynamic analysis. The in situ studies on AQDS reduction by c-Cyts in live cells would provide an in-depth understanding of the molecular-level mechanisms in the microbial quinone reduction. 2. Materials and methods 2.1. Cell suspension materials and preparation Shewanella oneidensis MR-1, an iron-reducing bacterium [22], was purchased from MCCC (Marine Culture Collection of China, China). The mutant strains of S. oneidensis MR-1 (mtrA, mtrC, mtrD, mtrF, omcA, and cymA) were provided by Prof. Haichun Gao in Zhejiang University, and the relevant information was provided with the references [23–26]. The humic analogue 9,10-anthraquinone-2,6-disulfonic acid (AQDS, 98%) was purchased from TCI (Japan). Equine heart cytochrome c, 4(2-hydroxythyl) piperazine-1-ethanesulfonic acid (HEPES) and piperazine-N,N-bis-2-ethanesulfonic (PIPES) were obtained from Sigma-Aldrich. Other chemicals used in the experiments were reagent grade or better. The MR-1 and the mutant strains were grown in Luria-Bertani (LB) medium, which consisted of NaCl (10 g L−1 ), yeast extract (5 g L−1 ), and tryptone (10 g L−1 ) with the following growth conditions: pH 7.0 ± 0.2 (25 ◦ C), oxic conditions, 30 ◦ C, shaking at 180 rpm for 16 h. The samples were harvested by centrifugation at 8000 × g for 10 min at 4 ◦ C, and the pellets were washed with sterile HEPES buffer (30 mM, pH 7.0) three times. Cells were then transferred into an anaerobic chamber (DG250, Don Whitley Scientific, England) with H2 :N2 (4:96), resuspended in the HEPES (30 mM, pH 7.0) buffer. Then, these steps are repeated two times to remove the residual LB medium as much as possible. A total of 30 mM of HEPES solution, with the pH adjusted to pH 7.0 with sodium hydroxide as otherwise indicated, was used as the buffer in all experiments. The AQDS stock solution (10 mM) was filtered with syringe filters (0.22 ␮m) to remove bacteria without cause any damage to the structure of AQDS. Stock solutions of sodium lactate (1.0 M) were also prepared with water from a Milli-Q ultraviolet (UV)-water system in an anaerobic chamber and used in the experiments. All stock solutions were stored at 4 ◦ C before used. 2.2. AQDS reduction by MR-1 and mutants The AQDS reduction was examined using S. oneidensis MR-1 with six types of c-Cyts deletion mutants (mtrA, mtrC, mtrD, mtrF, omcA, and cymA). The cell suspensions in HEPES buffer at a final concentration of 1.07 × 1012 cells mL−1 (OD600 = 1.0) were added to a rectangular quartz cuvette with an optical path length of 1.0 cm. Sodium lactate (20 mM) was added as an electron donor. AQDS (100 ␮M) was added as the sole electron acceptor. The cuvette was subsequently sealed and was removed from the anaerobic chamber. Once AQDS was added, spectra were measured using a DT-UV/Vis spectrophotometer by scanning at different intervals. AQDS was added as the sole electron acceptor at concentrations ranging from 20 ␮M to 120 ␮M. To investigate the effect of initial electron donor concentrations on AQDS reduction, different concentrations of sodium lactate (10 ␮M–20000 ␮M) were added. To investigate the effect of initial MR-1 cell density on AQDS reduction, different densities of MR-1 cells from 4.28 × 1011 cells mL−1 (OD600 = 0.4) to 1.60 × 1012 cells mL−1 (OD600 = 1.5) were added. The effects of different pH values (6.0–8.0) and temperature

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(20 ◦ C–40 ◦ C) on the AQDS-reducing capability of viable MR-1 cells were examined. The pH values were adjusted to 6.0, 6.3, and 6.7 using the sterile PIPES buffer (30 mM) and were adjusted to 7.0, 7.3, 7.6, and 8.0 using the HEPES buffer (30 mM). In all cases, except for the initial AQDS treatment, 100 ␮M of AQDS was added as a sole electron acceptor. 2.3. Quantification of c-Cyts in an intact cell and AQDS The equine heart cytochrome c was used as a standard [27] to quantify c-Cyts in living S. oneidensis MR-1 cell suspensions. Different concentrations of equine heart cytochrome c were measured with the same spectrum determination method as the above sections. Heme c was quantified by a difference in millimolar extinction coefficients (␧) of 21.4 mM−1 cm−1 for reduced and oxidized forms of meso-IX pyridine hemeochrome [27]. The accuracy of the quantification method was confirmed by comparison with the equine heart cytochrome c standards. The specific peaks are 328 nm for AQDSox and 552 nm for reduced cytochromes (cCytred ). The peaks at 385 nm and 408 nm observed in Fig. S1a are attributed to the AH2 QDS, AHQDS− , respectively [21]. A pseudofirst-order kinetic model can be applied to describe the kinetics of the AQDSox bio-reduction with MR-1. 2.4. Electrochemical measurements To observe the change in the UV/vis spectra of reduced AQDS and oxidized AQDS, dissolved AQDSox was reduced electrochemically at pH 6–8 in the presence of 0.1 M KCl in the same buffer as that used for kinetic studies (PIPES and HEPES) using Pt mesh working electrode, a Pt wire auxiliary electrode, and an Ag/AgCl reference electrode (all from Avantes, Netherlands). A potential of −0.8 V vs SHE was applied with an Autolab AUT85800 potentiostat (Metrohm, Germany). After approximately 60 min of reduction, the spectra of the reduced AQDS species were recorded using an Avalight-DHS (Netherlands). A potentiostat (CHI660D, Chenhua Co., Ltd., Shanghai, China) was used for the electrochemical measurements in this study. Under galvanostatic conditions, the electrochemical measurements were conducted in an anaerobic glass bottle with three electrodes on the top. The carbon cloth (2 × 6 cm), carbon felt (3.5 × 2 cm) and calomel electrode were used as the working electrode, counter electrode, and reference electrode, respectively. A suspension of MR-1 in logarithmic phase (1.07 × 1012 cells mL−1 ), AQDS (100 ␮M) in HEPES buffer (30 mM, pH = 7.0), and lactate (20 mM) were added to the reactor. The E-t curve was tested under 0.1 mA using the galvanostatic method, and the E values were recorded. Cyclic voltammetry (CV) measurements of AQDS at varying pH values (6.0–8.0) were performed using a potentiostat (CHI660D, Chenhua Co., 175 Ltd., Shanghai, China) [28]. 3. Results and discussion 3.1. Kinetics of AQDS reduction by c-Cyts in intact MR-1 cells 3.1.1. Spectral method for AQDS and c-Cyts The in situ kinetic study of AQDS reduction by c-Cyts in intact MR-1 cells was conducted with an initial AQDS concentration of 80 ␮M. The spectra in Fig. 1a clearly show changes in the peaks at 328 nm for AQDSox , which decreased substantially over time (Fig. 1b). AQDS can be reduced to different forms of AQDSred (AH2 QDS and AHQDS− ) (Fig. S1b). Two peaks at 385 nm and 408 nm appeared and increased gradually as the reaction proceeded. Compared to the electrochemically reduced AQDS standard at pH 7, the peaks at 385 nm and 408 nm were attributed to AH2 QDS

Fig. 1. (a) The in situ spectra of the intact S. oneidensis MR-1 cell suspensions with 80 ␮M AQDS under anoxic conditions. (b) Kinetic of AQDSox and Hemered in the intact S. oneidensis MR-1 cell suspensions with 80 ␮M AQDS under anoxic conditions. The symbols (䊏) and (䊐) represent [Hemered ] and [AQDSox ], respectively. Initial cell density of MR-1: 1.07 × 1012 cells mL−1 , sodium lactate 20 mM, pH 7.0, 30 ◦ C. The peak at 328 nm is attributed to AQDSox , and the peak at 552 nm is attributed to Hemered .

and AHQDS− , respectively. The peaks at 552 nm in Fig. 1a were attributed to Hemered , which refers to the reduced hemes that are present at the surface of Shewanella cells. The increase of Hemered refers to an increase of reduced cytochromes, and the decrease refers to the oxidation of reduced cytochromes present at the surface of Shewanella cells. It was found that Hemered transiently dropped to a low level at the very beginning (Stage 1: consumption of Hemered ) and then gradually increased to the initial level (Stage 2: recovery of Hemered ) (Fig. 1b). It was reported that the electrons can be accumulated in the periplasmic and outer-membrane cytochromes during growth of exoelectrogenic bacteria [29,30], so the Hemered was accumulated at the beginning. The rapid decrease of Hemered in the beginning of the traces in Stage (i) might attributed to the rapid redox reaction between AQDS and Hemered , as it was reported that the redox reaction between a quinone compound and Hemered occurred very fast (within 170 ms) [31]. Hence, it is feasible to directly measure AQDS and Hemered using the UV/Vis diffuse-transmittance spectral method. 3.1.2. AQDS reduction by MR-1 wild type and mutants To examine the electron transport chain of MR-1, the AQDS reduction was examined using MR-1 wild type (wt) and mutants including mtrC, mtrF, mtrA, mtrD, omcA, and cymA. The results in Fig. 2a and S2 show that while the k values for omcA and mtrF were close to the results for wt, the k values for the other mutants were substantially lower than that for wt. The k values were ranked in the following order: wt ≈ omcA ≈ mtrF > mtrD > mtrC > mtrA > cymA. cymA and mtrA exhibited distinctly low AQDS and Hemered reduction

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Fig. 2. The kinetics of the AQDS reduction by MR-1 wild type and the mutants mtrA, mtrC, mtrD, mtrF, omcA, and cymA. (a) AQDS reduction. The relationship between the pseudo-first-order reaction rate constants k and MR-1 wild type and different mutants (inset chart); (b) Hemered reduction. All experiments conducted with AQDS 100 ␮M, MR-1:1.07 × 1012 cells mL−1 , 20 mM sodium lactate, pH 7.0, 30 ◦ C.

rates (Fig. 2b), demonstrating their essential roles in controlling the electron transport chain for AQDS reduction. CymA, a member of the NapC/NirT family of hydroquinone dehydrogenases, can oxidize the quinol in the inner membrane and then transfer the electrons to MtrA through other periplasmic proteins and is essential for the respiratory reduction of AQDS [32,33]. The other periplasmic proteins are the tetraheme cytochromes STC and FccA [34,35], which are the important proteins in the extracellular electron transfer pathway of S. oneidensis MR-1. Deletion of CymA evidently hindered the ability of S. oneidensis MR-1 cell to use the terminal electron acceptors [36]. This is probably due to the inability of electron transfer to the outer-membrane cytochromes, which was confirmed by the lack of Hemered increase for cymA in Fig. 2b. MtrA was found to resemble a key periplasmic electron transfer component [37]. The MtrC and OmcA were found to be localized on bacterial cell surfaces [12]. The omcA mutant behaves as the wild-type strain, which suggests that OmcA is not important for the reduction of AQDS. The involvement of MtrC in extracellular reduction of AQDS suggests that MtrC probably serve as AQDS reductases [8,9]. MtrC was found to be required for extracellular reduction of AQDS [9]. The recovery of Hemered was severely attenuated after knocking out CymA and MtrA, moderately decreased after MtrC knock out, and changed only slightly when other proteins were knocked out. This result was similar to the AQDS reduction. It is probably correct that AQDS may penetrate into the OM and contact the periplasmic electron transfer proteins (CymA, MtrA, STC, and FccA), indicating that AQDS does not help to pump out sufficient electrons without

Fig. 3. The kinetics of AQDS reduction by c-Cyts in intact MR-1 cell suspension. (a) AQDS reduction. The relationship between the pseudo-first-order reaction rate constant k and the initial AQDS concentration (inset chart); (b) Hemered reduction. AQDS: 20 ␮M −120 ␮M, MR-1: 1.07 × 1012 cells mL−1 sodium lactate 20 mM, pH 7.0, 30 ◦ C.

CymA or MtrA. Hence, although we cannot exclude AQDS reduction via intracellular electron transfer, the extracellular electron transfer actually did play a dominant role in the AQDS reduction process. 3.2. Kinetics of AQDS and c-Cyts under various conditions 3.2.1. Effect of AQDS concentration The kinetics of AQDS reduction by c-Cyts in a living cell suspension were examined under different initial AQDS concentrations (20–120 ␮M). The AQDS reduction results (Fig. 3a and S3) showed that the pseudo-first-order rate constant (k) decreased gradually (from 0.34 min−1 to 0.12 min−1 ) with increasing AQDS concentration (from 20 ␮M to 120 ␮M). The initial AQDS reduction rate (r0 , ␮M min−1 ) can be calculated from k[AQDS]0 as 6.87, 6.18, 11.61, 13.69, and 14.99 ␮M min−1 for 20 ␮M to 40 ␮M, 80 ␮M, 100 ␮M, and 120 ␮M AQDS, respectively, so the AQDS reduction rates (r0 ) increase with increasing [AQDS]. Simultaneously, the Hemered changed with changes in the AQDS (Fig. 3b), showing that while the Stage (i) of all the treatments was similar, the recovery of Hemered in Stage (ii) became increasingly slow with increasing [AQDSox ]. The slow recovery rate of Hemered may account for the low AQDS reduction k values under high [AQDSox ]. 3.2.2. Effect of lactate concentration The kinetics of AQDS reduction and c-Cyts were examined under different initial lactate concentrations (10–20000 ␮M) with no electron donor as a control. The AQDS reduction results showed that

R. Han et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 520 (2017) 505–513

Fig. 4. The kinetics of AQDS reduction by c-Cyts in intact MR-1 cell suspension with different lactate concentrations from 0 ␮M to 20000 ␮M. (a) AQDS reduction. The relationship between the pseudo-first-order reaction rate constant k and lactate concentration (insert chart); (b) Hemered reduction. All experiments conducted with AQDS 100 ␮M, pH 7.0, 30 ◦ C.

the pseudo-first-order rate constant (k) increased gradually (from 0.0017 to 0.306 min−1 ) with the increase in initial lactate concentration (from 10 ␮M to 20000 ␮M) (Fig. 4a and S4). Shewanella cells with low lactate concentration (10 ␮M) cannot produce the necessary electrons to reduce excessive AQDS (100 ␮M) due to the lack of energy to grow. Obviously, the reduction rate of AQDS increased significantly when the concentration of sodium lactate increased to 200 ␮M. The Hemered in Stage (ii) showed a similar tendency with higher electron donor concentration inducing a higher Hemered recovery amount (Fig. 4b). This result indicates that the concentration of lactate may directly affect the MR-1 metabolic processes, followed by influencing the AQDS reduction rate. 3.2.3. Effect of cell density Five AQDS reduction treatment experiments with MR-1 were conducted with different cell densities from 0.4 to 1.5 (OD600 ). The kinetic results in Fig. 5a and S5 show that the k value of AQDS reduction increased proportionally with the increase in cell density, and the Hemered showed a similar tendency, i.e., that higher cell density induced higher Hemered recovery amount (Fig. 5b). The initial [Hemered ] was linearly correlated with the MR-1 cell suspension cell density, suggesting that Hemered played a dominant role in reducing AQDS in the living cell suspension. 3.2.4. Effect of temperature The AQDS reduction by MR-1 was also investigated under different incubating temperatures. The results in Fig. 6a and S6 show that the k value for AQDS reduction changed little when the temperature increased from 20 ◦ C to 30 ◦ C but then decreased dramatically

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Fig. 5. The kinetics of AQDS reduction by c-Cyts in intact MR-1 cell suspension in different MR-1 cell conditions from OD600 0.4 to OD600 1.5. (a) AQDS reduction. The relationship between the pseudo-first-order reaction rate constant k and the initial MR-1 cell concentration (insert chart); (b) Hemered reduction. All experiments conducted with AQDS 100 ␮M, sodium lactate 20 mM, pH 7.0, 30 ◦ C.

when the temperature increased from 30 ◦ C to 40 ◦ C. The Hemered recovery amounts under different temperatures (Fig. 6b) showed a similar pattern with the AQDS reduction, and the Hemered recovery amounts at 20 ◦ C to 30 ◦ C were higher than those at 35 ◦ C–40 ◦ C. This phenomenon may be related to the temperature-dependent cell physiological characteristics of MR-1, as it has previously been reported that biological activity of MR-1 is optimal at 30 ◦ C [38]. 3.2.5. Effect of pH To examine the effect of pH on AQDS reduction by MR-1, the kinetic investigation was performed under different pH values from 6.0 to 8.0. The results in Fig. 7a and S7 show that the k value for AQDS reduction increased substantially from 0.025 to 0.1 min−1 with increasing pH from 6.0 to 7.0 but then quickly decreased to 0.01 min−1 when the pH increased from 7.0 to 8.0. The recovery amounts of Hemered also increased at pH values from 6.0 to 7.0 but decreased very slightly with pH values from 7.0 to 8.0 (Fig. 7b). This phenomenon may be attributed to the pH-dependent cell growth and physiological characteristics of MR-1; a study by Han et al. [15] indicated that pH 7.0 was an optimal condition for cell growth. In addition to cell growth, pH is also a key parameter controlling the redox properties of AQDS, which may further influence AQDS reduction by MR-1. Previous studies have suggested that oxidized quinone can be transformed into hydroquinone after accepting two electrons, so the proton (H+ ) was involved in the transformation from quinone to hydroquinone [39]. This indicates that pH may directly influence the redox state of quinone. To obtain the UV/Vis spectra of AQDSox

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Fig. 6. The kinetics of AQDS reduction by c-Cyt in intact MR-1 cells at temperatures ranging from 20 to 40 ◦ C. (a) AQDS reduction. The relationship between the pseudofirst-order rate constant k and temperature (insert chart). (b) Hemered reduction. All experiments were conducted with 100 ␮M AQDS, MR-1: 1.07 × 1012 cells mL−1 , and sodium lactate 20 mM, pH 7.0.

and AQDSred under different pH conditions, dissolved AQDSox was reduced electrochemically at varying pH values (6.0–8.0). The results shown in Supplementary Material (SM) Fig. S1a indicate that the reduction products and reduction degrees of AQDSox were different and only the hydroquinone species (AH2 QDS− , AQDSH− ) were present under different pH conditions [21]. At pH 6.0–6.7, AQDSox was almost completely reduced, and the reduction products were mostly AH2 QDS− and a small amount of AHQDS− . At pH 7.0–7.3, AQDSox was partially reduced, and the reduction products were almost equal amounts of AH2 QDS and AHQDS− . At pH 7.6–8.0, the reduction products were mostly AHQDS− and a small amount of AH2 QDS. The pH value was also an important factor impacting AQDS reduction and the products by c-Cyts in MR-1 (SM Fig. S1b). When the reaction continued for 30 min, AQDS was only partially reduced to AH2 QDS at pH 6.0 and 6.3; AQDSox was completely reduced to AH2 QDS and AHQDS− at pH 6.7, 7.0, and 7.3; and the reduction products were almost all AHQDS− at pH 7.6 and 8.0. 3.3. Thermodynamic analysis of AQDS reduction by MR-1 3.3.1. Reactions involved in AQDS reduction by MR-1 It is well known that the OM c-Cyts (OmcA, MtrC, and MtrF) of S. oneidensis MR-1 were involved in electron transfer to the terminal electron acceptors via the Mtr pathway [24]. Based on the AQDS reduction results by mutant and wild type MR-1, the electron transfer pathway of AQDS reduction by MR-1 cells included intracellular electron transport (IET) and EET via inner membrane c-Cyts (In-Cyts) and outer membrane c-Cyts (Ex-Cyts), which can be concisely divided into the following three steps: (i) IET from electron donor to In-Cyts, (ii) IET from In-Cyts to Ex-Cyts or AQDS, and (iii) EET from OM c-Cyts to AQDS. In Step (i), the electron donor (lactate) can be utilized by MR-1, and concomitantly, IET then occurs

Fig. 7. The kinetics of AQDS reduction by c-Cyts in intact MR-1 cell suspension under different pH conditions from pH 6.0 to pH 8.0. (a) AQDS reduction. The relationship between the pseudo-first-order reaction rate constants k and pH (insert chart). (b) Hemered reduction. All experiments conducted with AQDS 100 ␮M, MR-1: 1.07 × 1012 cells mL−1 , sodium lactate 20 mM, 30 ◦ C.

from lactate to In-Cyts resulting in the redox transformation from In-Cytox to In-Cytred as Rxn. (1). − − + C3 H5 O− 3 + H2 O+4In-Cytox → 4In-Cytred + C2 H3 O2 + HCO3 + 5H

(1) From Rxn. (1), the reduction of In-Cyts was controlled by the utilization efficiency of lactate, which was influenced by incubation conditions, including lactate concentration, pH, cell density, and temperature. In Step (ii), In-Cytred can transfer electrons to Ex-Cyts, resulting in the formation of Ex-Cytred (Rxn. (2)). Because the AQDS may penetrate the cell membrane and contact the inner membrane and periplasmic electron transfer components (CymA and MtrA) [40], In-Cytred can also transfer electrons to AQDSox , resulting in the formation of AQDSred (Rxn. (3)). In-Cytred + Ex-Cytox → Ex-Cytred + In-Cytox

(2)

In-Cytred +AQDSox → AQDSred + In-Cytox

(3)

In Step (iii), the electrons from Ex-Cytred can be transferred to AQDSox which is directly in contact with OM, resulting in the reduction of AQDSox to AQDSred as Rxn. (4). Ex-Cytred + AQDSox → AQDSred + Ex-Cytox

(4)

The step-wise potential losses can drive electron flow from the electron donor via c-Cyts to AQDS, resulting in AQDS reduction [17].

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The specific theoretical redox potential of lactate (EC calculated by the Nernst equation shown in Eq. (1).



EC

− 3 H5 O3

= EC0

− 3 H5 O3

where E 0

C3 H5 O−

−





C2 H3 O− HCO− RT 3 2  ln 4F C3 H5 O−

H+

− 3 H5 O3

) can be

5 (1)

3

represents the standard redox potentials of lactate, R

3

is the ideal gas constant (8.3145 J mol−1 K−1 ), F is the Faraday constant (96,485C mol−1 e− ), and T is the temperature (298 K). From Eq. (1), when the concentration of lactate increased, the specific theoretical redox potential of lactate decreased gradually, resulting in the increase of AQDS reduction capacity. The electrons from oxidation of lactate can be transferred through the following IET and EET processes which were mediated by the In-Cyts and Ex-Cyts. Since the key electron transfer component of In-Cyts and Ex-Cyts is a heme with an iron centre, the half reaction for In-Cyts and Ex-Cyts transformation is shown in Rxn. (5): Hemeox + e− ↔ Hemered

(5)

The specific redox potentials of hemes (EHeme ) can be calculated by the Nernst equation (Eq. (2)): 0 EHeme = EHeme −

[Hemered ] RT ln F [Hemeox ]

(2)

where E 0heme represents the standard redox potentials of hemes. The driving force of the IET process (EIET ) can be indicated as Eq. (3). EIET = EHeme − EC

(3)

− 3 H5 O3

The electrons from Hemered in In-Cyts and Ex-Cyts can be transferred to AQDS resulting in the reduction into AHQDS− and AH2 QDS as Rxns. (6) and (7). AQDS + H+ + 2e− ↔ AHQDS−

(6)

AHQDS− + H+ ↔ AH2 QDS

(7) AHQDS−

The redox potentials of AQDS to (EQ/QH ) and AQDS to AH2 QDS (EQ/QH2 ) can be calculated from Eqs. (4) and (5). 0 EQ/QH = EQ/QH −

RT [AHQDS − ] ln 2F [AQDS][H + ]

0 EQ/QH2 = EQ/QH − 2

RT [AH2 QDS] ln 2F [AQDS][H + ]2

(4) (5)

0 where E 0Q/QH and EQ/QH are the standard redox potentials of AQDS 2

to AHQDS− and AQDS to AH2 QDS, respectively. The Rxns. (3) and (4) between In-Cyts/Ex-Cyts and AQDSox may be influenced by speciation of AQDS as indicated by Rxns. (6) and (7), which were strongly dependent on pH due to the pH-dependent redox potentials as indicated by Eqs. (4) and (5). While it is possible for AQDS to penetrate the cell membrane and react with In-Cytred , the mutant results suggested that the EET process played a dominant role in the AQDS reduction process. The driving force of the EET process (EEET ) is indicated in Eq. (6). EEET = (EQ/QH − EHeme )or(EQ/QH2 − EHeme )

(6)

The reaction rates and extent of AQDS reduction by MR-1 were determined by the driving forces for IET and EET (EIET and EEET ), which can be influenced by all the elementary reactions involved in the AQDS bioreduction processes based on the above thermodynamic analysis.

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3.3.2. Thermodynamic analysis of IET processes under various conditions Based on Eqs. (1)–(3), the driving force for IET processes (EIET ) was directly influenced by the standard redox potentials of lactate and hemes, the concentrations of lactate and its products, the ratios of reduced and oxidized hemes, and the H+ concentration as indicated by the solution pH. Hence, the incubation conditions, such as temperature, pH, cell density, and electron acceptor concentration, may influence IET processes via the metabolic rates of lactate, followed by influencing the bulk electron transfer rates to terminal electron acceptors. The incubation temperature was an important factor affecting the metabolic activity of cells. It has been well documented that the optimal physiological temperature of MR-1 was approximately 30 ◦ C [41,42]. Here, the k value for the AQDS reduction (Fig. 6) changed little at 20–30 ◦ C but decreased dramatically with the temperature from 35 ◦ C to 40 ◦ C, suggesting that the metabolic rate of lactate by MR-1 was slightly affected by the temperatures (20–30 ◦ C), but high temperature (35–40 ◦ C) may substantially reduce the driving force for IET processes (EIET ) by inhibiting the metabolic rates of lactate by MR-1, resulting in the low AQDS reduction rates. Similar to the case for temperature, pH was also an important factor affecting the metabolic activity of cells. It has been reported that the optimal pH for the cell physiological processes of MR-1 is 7.0 [22]. This study also confirmed the optimal pH to be 7.0. The k values for AQDS reduction (Fig. 7) also showed a maximum rate constant at pH 7.0, suggesting that the pH played a key role in controlling the EIET by affecting the metabolic rate of lactate by MR-1 and finally changed the AQDS reduction rate. Since slight decreases in OD600 at pH values from 7.3 to 8.0 were not well matched with the AQDS reduction rates, it can be speculated that some other factors may also contribute to the bulk electron transfer processes. MR-1 cells at a high density can consume much more lactate via cell metabolic processes, which can induce a high EIET . The presence of high concentrations of electron acceptor (AQDS) can also be favourable for the metabolic processes and enhance the IET reaction rates. The results in Figs. 3 and 5 for AQDS reduction show the same pattern, with variable AQDS concentrations and cell densities. Briefly, the high metabolic rates of electron donors can lead to high IET driving force rates as indicated by EIET . 3.3.3. Thermodynamic analysis of EET processes under various conditions Since the main reaction of the EET processes is the redox reaction between Hemered and AQDS as Rxn. (4), the effects of various conditions on the driving force for EET processes (EEET ) were determined by the thermodynamic properties of Hemered and AQDS. Based on the above discussion, the thermodynamic properties of hemes as indicated by the ratio of Hemered /Hemeox based on Rxn. (1) and Eq. (2) were mainly influenced by the metabolic rates of lactate in the IET processes, so the effects of various conditions on EEET were first caused by the changes in the redox status of hemes in IET processes, which was clearly illustrated above. In addition to hemes, the terminal electron acceptor (AQDS) may also be influenced by various incubation conditions, which concomitantly change the EEET and reaction kinetics. The redox properties of AQDS were mainly dependent on its speciation. During the redox cycling process of AQDS, it has been noted that the proton (H+ ) is also involved in the transformation from quinone to hydroquinone [33], indicating that pH may directly influence the quinone-mediated EET processes [43]. Hence, of all the incubation conditions (pH, temperature, cell density, and AQDS concentrations), pH is the most important factor influencing the thermodynamic properties of AQDS, as the speciation of AQDS was directly determined by pH, resulting in different redox properties.

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To clearly illustrate the effect of pH on the redox properties of AQDS, the CVs of AQDS and the OCVs of the reaction solution of MR-1 with AQDS were measured under the electrochemical workstation. The CVs of pure AQDS at different pH values (Fig. S8b) showed that symmetrical voltammetry results were observed for AQDS under different pH values, revealing that the redox transformation of AQDS was fully reversible. The midpoint potentials (Em ) of AQDS were plotted as a function of pH in Fig. S8d, which showed a linear relationship with a slope of −72 mV/pH. It’s close to the value of −60 mV/pH unit that expected for a 1e− :1H+ reaction. So, it suggests that the main species here are AQDS and AH2 QDS which based on the following 1e− :1H+ reaction. The OCVs of MR-1 with AQDS were plotted as a function of pH in Fig. S8a, which had a linear relationship with a slope at −84 mV/pH (Fig. S8c). The pH value can impact the general speciation of AQDS reduced by c-Cyts in MR-1 (Fig. S1b). The reduction product was AH2 QDS at low pH (6.0-6.3); AHQDS− began to appear and gradually increased with at increasing pH. At pH > 7.6, the reduction products were mainly AHQDS− . The results showed that just one electron is needed for AHQDS generation, while two electrons are needed for AH2 QDS generation, suggesting the electron-carrier capacity decreased with the increasing pH. Therefore, the effects of pH on the AQDS reaction by c-Cyts were attributed to different AQDS speciation and the involvement of protons. We investigated the reaction system OCVsuspension of AQDS reduction by MR-1 at varying pH values. A high correlation coefficient (R2 = 0.91) between OCVsuspension and pH was obtained, indicating that the change in pH caused the variation in the reaction system OCV (Figs. S8a and S8c). Based on CV testing under different AQDS conditions, we concluded that E’AQDS and pH were also negatively correlated (R2 = 0.99) (Figs. S8b and S8d). Therefore, it can be inferred that the higher the pH value is, the more likely the AQDS and MR-1 reaction is to occur. However, the results of reduction kinetics showed that the highest reduction rates of both AQDS and Hemered were obtained at pH 7.0 (Fig. 7a and b). Meanwhile, it is well-known that c-type cytochromes also present redox-Bohr effect (i.e. the reduction potential changes with pH), so the redox potential of the whole cytochromes is still pHdependent. However, it is reported that some of the hemes in electron transfer chain are also proton-coupled and some are not [44]. Furthermore, the redox potentials of MtrC and OmcA showed slight shift with the increasing of pH, indicating that the outermembrane cytochromes may be weakly affected by the changing of H+ . Thus, the EEET was probably mainly influenced by the potential of AQDS.

4. Conclusions AQDS and Hemered were directly measured in situ using the DT spectra to reflect the kinetics of AQDS reduction by c-Cyts in intact cells, which provides a useful approach to understanding the behaviour of the MR-1 outer-membrane enzymes under non-invasive conditions. In the presence of AQDS, the reduced cCyts initially rapidly decreased and then slowly recovered under all tested incubation conditions. Compared with the wt, cymA and mtrA exhibited distinctly low AQDS reduction rates of and Hemered regeneration. The highest AQDS reduction rate and fastest c-Cyts recovery were obtained at 30 ◦ C and pH 7.0, with sodium lactate serving as an electron donor. These conditions may be optimal due to the resemblance to suitable physiological conditions, which are favourable for metabolism and Hemered recovery. Thermodynamic analysis on the effects of various incubation conditions on AQDS reduction by c-Cyts in intact cells showed the most suitable temperature and pH might improve the metabolism of cells, thus resulting in a high lactate consumption rate and high electron out-

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