전기화학적 효소반응에 의한 TNT 분해

이기범, 구만복, 문승현

광주과학기술원 환경공학과

광주광역시 북구 오룡동 1 번지 (500-712)

Degradation of trinitrotoluene by a novel electroenzymatic method

KiBeom Lee, Man Bock Gu and Seung-Hyeon Moon

Dept. of Environmental Science and Engineering

Kwangju Institute of Science and Technology (K-JIST)

e-mail:shmoon@kjist.ac.kr

Introduction

TNT (2,4,6-trinitrotoluene) is the primary explosive used in munitions production. The production and use of TNT resulted in contamination of soil and surface water in the vicinity of several munitions and munition plants. TNT is a suspected toxic and mutagenic agent and is relatively stable in the environment. Due to its toxicity and mutagenic effects, their release to the environment is strictly controlled and must be remedied. Much research has been conducted to remove TNT from contamination of soil and surface water. Recently degradation via bioremediation was investigated under aerobic and anaerobic conditions instead of physical or chemical treatment because of no second effluent. However, whole cell cultures have limited use for the bioremediation of toxic chemicals on a large scale, due to the time required for cell cultivation. In addition, partial or complete inhibition of bioremediation effectiveness has been found due to the toxicity of chemical. These disadvantages have led to the thought that an enhanced enzymatic method is used for degradation of TNT.

In this study, a novel electro-enzymatic method hybridizing electrochemical and enzymatic reactions using lignin peroxidase for treatment of TNT was developed.

Basic concept of the electroenzymatic process

The electroenzymatic method uses an electrode reaction as a means of producing H2O2, followed by an oxidation reaction sequence catalyzed by the enzyme as following.

at the anode: 2H2O = 4e- + 4H+ + O2

at the cathode: O2 + 2H+ + 2e- = H2O2

H2O2 + Ligninase + Veratryl alcohol + TNT => TNT degradation

Materials and Methods

Enzyme preparation and chemicals

Phanerochaete chrysosporium (ATCC 24725) was cultured in the same manner as reported by Tien and Kirk1). Phanerochaete chrysosporium spores were immobilized in polyurethane foam and used for the production of lignin peroxidase in repeated batch cultures using carbon-limited medium. Technical- and analytical-grade TNT were purchased from Chem Service Ind., LTD (USA). Other chemicals used in this work were of reagent grade.

Electrochemical reactor and assays

The apparatus used for the studies is shown in Figure 1. The reactor used in this study was divided into an anode and a cathode compartment (each volume: 30cm3) with a cation-exchange membrane. Two 4 x 2cm conductive titanium with platinum coating and reticulated vitreous carbon plates were used as a counter and a working electrode. The potential of the working electrode was measured with respect to a saturated Ag/AgCl reference electrode. One mole per liter H2SO4 and 50 mM sodium acetate, sodium phosphate, and 100 mM potassium nitrate at pH 3 were used as the anolyte and catholye, respectively. Pure O2 gas was sparged into the catholyte at a flow rate of 25 ml/min. Electrolysis was carried out over a period of 60 min at a constant potential during batch experiments by using a potentiostat (263A EG&G Co.) at 25oC. In the electroenzymatic treatment, the electrolysis was performed at +0.1 V with a Ag/AgCl reference electrode. Lignin peroxidase (100 U/L), veratryl alcohol (0.5mM) and 2,4,6-trinitrotoluene (44□M) were added to the catholyte. During the experiments, samples were taken periodically to measure the concentration of TNT by  HPLC at 254 nm. H2O2 was analyzed colorimetrically by the method of Zhen-Yue et a12). Analysis of the nitrite ion concentration was performed by reverse phase high performance liquid chromatography with a pre-column reaction3).

Results and Discussion

H2O2 formed by electrochemical reaction

The rates of H2O2 formation were examined in the range of +0.2 to -0.4 V. As shown in Figure 2, the H2O2 formation increased with an increase in the potentials. H2O2 is essential for lignin peroxidase catalysis, but H2O2 concentrations of over 0.3 mM are not conducive for the enzyme activity and caused a decrease in the reaction rate4).

Effect of applied voltage

A set of experiments were performed to examine the effects of the applied voltage on the TNT degradation without enzyme (Figure 3). The TNT degradation rate increased with an increase in the voltage. To prevent ligninase denaturation by the electrode reaction, +0.1 V was determined to be the optimal potential for the following experiment with ligninase.

Proof of concept : electroenzymatic degradation of TNT

The degradation of TNT under different conditions is shown in Figure 4. TNT was not degraded without an applied potential due to the absence of H2O2. It was degraded slightly at a potential of 0.1 V against a Ag/AgCl electrode. When veratryl alcohol was added with ligninase, TNT was rapidly degraded, implying that veratryl alcohol plays a significant role in the electroenzymatic oxidation reaction. Also, the results show that the rapid degradation of TNT takes place when the enzyme is activated by H2O2 under an electric field.

TNT degradation and nitrite generation

The TNT degradation and nitrite ion release rates are presented in Figure 5. Complete removal of TNT was obtained within 60 min, while this was not reached without lignin peroxidase and veratryl alcohol. It was observed that the nitric ion released was proportional to the TNT degraded. The mole ratio of the nitrite generated to the TNT degraded indicates that denitrification occurs primarily during the degradation of TNT.

Energy consumption

Figure 6 shows the energy consumption during the each electrolysis treatment. In the electroenzymatic treatment of TNT, a significant power reduction was seen  compared to electrochemical oxidation at -0.2 and -0.4 V. Thus, for 30 ml of 44 □mole TNT, the energy consumption was measured as 5.72 x 10-7 kWh for electroenzymatic reaction using a +0.1 V and 9.21 to 17.15 x 10-7 kWh for the electrochemcial reaction at -0.2 V and -0.4 V, respectively. These results imply that electroenzymatic process improved the efficiency of electrolysis.

Conclusions

The present work has demonstrated the feasibility of electroenzymatic degradation of TNT using lignin peroxidase. The results showed a reasonable reaction rate for the electroenzymatic oxidation without H2O2 addition.

References

1. Tien, M. and Kirk, T. K. Methods Enzymol. 1988, 161, 238-249.

2. Zhen-Yue Jiang., Hunt, J. V. and Wolf, S. P. Analytical Biochemistry. 1992, 202,    384-389.

3. Krishna, K. V. and Archana. Analytical letters. 1992, 25(11), 2083-2093.

4. Michael, D. A., Rajagopalan, V. and Robert, L. I. Wat. Res. 1989, 23(4), 443-450.

Acknowledegement

This work was supported in part by Brain Korea 21 from the Ministry of Education through the Graduate Program for Chemical & Environmental Engineering at Kwangju Institute of Science & Technology.

Fig.1. Diagram of                      Fig. 2. H2O2 formation

an electroenzymatic reactor              in an electrochemical cell

Fig. 3. Effect of the applied voltage      Fig. 4. Proof of concept :

on the degradation rate                  electroenzymatic degradation of TNT

Fig. 5. nitrite generation                 Fig. 6. Energy consumption

during degradation