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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Abstract

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Introduction

Electrochemical techniques to directly characterize a redox protein in an intact bacterial cell have recently emerged since the discovery of metal-reducing microbial strains, such as S. oneidensis MR-1 or Geobacter sulfurreducens PCA, which have outer membrane c-type cytochrome complexes (OM c-Cyts) exposed to the cell exterior1,2,3,4,5. The OM c-Cyts mediate electron transport from the respiratory chain to solid substrates located extracellularly. This transport is referred to as extracellular electron transport (EET)1,6 and is a critical process for emerging biotechnologies, such as microbial fuel cells6. Therefore, to understand the underlying EET kinetics and mechanisms, and its link to microbial physiology, OM c-Cyts have been investigated using whole-cell electrochemistry4,7, combined with microscopy8,9, spectroscopy10,11, and molecular biology2,4. In contrast, methods to investigate the impact of EET-associated cation transport, e.g., protons, on EET kinetics in living cells have been scarcely established, despite proton transport across bacterial membranes having a critical role in signaling, homeostasis, and energy production12,13,14. In the present study, we describe a technique to examine the impact of proton transport on EET kinetics in the S. oneidensis MR-1 cell using whole-cell electrochemical measurements, which requires the identification of the rate-limiting step in microbial current production15.

One direct way to evaluate the contribution of proton transport on the associated EET is the deuterium kinetic isotope effect (KIE). The KIE is observable as the change in electron transfer kinetics upon the replacement of protons with deuterium ions, which represents the impact of proton transport on electron transfer kinetics16. The theory of KIE itself has been well established using electrochemical measurements with purified enzymes17. However, since current production in S. oneidensis MR-1 results from multiple, diverse, and fluctuating processes18, one cannot simply identify EET as the rate-limiting process. To observe the KIE on proton transport processes coupled with EET, we need to confirm that the microbial current production is limited by electron transport via OM c-Cyts to the electrode. For this purpose, we replaced the supernatant solution with fresh medium containing a high concentration of lactate as an electron donor at the optimal pH and temperature conditions before KIE measurement; this replacement served two roles: (1) it enhanced the rate of the upstream metabolic processes compared to the EET, and (2) omitted the swimming cells in the supernatant released from the monolayer biofilm of S. oneidensis MR-1 on the working electrode (indium tin-doped oxide (ITO) electrode). The presented detailed protocol is intended to help new practitioners maintain and confirm that the EET process is the rate-determining step.

Protocol

1. Formation of a Monolayer Biofilm of S. oneidensis MR-1 on an ITO Electrode (Figure 1)

NOTE: To prevent the contamination of the electrochemical reactor with other microbes, all the media, implements, and components of the electrochemical reactor should be sterilized in advance. When using S. oneidensis MR-1 cells and constructing the electrochemical reactors, all the procedures should be conducted on a clean bench.

  1. Cultivation of S. oneidensis MR-1 cells
    NOTE: A monolayer biofilm of S. oneidensis MR-1 was formed on an ITO electrode following the conditions reported previously4.
    1. To grow S. oneidensis MR-1 cells, add one colony of S. oneidensis MR-1 grown on an agar plate comprising Luria-Bertani (LB) (20 g/L) and bacto agar (15 g/L) into 15 mL of LB medium (20 g/L) at 30 °C for 24 h in aerobic conditions with shaking at 160 rpm.
    2. Centrifuge the cell suspension at 6,000 × g for 10 min, and resuspend the resultant cell pellet in 15 mL of defined medium (pH 7.8) (DM: NaHCO3 [2.5 g/L], CaCl2·2H2O [0.08 g/L], NH4Cl [1.0 g/L], MgCl2·6H2O [0.2 g/L], NaCl [10 g/L], and 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid [HEPES; 7.2 g/L]), supplemented with 10 mM lactate and 0.5 g/L yeast extract as the source of carbon, and trace elements for S. oneidensis MR-1, respectively.
    3. Further cultivate the cell suspension aerobically at 30 °C for 12 h with shaking at 160 rpm and centrifuge again at 6,000 × g for 10 min. Wash the resultant cell pellet twice with DM medium by centrifugation for 10 min at 6,000 × g before the electrochemical experiment.
  2. Construction of a three-electrode electrochemical reactor (Figure 1)
    1. Put an ITO substrate as the working electrode at the bottom of the reactor.
    2. Subsequently, insert a glass cylinder (diameter of 2 cm) and a polytetrafluoroethylene (PTFE) cover. Then insert Ag/AgCl (KCl saturated) and a platinum wire into the reactor as the reference and counter electrodes, respectively.
      NOTE: To prevent the leakage of air and solution, insert a butyl rubber sheet between each component.
    3. Add 4.0 mL of DM supplemented with 10 mM lactate and 0.5 g/L yeast extract into the electrochemical reactor.
    4. After confirming that there is no leakage from the electrochemical reactor, flow the nitrogen gas into the electrochemical reactor over 20 min to maintain anaerobic conditions inside the electrochemical reactor.
      NOTE: To prevent contamination with other microbes, the gas should be filtered before it flows into the electrochemical reactor.
    5. Connect the electrochemical reactor to a potentiostat and apply +0.4 V (versus standard hydrogen electrode, SHE) to the ITO electrode, keeping the temperature of the electrochemical reactor at 30 °C using an external water circulation system.
      NOTE: To prevent the effect of an external electric field, the electrochemical reactor should be placed in a Faraday cage.
  3. Electrochemical cultivation of S. oneidensis MR-1 cells (Figure 1 and Figure 2)
    1. Adjust the cell density of the suspension obtained in step 1.1 to an optical density of 1.43 at 600 nm (OD600) with DM supplemented by 10 mM lactate and 0.5 g/L yeast extract.
      NOTE: To obtain the correct OD600, apply the cell suspension of OD600 ≤ 0.8 to a UV-vis spectrometer prior to adjusting the OD600 to 1.43.
    2. Add 0.3 mL of the cell suspension into the electrochemical reactor through the injection port using a syringe: the OD600 in the reactor changes to 0.1.
      NOTE: The addition of 0.3 mL cell suspension with OD600 = 1.43 into electrochemical reactor containing 4.0 mL medium results in 4.3 mL of solution with an OD600 = 0.1. When using other reactors with different volumes, the calculation of the cell density is required.
    3. Continue the potential application at +0.4 V (versus SHE) to the ITO electrode for 25 h.
      NOTE: For the formation of a monolayer biofilm on an ITO electrode, ensure that the produced current exhibits a deviation less than 50% from Figure 2.

2. Replacement of the Supernatant with Fresh DM Medium with 10 mM Lactate (Figure 3)

  1. Stop the potential application and disconnect the electrochemical reactor from the potentiostat and the water circulation system.
  2. Replacement of the supernatant with fresh DM medium with 10 mM lactate
    1. Flow the nitrogen gas into a bottle containing DM with 10 mM lactate over 20 min to remove oxygen from the medium.
    2. Slowly remove all the supernatant inside the electrochemical reactor using a syringe, under flowing nitrogen gas (Figure 3a, b).
      NOTE: To avoid breaking the biofilm on the ITO electrode by nitrogen gas, the gas should flow above the liquid surface.
    3. Add 4.0 mL of fresh DM containing 10 mM lactate using a syringe (Figure 3c).
      NOTE: To avoid breaking the biofilm on the ITO electrode, slowly inject the medium along the wall of the electrochemical reactor. To keep the biofilm wet, the medium should be added immediately after the removal of supernatant. Injection of the medium upto 1 min after the removal of supernatant does not impact the current production from the biofilm of S. oneidensis MR-1.
    4. Slant the electrochemical reactor to remove all of the supernatant attached to the wall of the electrochemical reactor.
    5. Repeat the steps 2.2.2-2.2.4 three times in total.
  3. Stop the gas flow and connect the electrochemical reactor to the potentiostat again, applying +0.4 V (versus SHE) to the ITO electrode at 303 K.

3. Addition of Deuterium Water to Measure the KIE on the EET Process (Figure 4)

  1. Confirm that the current production from a monolayer biofilm of S. oneidensis MR-1 is stable and does not increase rapidly. If the current increases steeply, wait until the current stabilizes within a 5% increase over 10 min.
    NOTE: The formation of a monolayer biofilm on an ITO electrode without contamination of other microbial strains was confirmed by rDNA sequences and a scanning electron microscopic image of the biofilm on an ITO electrode, as reported previously4. For confirmation of the rate limitation by the EET process, monitor the effect of adding shuttling electron mediators such as 100 µM anthraquinone-1-sulfonate ( α-AQS). See the section of Representative Results and reference15 for further details.
  2. Add 40 µL of anoxic 50% (v/v) D2O into the electrochemical reactor using a syringe such that the concentration is 0.5% (v/v) D2O in the reactor.
    NOTE: To prevent damage to the biofilm by D2O addition, inject the D2O drop-wise.
  3. Wait for the current to stabilize, and subsequently add the D2O up to 4.0% (v/v).
    NOTE: To obtain the KIE value (the ratio of current production in the presence of D2O and H2O), check the effect of the same volume of H2O addition on the current production.

Results

After 25 h of potential application at +0.4 V (versus SHE), a monolayer biofilm was formed on the working electrode of ITO glass, which was previously confirmed by either a scanning electron microscopy or a confocal microscopy4. The representative time course of current production from the S. oneidensis MR-1 during the formation of a monolayer biofilm is shown in Figure 2. Although the current alters in every measurement, the ...

Discussion

Our whole-cell electrochemical assay has several technical advantages compared with protein electrochemistry. While protein purification requires multi-step time-consuming procedures, our whole-cell method takes one day of self-organized biofilm formation after cell culture. To achieve a stable interaction between OM c-Cyts and the electrode, we need only sterilization and cleaning of the electrode surface; it does not require electrode modification for organizing the orientation of proteins4

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was financially supported by a Grant-in-Aid for Specially Promoted Research from the Japan Society for Promotion of Science (JSPS) KAKENHI Grant Number 24000010, 17H04969, and JP17J02602, the US Office of Naval Research Global (N62909-17-1-2038). Y.T. is a JSPS Research Fellow and supported by JSPS through the Program for Leading Graduate Schools (MERIT).

Materials

NameCompanyCatalog NumberComments
Glass cylinderN/AN/ACustom-made, used as the electrochemical reactor
PTFE cover and baseN/AN/ACustom-made, used as a cover and a foundation of the electrochemical reactor
Buthyl rubberN/AN/ACustom-made, inserted between each component of electrochemical reactor
SeptaGL Science3007-16101Used as an injection port of electrochemical reactor
Indium tin-doped oxide (ITO) electrodeGEOMATECNo.0001Used as a working electrode, 5Ω/sq
Ag/AgCl KCl saturated electrodeHOKUTO DENKOHX-R5Used as a reference electrode, Φ0.30mm
Platinum wireThe Nilaco CooporationPT-351325Used as a counter electrode
Luria-Bertani (LB) Broth, MillerBecton, Dichkinson and Company244620Medium for precultivation of S. oneidensis MR-1
Bacto agarBecton, Dichkinson and Company214010
Anthraquinone-1-sulfonate (α-AQS)TCIA1428
Flavin mononucleotide (FMN)Wako184-00831
NaHCO3Wako191-01305Used for defined medium (DM)
CaCl2 · 2H2OWako031-00435Used for DM
NH4ClWako011-03015Used for DM
MgCl2 · 6H2OWako135-00165Used for DM
NaClWako191-01665Used for DM
2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)DOJINDO346-08235Used for DM
Sodium Lactate SolutionWako195-02305
Bacto Yeast ExtractBecton, Dichkinson and Company212750
Deuterium oxide (D, 99.9%)Cambridge Isotope Laboratories, Inc.DLM-4-PKAdditive for kinetic isotope effect experiments
IncubatorTOKYO RIKAKIKAI CO. LTD.LTI-601SDUsed for precultivation
ShakerTAITECNR-3Used for precultivation
Autoclave machineTOMY SEIKO CO. LTD.LSX-500Used for sterilization of the electrochemical reactor and the medium
Clean benchSANYOMCV-91BNFUsed to prevent the contamination of the electrochemical reactor and the medium with other microbes
Centrifuge separatorEppendorf5430RRotational speed upto 6000×g is required
Nitrogen gas generatorPuequ CO. LTD.PNTN-2Nitrogen gas cylinder can also be used instead of gas generator
UV-vis spectrometerSHIMADZUUV-1800Used for optimization of cell density
PotentiostatBioLogicVMP3Used for biofilm formation and kinetic isotope effect experiments
Thermal water circulatorAS ONETR-1AUsed for maintanance of temperature of electrochemcial reactor
Faraday cageHOKUTO DENKOHS-201SUsed for electrochemical experiments

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