Process Biochemistry 37 (2002) 1367 1373 www.elsevier.

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Biodegradation of phenol and chlorophenols with dened mixed culture in shake-asks and a packed bed reactor
Jung-Hwa Kim a, Kyung-Keun Oh b, Sung-Taik Lee c, Seung-Wook Kim d, Suk-In Hong d,*
Department of Biotechnology, Graduate School of Biotechnology, Korea Uni6ersity, Seoul, Republic of Korea b Department of Industrial Chemistry, Dankook Uni6ersity, Chonan, Republic of Korea c Department of Biological Science, KAIST, Daejeon, Republic of Korea d Department of Chemical and Biological Engineering, Korea Uni6ersity, 5 -ka, Anam-dang Sungbuk-ku, Seoul 136 -701, Republic of Korea Received 29 June 2001; received in revised form 12 December 2001; accepted 28 December 2001
a

Abstract Pseudomonas testosteroni CPW301 degraded phenol and 4-chlorophenol simultaneously, but degradation rates of these compounds were affected by 4-chlorophenol. Phenol increased the cell concentration and therefore the degradation efciency of 4-chlorophenol was improved. Pseudomonas solanacearum TCP114 could degrade only 2,4,6-trichlorophenol. A dened mixed culture of P. testosteroni CPW301 and P. solanacearum TCP114 could treat phenol, 4-chlorophenol, and 2,4,6-trichlorophenol completely and overcome the inhibition of substrates to other microorganisms. The degradation capacity of the packed bed reactor (PBR) was higher than that of the continuous stirred tank reactor, but the PBR was unsuitable for oxygen-sensitive microorganisms. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Pseudomonas testosteroni CPW301; Pseudomonas solanacearum TCP114; Phenol; 4-chlorophenol; 2,4,6-Trichlorophenol; Biodegradation; Packed bed reactor

1. Introduction Phenolic compounds are used for various purposes in many areas, for example chlorinated phenolic compounds are specically utilized as preservatives of paint, leather, textile goods and as antimicrobial agents. They are produced in petrochemical plants and processes of chlorine bleaching of excessive pulp [1]. Because of improper treatment of these materials, they have widely contaminated soil and groundwater and their toxicity seriously affects living organisms. They are suspected carcinogens and well-known as precursors of dioxin [2]. To treat phenolic compounds, biological methods are preferable because this is economical, and there is a low possibility of the production of byproducts. Many papers support the biological treatment of waste or ground water. The microorganisms used are usually
* Corresponding author. Tel.: + 82-2-3290-3294; fax: + 82-2-9266102. E-mail address: sihong@korea.ac.kr (S.-I. Hong).

aerobes, including Pseudomonas sp. [46], Alcaligenes sp. [3,7], Azotobacter sp. [8], Rhodococcus sp. [9,10], Phanerochaere sp. [11,12], and Cryptococcus sp. [13]. These aerobes are more efcient at degrading toxic compounds because they grow faster than anaerobes and usually transform organic compounds to inorganic compounds (CO2, H2O). The difference of structure and toxicity among phenolic compounds requires that various bacteria have specic qualities to degrade each compound and a mixed culture may be applied. Mixed culture means that several kinds of microorganisms are cultured simultaneously. Mixed culture is divided into undened and dened types. The activated sludge process is one example of an undened mixed culture. Activated sludge is a complex group of microorganisms that can oxidize wastewater under aerobic conditions. To treat special wastewater, including very toxic and recalcitrant compounds by activated sludge, adaptation is generally needed. If microorganisms that can degrade these materials are added, the adaptation time might be reduced

0032-9592/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 0 0 7 - 9

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and the efciency may increase. To do this, a study of pure cultures or dened mixed cultures is important. A dened mixed culture is a miniature of complex microorganisms in nature. Through the study of this, various interactions between microorganisms in nature may be analyzed. In fact, many papers concerned with mixed culture have been reported. Morsen and Rehm [13] studied a mixed culture of Psuedomonas putida and Cryptococcus elino6ii to degrade phenol, and Oh et al. [14] reported a mixed culture of known bacteria (Fla6obactrium sp. BEN2, Acinetobacter sp. GEM63 and GEM2) and sludge to treat industrial wastewater. Also, Wiesel et al. [15] used ve kinds of bacteria to treat polycyclic aromatic hydrocarbons. Although there are suitable bacteria groups to do this research, a proper kind of reactor to promote treatment is also necessary. Generally, a stirred tank reactor is used. The advantage of this reactor is that operating it and adjusting its retention time is simple. On the other hand, it is vulnerable to shock and washout and it takes a long time to recover from perturbation. A packed bed bioreactor can recover from shock quickly because active microorganisms are held on a stationary surface. For this reason, they can be resistant to washout and shock [16]. Holladay et al. [16] mentioned the severe disadvantage of a packed bed reactor (PBR), however, as that it produced excess biomass resulting in severe ow stoppage. But Beg and Hassan [17] however, reported that microbial growth was reduced through endogenous respiration, resulting in a promising biochemical treatment process with respect to economic considerations and simplicity of operation. The objectives in this work were, (i) to degrade phenol, 4-chlorophenol (4-CP), and 2,4,6-trichlorophenol (2,4,6-TCP) using Pseudomonas testosteroni

CPW301 and Pseudomonas solanacearum TCP114, (ii) to compare the reactors efciency between the continuous stirred tank reactor (CSTR) and the PBR.

2. Materials and methods

2.1. Microorganisms
P. testosteroni CPW301 was used to degrade 4chlorophenol (4-CP) and phenol. P. solanacearum TCP114 was also used to degrade 2,4,6-trichlorophenol (2,4,6-TCP). These bacteria were provided by the Laboratory of Environmental Microbiology in the Department of Biological Science, KAIST.

2.2. Media and culture conditions

Microorganims were precultured in a 500 ml bafed ask with a working volume of 100 ml. Media consisted of 5.0 g peptone, 3.0 g beef extract in 1 l and 50 mg phenol or chlorophenol in 1 l. After the seed culture, cells were centrifuged at 10 000 rpm for 10 min and transferred into minimal salt medium (MSM) containing phenolic compounds. MSM contained 1 g K2HPO4, 0.6 g NaH2PO4, 1 g NH4NO3, 0.2 g MgSO47H2O, 0.2 g KCl, and 1 ml trace elements of 1 l and 0.002% (w/v) yeast extract. Trace elements contained 0.3 mg CuSO45H2O, 0.16 mg ZnSO4H2O, 0.1 mg CoSO47H2O, 0.1 mg Na2MoO47H2O, 0.05 mg H3BO3, and 0.2 mg CaSO41/2H2O in 1 ml [18]. Initial pH was 7.2 (9 0.02) and the culture condition was adjusted to 30 C and 120 rpm. Phenolic compounds were the sole carbon and energy sources and were purchased from the Dongkyung Chemical Co., Japan.

Fig. 1. Schematic diagram of PBR packing with Celite R-635 and with aeration tank. 1 Packed bed reactor, 2 peristaltic pump, 3 aeration tank, 4 air pump.

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2.3. Reactor and operation conditions

A PBR was used with an aeration tank (Fig. 1). It was made of Pyrex glass. The diameter was 6.0 cm and the height was 40 cm. The working volume was 1.0 l with the aeration tank. Celite R-635 was used as carrier material (Celite Korea Co.) This material is thermally and chemically stable, resistant to microbial degradation, and can be reused and so is considered to be good immobilization material [19]. The reactor was streamed by upow and recycled with a retention time of 30 min in the column to increase the degradation efciency. A peristaltic pump (Cole-Parmer, Masterex) was used to adjust the ow rate. Celite was packed at 70% of the working volume. Microorganisms were adsorbed onto Celite for 24 h using MSM and phenolic compounds as carbon and energy sources. The temperature was maintained at 30 C with a waterbath (Vision Scientic. Co.). A CSTR was used with a bottom-driven agitation system, pH, and DO meter. The CSTR was operated at 300 rpm and 1 vvm. The temperature was maintained at 30 C.

Fig. 2. Adsortion of P. testosteroni CPW301 on Celite R-635 in a PBR. Co: initial optical density of cell, C: optical density of cell according to time.

2.4. Analytical methods

Phenolic compounds were analyzed by HPLC with a m Bondapak C18 column (3.9300 mm2) and a UV detector (Younglin Co.) at 280 nm. The solvent was methanol, water, acetic acid in a ratio of 400:300:6. Cell concentration was determined by optical density at 600 nm with a VIS spectrophotometer (Spectronics 20D). Chloride ion concentration in broth was determined by colorimetric assay [20]. Dry cell mass adsorbed into Celite was measured as follows: dry weight of Celite on which microorganisms were adsorbed was measured, then microorganisms on Celite were washed in 6 M NaOH solution and then water. After this process, the dry weight of Celite was measured again. The difference in dry weight of these Celites was the dry cell mass. 3. Results and discussion

3.1. Comparison of degradation ability between CSTR and PBR

Phenol and 4-CP were metabolized by the same degradation pathway in P. testosteroni CPW301 [21]. Bae et al. [21] showed that CPW301 could take up these compounds simultaneously and there could be interactions between the two compounds. Although P. testosteroni CPW301 degraded phenol and 4-CP simultaneously, the degradation rate of these compounds was determined by 4-CP. In this study, the PBR and CSTR were employed for efcient degradation of phenol and 4-CP.

Before the operation of the PBR, two pre-experiments were performed. The rst experiment was performed to test the oxygen sensitivity of CPW301, as there might be oxygen limitation during PBR operation because air was not supplied directly. To establish this, the method described by Wang et al. [22] was used. The oxygen absorption rates of shake-asks were quantied according to media volume. When a 500 ml unbafed ask was used at 400 rpm, the absorption rate of oxygen declined logarithmically as the volume increased. In this study, the working volume in the 500 ml bafed ask was raised from 100 to 400 ml step by step and the degradation of phenol and 4-CP was observed. Despite the different working volumes, the degradation rates of phenol and 4-CP were constant. Based on this result, it could be concluded that CPW301 was not sensitive to oxygen concentration. In order to conrm cell adsorption on to Celite R-635, CPW301 was precultured, harvested, and inoculated into a PBR without broth. The column was lled with phosphate buffer adjusted to pH 7.2, nutrient was not included in order to prevent cell growth. During the recycle operation, a change of optical density was observed. The retention time in the column section was 30 min and a rapid decline of OD was observed after the duration of the rst recycle. OD decreased exponentially for 4 h, and then the value became constant. The difference of OD between the initial and nal state is the amount of CPW301 attached to Celite. Thus, cells adsorbed to Celite (or other carrier material) initially and then grew there using the nutrient supplied (Fig. 2). The PBR was operated with a batch system. Phenol and 4-CP were each fed at 20 mg/l in the rst stage and took 10 h for complete degradation. After the batch reaction ended, a semicontinuous system was applied. The broth was drained and washed with phosphate buffer adjusted to pH 7.2 and the next reaction was

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begun with 20 mg/l of 4-CP and phenol. Fig. 3 shows that the degradation time during each batch system was almost constant. It can be concluded that the PBR system was stable and sufciently effective to be applied to degradation. After the fth batch culture, the substrate was fed continuously with 1 ml/min (dilution rate 0.06 per hour). The dilution rate was changed incrementally as required. Until the dilution rate reached 0.36 per hour, the concentration of phenol and 4-CP in efuent was zero, but an unknown intermediate began to be produced at 0.36 per hour. When the dilution rate was increased to 0.6 per hour; phenol and 4-CP were not degraded completely. A dilution rate of 0.36 per hour is higher than the maximum specic growth rate on phenol, thus a PBR can overcome the limitation of washout in CSTR without immobilization (Fig. 4). Celites were sampled after these experiments and the dry weight of attached cell mass measured. The value was 0.3 g dry weight of CPW301/g Celite. The CSTR with free cells was operated in batch. The initial concentration of each compound was 20 mg/l and total degradation was completed within about 16 h (Fig. 5). During the batch system, the cell concentration decreased as substrates exhaust. Yields of phenol and 4-CP were 0.69 and 0.11. Thus, only 16 mg/l of the microorganism can be produced, and this is very low compared with the initial cell concentration of 66.10 mg/l. Continuous operation began with an incremental dilution rate. In this case, to prevent wall growth, the agitation speed was maintained at 300 rpm up to a dilution rate of 0.24 per hour, phenol and 4-CP were degraded completely, but washout occurred at a dilution rate of 0.36 per hour. When the abilities of degradation in PBR and CSTR were compared with respect to the dilution rate, the tolerance in the PBR was better than in the CSTR. The phenolic compounds were degraded completely in the

Fig. 4. Continuous degradation of 4-CP (20 mg/l) and phenol (20 mg/l) by P. testosteroni CPW301 in a PBR with different dilution rate.

PBR until 0.36 per hour, but washout was observed in the CSTR at a dilution rate, 0.36 per hour.

3.2. Effect of phenol and 4 -CP on the degradation of 2,4,6 -TCP by TCP114
Fig. 6 shows the effects of phenol and 4-CP on the degradation of 2,4,6-TCP with a pure culture of

Fig. 3. Simultaneous degradation of phenol and 4-CP in semicontinuous cultivation in PBR by P. testosteroni CPW301.

Fig. 5. Continuous degradation of phenol (20 mg/l) and 4-CP (20 mg/l) in CSTR with different dilution rate.

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a mixed culture, the severe effects of one compound could be relieved. Fig. 8b shows the effects of phenol and 4-CP on the degradation of 2,4,6-TCP by TCP114. The result was in accord with Fig. 6. Although phenol and 4-CP were mixed with 2,4,6-TCP simultaneously, the degradation time of 2,4,6-TCP was the same as the case in which only 4-CP was added. From these data, it can be concluded that a mixed culture is useful for the treatment of wastewater contains many kinds of compounds.

3.4. Dened mixed culture of CPW 301 and TCP114 in a PBR

The oxygen sensitivity of TCP114 was tested before the use of PBR. This method was the same as that of CPW301, and the working volume was increased from 100 to 300 ml incrementally. Although 2,4,6-TCP degradation time did not vary, the lag time was prolonged at 300 ml, and yields were lowered as the working volume was increased. Thus, there might be a signicant oxygen effect as reaction time was extended, although the initial phase of degradation was not affected. The reason is that the activity and concentration of cells were diminished.

Fig. 6. Effect of phenol and 4-CP on the degradation of 2,4,6-TCP by P. solanacearum TCP114; (a) 2,4,6-TCP only, (b) 20 mg/l phenol added, (c) 20 mg/l 4-CP added.

TCP114. The degradation of 2,4,6-TCP was not altered by phenol. 2,4,6-TCP degradation time was constant when phenol was added. Bae [23] reported that TCP114 could not degrade phenol in the presence of TCP, but an enzyme degrading phenol was induced after complete degradation of TCP. Thus, it can be thought that metabolic pathways between 2,4,6-TCP and phenol are different, and there is no signicant effect on TCP114 by phenol because toxicity of phenol is lower than 2,4,6TCP. There was an alteration of 2,4,6-TCP degradation by 4-CP. Fig. 6 shows the degradation of 2,4,6-TCP with and without the addition of 4-CP. When 4-CP was added, the degradation rate was lowered.

3.3. Dened mixed culture of CPW 301 and TCP114

Fig. 7b shows the effects of 2,4,6-TCP on the degradation of phenol and 4-CP by CPW301. CPW301 was seriously inhibited by 2,4,6-TCP. Total degradation time was prolonged four times compared with the case without 2,4,6-TCP. When TCP114 was added, the concentration of 2,4,6-TCP decreased and the degradation of phenol and 4-CP began. The toxicity of 2,4,6-TCP affected the degradation capacity of CPW301. By using

Fig. 7. Comparison of pure culture of P. testosteroni CPW301 and mixed culture of P. testosteroni CPW301 and P. solanacearum TCP114 in ask at 30 C and 120 rpm. (a) Degradation of phenol and 4-CP by CPW301, (b) degradation of phenol, 4-CP, 2,4,6-TCP by CPW301, (c) degradation of phenol, 4-CP, 2,4,6-TCP by CPW301 and TCP114.

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Fig. 10. Continuous degradation of phenol, 4-CP, and 2,4,6-TCP by 20 mg/l simultaneously by mixed culture of P. testosteroni CPW301 and P. solanacearum TCP114 in PBR with different dilution rate. Fig. 8. Comparison of pure culture of P. solanacearum CPW114 and mixed culture of P. testosteroni CPW301 and TCP114 in ask at 30 C and 120 rpm. (a) Degradation of 2,4,6-TCP by TCP114, (b) degradation of phenol, 4-CP, 2,4,6-TCP by TCP114, (c) degradation of phenol, 4-CP, and 2,4,6-TCP by CPW301.

After the immobilization of TCP114 and CPW301 for 24 h, the batch system was operated, and semicontinuous operation was started (Fig. 9). In the rst batch, the total degradation time was 20 h-longer than that of the batch operation in the ask. Phenol and 4-CP were degraded faster than 2,4,6-TCP. This can be explained because CPW301 was protected from 2,4,6TCP by immobilization, and TCP114 was affected by decient oxygen in the column. The concentration of

oxygen in the upper part of the column was 0.21 mg/l. This is almost an anaerobic condition. The duration of total degradation was shortened after the rst batch. Fig. 10 shows the results of continuous degradation of phenolic compounds in a PBR. 2,4,6-TCP was not degraded completely. CPW301 was affected by accumulated 2,4,6-TCP, so phenol and 4-CP were not degraded at the dilution rate of 0.36 per hour.

4. Conclusions Since P. testosteroni CPW301 degraded phenol and 4-CP with the same degradation pathway (meta-cleavage pathway), the degradation capacity of CPW301 was determined by 4-CP. Although CPW301 did not distinguish phenol and 4-CP, the degradation rate of these compounds was affected by 4-CP concentration. This fact shows that the toxicity of 4-CP was more than that of phenol and it affected cell activity. The material with less toxicity and no inhibition of the degradation pathway can encourage growth of the bacteria and improve the degradation capacity with cell mass augmentation. The packed bed bioreactor (PBR) was applied to degrade phenolic compounds with the immobilized carrier, Celite R-635. The degradation rate was accelerated and the microorganisms immobilized on Celite were reused several times without any loss of activity. The degradation capacity of the PBR was higher than a CSTR with free cell with respect to the dilution rate.

Fig. 9. Simultaneous degradation of phenol, 4-CP, and 2,4,6-TCP by mixed culture of P. testosteroni CPW301 and P. solanacearum TCP114 in semi-continuous cultivation in a PBR.

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The threshold dilution rate of PBR was higher than the maximum specic growth rate of phenol. This means that CPW301 absorbed into Celite can resist a higher load of phenolic compounds. PBR is an effective reactor in this case. P. solanacearum TCP114 could only degrade 2,4,6TCP. When phenol or 4-CP was added, TCP114 was not affected by phenol, but the degradation rate of 2,4,6-TCP was prolonged by 4-CP. The toxicity of 4-CP may have affected TCP114. Without TCP114, the degradation of phenol and 4-CP by CPW301 was seriously affected by 2,4,6-TCP. To relieve the effect of 2,4,6-TCP, a dened mixed culture of CPW301 and TCP114 was applied. As the concentration of 2,4,6TCP decreased, the degradation of phenol and 4-CP proceeded more effectively than when CPW301 only was applied. If there are many compounds, which inhibit the degradation of other materials, a mixed culture, which contains suitable microorganisms for each compound can solve this problem. When the packed bed bioreactor was applied to degrade phenol, 4-CP, and 2,4,6-TCP, 2,4,6-TCP was not degraded effectively in comparison to ask culture. This may be explained by the effect of oxygen on TCP114. The cell mass of TCP114 was reduced in an oxygen decient condition, so the activity of TCP114 was reduced with time. The degradation of 4-CP and phenol was affected by nondegraded 2,4,6-TCP. Thus, the PBR was not suitable for oxygen-sensitive bacteria and another reactor system, which can supply sufcient oxygen should be used to treat 2,4,6-TCP by TCP114.

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