Conversion of Carbon Dioxide to Metabolites by <i>Clostridium acetobutylicum</i> KCTC1037 Cultivated with Electrochemical Reducing Power

doi:10.4236/aim.2012.23040

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Advances in Microbiology, 2012, 2, 332-339

http://dx.doi.org/10.4236/aim.2012.23040 Published Online September 2012 (http://www.SciRP.org/journal/aim)

Conversion of Carbon Dioxide to Metabolites by

Clostridium acetobutylicum KCTC1037 Cultivated with

Electrochemical Reducing Power

Bo Young Jeon1, Il Lae Jung2, Doo Hyun Park1*

1Department of Biological Engineering, Seokyeong University, Seoul, South Korea

2Department of Radiation Biology, Environmental Radiation Research Group,

Korea Atomic Energy Research Institute, Daejeon, South Korea

Email: *[email protected]

Received June 21, 2012; revised July 29, 2012; accepted August 6, 2012

ABSTRACT

In this research, metabolic fixation of CO2 by growing cells of C. acetobutylicum cultivated with electrochemical re-

ducing power was tested on the basis of the metabolites production and genes expression. In cyclic voltammetry, elec-

trochemical oxidation and reduction reaction of neutral red (NR) immobilized in intact cells of C. acetobutylicum was

stationarily repeated like the soluble one in the condition without CO2 but the electrochemical reduction reaction was

selectively increased by addition of CO2. In electrochemical bioreactor, the modified graphite felt cathode with NR

(NR-cathode) induced C. acetobutylicum to generate acetate, propionate, and butyrate from CO2 in defined medium.

When H2 and CO2 were used as an electron donor and an electron acceptor, respectively, C. acetobutylicum also pro-

duced the same metabolites in a defined medium. C. acetobutylicum was not grown in the defined medium without sub-

stituted electron donors (H2 or electrochemical reducing power). C. acetobutylicum cultivated with electrochemical re-

ducing power produced more butyrate than acetate in complex medium but produced more acetate than butyrate in de-

fined medium. The genes of encoding the enzymes catalyzing acetyl-CoA in C. acetobuylicum electrochemically culti-

vated in defined medium than conventionally cultivated in complex medium. These results are a clue that C. acetobu-

tylicum may metabolically convert CO2 to metabolites and produce free energy from the electrochemical reducing power.

Keywords: C. acetobutylicu m ; CO2-Assimilation; Electrochemical Reducing Power; Coupling Redox Reaction

1. Introduction

Chemoautotrophs that regenerate reducing power and

produce free energy in coupling with oxidation of H2 can

more effectively fix CO2 than ammonium, nitrite, and

ferrous-oxidizing bacteria because redox potential of

H2/2H+ (–0.42 V vs. NHE) is lower than NAD+/NADH

(–0.32 V vs. NHE) [1]. The reducing power generated in

coupling with oxidation of ammonium, nitrite, and fer-

rous ion can’t induce regeneration of NAD(P)H without

a reverse electron transport system coupled to consump-

tion of external energy [2-5]. Experimentally measured

redox potential of NR is –0.325 V (vs. NHE), which is

theoretically enough to mediate generation of electron-

driving force from electrode to NADH [6]. Practically,

electrochemically reduced NR catalyzes NADH regen-

eration by non-enzymatic catalysis [7]. Theoretically,

electrochemically reduced NR may be more effective in

reducing power than H2 on the basis of non-enzymatic

catalysis of NADH regeneration.

C. acetobutylicum is a typical fermentation bacterium

that produces acetate, propionate, and butyrate by the

coupling redox reaction of reducing power and ace-

tyl-CoA generated from metabolic oxidation of glucose,

and also autotrophically produces acetyl-CoA from the

coupling redox reaction of H2 and CO2 in defined me-

dium with organic nitrogen nutrient [8,9]. The acetyl-

CoA is metabolically oxidized to acetate coupled to re-

generation of NADH or reduced to butyrate coupled to

oxidation of NADH [10]. Biological production of or-

ganic polymers, fatty acids and alcohols from H2 and

CO2 has been studied in order to decrease global warm-

ing and produce renewable energy and useful biomass

[11,12]. H2 is the most effective electron donor to bacte-

riologically fix CO2 on the basis of its redox potential but

is less practical to apply to biological system owing to

very low solubility in water and possible explosiveness

in the process of use, transport and storage. The cost for

production of H2 is not economical in comparison of

glucose price. In order to solve the problem caused by

the H2 production cost, an alternative technology has

*Corresponding author.

B. Y. JEON ET AL. 333

been developed [13,14].

Electricity generated from the solar cell can be directly

converted to biochemical reducing power in coupling

with the redox reaction of NR immobilized in bacterial

cell or graphite felt electrode [6]. Bacterial CO2 fixation

induced by biochemical reducing power electrochemi-

cally regenerated by the solar cell electricity may corre-

spond to the photosynthesis because O2 is generated from

anode compartment and CO2 is biochemically assimi-

lated into biomass and converted to metabolites in cath-

ode compartment [15]. Covalently immobilized NR in

graphite felt electrode can function as a catalyst for NADH

regeneration and a redox carrier for electron transfer

from electrode to bacterial cell [16]. Redox potential of

NR is –0.325 volt (vs. NHE), which is 0.05 volt lower

than NAD+. The electrochemical redox reaction of NR

can be coupled to biochemical redox reaction as follows:

[NRox + 2e– + 1H+ → NRred; NRred + NAD+ → NRox +

NADH]. Commonly, NRox and NAD+ are reduced to NRred

and NADH, respectively by accepting two electrons from

electrode and NRred (ox: oxidation; red: reduction).

In this study, electrochemical reducing power was

charged to C. acetobutylicum culture using the NR-

cathode to induce autotrophic production of acetate and

butyrate from CO2 in a defined medium and increase of

butyrate production in a complex medium. The NR-

cathode, to which –2 V of DC-electricity was charged,

may be an optimized habitat for strict anaerobes because

the lower oxidation-reduction potential than –300 mV (vs.

NHE) is electrochemically generated and the electro-

chemically reduced NR may catalyze bacterial NADH

regeneration.

2. Materials and Methods

2.1. Medium

Reinforced clostridial (RC) medium (Tryptone 10 g/L,

Sodium chloride 5 g/L, Beef extract 10 g/L, Yeast extract

3 g/L, Glucose 20 g/L, Starch 1 g/L, L-cystein hydro-

chloride 0.5 g/L, Sodium acetate 3 g/L) was used as a

complex medium and for successive cultivation of C.

acetobutylicum. M9 mineral medium (Disodium phos-

phate 6.8 g/L, Monosodium phosphate 3 g/L, Ammo-

nium chloride 5 g/L, Sodium chloride 0.5 g/L, Magne-

sium sulfate 0.246 g/L, Calcium chloride 0.0147 g/L)

supplemented with sodium bicarbonate (25 mM) and

yeast extract (3 g/L) was used as a defined medium. Fifty

ml of RC or defined medium was prepared in anaerobic

serum vials (total volume 165 ml) whose headspace was

filled with 2 atmospheres of oxygen-free N2 or H2.

2.2. Electrochemical Bioreactor

An electrochemical bioreactor that was designed for con-

tinuous culture in previous research [16] was partially

modified for cultivation of strict anaerobic bacterium in

batch culture, as is shown in Figure 1. The electro-

chemical bioreactor (inner diameter, 80 mm; height, 200

mm; medium volume, 500 ml; electrode volume, 250 ml;

total volume, 1000 ml; Pyrex, USA) with a built-in anode

compartment was designed to equalize distance between

anode and all round of cylindrical cathode. A sintered

glass filter (diameter, 50 mm; thickness, 5 mm; pore, 1 -

1.6 μm, Duran, Germany) that was modified with cellu-

lose acetate film (35 μm thickness, Electron Microscopy

Sciences, USA) was fixed at the bottom end of the

tube-type anode compartment (inner diameter 20 mm;

height, 150 mm; working volume, 50 ml). Cellulose ace-

tate film attached to the sintered glass functions as a

semipermeable membrane capable of selectively trans-

ferring water, gas, and proton. Five hundred ml of the

media was prepared in the electrochemical bioreactor

(Figure 1) to which O2-free CO2 (50 ml·min–1) was con-

tinuously supplied during cultivation. Inoculation ratio

was adjusted to 5% (w/w) of medium volume. DC –2 V

of electricity was charged to NR-graphite felt cathode to

induce electrochemical reduction reaction of NR for C.

acetobutylicum. The defined medium was used as ano-

lyte to avoid generation of osmosis between anode and

cathode compartment.

2.3. Electrode

Graphite felt (thickness, 10 mm; height, 200 mm; length,

Figure 1. Electrochemical bioreactor composed of graphite

felt modified with NR (NR-cathode), glass filter membrane

modified with cellulose acetate film, and platinum anode.

B. Y. JEON ET AL.

334

500 mm; Electrosynthesis, USA) was rolled up to be a

cylinder type (internal diameter, 40 mm; external diame-

ter, 75 mm). Neutral red was immobilized to thegraphite

felt (10 × 200 × 500 mm, Electrosynthesis, USA) by the

covalent bond between neutral red and polyvinyl alcohol

(mean molecular weight, 80,000, Sigma, USA) according

to the technique used in previous research [16]. The

graphite felt modified with NR was used as a cathode

and a platinum wire (thickness 0.5 mm, length 150 mm)

was employed as an anode. Electric potential charged to

NR-cathode was precisely adjusted to –2 V.

2.4. Analysis of Electrochemical Reaction of

C. acetobutylicum

The cyclic voltammetry was employed in order to ana-

lyze electrochemical redox reactions between electrode

and intact cell of C. acetobutylicum. The cyclic voltam-

metry was conducted using a voltammetric potentiostat

(BAS model CV50W, USA) linked to a data acquisition

system. Aglassy carbon electrode (5mm diameter, Elec-

trosynthesis, USA), a platinum wire, and an Ag/AgCl

electrode (redox potential, +0.2 V vs. NHE, Electrosyn-

thesis, USA) were utilized as a working electrode,

counter-electrode, and reference electrode, respectively.

The reactant was composed of 25 mM Tris-HCl buffer

(pH 7.5) containing 5 mM NaCl and 100 μM NR. C.

acetobutylicum that was anaerobically cultivated in the

modified M9 medium for 48 hr under H2 atmosphere was

anaerobically centrifuged at 1500 × g and 4˚C for 60 min.

The precipitated bacterial cells were suspended in 0.05

volume of the oxygen-free reaction mixture, in which NR

was spontaneously immobilized in bacterial cells. Prior

to and during the cyclic voltammetry measurement, ar-

gon (99.999%) was sparged into headspace of reaction

beaker in order to protect contamination of the reaction

mixture by oxygen. The scanning rate was 25 mV·s–1 over

a range of 0 to –1200 mV. During cyclic voltammetry for

NR dissolved in reactant or immobilized in C. acetobu-

tylicum, the variations of upper voltammetric peaks (an

indicator for electron transfer from electrode to bacterial

cells through NR) and lower voltammetric peaks (an in-

dicator for electron transfer from bacterial cells to elec-

trode through NR) by addition of CO2 were recorded.

2.5. Analysis of Metabolites

Bacterial metabolites were analyzed using a Gas Chro-

matography/Mass Spectrometry (Clarus 600 series +

TurboMatrix HSS Trap, PerkinElmer, USA) equipped

with Elite-FFAP column (ID 0.25 μm, OD 0.32 μm,

length 30 m) and electron ionization system. Bacterial

culture was centrifugation at 10,000  g and 4˚C for 30

min and filtered with membrane filter (pore 0.22 μm),

and then directly injected into the GC/MS injector. Con-

centration of metabolites was determined based on peak

area of standard compounds and chemical species was

determined based on mass profile database.

2.6. Microarray of mRNA

C. acetobutylicum was cultivated in the electrochemical

bioreactor using the defined medium under strict anaero-

bic CO2 atmosphere and in the complex medium under

strict anaerobic N2 atmosphere for 5 days. Total RNA

was isolated and purified from harvested bacterial cells

using a RNA purification kit (Total RNA, spin-column

format, Oligotex mRNA mini kit, Qiagen Korea, Seoul).

Microarray analysis of mRNA was conducted at Ge-

nomictree (Daejeon, Korea) using the systems, kits,

DNA chips, and analysis software offered by Agillent

Technologies (Korea branch, Seoul) via a turnkey-based

analyzing order. The significantly expressed genes that

are concerned with CO2 fixation and energy metabolism

were selectively analyzed to compare the relationship

between metabolic pathway related with CO2 fixation

and cultivation conditions.

3. Results

3.1. Electrochemical Redox Reaction in Coupling

with CO2

Cyclic voltammetry is a useful technique to measure

electrochemical coupling redox reaction of electron me-

diator immobilized in bacterial cells. In the cyclic volt-

ammetry without bacterial cells, the electrochemical re-

dox reaction of NR was measured to be –0.52 V (vs.

Ag/AgCl), which is very similar to the experimental

value –0.525 V (vs. Ag/AgCl) measured in standard con-

dition. Both the upper and lower voltammetric peaks

were not altered by addition of CO2 as expected; in con-

trast, the upper voltammetric peak generated by NR im-

mobilized in C. acetobutylicum was shifted upward from

2.4 to 2.9 μA and rightward from –0.52 to –0.55 V by

addition of CO2 as shown in Figure 2. Increase of 0.4 μA

of current indicates that electrons are transferred from

electrode to bacterial cells coupled to redox reaction of

NR. Increase of –0.3 V of redox potential is a clue that

electrons are transferred from electrode to NR by lower

electrode (working electrode) potential than intrinsic

redox potential of NR. Relatively higher electron-driving

force (electrode potential) may be required for electrons

to move through the electric resistance generated be-

tween electrode and NR immobilized in bacterial mem-

brane. Meanwhile, other upper voltammetric peak (bold

arrow mark) located at –0.9 V (vs. Ag/AgCl) was also

shifted upward from 2.7 to 3.0 μA and rightward from

–0.9 V to –0.93 V by addition of CO2. It seems possible

that electrons are transferred from the electrode via one

of the electron carriers located in the bacterial membrane,

B. Y. JEON ET AL.

335

allowing for current and potential increase by addition of

CO2. CO2 could act as an electron acceptor to induce

biochemical oxidation of NADH that may be electro-

chemically regenerated, by which electrons may be

transferred from electrode to bacterial cells via the cou-

pling redox reaction of NR and NAD+.

3.2. Growth and Metabolite Production of

C. acetobutylicum

C.acetobutylicum did not grow and didn’t produce me-

tabolites in the defined medium under N2 atmosphere

(DM-N2) but grew and produced acetate, propionate, and

butyrate under H2 atmosphere (DM-H2), as shown in

Table 1. The metabolites detected in the chromatography

for culture fluid of C. acetobutylicum cultivated in the

DM-N2 may have originated from the metabolites con-

tained in the inoculum. The electrochemical reducing

power generated from NR-cathode (reduced NR) is con-

verted to the biochemical reducing power (NADH),

which can be presumed on the basis of the growth and

metabolite production of C. acetobutylicum cultivated in

the DM-ER. C. acetobutylicum cultivated with electro-

chemical reducing power produced more acetate than

butyrate in DM-ER but more butyrate than acetate in

CM-ER. These are more clues that biochemical reducing

power (NADH) may be regenerated by electrochemical

reducing power generated from –2 V of NR-cathode and

the high balance of NADH/NAD+ may induce metabolic

conversion of CO2 to metabolites in coupling with free

energy synthesis.

3.3. Quantitative and Qualitative Verification of

Metabolites

Figure 2. Cyclic voltammetry for NR dissolved in reactant

and immobilized in C. acetobutylicum during cyclic potential

scanning from 0 mV to –1200 mV. Upper and lower volt-

ammetric peaks for dissolved NR were not altered but for

immobilized NR were shifted upward and rightward, re-

spectively, by addition of CO2. Other upper peak (bold ar-

row mark) also was shifted upward and rightwar d by addi-

tion of CO2.

Metabolites generated by C. acetobutylicum in different

cultivation conditions were quantitatively and qualita-

tively analyzed by a specially trained expert, and found

to be acetate, propionate, and butyrate as shown in Fig-

ure 3, as expected. This analytical process is absolutely

Table 1. Growth and metabolite production of C. acetobutylicum cultivated in complex medium (CM) and defined medium

(DM) under 2 atm of N2 atmosphere, 2 atm of H2 atmosphere, and electrochemical reduction condition (ER) for 5 days.

Metabolites (mM)

Cultivation conditions Growth at OD660 (initial-final)

Acetic acid Propionic acid Butyric acid

DM-N2 0.08 - 0.06 0.6 ± 0.04 0.1 ± 0.01 0.5 ± 0.02

DM-ER 0.08 - 0.36 9.2 ± 0.2 0.8 ± 0.03 6.4 ± 0.3

DM-H2 0.08 - 0.38 9.8 ± 0.3 0.9 ± 0.02 6.0 ± 0.3

CM-N2 0.08 - 1.28 35.8 ± 1.4 2.8 ± 0.1 21.6 ± 1.1

CM-ER 0.08 - 1.06 19.6 ± 0.8 4.8 ± 0.2 38.4 ± 0.9

CM-H2 0.08 - 1.21 24.4 ± 0.9 4.2 ± 0.2 36.6 ± 1.3

B. Y. JEON ET AL.

336

Figure 3. Mass spectrometer profiles of three major peaks detected in gas chromatography of volatiles contained in the cul-

ture fluid of C. acetobutylicum.

required because some metabolic intermediates derived

from amino acids (yeast extract) may be produced by

bacterial cells cultivated in the DM-H2 and DM-ER con-

dition.

3.4. Analysis of Genes Induced by

Electrochemical Reducing Power

Significant genes (higher than twice the signal intensity)

commonly expressed in C. acetobutylicum that was elec-

trochemically cultivated in the defined medium under

CO2-atmosphere and in the complex medium under

N2-atmosphere numbered in 318. All of the fundamental

genes related to CO2-fixation were not detected; however,

the genes of encoding the enzymes catalyzing acetyl-

CoA synthesis from CO2, ATP synthesis, and butyrate

production were quantitatively analyzed and compared as

shown in Table 2. The genes encoding the enzymes

catalyzing acetyl-CoA generation from CO2 and ATP

synthesis in pathway from acetyl-CoA to acetate were

more expressed but those catalyzing NADH regeneration

coupled to oxidation of substrates (metabolic intermedi-

ates) and butyric acid production were less expressed in

C. acetobuylicum cultivated in DM-ER condition than

CM-N2condition (Table 1).

4. Discussion

Electron transfer from electrode to bacterial cells can be porarily function in proportion to physiological activity

generated by the simultaneous contact of an electron me-

diator with both electrode and bacterial cells. Contact of

an electrode with the electron mediator (NR) immobi-

lized in bacterial cell or contact of bacterial cells with the

electron mediator immobilized in an electrode is a unique

way to induce electron transfer between bacterial cells

and electrode [17-19]. Patterns of cyclic voltammetry of

NR immobilized in bacterial cells were identical to those

of NR dissolved in reactant in the condition uncoupled to

the external redox reaction, because the redox reaction is

proportional to the concentration of NR contacted stably

with the electrode (Figure 2). Some NRs immobilized in

C. acetobutylicum are electrochemically reduced and

biochemically oxidized coupled to NADH regeneration

[6]. This electrochemical and biochemical coupling re-

dox reaction of NR and NAD+ may be continuously re-

peated in this condition with both electron donor and

acceptor. The electrode may be an electron donor and

CO2 may be an electron acceptor in the cyclic voltam-

metry for the modified C. acetobutylicum with NR, con-

sidering that addition of CO2 induced electron transfer

from electrode to bacterial cells via NR immobilized in

bacterial cells or other bacterial electron carrier (upper

voltammetric peaks in Figure 2) can be quantitatively

analyzed based on the increase of current (electron num-

ber) and variation of redox potential (electron-driving

force). The NR immobilized in bacterial cells can tem-

B. Y. JEON ET AL. 337

Table 2. Comparison of genes expressed in C. acetobutylicu

saturated defined medium and cultivated with glucose in the c

m cultivated with electrochemical reducing power in the CO2-

omplex medium.

Signal intensity for specific genes expressed in C. acetobutylicum

Ratio of A/B Gene products (Functions) Electrochemically

cultivated (A)

Cultivated in

complex medium (B)

4.87 Carbon monoxide n from CO2) 7764 dehydrogenase (CO formatio1595

4.71 Biotin-acetyl-CoA-carboxylase (Malonyl-CoA production) 2846 604

31.7 Formyl-H2folate synthase (Methyl-formation from CO2) 317 10

2.89 Formyl-H2folate cyclohydrolase (Methyl-formation from CO2) 772 267

4287

244.8 Phosphl-CoA)

3.24 Putative

methyltransferase (Methyl-formation from CO2) 13,908

otransacetylase (Formation of acetyl-Pi from acety2448 10

212.4 Acetate kinase (Acetate production coupled to ATP synthesis) 2124 10

0.74 NAD-dependent dehydrogenase (NADH regeneration

coupled to substrate oxidation) 1856 524

0.21 Acetyl-CoA acetyltransferase (Butyric acid production) 4179 19,492

of tcte

zed in the graphite felt cathode can semipermanently

de, the electron transfer from electrode to bac-

fer from elec-

tro

coupled to Ndation in C. acetum grown

in glucose. Accordingly, higher production of butyric

O fixa-

tio

he immobi-

he baria modified with NR; however, NR immobi-

function as long as it is contacting with intact cells of

bacteria.

In the electrochemical bioreactor equipped with the

NR-catho

rial cells can’t be quantitatively analyzed because the

number of electrons (current) transferred from power

supply to NR-cathode is not proportional to the meta-

bolic reduction reaction catalyzed by bacterial cells, and

–2 V of electrode potential charged to the bioreactor was

stronger than the redox potential of NR. This can gener-

ate electron-driving force to transfer electrons via NR

immobilized in electrode to bacterial cells but may in-

duce electrochemical reduction of medium ingredients

and other organic compounds. The –2 V of electrode

potential may be too strong to induce the electrochemical

reduction of NR (–0.325 V vs. NHE) but is required to

induce electron transfer from electrode to bacterial cells

through the electron barrier (cytoplasmic membrane)

because electric resistance may be generated by reactor

membrane between anolyte and catholyte, connecting

error between NR and bacterial cells, and structural

mismatch between NR and NAD+ [20].

It is unquestionable that the NR immobilized in graph-

ite felt electrode mediated electron trans

de to bacterial cells and catalyzed regeneration of

biochemical reducing power on the basis of acetic and

butyric acid production from CO2 and production of

more butyric acid than acetic acid from glucose. This

result is very similar to that obtained from C. acetobu-

tylicum culture using H2 and CO2 as an electron donor

and acceptor. Metabolic production of acetic acid is cou-

pled to NADH regeneration but that of butyric acid is

acid than acetic acid by C. acetobutylicum cultivated in

CM-ER and CM-H2 is another clue that H2 and electro-

chemically reduced NR may be an additional reducing

power to increase ratio of NADH/NAD+ [21].

Metabolic conversion of CO2 to metabolites is coupled

to oxidation of biochemical reducing power regenerated

by the electrochemically reduced NR or H2; however, the

metabolic pathways related to the autotrophic C

ADH oxiobuylic

n can be assumed only by the metabolites produced by

C. acetobutylicum cultivated in different media and un-

der different conditions. Microarray analysis of mRNA is

effective to analyze variations of metabolic pathway and

free energy production related to autotrophic CO2fixation

or heterotrophic growth. Practically, the specific genes

related to the CO2 fixation and energy metabolism ex-

pressed in C. acetobutylicum electrochemically or con-

ventionally cultivated are a clue that the metabolic con-

version of CO2 to metabolites in coupling with the free

energy production and redox reaction of reducing power

may be generated by the electrochemical reducing power.

Theoretically, –2 V of electricity charged to the NR-

cathode located in culture medium may induce H2 gen-

eration by electrolysis of H2O. However, H2 was not de-

tected in the electrochemical bioreactor even by precision

analysis. Accordingly, the NR-cathode may directly

transfer electrons from electrode to intact cells of C.

acetobutylicum and induce catalyzing of NADH regen-

eration in metabolism of C. acetobutylicum.

5. Conclusion

The electrochemical redox reaction of NR, the catalytic

function of NR for NADH regeneration, and t

B. Y. JEON ET AL.

338

lization technique of NR in the electrode permit C. ace-

& Renewabl

ergy of the Korea Institute of Energy Technology

) grant funded by the Korea

. Jungermann and K. Decker, “Energy Con-

servation in Chemotrophic Anaerobic Bacteria,”

riological Rev, pp. 100-180.

, No. 2, 1979, pp. 177-182.

tobutylicum KCTC1037 to grow and produce metabolites

using electrochemical reducing power. Mixed acid fer-

mentation bacteria produced the relatively reduced me-

tabolite (butyrate) or oxidized metabolite (acetate) de-

pending on balance of NADH/NAD+. In autotrophic mi-

crobes, CO2 can be reduced to CO by catalysis of carbon

monoxide dehydrogenase in coupling with oxidation of

biochemical reducing power (NADH or NADPH). Prac-

tically, C. acetobutylicum produced more butyrate than

acetate from glucose and more acetate than butyrate from

CO2, reasonable on the basis of metabolic pathway for

ATP regenerations. C. acetobutylicum cultivated hetero-

trophically with glucose synthesizes ATP by substrate-

level phosphorylation and regenerates NADH in both

glycolysis and pathway from pyruvate to acetate but that

cultivated autotrophically with electrochemical reducing

power and CO2 synthesizes ATP in the pathway from

acetyl-CoA to acetate and regenerates NADH coupled to

electrochemical redox reaction of NR.

6. Acknowledgements

This work was supported by the New e En-

Eva-

luation and Planning (KETEP

governmental Ministry of Knowledge Economy (2012-

T1001100334).

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