Developing a Bacterial Monolayer Biofilm Using an Electrochemical Reactor

Abstract

Source: Tokunou, Y. et. al., Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1. J. Vis. Exp. (2018)

This video demonstrates the assembly and operation of an electrochemical reactor model designed to simulate biofilm formation on electrode surfaces. Controlled electrochemical stimulation promotes microbial adhesion, leading to the gradual development of a bacterial monolayer biofilm.

Protocol

1. Construction of a three-electrode electrochemical reactor (Figure 1)
   1. Put an indium tin-doped oxide (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 silver/silver chloride (Ag/AgCl) (potassium chloride, 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 defined medium (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.
2. 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 an 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.

Results

Figure 1: Electrochemical reactor used in this study. Photograph of the electrochemical cell before construction...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass cylinder	N/A	N/A	Custom-made, used as the electrochemical reactor
PTFE cover and base	N/A	N/A	Custom-made, used as a cover and a foundation of the electrochemical reactor
Buthyl rubber	N/A	N/A	Custom-made, inserted between each component of electrochemical reactor
Septa	GL Science	3007-16101	Used as an injection port of electrochemical reactor
Indium tin-doped oxide (ITO) electrode	GEOMATEC	No.0001	Used as a working electrode, 5Ω/sq
Ag/AgCl KCl saturated electrode	HOKUTO DENKO	HX-R5	Used as a reference electrode, Φ0.30mm
Platinum wire	The Nilaco Cooporation	PT-351325	Used as a counter electrode
Luria-Bertani (LB) Broth, Miller	Becton, Dichkinson and Company	244620	Medium for precultivation of S. oneidensis MR-1
Bacto agar	Becton, Dichkinson and Company	214010
NaHCO₃	Wako	191-01305	Used for defined medium (DM)
CaCl₂·2H₂O	Wako	031-00435	Used for DM
NH₄Cl	Wako	011-03015	Used for DM
MgCl₂·6H₂O	Wako	135-00165	Used for DM
NaCl	Wako	191-01665	Used for DM
2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)	DOJINDO	346-08235	Used for DM
Sodium Lactate Solution	Wako	195-02305
Bacto yeast extract	Becton, Dichkinson and Company	212750
Deuterium oxide (D, 99.9%)	Cambridge Isotope Laboratories, Inc.	DLM-4-PK	Additive for kinetic isotope effect experiments
Incubator	TOKYO RIKAKIKAI CO. LTD.	LTI-601SD	Used for precultivation
Shaker	TAITEC	NR-3	Used for precultivation
Autoclave machine	TOMY SEIKO CO. LTD.	LSX-500	Used for sterilization of the electrochemical reactor and the medium
Clean bench	SANYO	MCV-91BNF	Used to prevent the contamination of the electrochemical reactor and the medium with other microbes
Centrifuge separator	Eppendorf	5430R	Rotational speed upto 6000×g is required
Nitrogen gas generator	Puequ CO. LTD.	PNTN-2	Nitrogen gas cylinder can also be used instead of gas generator
UV-vis spectrometer	SHIMADZU	UV-1800	Used for optimization of cell density
Potentiostat	BioLogic	VMP3	Used for biofilm formation and kinetic isotope effect experiments
Thermal water circulator	AS ONE	TR-1A	Used for maintanance of temperature of electrochemcial reactor
Faraday cage	HOKUTO DENKO	HS-201S	Used for electrochemical experiments