WO2002097106A1

WO2002097106A1 - Electrochemical preparation of acetic acid

Info

Publication number: WO2002097106A1
Application number: PCT/KR2001/001077
Authority: WO
Inventors: Woon-Sup Shin
Original assignee: Bioneer Corp
Current assignee: Bioneer Corp
Priority date: 2001-05-30
Filing date: 2001-06-22
Publication date: 2002-12-05
Anticipated expiration: 2003-11-30

Abstract

The present invention relates to an electrochemical preparation process for acetic acid. More particularly, the present invention is directed to electrochemical processes for the preparation of acetic acid from carbon dioxide and to electrodes employed for the processes.

Description

ELECTROCHEMICAL PREPARATION OF ACETIC ACID

Technical Field

The present invention relates to an electrochemical preparation process for acetic acid. More particularly, the present invention is directed to electrochemical processes for the preparation of acetic acid from carbon dioxide and to electrodes employed for the processes. Background Art

Carbon dioxide is a major compound that causes green house effect and therefore, is under the control of international rules and negotiation as like the environmental round. The method for treating and reducing the generation of carbon dioxide is laid on the current emergency.

Particularly, a new method for reducing the generation of carbon dioxide or for disposing carbon dioxide has been required seriously for the countries under development such as South Korea.

Therefore, various methods which converts carbon dioxide into methanol or fuel through chemical process (Masuda, S. Energy Conv. Mgmt. 1995, 36, 567-572; Kakunoto, T. Energy Conv. Mgmt. 1995, 36, 661-664; Ando, H.; Fujiwara, M, ; Kieffer, R. Energy Conv. Mgmt. 1995, 36, 593-596) and into organic materials through biological process, has been studied actively all over the world (Michiki, H. Energy Conv. Mgmt. 1995, 36, 701- 705;Murakami, M. Ikenouchi. M. Energy Conv. Mgmt. 1997, 36, S493-S497) .

Also, some another methods which converts carbon dioxide into useful organic compound through electrochemical reduction process which employs enzymes and catalysts. One of them is a method that synthesizes isocitric acid by isocitrate dehydrogenase (ICDH) with carbon dioxide and oxoglutaric acid (Sugi ura, K. ; Kuwabata, S.; Yoneyama, H. J. Amer. Chem . Soc. 1989, 111 , 2361-2362) . Pyruvate dehydrogenase (PDH) was used to make pyruvic acid whth carbon dioxide and acetyl coenzyme A (Kuwabata, S.; Morishita, N.; Yoneyama, H. Chem . Lett . 1990, 1151-1154), formate dehydrogenase (FDH) and methanol dehydrogenase (MDH) were used together to synthesized formate, formaldehyde, or methanol from carbon dioxide (Kuwabata, S.; Tsuda, R.; Yoneyama, H. J. Amer. Chem . Soc. 1994, 116, 5437-5443) . Carbon monoxide could be generated selectively by using carbon monoxide dehydrogenase (CODH, Jun Won, Shin. Sogang Univ. 1998. Sang Hee, Lee. Sogang Univ. 1997).

However, the methods explained above, are not used widely in industry but has been studied only in academic field and a method for converting carbon dioxide into acetic acid directly through electrochemical process which employs, enzymes has not yet known to public. To the present, among the industrial process for making acetic acid, Monsanto process which employs methanol carbonylation, has been known as the most successful process in view of economical aspect (Forster, D. J. J. Am. Chem. Soc. 1976, 98, 846-848; Forster, D. J. Adv. Organomet . Chem. 1979, 17, 255-266). However, an improvement which can reduce loss of resources and/or utility and can also recycle raw materials, has been required due to the high price of rhodium catalyst and the side effect of hydroformylation. Therefore, various modifications of Monsanto process are now being studied widely, such as researches regarding to the activity and stability of rhodium catalyst for the methanol carbolyation (Blasio, N. D.; Tempesti, E. ; Kaddouri, A. ; Mazzocchia, C. ; Cole Hamilton, D. J. J. Organomet, Chem. 1998, 551, 229-234 ; Protzmann, G. ;^' Luft, G. Allp. Cat. A; Gen. 1998, 173, 159-163), electrochemical carbonylation of methanol by CuO electrode (Kiyoshi, 0.; Toshikazu, Y. ; Ichiro, Y. J. Electrochem. Soc. 1995. 142, 130-135), and using nickel complex (Kellar, A. A.; Ubale, R. S. ; Deshpande, R. M. ; Chudhari, R. V. J. Cat. 1995, 156, 290-294 ; Moser, W. R. ; Marshik-Guerts, B. J. ; Okrasinski, S. J. J. Mol. Cat. A; Chem. 1999, 143, 71- 83)

Some methods for the preparation of acetic acid through biological process by using Clostridium bacteria. Acetic acid can be made from waste gas or waste biomass. Other methods which employ more efficient fermentation process, well-adapting mutation (Gaddy. J. L. US Patent. 5,593,886, January 14, 1997; Grady. J. L.; Chem. G. J. US Patent. 5,821,111, October 13 1998; Gaddy. J. L. US Patent. 5,807,722, September 15, 1998; Schwartz. R. D. US Patent. 4,371,619, February 1, 1983; Brumm. P. J. ; Datta. R. US Patent. 4,814, 273, March 21, 1898; Gaddy. J. L.; Clausen. El Cl US Patent. 5,173,429, December 22, 1992) have been developed. Clostridium thermoaceticum is known to produce acetic acid from carbon monoxide or carbon dioxide and hydrogen mixture. However, to the present, a process for converting carbon dioxide directly into acetic acid without supplying hydrogen has never been disclosed.

Therefore, a development of novel process for converting carbon dioxide, green house gas, into acetic acid which is very useful in industry, has been anticipated and required in this technical field. Disclosure of Invention

Therefore, it is an object of the present invention to provide a process for producing acetic acid from carbon dioxide. Another object of the present invention is to provide bacteria combined with electron carrier through covalent bond, which can be employed in the electrochemical process for converting carbon dioxide into acetic acid without causing environmental problem. Yet another object of the present invention is to provide process for combining bacteria with electron carrier to produce bacteria/electron carrier complex catalyst used in electrochemical process for converting carbon dioxide into acetic acid. A still another object of the present invention is to provide an electrode on which bacteria is immobilized, used in electrochemical process for converting carbon dioxide into acetic acid.

A still further object of the present invention is to provide an electrode on which bacteria/electron carrier complex is immobilized, used in electrochemical process for converting carbon dioxide into acetic acid.

A still further object of the present invention is to provide a method for converting carbon dioxide into acetic acid through electrochemical process which employs bacteria-immobilized electrode.

A still further object of the present invention is to provide a method for converting carbon dioxide into acetic acid which employs bacteria/electron carrier/glassy carbon complex electrode.

The above objects are carried out through electrochemical process which is represented by

2C0₂ + 8H⁺ ► CH₃COOH + 2H₂0 which employs electrode on which bacteria or bacteria/electron carrier complex.

The starting material of the process of the present invention is carbon dioxide and catalyst used therein is enzymes containing CODH in anaerobic bacteria. A proper electron carrier is used and electron is provided through electrodes. The catalyst used in the present invention, CODH can be isolated from various microorganisms. Preferably, a CODH isolated from Clostridium thermoaceticium, is employed as a catalyst.

Clos tridium thermoa cetici um is anaerobic homoacetogen which produces acetic acid only. The organism can converts one molecule of glucose (C6) into three molecule of acetic acid (C2); At first, glucose (C6) is degradated into two molecules of pyruvic acid and then, pyruvic acid is converted into one molecule of acetic acid(C2) and one molecule of carbon dioxide (Cl). Then two molecules of carbon dioxide are combined to one molecule of acetic acid by acetyl-CoA pathway.

Anaerobes such as Clostridium thermoaceticium, have a acetyl-CoA pathway, so called Wood pathway, which produces acetic acid through reduction of two molecules of carbon dioxide and the formation of carbon-carbon bond. In this pathway, the key enzyme is carbon monoxide dehydrogenase (CODH) containing Ni, Fe clusters as active sites. CODH not only reduces carbon dioxide to carbon monoxide but also combines methyl moiety generated by various other enzymes and carbon monoxide reduced from carbon dioxide, with CoA, to produce acetyl-CoA which is precursor of acetic acid. So it is called as acetyl-CoA synthase (ACS, Ragsdale S. W.; Clark, J. E. ; Ljungdahl, L. G. ; Drake, H. L. J. biol. Chem. 1983, 258, 2364-2369 ; Diekert, G. ; Ritter, M. FEBS Lett. 1983, 151, 41-44 ; Pezacka, E. ; Wood, H. G. J. Biol. Chem. 1988, 263, 16000-16006 ; Ragsdale, S. W. Biochem. & Mol. Biol. 1991, 26, 261-300 : Barondeau, D. P. ; Lindahl, P. A. J. Am. Chem. Soc. 1997, 119, 3959-3970) . Clostridium thermoaceticium or carbon monoxide dehydrase (CODH) are stable at 50 to 60 °C, and they reveal good catalytic activities even at high temperature and not affected by the heat generated in the electrical reactions. In addition, since Clostridium thermoa cetici um and the enzymes isolated therefrom can resist against sulfur compound contained in waste gas from factories, which inactivates common catalysts very easily, they can be used properly for treating carbon monoxide or carbon dioxide discharged together with sulfur compounds .

In addition, Clostridium thermoaceticium have a good activities toward for CO or H₂, so it can be employed in treating gas discharged in the form of mixture of CO, C0₂ and H₂ without additional separation process .

The electron carrier used in the present invention plays a role of facilitating electron transfer between electrode and biological catalyst, and selected from compounds which can be oxidized or reduced reversibly at electric potential of -300 mV to -700 mV (NHE based on Normal Hydrogen Electrode) . Preferably, compound of which reduction potential is similar with that of C0₂, such as alkylviologen which includes methyl viologen, N,N-dimethryl-4, 4' -bipyridyl and derivatives thereof. The compound which can be used as electron carrier in the present invention are tetramethyl viologen, N, N-diethyl-4, 4' -bipyridyl, N,N- diisopropylyl-4, 4' -bipyridyl, and triquat etc., which are 4 , 4' -bipyridyl compound which contains alkyl moiety. Methyl viologen (MV²⁺) is reduced upon receiving electron from the electrode to form MV⁺, and then MV^"1" transfers electron to Clostridium thermoaceticium or enzymes which contain oxidized CODH and is changed to MV²⁺" The reduced enzymes can reduce C0₂ to CO to produce acetic acid finally.

Brief Description of the Drawings

The objects and other advantages of the present invention will become more apparent by describing the preferred embodiments thereof in detail with reference to the attached drawings, in which;

FIG.l of A is acetyl-CoA or Wood pathway showing synthesis of bacterial acetic acid, and FIG. 1 of B is showing the concept of the present invention of reduction pathway of electrical of carbon dioxide to acetic acid.

FIG. 2 is an overview of the apparatus of electrochemical experiments and product analysis of the present invention.

FIG. 3 is an overview of the electrochemical cell for electrolysis and cyclic voltammogram of the present invention. FIG. 4 is an overview showing the concept of bonding Clostridium thermoaceticium to DAPV.

FIG. 5 is a graph of cyclic voltammogram in the presence of the DAPV bonded to C. t at 55°C. , ^•

FIG. 6 is a graph of electrolysis in the presence of DAPV bonded to Clostridium thermoaceticium at 55°C.

FIG. 7 is a graph of reduction of C0₂ by CODH- active DEAE fraction at room temperature.

FIG. 8 is a graph of preparation of acetic acid by an example of the present invention at room temperature .

FIG. 9 is a graph of cyclic voltammogram of reduction of C0₂ by CODH-active DEAE fraction at 55°C.

FIG. 10 is a graph of preparation of acetic acid by an example of the present invention at 55°C. FIG. 11 is a graph of cyclic voltammogram of reduction of C0₂ by CODH-active DEAE fraction at 55°C.

FIG. 12 is a graph of the preparation of acetic acid by an example of of the present invention in other condition at 55°C. FIG. 13 is cyclic voltammogram for reduction of C0₂ by crude extracts of Clostridium thermoaceticium .

FIG. 14 is a graph of preparation of acetic acid electrolyzed C0₂ in crude extracts of Clostridium thermoa ceticium at 55 °C FIG. 15 is cyclic voltammogram for reduction of C0₂ by Clostridium thermoaceticium at 55°C. FIG. 16 is a graph of electrolysis of C0₂ by an example of the present invention at 55°C.

FIG. 17 is schematic diagram of immobilization process of Clostridium thermoaceticium cell on electrode by using cellulose acetate. . >

FIG. 18 is Cyclic vlotammogram of C0₂ reduction in the presence of 1.0 mM MV in pH 7.0 phosphate buffer on Clostridium thermoaceticium immobilized glassy carbon electrode by cellulose acetate, at 55^°C. FIG. 19 is electrolysis of C0₂ reduction in the presence of l.OmM MV in pH 7.0 phosphate buffer on Clostridium thermoaceticium immobilized glassy carbon electrode by cellulose acetate, at 55°C.

FIG. 20 is a schematic diagram of immobilization process of DAPV linked cell on electrode by cellulose acetate.

FIG. 21 is Cyclic vlotammogram of C0₂ reduction by DAPV linked Clostridium thermoaceticium immobilized copper electrode by cellulose acetate, at 55^°C. FIG. 22 is graph of electrolysis of C0₂ by DAPV linked Clostridium thermoacetium immobilized copper electrode by cellulose acetate, at 55°C.

FIG. 23 is a schematic represent of preparation of carboxyl-terminated glassy carbon electrode and covalent attachment of DAPV and clostridium thermoaceticum on the electrode.

FIG. 24 is cyclic vlotammogram of reduction of

C0₂ by using clostridium thermoaceticum/DA^'PV/glassy carbon electrode complex. FIG. 25 is electrolysis of C0₂ clostridium thermoaceticum covalently linked to DAPV-modified glassy carbon electrode (phosphate buffer, PH 7.0), at 55°C.

FIG. 26 is stability of cell immobilized by cellulose acetate on gold, copper, GC electrode upon storage in glove box.

FIG. 27 is stability of cell immobilezed with DAPV by cellulose acetate on gold, GC electrode upon storage in glove box

Best Mode for Carrying Out the Invention Hereinafter, the present invention will ^' be described in more detail with reference to the accompanying drawings .

Activity of Clostridium thermoaceticium or enzyme contained in the Clostridium thermoaceticium such as CODH reducing CO electrochemically is seriously damaged by a small amount of oxygen. Therefore, all electrochemical experiments involved in this method were carried out in the glove box (Vacuum Atmosphere Co. HE-234-2, MO-20-SSG purifier. FIG.2) . 2-compartment cell having working and reference electrode separated from counter electrode by vycor tip glasses used as an electrolysis cell to minimize the effect of contaminants. (FIG. 3)

As indicated in FIG. 3, electrode was linked to the cell by using adaptor with O-ring in order to prevent leakage and influx of the gas. C0₂ was saturated through septum shown in the figure using injection needle. The pH of the solution dropped from 7.0 to 6.3 due to the melted C0₂ when it was saturated with C0₂.

Ag/AgCl(3M NaCl, BAS) was used as a reference electrode which was +0.200 V vs. NHE. All potential values described in the present invention are reported vs. NHE.

When LC (liquid chromatography) was used in order to analyze compounds produced in solution such as acetic acid and organic acid, air tight syringe was used in the amount of 20μ. and analyzed quantitatively by using propionic acid as internal reference.

Proteins were removed from the reagent by using injector filter (Whatman, PVDF) . While this process was carried out, LC used was Youngln Model 910, Shodex, Rspak KC-811(7.8 x 300 mm) column adjusted to 40 °C UV detector (215 nm, Youngln M-720) was used and extraction rate was 1.0 mL/ in in the 0.05 M H₂S0₄ solution. Meanwhile, acetic acid was also analyzed quantitatively by using GC (gas chromatography; HP5890, FFAP column, FI detector) and the result was similar to that of LC. Also, gas phase reactants and products was analyzed by using GC ( Varian 3700, Pora Q column, TC detector) . After reagents were took out of the glove box by using gas airtight injector by 100~200,«.K, they were injected to GC. In order to analyze quantitatively, previously adjusted CO-checking tool was used.

Hereinafter, the present invention will be described in greater detail with reference to the following examples. The examples are given for illustration of the invention and not intended to be limiting the present invention.

Example 1.

The cultivation of Clostridium thermoaceticium and isolation of CODH active fractions. Clostridium thermoaceticium was cultivated in the well-known method that was used widely for cultivating anaerobes (Lundie, L. L. Jr. ; Drake, H. L. J. Bacteriol . 1984, 159, 700-703) and when collecting bacteria, centrifuge bottle and glove box was used to prevent oxygen from damage.

Isolation of enzyme was carried out through the method that explained previously (Ragsdale, S. W. ,

Clark, J. E., Ljungdahl, L, G., Drake, H. L. J. Biol. Chem. 1983, 258, 2364-2369 ; Shin, W., Lindahl, P. A.

Biochim. Biophys. Acta 1993, 1161, 317-322)

After the collected bacteria were homogenized by using ultrasonication in the glove box, crude extract were obtained and the enzymes were isolated by using DEAE Sephacel column. The CODH and other enzymes used in this example were the fractions collected by using DEDA Sephacel column having CODH activity.

Example 2. The reduction of carbon dioxide by using CODH active DEAE fractions at room temperature.

CV data were compared to see CODH-active DEAE fractions worked as catalyst for the electrochemical reduction (FIG. 7). In the nitrogenous condition, only electron carrier, MV²⁺, carried out typical reversible oxidation-reduction reaction. The current increasing from -600 mV is background current that water reduces into the hydrogen. When this solution was saturated with C0₂, reducing current increased in a small amount. This result was because of the phenomena of mixture of 1) current that C0₂ reduces by enzyme such as CODH. 2) decrease in pH of solution according to the saturation with C0₂(in nitrogen condition, pH is 7.0 and if it is saturated with C0₂, pH decreases to 6.3).

The second reaction, the formation of H₂, went well in the condition of low electric potential (more negative) and at the -500 to -600 V reduction of C0₂ occurs without forming H₂. As explained above, at -600 mV below the side effect, the formation of H₂, mainly occurred; therefore, the voltage range occurring only reduction of C0₂, i.e., -550 to -600 mV was selected.

The reduction of C0₂ by using enzyme was the electron transfer reaction mediated by methyl viologen so after enough amount of MV²⁺ became reduced into MV⁺, CODH could be reduced and initial charge was included to the reduction of MV²⁺ which was not related to the reduction of C0₂. Therefore, because initial charge was used in the reaction of MV²⁺, the current efficiency of the formation of acetic acid was calculated after calibrating initial charge used in the reduction of MV²⁺. While electrolysis was being carried out, the amount to change passed was measured and those results were shown in the FIG. 8.

The increase of the concentration of acetic acid could be observed proportional to the change passed when C0₂ was reduced. The products by other side effect except acetic acid were not detected. The efficiency calculated from the change passed and acetic acid formed was very high in the range of 95 to 100%. This data shows that CODH-active DEAE fractions is a good catalytic system, which selectively reduces C0₂ into acetic acid. The quantitative result such as the calculation of current efficiency was shown in table 1. The formation of acetic acid increased linearly in about 1 hour, reaching concentration of 7 mM, but it did not increase after reaching that concentration. This is from the loss of activity of the enzyme because CODH playing the most important role became unstable in the reducing condition. At room temperature, for first 30 minutes 4.3 mM acetic acid was formed ; therefore, the rate of formation of acetic acid was 1.0 umol/min(4.3 mM x 7.0 mL / 30 min)

Table 1.

The amount of acetic acid and current efficiency calculated from it and charge used when C0₂ ^■ was electrolyzed electrochemically at the condition of -550 mV vs. NHE at room temperature by using 1.0 M MV²⁺ and 12 mg/mL CODH-active DEAE compartment protein in 0.1 M phosphate buffer solution.

Example 3 .

The reduction of C0₂ by using CODH active DEAE fractions at 55°C .

Clostridium thermoaceticium is a thermophile and because its optimal growing condition is 55°C, experiment of the example 2 was compared repetitively at this temperature.

On the CV data, the reducing current of MV²⁺ increased at the room condition and although it is hard to compare it quantitatively, the increase of catalytic current by the reduction of C0₂ could be verified. (FIG.

9)

The electrolysis was carried out and the amount of acetic acid formed by the reduction of C0₂ was compared. (FIG. 10, Table 2) acetic acid' was selectively formed at the efficiency of 95~100% and the amount of acetic acid formed was about 25 mM, which was over three times as much as at room temperature. Since 14.3 mM of acetic acid is formed for first 30 min, the rate of the formation of acetic acid is 3.3 umol/min (14.3 mM x 7.0 mL/30 min), which was about 3 times as much as at room temperature.

Table 2. The amount of acetic acid and current efficiency calculated from it and charge used when C0₂ is electrolyzed at -550 mV vs. NHE at 55°C by using 1.0 mM

MV 2+ and 12 mg/mL CODH-active DEAE fractions in 0.1 M phosphate buffer solution,

Example .

The reduction of CQ₂ by using CODH active DEAE fractions at 55 °C, 0. ImM MV²⁺

Example 4 was similar to exmaple 3 except using 0.1 mM methyl viologen in order to prevent methyl viologen from being unstabilized.

The flow of catalytic current according to reduction of C0₂ could be verified through CV but amount of current was small in the extent of one tenth. (FIG.11) Also, electrolysis was carried out at -550mV, flowing current was only one fifth of previous experiment, which is the evident that electron transfer was less effective than that of previous case. (FIG. 12, Table 3)

As result of data, although the stability of enzyme increased more or less when using low concentration of MV²⁺, the rate of increase was not remarkable after 1 hour.

Table 3.

The amount of acetic acid and current efficiency calculated from it and charge used when C0₂ is electrolyzed at -550 mV vs. NHE at 55°C by using 1.0 mM MV₂+ and 12 mg/mL CODH-active DEAE fractions in 0.1 M phosphate buffer solution.

Example 5.

The reduction of C0₂ by using crude extracts at room temperature.

The probability of the formation of acetic acid by the reduction of C0₂ was verified in case of using crude extracts of Clostridium thermoaceti cium . Whether the catalytic current flowed or not could not be confirmed but quantitative formation of acetic acid could be confirmed when carrying out at -550 mM. (FIG. 14, Table 4) In this case, initial concentration of acetic acid was 32.5 mM, which was because that even though crude extracts was made by sonication after substituting the solution with buffer solution by ^'using centricon, acetic acid located in the cell got out of the cell

Table 4.

The amount of acetic acid and current efficiency calculated from it and charge used when C0₂ was electrolyzed at -550 mV vs. NHE at 55 °C by using crude extracts of 14 mg/mL of Clostridium thermoaceticium at the 7.0 mL of 0.1 M phosphate buffer solution.

Example 6.

Transformation of acetic acid from carbon dioxide by using electron carrier and solution of Clostridium thermoaceticium.

Clostridi um thermoaceticium was distributed into centricon (Amicon, Inc., Centricon YM-30, cut off 30,000) and washed several times with 0.1 M phosphate buffer (pH 7.0). When this solution containing Clostridium thermoaceticium was made homogeneous with a small amount of 0.1 M phosphate buffer, optical density (O.D) was 1.8 at 680 nm, which meant that there were 0.61 g/mL Clostridium thermoaceticium in solution. 1.00 mL of this solution was put into the electrolytic cell and after making solutions well mixed by adding buffer containing methyl viologen to Clostridium thermoaceticium under the condition of 4.00 mL of C0₂ in total, CV was observed (FIG. 15) . When electrolysis was carried out for 4 hours at -570 mV under this condition, the flow of charge was 45 C(FIG 16) and it was observed that the amount of acetic acid formed was 72 M and current efficiency was 92 %.

Exampl 7. Electrochemical transformation of carbon dioxide to acetic acid and preparation for the complex of electron carrier and Clostridium thermoaceticium

Methyl viologen used as electron carrier in this experiment was often used as experiment, not only will the pollution of environment be caused, but the activity of Clostridium thermoaceticium also seriously dropped. To minimize these problems, methyl viologen was linked to Clostridium thermoaceticium . If there was any carboxyl group in the surface of

Clostridium thermoaceticium and any amine group at the end of the electron transfer, amide bond would be able to be formed and electron carrier would be linked to

Clostridium thermoaceticium more easily (FIG. 4) . Therefore, in this experiment, di-3-aminprophy-viologen

(DAPV) with amine group at the end of the electron carrier was used. DAPV was synthesized according to a method below.

Reaction 1

CH3CN, Reflux, 12h

The method described above is that methylaminopropylviologen was synthesized (Katz, E. ; de Lacey, A. L. ; Fierro, J. L. G. ; ralacios, J. M. ; Fernandez, V. M. J. Electroanal. Chem. 1993, 358, 247- 259). 10 mmol of 4 , 4' -bipyridine was refluxed with 22mmol of bromoprophylamine hydrobromide in 30 mL of acetonfitrile solution for 12 hours. DAPV crystals obtained after removing suction from solutions were recrystalized three times after adding a small amount of ethanol .

To link DAPV to Clostridium thermoaceticium, several steps were required; First, Clostridium thermoaceticium was washed with 0.1 M phosphate buffer solution by using centricon. Next, to activate carboxyl group of Clostridium thermoaceti cium, N-(3'- dimethylaminoprophy) -N' -ethyl carbodiimide (EDC) was added to solution and then stirred for 30 min. Finally, DAPV was added to solution to make solution homogeneous and then stirred for 30 min.

Clostridium thermoaceticium linked by DAPV through procedures explained above oxidized and reduced electrochemically, which was confirmed by using CV (FIG. 5) . The oxidation-reduction reaction was observed by DAPV linked to bacteria but diffusion-limited current was caused by bacteria itself. Electrolysis was carried out at the condition of -610 mV and the change passed was 67 C (FIG. 6) . According to the result of LC, 20 mM of acetic acid was formed. Therefore, acetic acid could be formed even though both Clostridium thermoaceticium and electron carrier were immobilized on the electrode.

Example 8.

Immobilization of Clostridium thermoaceticium on the surface of electrode by using cellulose acetate and its use for electrochemical transformation of carbon dioxide to acetic acid Method for immobilizing enzyme was used to prepare bio-sensor by using glucose oxidase (Maines, A.; Ashworth, D. ; Vadgama, P. Amal. Chim. Acta 1996, 333, 223-231), lactate dehydrogenase, NAD⁺ (sprules, S. d. ; Hart, J. P. ; Wring, S. A. ; Pittson, R. Anal. Chim. Acta 1995, 304, 17-24), and in the case of method for immobilization of bacteria by using CA, Pseudomonas putida was used ( Chung, T. S.; Loh, K, -C. ; Goh, S. K. J. Appl. Polym. Sci. 1998, 68, 1677-1688) and basically, this method was a physical method for immobilization through trapping by using macromolecules. Also, it was being reported that immobilization was carried out through physical absorption of bacteria in carbon electrode (Ikeda, T, ; Kato, K, ; Maesa, M. ; Tatsume, H. ; Kano, K. ; Mazunobu, K. J. Electroanal. Chem. 1997, 430, 197-204 ; tatsu i, H. ; Takagi, K. ; fujita, M. ; Kano, K. ; Ikesa, T. Anal, Chem. 1999, 71, 1753-1759)

Clostridium thermoaceticium was immobilized on electrode by using CA (cellulose acetate) as a physical immobilization method through trapping which uses macromolecules described above. First, 0.04g CA was added to 4.0 mL toluene/acetone solution (1 : 1 (v/v) ) to make 1% solution. After the solution was dropped on the electrode, it was spreaded and dried for 20-30 min at room temperature. After 0.2 ul of Clostridium thermoaceticium was added on the electrode, 1% CA was added again to prevent bacteria from coming out (FIG. 17) .

Clostridium thermoaceticium could be immobilized on the gold, glassy carbon, copper electrode and CV picture was examined in the presence of carbon dioxide. And then, after experiment for formation of acetic acid was carried out, current, the amount of acetic acid formed, and current efficiency were calculated. In the case of glass carbon, reduction current of carbon dioxide was observed (FIG. 18) . As the result of electrolysis at -560 mV, charges consumed during electrolysis for 4 hours were 0.4 C. The amount of acetic acid formed was 0.4 mM and current efficiency was 78% (FIG. 19). In the case of gold and copper electrode, the result was the same as that of described above .

Example 9.

Simultaneous immobilization of Clostridium thermoaceticium and electron carrier on the surface of electrode by using cellulose acetate and electrochemical transformation of carbon dioxide to acetic acid

In this example, both bacteria and electron carrier were immobilized on the surface of electrode at the same time According to ■ Example 7, Clostridium thermoaceticium liked to electron carrier was prepared first, after 0.1 ul of 1% CA was spread on the electrode and dried for 20-30 min, lul of Clostridium thermoaceticium linked to electron carrier was spread on that electrode. This procedure was carried out twice. Finally, 0.2 ul of 1% CA was spread again to prevent bacteria from coming out (FIG. 20) . The electrode was put into the 0.21 M phosphate buffer saturated with C0₂ and CV was taken (FIG 21) . And the electrolysis was carried out at -550 V. The charge consumed for 11 hours was 63 C, the amount of acetic acid formed was 25 mM, and current efficiency was 94% (FIG. 22) . The result was the same for gold, copper, glassy carbon electrode.

Example 11. Clostridium thermoaceticium immobilization by covalent bonding through electron carrier on the surface of glassy carbon electrode and electrochemical transformation of carbon dioxide to acetic acid

In this experiment, after preparing carboxyl group on the surface of electrode, amine group of one side of electron carrier having amine group at each end of the carrier was bonded to carboxyl group on the surface of the electrode, the other amine group was bonded to carboxyl group at the end of bacteria, so both bacteria and electron carrier were bonded to the surface of the electrode at the same time.

The method for preparing carboxyl group on the surface of the electrode was already described (Matsuo, M.; Yuji, Y.; Masashi, Y. ; Hidenobu, 0. Anal. Sci. 1995, 11, 947-952; Mizutani, F. ; Yabuki, S. Trans. IEE Jp. 1999, 119-E, 554-559). After introducing carbonyl ^■ group, to the electrode, DAPV was bonded by activating carboxyl group by adding N- (3' -dimethylaminopropyl) -N- -ethyl carbodiimide (EDC) . The amine group of only one side of DAPV was liked to electrode and the other side was exposed out of electrode. Next, after activation carboxyl group of Clostridium thermoaceticium by using EDC, it was reacted with DAPV-liked electrode and immobilized.

In CV of this electrode (FIG- 14), there was a small peak of oxidation/reduction around -400mV and this indicated that DAPV was bonded and electron transfer can be occurred. Also, the bell-shaped peak indicates that this reaction is the oxidation/reduction of the absorbed materials. Electrolysis was carried out at -550 mV and the charge consumed was about 61 C, the amount of acetic acid formed was 20 mM, and current efficiency was 93% (FIG. 25).

Example 12.

The measurement of storage life time of Clostridium thermoaceticium immobilized on the surface of electrode

By measuring the life span of storage of bacteria linked to electrode, the efficiency of immobilization of bacteria linked to electrode was measured by using

Clostridium thermoaceticium immobilized electrode by CA. After a week, two weeks, four weeks, after storing the electrodes in glove box the experiment for formation of acetic acid was carried out and charge, the amount of acetic acid formed, and current efficiency were measured (electrode was stored in glove box in dried state after washing with 0.1 M phosphate buffer. FIG. 26) . As shown in FIG. 26, current and the amount of acetic acid formed decreased and after two weeks, a half of initial amount was formed. But a small amount of change of current efficiency was changed and this result indicates the fact that although some of bacteria lose their activity but the others can form acetic acid.

The life span of storage of Clostridium thermoaceticium immobilized with DAPV covalently on GC (example 11.) was measured (FIG. 27) and the result was similar.

Industrial Applicability

The advantage of the present invention is; Because biological catalyst is used in order to lower activation energy required when carbon dioxide is reduced into acetic acid, carbon dioxide not only can be selectively transformed to acetic acid, but also pollution of environment can be minimized more effectively.

If acetic acid is manufactured by using Clostridium thermoaceticium itself, breakage of cells and purification step through column are not required. Therefore, when acetic acid is manufactured in industrial scale, the present invention will help a lot in economical aspect.

Claims

What is claimed is:

1. A process for preparing acetic acid from carbon dioxide, wherein bacterial extract which contains carbon monoxide dehydrogenase (CODH) and electron carrier are used as catalysts.

2C0₂ + 8H⁺ + 8e^~ ► CH₃COOH + 2H₂0

2. The process for preparing acetic acid according to Claim 1, wherein said extract is extracted from Clostridium thermoaceticium .

3. The process for preparing acetic acid according to Claim 1, wherein said electron carrier is oxidized or reduced reversibly at electric potential of -300 mV to -700 mV.

. The process for preparing acetic acid according to claim 1, wherein said electron carrier is selected from the group consisting of methyl viologen, tetramethyl viologen, N, N-diethyl-4 , 4' -bipyridyl, N,N- diisoprophylyl-4, 4' -bipyridyl, 4,

4' -bipyridyl and mixtures thereof.

5. The process for preparing acetic acid according to Claim 4, wherein said electron carrier is methyl viologen.

6. The process for preparing acetic acid according to Claim 1, wherein said process is carried out at 20°C to 70°C .

7. The process for preparing acetic acid according to Claim 1, wherein said process is carried out at electric potential of -300 mV to -700 mV.

8. The process for preparing acetic acid according to Claim 1, wherein said process is carried out at pH6 to pH9.

9. A process of preparing acetic acid from carbon dioxide, wherein any one selected from the group consisting of Clostridium thermoaceticium, Clostridium thermoaceticium, Clostridium thermoautotrophium,

Acetobacterium woodii , Acetogeium kivui , Clostridium aceticum, Clostridium Ijungdahlii , Eubacterium limosum, and electron carrier are used as catalysts.

2C0₂ + 8H⁺ + 8e^~ ► CH₃C00H + 2H₂0

10. The process for preparing acetic acid according to Claim 9, wherein said electron carrier is selected from the group consisting of methyl viologen, tetramethyl viologen, N,N-diethyl-4, 4' -bipyridyl, N,N- diisoprophylyl-4, 4' -bipyridyl, 4, 4' -bipyridyl and mixtures thereof.

11. The process for preparing acetic acid according to Claim 10, wherein said electron carrier is methyl viologen.

12. A process for preparing acetic acid from carbon dioxide, wherein Clostridium thermoaceticium combined with di- (3-aminopropyl) -viologen is used as catalyst.

13. A complex of di- (3-aminopropyl) -viologen and Clostridium thermoaceticium, wherein carboxy group of Clostridium thermoaceticium is combined with amine group of di- (3-aminopropyl) -viologen through covalent bond.

14. A process for binding di- (3-aminopropyl) -viologen on the surface of Clostridium thermoaceticium through covalent bond, which comprises: i)a step for adding Clostridium thermoaceticium into the solution of N- (3' -dimethylaminopropyl) -N' -ethyl carbodiimide; ii) a step for adding di- (3-aminopropyl) -viologen into the solution obtained in step i)

15. A bacteria-immobilized electrode used in electrochemical process for converting carbon dioxide into acetic acid, wherein bacteria which contain carbon monoxide dehydrogenase (CODH) is immobilized on the surface of electrode.

16. The bacteria-immobilized electrode according to Claim 15, wherein said bacteria is Clostridium thermoa ceti ci urn .

17. The bacteria-immobilized electrode according to Claim 15, wherein said electrode is selected from the group consisting of gold electrode, copper electrode and glassy carbon electrode.

18. The bacteria-immobilized electrode according to Claim 15, wherein said bacteria is immobilized on the surface of the electrode by using of cellulose acetate.

19. The bacteria-immobilized electrode for preparing acetic acid according to Claim 18, wherein said bacteria immobilized electrode is further coated with cellulose acetate.

20. A bacteria/electron carrier complex-immobilized electrode used in electrochemical process for converting carbon dioxide into acetic acid, wherein bacteria containing carbon monoxide dehydrogenase (CODH) is combined with electron carrier through covalent bond , is immobilized on the surface of electrode.

21. The bacteria/electron carrier complex-immobilized electrode according to Claim 20, wherein said bacteria is Clostridium thermoaceticium .

22. The bacteria/electron carrier complex-immobilized electrode according to Claim 20, wherein said electron carrier is viologen derivatives which contain terminal amine group.

23. The bacteria/electron carrier complex-immobilized electrode according to Claim 20, wherein said electron carrier is di- (3-aminopropyl) -viologen (DAPV) .

24. The bacteria/electron carrier complex-immobilized electrode for preparing acetic acid according to Claim

20, wherein said complex of bacteria/electron carrier are formed through amide bond between carboxy moiety of bacteria and amine of electron carrier.

25. The bacteria/electron carrier complex-immobilized electrode according to Claim 20, wherein said electrode is selected from the group consisting of gold electrode, cupper electrode and glassy carbon electrode.

26. The bacteria/electron carrier complex-immobilized electrode according to Claim 20, wherein said complex of bacteria/electron carrier are immobilized on the surface of the electrode by using of cellulose acetate.

27. A bacteria/electron carrier complex -immobilized electrode used in electrochemical process for converting carbon dioxide into acetic acid, wherein bacteria, electron carrier and electrode are combined with each other through covalent bond.

28. The bacteria/electron carrier complex-immobilized electrode according to Claim 27, wherein said bacteria is Clostridium thermoaceticium .

29. The bacteria/electron carrier complex-immobilized electrode according to Claim 27, wherein said electron carrier is viologen derivatives which contain terminal amine group.

30. The bacteria/electron carrier complex-immobilized electrode according to Claim 29, wherein said electron carrier is di- (3-aminopropyl) -viologen (DAPV) .

31. The bacteria/electron carrier complex-immobilized electrode according to Claim 27, wherein said electrode is glassy carbon electrode.

32. The bacteria/electron carrier complex-immobilized electrode according to Claim 27, wherein said complex is formed through amide bond between carboxy moiety of bacteria and amine terminal of electron carrier and through amide bond between carboxy group of electrode and amine terminal of electron carrier.

33. A process for preparing acetic acid from carbon dioxide, wherein Clostridium thermoaceticium- immobilized electrode and electron carrier are used as catalysts .

2 C0₂ + 8H⁺ + 8e^~ ► CH₃COOH + 2H₂0

34. The process for preparing acetic acid according to Claim 33, wherein said electron carrier can be oxidized or reduced reversibly at electric potential of -300mV to -700mV.

35. The process for preparing acetic acid according to Claim 33, wherein said electron carrier is selected from the group consisting of methyl viologen, tetramethyl viologen, N,N-diethyl-4, 4' -bipyridyl, N,N- diisoprophylyl-4, 4' -bipyridyl, 4, 4' -bipyridyl and the mixtures thereof.

36. The process for preparing acetic acid according to Claim 33, wherein said process is carried out at 20°C to 70°C.

37. The process for preparing acetic acid according to Claim 33, wherein said process is carried out at electric potential of -300mV to -700mV.

38. The process for preparing acetic acid according to Claim 33, wherein said process is carried out at pH4 to pH9.

39. A process for preparing acetic acid from carbon dioxide, wherein an electrode which Clostridium thermoaceticium and electron carrier are immobilized is used.

2 C0₂ + 8H⁺ + 8e^" ► CH₃C00H + 2H₂0

40. The process for preparing acetic acid according to Claim 39, wherein said electron carrier is viologen derivatives which contain terminal amine group.

41. A process for preparing acetic acid from carbon dioxide, wherein Clostridium thermoaceticium, electron carrier and glassy carbon complex electrode is used.

2 C0₂ + 8H⁺ + 8e^~ ► CH₃COOH + 2H₂0

42. The process for preparing acetic acid according to Claim 41, wherein said electron carrier is viologen derivatives which contain terminal amine group.