Principal Research Results

Stimulation of Microbial Growth by Regulation of Redox Potential

Background
Microorganisms gain chemical energy for sustenance through electron-transfer reactions such as respiration, fermentation,and photosynthesis. We previously proposed an electrochemical cultivation method to increase the cell density of specific bacteria(e.g., iron-oxidizing bacteria); this method involved the supply of electrons as the energy source from an electrode to the bacteria. In
general, since electron-transfer reactions depend on redox potential1), it has been estimated that every microorganism has the optimal
redox potential required for its growth. However, no electrochemical cultivation method has been proposed so far to control growths
and activities of microorganisms by regulating and maintaining the redox potential in culture media. Therefore, the requirement toregulate the redox potential in culture media by electron transfer between an electrode and an electron mediator led us propose thenovel electrochemical cultivation system for stimulating microbial growth by creating the optimal redox potential required for growth
without electron supply to the microorganism as proposed previously (Fig1).

Objective
We aim to develop a novel method to stimulate the growth of microorganisms by regulating the electrochemical redox
potential.

Principal Results
1. Confirmation of decrease in redox potential with the growth of sulfate-reducing bacterium
Sulfate-reducing bacterium2) “Desulfovibrio desulfuricans” was used as the representative of environmental microorganisms. It was confirmed that the redox potential (E) in the culture medium decreased from -0.1 V to -0.5 V (vs. Ag/AgCl) with thegrowth of the sulfate-reducing bacterium, without any electrochemical regulation.
2. Effect of the regulation of electrochemical redox potential on the growth of sulfate-reducing bacterium
Electrochemical cultivation of the sulfate-reducing bacterium was performed by constantly maintaining the redox potential
between +0.4 V and -0.8 V using quinone compound as the electron mediator (Fig2). When the redox potential was maintained above-0.45 V, the growth curve was quite similar to the one without the regulation of the electrochemical redox potential. However, it wasrevealed that maintenance of the electrochemical redox potential below -0.6 V enabled early initiation of the growth of the sulfatereducing bacterium. These results suggested that regulation of redox potential can preferentially stimulate the growth of specificmicroorganisms. This is the first report that clarifies that optimal redox potential facilitates microbial growth.
3. Selective cultivation of specific bacteria by the regulation of electrochemical redox potential
Electrochemical cultivation of various types of bacteria (mixed culture) was performed at E = +0.4 V, -0.1 V, and -0.6 V. It
was clarified by denatured gradient gel electrophoresis (DGGE) that dominant species of bacteria changed by varying applied potential (Fig3). Furthermore, the sulfate-reducing bacteria grew preferentially and became dominant when the electrochemical redox
potential was maintained at -0.6 V, as clarified in Fig2.
On the basis of these results, we propose a novel cultivation method, which is different from the conventional cultivation
method, for stimulating the growth of specific bacteria by regulating the electrochemical redox potential in culture media.

Future Developments
The principle that the regulation of redox potential affects microbial growth will be clarified.
1) Redox potential: a measure of the tendency of a given system to donate or accept electrons
2) Sulfate-reducing bacterium: a category of strictly anaerobic bacteria that utilize sulfate for respiration
Activities of these bacteria are responsible for biotechnological removal of pollutants from the environment.
Main Researcher: Shin-ichi Hirano, Ph.D.,
Research Scientist, Biotechnology Sector, Environmental Science Research Laboratory
Reference
S. Hirano, et al., 2008, “Electrochemical control of bacteria (Part XI) -Regulation of sulfate-reducing bacteria by redox control-”
CRIEPI report (V07018) (in Japanese)402. Environment

Electrochemical cultivation (this study)
Electrochemical cultivation(conventional)

Redox potential regulation
by electrolysis

Electron transfer from electrodeto microorganisms

e-Microorganisms
e.g., Iron-oxidizing bacteria..

electrodeMetale-Controlled redox potentiale-

電極

Stimulation ofelectron transfer

Electron mediator(Reduced)Electron mediator(Oxidized)

e-Not suitableSuitable

The ratio of oxidized form
and reduced form of themediator is maintained byelectrolysis

Growth suppression

2Growth enhancement

Increased cell density

Fig.1 Stimulation of bacterial growth by regulation of redox potential using electrolysis
The cultivation method in this study establishes an optimal redox potential for the growth of a specific microorganism by electrontransfer between an electrode and an electron mediator in culture medium. This method is based on a new concept to stimulate
microbial growth by regulating the redox potential by electrolysis.

about 10hr
Withoutelectrolysis
Oxidizedcondition

No effect ofelectrolysis

0.45 V–0.6 V

Positiveeffect–0.7 V–0.8 V

Cell density (cells/ml)+0.4 V

109

108

107
109

Reducedcondition

108

107
01020304050
Incubation time (h)6070Fig.2 Effect of the regulation of redox potential on the growth of sulfate-reducing bacterium
There is no effect of electrolysis on microbial growth when the redox potential is maintained between +0.4 V and –0.45V (topright). The growth of the sulfate-reducing bacteria initiated approximately 10 hours earlier when the redox potential in the culture
medium was maintained below –0.6 V than when the electrochemical redox potential was not regulated, in which case growth
initiation takes approximately 37 hours.

Fig.3 Enrichment of specific bacteria by the regulation ofredox potential

Sulfate reducing
bacteria

Mixed cultures containing various types of environmental
microorganisms were cultivated at controlled redox potentials (+0.4 V,–0.1 V, and –0.6 V). Depending on the applied redox potential, density ofexisting microbial species varied, and sulfate-reducing bacteria wereenriched dominantly at –0.6 V (red arrow), which is the optimal redox
potential for their growth.
[Each band refers to individual species of microorganism]

Without +0.4 V –0.1 V –0.6 V
electrolysis

Oxidizedcondition

Reducedcondition

41