Chinese Journal of Chemical Engineering, 16(5) 796800 (2008)

Biodegradation of Phenol and 4-Chlorophenol by the Mutant Strain CTM 2*

JIANG Yan ()1,2,3,**, REN Nanqi ()2, CAI Xun ()1, WU Di ()1, QIAO Liyan ()1 and LIN Sen ()1
1 2 3

Department of Chemical Engineering, Daqing Oilfield Engineering Limited Company, Daqing 163712, China School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China School of Life Sciences and Chemistry, Harbin University, Harbin 150016, China

Abstract The biodegradations of phenol and 4-chlorophenol (4-cp) were studied using the mutant strain CTM 2 obtained by the He-Ne laser irradiation on wild-type Candida tropicalis. The results showed that the capacity of the CTM 2 to biodegrade 4-cp was increased up to 400 mgL1 within 59.5 h. In the dual-substrate biodegradation, both velocity and capacity of the CTM 2 to degrade 4-cp increased with low-concentration phenol. A total of 400 mgL1 1 4-cp was completely degraded within 50.5 h in the presence of 300 mgL phenol. The maximum 4-cp biodegradation could reach 440 mgL1 with 120 mgL1 phenol. Low-concentration 4-cp caused great inhibition on the CTM 2 to degrade phenol. In addition, the kinetic behaviors were described using the kinetic model proposed in this lab. Keywords biodegradation, phenol, 4-chlorophenol, the mutant strain CTM 2

INTRODUCTION

Phenol and its derivatives are ubiquitous pollutants in aquifers and wastewaters [1]. Chlorinated organic compounds are one of the most important groups of xenobiotic chemicals. Toxic low molecular weight chlorophenols are persistent in the environment, which increases risk to health [2-4]. 4-cp has been extensively used in the chemical industry as intermediate product in herbicide, insecticide, and fungicides [5-7]. They have been characterized as first-priority pollutants by both the European Union (EU) and the United State Environmental Protection Agency (US EPA) [8, 9]. Because of the toxic properties of both phenol and 4-cp, the effective removal of those compounds from industrial aqueous effluents is of great practical significance for environmental protection. At present, some researches on removal of phenol and chlorophenol have been reported, and consequently, the search for the approaches to completely get rid of such kinds of pollutants has become a hot topic in the environmental science [10-12]. Biotechnology plays a key role for the removal of phenolic compounds [13, 14]; hence, their biodegradations have been widely studied, for example, using bacterial and filamentous pure cultures, mixed cultures, and yeast cultures [15-18]. Fialov et al [19]. reported that the yeast Candida maltosa had the potential to degrade phenol in the concentration of up to 1700 mgL 1. Zouari et al [20]. reported that the fungus Phanerochaete chrysosporium had the potential to degrade 4-cp in the concentration of up to 300 mgL 1. Among those studies, C. tropicalis was given high importance because of its strong phenol-degrading capacity and wide existence in the sites with phenol [21-23]. In our lab, a wild strain C. tropicalis was isolated from acclimated activated sludge, which was

capacity to degrade phenol up to 2000 mgL 1 [24], and He-Ne laser was then used to irradiate the wild C. tropicalis, and consequently the mutant strain CTM 2 was obtained [25]. The objective of the present study was to investigate biodegradation behaviors of phenol and 4-cp as single and dual substrates by the mutant strain CTM 2. 2 2.1 MATERIALS AND METHODS Microorganism and cultivation conditions

Wild-type C. tropicalics was isolated from acclimated activated sludge taken from Tianjin Gasworks in China. He-Ne laser (Model HN-1000, made in Guangzhou Laser Technology Applied Institute) was used for the irradiation of wild C. tropicalis. The mutant strain CTM 2 was obtained based on its stability in the continuous transfers and the maximum potential for phenol biodegradation after repeated screens [25]. The CTM 2 and its parent strain were maintained in YEPD medium (yeast extract, 10 gL 1; peptone, 20 1 1 1 gL ; dextrose 20 gL ; agar, 18 gL ) [26]. A mineral salt medium supplemented with phenol and 4-cp was applied for the biodegradation studies [24]. All the cultivation was conducted at 30C, and the shaking flasks were incubated in a rotary shaker at the speed of 200 rmin 1. 2.2 Biodegradation biodegradations of phenol and 4-cp

The experiments were strated with inoculation of C. tropicalis from nutrient agar slants into 10 ml of YEPD medium. After 16 h of incubation, 2 ml of this cell culture was added to 500 ml shaking flasks with

Received 2008-01-24, accepted 2008-05-15. * Supported by the Science and Technology Innovative Talents Foundation of China (2006RFQXS070), the Youth Academic Cadreman Project of Heilongjiang Province (1152G068), Scientific Research Fund of Heilongjiang Province (11523063) and the Science Foundation for Post Doctorate of China (20070410268). ** To whom correspondence should be addressed. E-mail: yjiang@hit.edu.cn

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100 ml fresh YEPD medium. Cells grown in late phase of the growth curve were harvested as inoculum. Five percent of the subculture (OD6001.3) was inoculated into the mineral salt medium with varying initial phenol and/or 4-cp at an interval. In the process of batch culture, all samples were periodically measured for the biomass and substrate concentrations. All the experiments were repeated three times. 2.3 Analytical methods

Cell density was monitored spectrophotometrically by measuring the absorbance at wavelength 600 nm [27]. Biomass concentrations based on dry mass were then measured by filtering cell suspension with the filler and drying the filter paper and cells to a constant mass for 24 h at 105C. To measure concentration of residual substrate, right after measurements of optical density, samples of suspended culture were centrifuged at 7500 rmin 1 for 10 min. The free cell supernatants were used to determine the substrate concentration by high performance liquid chromatography using a LabAlliance (model SeriesIII) system, with a C18 column (250 mm4.6 mm, LabAlliance, U.S.A). Elution was performed with 400/300 (volume ratio) methanol/water at a flow rate of 1.0 mlmin 1, and detection was realized using a UV detector (Model 500, LabAlliance, U.S.A.) at 280 nm. The retention time for phenol was 4.89 min and for 4-cp was 8.98 min. 3 3.1 RESULTS AND DISCUSSION 4-cp biodegradation

3.1.2 Maximum 4-cp biodegradation The maximum phenol biodegradation has been previously studied, and the CTM 2 possessed the ca pacity to degrade 2600 mgL 1 phenol [25]. The biodegradation behavior of CTM 2 in the utmost 4-cp concentration from 360 mgL 1 to 400 mgL 1 at an 1 interval of 20 mgL is described in Fig. 2. With the increased concentration of 4-cp, cell growth lag stage became longer and the specific degradation rate de creased. A total of 400 mgL 1 4-cp was used within 59.5 h by the mutant with the larger capacity than wild strain (350 mgL 1). It was impressive because there were few reports on the 4-cp biodegradation beyond 300 mgL 1.

Figure 2 Cell growth and substrate degradation of the 1 4-cp CTM 2 for 360 -400 mgL 1 1 360 mgL ; 380 mgL ; 400 mgL1

3.2

Phenol and 4-cp dual-substrate biodegradation

3.1.1 Comparison of biodegradation between wild and mutated C. tropicalis The maximum 4-cp biodegradation of wild C. tropicalis occurred at 350 mgL 1. Comparison between the wild-type C. tropicalis and its mutant CTM 2 to degrade 350 mgL 1 4-cp is shown in Fig. 1. It is obvious that the mutant strain CTM 2 grew faster and possessed the higher velocity to degrade 4-cp than its parent strain. It took 4.5 h less for the mutant CTM 2, and the final concentration was a little higher than wild strain (8.25 mgL 1), which was because 4-cp inhibition on the mutant CTM 2 was smaller than that on its parent strain.

3.2.1 Effect of phenol on 4-cp biodegradation velocity Figure 3 shows the effect of different concentra tions of phenol on the CTM 2 to degrade 400 mgL 1 4-cp. In dual-substrate system, it took the CTM 2 the longer time to completely degrade phenol with the increase of phenol concentration. However, the most effective 4-cp biodegradation with the same concen tration occurred in the presence of 300 mgL 1 phenol, 1 and 400 mgL 4-cp was completely degraded within 50.5 h. It can be observed from the figure that 4-cp biodegradation velocity increased with the phenol concentration from 0-300 mgL 1 and then decreased. Even as a kind of toxic compound for cell, in dualsubstrate system, phenol took the effect to provide

Figure 1 Comparison of biodegradation between wild-type C. tropicalis and its mutant CTM 2 for 350 mgL1 4-cp wild; mutant

Figure 3 Effect of phenol on 400 mgL1 4-cp biodegradation behavior by the CTM 2 with different initial phenol concentrations from 0 to800 mgL1 1 1 1 1 0 mgL ; 100 mgL ; 200 mgL ; 300 mgL ; 500 mgL1; 800 mgL1

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carbon and energy for cell, which took the domination in this stage and lasted with the phenol concentration of 0-500 mgL 1. Only when the concentration of phenol was more than 500 mgL 1, its toxic property prevailed and weakened its acceleration and substrate inhibition limited cell growth, thus cell underwent the long lag and therefore, 4-cp biodegradation slowed down. At such phenol concentration, although the strong toxicity existed in dual-substrate system, biodegradation could still proceed. However, it would take the longer time than single-substrate biodegrada tion of 400 mgL 1 4-cp. The two effects of phenol, both inhibition as a toxic compound and acceleration as a carbon, simultaneously existed in dual-substrate system. They competed with each other, which resulted in the optimal phenol concentration (300 mgL 1). Once phenol concentration was more than 500 mgL 1, inhibition began to prevail, and it took the CTM 2 a longer period of time to degrade all the substrates than to degrade single 4-cp. In the experiments, when higher-concentration phenol (over 800 mgL 1) was introduced into the medium, neither phenol nor 400 mgL 1 4-cp was used by the CTM 2. 3.2.2 Effect of phenol on 4-cp biodegradation capacity Phenol could not only accelerate the velocity but also increase the capacity for 4-cp biodegradation. Fig. 4 describes the biodegrading behavior of 440 mgL 1 4-cp with changing phenol concentrations from 60 mgL 1 to 180 mgL 1. A total of 440 mgL 1 4-cp could be degraded in the presence of 80-160 mgL 1 1 phenol. In 120 mgL phenol solution, after the initial biodegradation for 4 h, 4-cp began to degrade without any residual phenol in dual-substrate system. Eventually, the CTM 2 possessed the maximum biodegradation velocity, and all substrates could be completely degraded within 63 h. It was also observed in the samples with 60 mgL 1 and 180 mgL 1 phenol that 4-cp could not be used by the CTM 2. Despite the same experimental results, the biodegrading behaviors were different. From Figs. 4 and 5, it can be observed that 60 mgL 1 phenol was degraded very quickly but cell concentration only increased a little, whereas 180 mgL 1 phenol could not be used by cell all along and cell concentration was kept as a constant. The phenol

Figure 5 Cell concentrations in dual-substrate system with 440 mgL 1 4-cp and different initial phenol concentrations before complete degradation of phenol 1 1 1 60 mgL ; 80 mgL ; 100 mgL ; 120 mgL1; 1 1 1 140 mgL ; 160 mgL ; 180 mgL

concentration of 60 mgL 1 was consumed to synthesize new cells and overcome the substrate inhibition. However, after phenol was consumed, cell growth ter minated, and the total biomass (25.32 mgL 1) could not overcome the inhibition of 4-cp, and the concentration of 4-cp was kept as constant during the long-time determination. Thus, although 60 mgL 1 phenol was used by cells, 4-cp biodegradation could not occur. It was further proved that phenol provided carbon for the initiation of the biodegradation and increased the metabolism velocity and capacity of the strain. 3.2.3 Effect of 4-cp on phenol biodegradation The degradtion of 2500 mgL 1 phenol by CTM 2 in the presence of 4-cp from 0 to 30 mgL 1 is shown in Fig. 6. Obviously, very little 4-cp could create inhibition on phenol biodegradation for the CTM 2. In dual-substrate system, even 30 mgL 1 4-cp could bring about the termination of phenol biodegradation. In the two samples of 10 mgL 1 and 20 mgL 1 4-cp, cells underwent very long lag phase, and 2500 mgL 1 phenol was completely degraded within 74 h and 79 h, respectively, which was evidently longer than single-substrate biodegradation. It was worth noticing that regardless of the concentration of phenol and 4-cp, the mutant strain CTM 2 always preferentially used phenol, and 4-cp biodegradation always occurred at

Figure 4 Effect of phenol on 440 mgL 1 4-cp biodegradation behavior by the CTM 2 with different initial phenol concentrations from 60 to180 mgL 1 1 1 1 60 mgL ; 80 mgL ; 100 mgL ; 120 mgL1; 1 1 1 140 mgL ; 160 mgL ; 180 mgL

Figure 6 Effect of 4-cp on the CTM 2 for phenol biodegradation behavior with 2500 mgL1 phenol and different initial 4-cp concentrations 1 1 0 mgL ; 10 mgL ; 20 mgL1; 30 mgL1

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the end stage of phenol biodegradation. That is, in phenol and 4-cp dual-substrate biodegradation system, the biodegradation time was completely determined by 4-cp biodegradation. This is proven in Figs. 3 and 4. 3.3 Phenol and 4-cp dual-substrate biodegradation kinetics Because of the phenol inhibition on cell growth, the Haldanes equation was selected for assessing the dynamic behavior of the CTM 2 grown on phenol [24, 28]. However, 4-cp imposed a stronger inhibition than phenol on the cells; the Haldanes equation was thus not suitable to describe the 4-cp biodegradation. Therefore, inhibition constant for cell growth ( K i1 ) was appended in the Eq. 1. m2 S2 X = (1) 2 3 S2 S2 KS2 + S2 + + Ki2 Ki2 Kinetic equations of the CTM 2 were obtained by regression using MATLAB software: 5.28S2 Cell growth: X = 3 S2 S2 1064.6 + S2 + 2 + 5.10 1863.1 The value of the root mean square of the residuals at these parameters was small (0.084). Obviously, max was higher than wild strain (max2.78 h 1). Substrate biodegradation behavior was described using the Eq. (2).

S2 S3 S S2 = max 2 S2 KS2 + S2 + 2 + 2 + 1 + 1 + Ki2 Ki2 f fKi1

1 1 1 2 KS1S2 + MS12 S2 + QS1S2 f f f

(4)

Equations (3) and (4) are cell growth kinetic equations of phenol and 4-cp biodegradation in dual-substrate system, respectively. On the basis of the Eqs. (3) and (4) mentioned above, the total cell specific growth rate could be obtained as

X = X1 + X2

(5)

) ( max1 , KS1, Ki1) and ( max 2 , KS2, Ki2, Ki2 could be obtained separately from the kinetics of the individual cell growth on the phenol alone and 4-cp alone, respectively. Using the experimental data, the parameter values of cell growth kinetic equation were obtained as follows: f = 3.8 105 , K = 4.2 109 , M = 3.9 107 ,

Q = 2.6 105 . The specific degradation rates of phenol and 4-cp in phenol and 4-cp dual-substrate system could be represented based on the experimental data. For phenol:

S1 = 5.13X1 + 0.026
For 4-cp:

( R 2 = 0.970)

S = AX + B

(2)

S2 = 0.714X2 + 0.038

( R 2 = 0.959 )

0 0 Thus, 4-cp biodegradation: S2 = 0.502 X2 +

0.097 ( R 2 = 0.967) It was clear that the simulated values of the growth and degradation kinetics agreed well with the experimental data. The parameter values indicated that it was easier for 4-cp to be metabolized by the mutant cells than by the wild strain. On the basis of the experimental results (both substrate inhibition on the cells and mutual inhibition of phenol and 4-cp) and the fact that the inhibitory effect of 4-cp on cell growth was larger than that of phenol, by quasi steady state approximation, the dual-substrate kinetic equations of the strain for the phenol and 4-cp biodegradation were obtained: k X X1 = X1 = +2 XT XT
S2 fS 2 = max1S1 KS1 + S1 + 1 + fS2 + 2 + Ki1 Ki2
3 fS2 2 + KS1S2 + MS12 S 2 + QS1S2 Ki2 1

According to R2, it was concluded that the regression curve was very well consistent with the experimental data. The kinetic equations were suitable to describe the biodegradation behavior of the CTM 2 in dual-substrate system.
4 CONCLUSIONS

(3)

and

X2 =

X2
XT

2 X k+ XT

The mutant strain CTM 2 possessed the strong capacity to degrade phenolic compounds. In dualsubstrate biodegradation system, low-concentration phenol could enhance 4-cp biodegradation of the CTM 2. Phenol always played two roles: to accelerate 4-cp biodegradation as a carbon and to inhibit cell growth as a toxic compound. Different phenol quantity decided its dominancy. However, regardless of the ratio of phenol and 4-cp concentration, the CTM 2 always used phenol first, and 4-cp biodegradation occurred at the end phase of the phenol biodegradation. The biodegradation time completely depended on the consumption time of 4-cp biodegradation. Very little 4-cp could greatly inhibit phenol biodegradation. In addition, the kinetic models for the specific growth and degradation rates of phenol and 4-cp as the single and dual substrates were obtained, and the simulated values of these models agreed well with the experimental data.

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NOMENCLATURE
A B f K, M, Q Ki1 Ki2 growth associated constant for substrate consumption non-growth associated constant for substrate consumption, h 1 substrate interaction coefficient substrate interaction coefficient, (mgL 1) 1 self-inhibition constant of phenol, mgL 1 1 self-inhibition constant of 4-cp, mgL self-inhibition constant of 4-cp, (mgL 1)2 saturation constant for cell growth on phenol, mgL 1 saturation constant for cell growth on 4-cp, mgL 1 initial substrate concentration, mgL 1 time, h biomass concentration, mgL 1 substrate degradation rate, mgLh 1 maximum specific cell growth rate on phenol (max1) or on 4-cp (max2), h 1 specific substrate degradation rate, h 1 specific degradation rate of phenol in dual substrates, h 1 specific degradation rate of 4-cp in dual substrates, h 1 overall specific growth rate in dual substrates, h 1 specific growth rate on phenol in dual substrates, h 1 specific growth rate on 4-cp in dual substrates, h 1 single growth substrate growth substrate, phenol growth substrate, 4-cp 19 11

12

K i2
KS1 KS2 S t X

13

14

max

S S1 S2 X X1 X2
0 1 2

15 16

17

Superscripts Subscripts
18

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