Kinetics of cometabolic degradation of 2-chlorophenol and phenol by Pseudomonas putida

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1 Water Siene and Engineering, 2009, 2(3): doi: /j.issn inetis of ometaboli degradation of 2-hlorophenol and phenol by Pseudomonas putida Xing-ping LIU* 1, 2 1. ey Laboratory of Integrated Regulation and Resoure Development of Shallow Lakes of Ministry of Eduation, Hohai University, Nanjing , P. R. China 2. College of Environmental Siene and Engineering, Hohai University, Nanjing , P. R. China Abstrat: In order to address the omplex ometaboli degradation of toxi ompounds, bath experiments on the biodegradation of 2-hlorophenol (2-CP) and phenol by Pseudomonas putida were arried out. The experimental results show that 2-CP has an inhibitory effet on ell growth and phenol degradation, whih demonstrates that the interation between substrates affets ell growth and substrate degradation. A kineti model of ell growth and substrate transformation was also developed. The square of the orrelation oeffiient from the experiment was 0.97, indiating that this model properly simulates the ometaboli degradation of 2-CP and phenol. ey words: ometabolism; kineti model; 2-hlorophenol; inhibition 1 Introdution As a group of hemials with signifiant eonomi value, hlorophenols are widely used in herbiides, insetiides, fungiides, and preservatives. Beause of their strong toxiity, non-degradability, ready biologial aggregation, and strong arinogeniity, the hlorophenols that universally exist in the soil and water are attrating more and more attention. The U. S. has listed them as priority pollutants (Contreras et al. 2003; Sahinkaya and Dilek 2007; Jiang et al. 2007). The ometaboli biodegradation method of removing these toxi ompounds with suitable arbon and energy soures is one of the fouses of environmental pollution ontrol researh today. The key ingredients in ometaboli degradation of hlorophenoli organi ompounds are proper organi ompounds that an be used as the growing substrates (Atuanya and Chakrabarti 2004) and suitable ometaboli degrading bateria. Generally speaking, it is possible to find degrading bateria in soil ontaining hlorophenoli pollutants (Chang and Alvarez-Cohen 1995; Little et al. 1988; Mu and Sow 1994). However, beause of their toxiity and non-degradability, hlorophenols, the non-growing substrates, may inhibit the growth of ells during the ometaboli degradation proess and hinder degradation This work was supported by the Chinese ey Laboratory of Integrated Regulation and Resoure Development of Shallow Lakes of China (Grant No. 2007J006). *Corresponding author ( liuxingping@hhu.edu.n) Reeived Feb. 25, 2009; aepted Jul. 29, 2009

2 (Fakhruddin and Quilty 2005; Polymenakou and Stephanou 2005). From the perspetive of engineering ontrol, it is neessary to find more suitable onditions to improve the speed and effiieny of ometaboli degradation. A lot of researh has been done on ometaboli degradation kinetis, espeially on the ometaboli degradation of trihloroethylene. Different kineti models have been developed in order to desribe different phenomena, suh as ompetitive inhibition (Aziz et al. 1999), produt toxiity (Alvarez-Cohen and MCarty 1991; Zhang and Bajpai 2000), the deay and death of miroorganisms (Chang and Criddle 1997), the deativation and repair of enzymes (Ely et al. 1995), and the main fators in ometaboli degradation (Chang and Alvarez-Cohen 1995; Polymenakou and Stephanou 2005). These models play different roles in different ometaboli degradation systems. Whatever the ase is, the growth of ells is diretly related to the ometaboli degradation. The growing and non-growing substrates oexist in the ometaboli degradation system, whih makes the ometaboli degradation proess rather omplex. The objetives of this study were to investigate the biodegradation of phenol and 2-hrophenol (2-CP) as a single substrate by Pseudomonas putida, to investigate the interation of phenol and 2-CP in a dual-substrate system, and to researh the ell growth and substrate degradation kinetis of Pseudomonas putida in a dual-substrate biodegradation system with high substrate onentration. 2 Materials and methods 2.1 Strain soures The strains were fed from the ativated sludge in the aeration tank of the wastewater treatment system of the Sinope Yangzi Petrohemial Company. Using phenol as the only arbon soure, baterial flora that an degrade 2-CP was obtained through four months of ultivation and domestiation. The extrated miroorganisms were preserved in the fridge at 4 for future use. The strains and ulture onditions have been desribed in previous studies (Liu 2008). 2.2 Culture medium A 300-mL onial flask with a stopper was filled with 150 ml of ulture medium. The ulture medium was an inorgani salt solution ontaining phenol, in whih the inorgani omponents were 3.8 g/l Na 2 HPO 4, 1 g/l H 2 PO 4, 1 g/l NaCl, and 0.2 g/l MgSO 4. A 1-mL quantity of trae element solution was added to the onial flask. The trae element omponents were 2 g EDTA-2Na, 0.2 g FeSO 4 7H 2 O, 0.1 g MnCl 2 4H 2 O, 0.1 g H 3 BO 3, 0.1 g CoCl 2 6H 2 O, 0.1 g ZnCl 2, 0.02 g Na 2 MoO 4 2H 2 O, 0.02 mg NiCl 2 6H 2 O, 0.01 g CuCl 2 2H 2 O, and ml distilled water. To derease pollution as muh as possible, instruments suh as the sukers and onial flasks were autolaved before use, and the transferring and sampling of the ulture medium Xing-ping LIU Water Siene and Engineering, Sep. 2009, Vol. 2, No. 3,

3 were both undertaken in a bateria-free environment. Before inoulation, the bateria grew in the inorgani salt solution, whih ontained agar gel and a ertain amount of phenol (200 mg/l) as the only arbon soure. 2.3 Methods A quantity of 1.5 ml of bateria solution in the logarithmi growth phase was poured into eah onial flask, and then different onentrations of phenol and 2-CP were added. Later, these solutions were ultivated in a onstant-temperature shaking table (at 30 and 160 r/min). This experiment studied the effets of the different onentrations of phenol on the removal of 2-CP. It also provided relevant ontrol systems in whih phenol and 2-CP were removed by the miroorganisms separately. The sample flask was plaed in the shaking table at a temperature of 30, and sampling analyses of the ontents of phenol and 2-CP were made over a ertain period. The onentrations of phenol and 2-CP were analyzed using a gas hromatograph (Aglient6890, U. S.), inluding HP-5 (5% phenyl and 95% polymethyl siloxane), a apillary olumn (30 m 0.32 mm 0.25 μm) and a flame ionization detetor (FID). The olumn flow rate was 3.0 ml/min. The temperatures of the olumn, vaporizing hamber, and detetor were 150, 200, and 270, respetively. The split ratio was 1:5. Dry ell weight was measured with the onstant weight method after entrifugation. The turbidity of the bateria solution was examined with the turbidimetri method, mainly using a UV-1200V spetrophotometer (Shimazu Company, Japan). A 5-mL sample was taken from eah onial flask, and the optial density (OD) value of the growth of the degrading bateria was measured through spetrophotometri olorimetry (the wave length was 600 nm). Taking the effets of deep olor substanes on the OD value into onsideration, after the transformation of 2-CP, the atual onentration of the miroorganisms was determined by testing the OD value of the sample before and after the sample was filtered through the 0.45-μm mesh. The experimental plan is shown in Table 1. Table 1 Cometaboli degradation of phenol and 2-CP experimental plan Serial number Initial onentration of substrates Phenol (mg/l) 2-CP (mg/l) A1, A2, A3, A4, A , 20, 30, 40, 50 B1, B2, B3, B4, B , 20, 30, 40, 50 C1, C2, C3, C4, C , 20, 30, 40, 50 D E Model setup In the system for ometaboli degradation of miroorganisms, enzymes an sometimes degrade primary and seondary substrates simultaneously. Thus, ompetition exists and the 112 Xing-ping LIU Water Siene and Engineering, Sep. 2009, Vol. 2, No. 3,

4 two substrates mutually inhibit eah other s degradation. The inhibitory effet should be onsidered in the ometaboli kinetis. An inhibition onstant i was introdued into the Monod equation (Eq. (1)) (Tang et al. 2004) and the Andrews equation (Eq. (2)) was obtained (Andrews 1968): S (1) Xt d s + S S (2) 2 Xt d + S+ S s where S is the substrate onentration, i is the self-inhibition onstant, X is the biomass onentration, s is the saturation onstant, t is time, and is the maximum substrate utilization ratio. When i goes to infinity, Eq. (2) beomes the Monod equation. Neufeld et al. (1986) proposed the following equation by introduing the inhibition exponent n into Eq. (2): S (3) n Xt d + S + S S s i ( ) A larger inhibition exponent n indiates stronger inhibition and a slower rate of degradation. When n is equal to 1, this equation beomes the Andrews equation. Therefore, this equation is suitable for desribing the inhibition reation at different inhibition levels. Using the two onstants n and i to ontrol the inhibition level gives this equation a wider range of appliation and a more aurate result. Gupta et al. (1996) proposed to analyze ometabolism more thoroughly. When the primary substrates are diretly provided by the outside world, the utilization veloity of seondary substrates is related to the onsumption of primary substrates, but when the outside world does not provide any primary substrates diretly, the utilization veloity of seondary substrates is related to the self-oxidation of miroorganisms during the lag phase. When there are external primary substrates, the utilization veloity of seondary substrates aused by these external primary substrates is muh larger than that aused by the self-oxidation of miroorganisms during the lag phase. Thus, the utilization veloity aused by the self-oxidation of miroorganisms during the lag phase an be negleted. However, when the seondary substrates or their intermediates have a toxi effet on miroorganisms, they will, to a large extent, inhibit the degradation of primary substrates and greatly redue their utilization veloity. At this time, the utilization veloity aused by the self-oxidation of miroorganisms during the lag phase annot be negleted. The total utilization veloity of the seondary substrate an be expressed as S S + (4) n Xt d s + S + S( S i) s + S where is the maximum substrate utilization ratio of the seondary substrate, S is the utilization veloity of the seondary substrate aused by the n + S + S S s ( ) i i Xing-ping LIU Water Siene and Engineering, Sep. 2009, Vol. 2, No. 3,

5 primary substrate, and S + S s is the utilization veloity of the seondary substrate aused by the self-oxidation of miroorganisms during the lag phase. The maximum substrate utilization ratios of the two substrates, and, are different, but the saturation onstants s are the same. Wang and Loh (1999) made use of Pseudomonas putida to degrade hlorophenols. They did a lot of researh using the models desribed above, and onluded that the degradation of phenol was in aordane with Eq. (5) and the degradation of 2-CP was in aordane with Eq. (6): S (5) Xt d ( S ) 2 0 S s + S + Xt d s S S + S + where S is the original onentration of phenol, S is the onentration of 2-CP, is the self-inhibition onstant of 2-CP, and s 0 is the saturation onstant of 2-CP. i 2 i (6) is the maximum substrate utilization ratio of 2-CP, By onsidering the ompetitive mutual inhibition of phenol and 2-CP in the ometaboli degradation and the introdution of the inhibitor into Eq. (5) aording to Eq. (3), we an obtain the degradation kinetis equation of phenol: S (7) Xt d S ( S ) 2 0 S s 1+ + S + p i S where p is the oeffiient of inhibition of phenol by 2-CP, and is the inhibitor. We an also obtain the following degradation speed equation of 2-CP after orretion of Eq. (6): S (8) Xt d 2 S S S s S + p i where is the oeffiient of inhibition of 2-CP by phenol, and is the toxiity p oeffiient of self-inhibition In the ometaboli degradation model, Gu and orus (1995) found that the speifi growth rate of ells μ was in aordane with the following equation: μms0 S0 μ = 2 d0 exp S0 S 0 (9) d S + s 1+ + s i p i 114 Xing-ping LIU Water Siene and Engineering, Sep. 2009, Vol. 2, No. 3,

6 where S0 is the initial onentration of 2-CP, is the endogenous deay oeffiient, and d μ m is the maximum speifi growth rate, d0 is the toxiity oeffiient. In this study, it is assumed that the inhibition oeffiient of phenol to 2-CP and of 2-CP to phenol is the same: p =. p 4 Results and disussion 4.1 Cometaboli degradation of 2-CP and phenol Through the bath experiments on the ometaboli degradation of 2-CP and phenol, the ell growth and substrate degradation were studied and the possible modes of degradation were determined. Based on the seletion of parameters for the kineti model and omparison of the experimental data with the model alulation data, an appropriate dual-substrate ometaboli degradation model was developed with onsideration of the ompetition and self-inhibition effet. Figs. 1(a) and 1(b) are the urves of phenol and 2-CP degradation in experiments B2 and B5, respetively. With the inrease of the onentration of 2-CP, the time needed for omplete degradation of phenol inreased: it was 9.5 hours for experiment D, 13 hours for B2, and about 20 hours for B5. The average degradation rates of phenol were 40 mg/(l h) for experiment D, 28 mg/(l h) for B2, and 23 mg/(l h) for B5. Fig. 1 also indiates that, with the inrease of the onentration of 2-CP, the degradation of phenol was inhibited. High substrate onentration brought about strong substrate inhibition, whih is quantitatively demonstrated by the semi-log graph of ell growth and the substrate degradation urve. Only due to the small hange of 2-CP onentration did the undistinguished hanges our. Figs. 1(a) and 1(b) show that the degradation rate of 2-CP inreased after the phenol was signifiantly degraded, whih indiates that there is a signifiant mutual inhibitory effet between phenol and 2-CP. When the onentration of 2-CP inreased to 50 mg/l, only 35% of 2-CP was degraded, as shown in Fig. 1(b). The reason for the inomplete degradation is that the 2-CP has a toxi effet on the growth of ells, whih auses the weakening of the degradation ability of the miroorganisms. Phenol still played a role in supplying the neessary arbon for the biodegradation to begin, beause of its readier utilization by the miroorganisms. The onentration of phenol also influenes the degradation of 2-CP by the miroorganisms. When the onentrations of 2-CP and phenol were 50 mg/l and 300 mg/l, respetively, the degradation rate of 2-CP was 50%; when the onentrations of 2-CP and phenol were 50 mg/l and 150 mg/l, respetively, the degradation rate of 2-CP was 35%. The reason may be that the inrease of the onentration of phenol provides more growing substrates for the degradation of 2-CP. This doesn t mean, however, that the degradation rate of 2-CP an be endlessly inreased by inreasing the onentration of phenol, beause the toxi effet of phenol on the miroorganisms also inreases with the onentration of phenol, whih auses a derease in the growing speed of the ells and the degradation rate of 2-CP. Xing-ping LIU Water Siene and Engineering, Sep. 2009, Vol. 2, No. 3,

7 Fig. 1 Degradation urves of phenol and 2-CP Table 2 presents the μ m values of the ometaboli degradation experiment of phenol and 2-CP. Fig. 2 shows the effet of 2-CP on phenol degradation when the onentration of phenol was 200 mg/l. As shown in Table 2 and Fig. 2, the maximum speifi growth rate of the ells dereases with the inrease of the onentration of 2-CP. This is beause the toxi effet of 2-CP on the miroorganisms and the inhibitory effet of 2-CP on phenol inrease. Table 2 μm values of ometaboli degradation of phenol and 2-CP Original substrate ontent Original substrate ontent Serial number Phenol 2-CP μ m (h -1 Serial ) number Phenol 2-CP (mg/l) (mg/l) (mg/l) (mg/l) μ m (h -1 ) A B A C A C A C A C B C B D B E B Fig. 2 Effet of onentration of 2-CP on phenol degradation when onentration of phenol is 200 mg/l 4.2 Degradation kinetis of dual-substrate system Equation of ell growth kinetis In the dual-substrate system of the ometaboli degradation of phenol and 2-CP, the growth kinetis of ells are simulated with Eq. (9). Related literature (Annadurai et al. 2002; Chung et al. 2003) provided the parameters for this equation, whih were μ m = 0.9 h -1, s = 116 Xing-ping LIU Water Siene and Engineering, Sep. 2009, Vol. 2, No. 3,

8 6.93 mg/l, and i = 284 mg/l. d0 is equal to 0.002, small enough to be negleted (Haller and Finn 1979). The parameters and are determined by experiments A1 through A5 s d and C1 through C5, and a fitting urve of the degradation of 2-CP. The values used in this 2 study were = 5.62 mg/l and = 17.7 mg/l. The square of the orrelation oeffiient R s d was equal to The data from experiments B1 through B5 were used to validate the parameters. Based on the seleted parameters, Eq. (9) an be simplified as follows: 0.9S0 S0 μ = 0.002exp 2 (10) S0 S S Fig. 3 shows a omparison of measured and simulated speifi growth rates of ells. It an be seen that the experimental data are onsistent with the model alulation results, and the seletion of parameters is appropriate. The value of eah parameter was determined through the Matlab program and urve fitting of the data from experiments A1 through A5 and C1 to C5. These parameters were validated with data from experiments B1 through B5. Fig. 3 Comparison of measured and simulated speifi growth rates of ells Degradation kinetis equation of substrate In the dual-substrate system, the degradation kinetis equation of phenol an be expressed by Eq. (7). Wang and Loh (1999) have onduted systemati researh on the degradation of hlorophenols by Pseudomonas putida, and the parameters they seleted were s = 2.19 mg/l, = mg/(mg h), and i = 810 mg/l, so Eq. (7) an be written as 0.819S (11) Xt d S ( S ) 2 0 S S + p 810 The degradation kinetis equation of 2-CP in the system an be expressed by Eq. (8). The parameters were determined through the Matlab program and urve fitting of two groups of experimental data (B1-B5 and C1-C5). In this study their values were = 4.40 mg/l (±1.08), = mg/(mg h) (±0.0075), s = 2.83 mg/l (±0.53), p = p = 3.35 mg/l (±1.06), and i = 117 mg/l (±10). Substituting these parameter values into Eq. (8) leads to Eq. (12): Xing-ping LIU Water Siene and Engineering, Sep. 2009, Vol. 2, No. 3,

9 0.0325S Xt d 2 S S S S Fig. 4 and Fig. 5 show the onentration-time urves of phenol and 2-CP during the degradation proess of experiments B1 and B4, respetively. (12) Fig. 4 Comparison of experimental and simulated data in experiment B1 Fig. 5 Comparison of experimental and simulated data in experiment B4 The square of the orrelation oeffiient R 2 was The data show that the model suessfully simulates the degradation in the dual-substrate system. 5 Conlusions The following onlusions an be drawn: (1) The experiment produed a quantitative desription of the mutual inhibitory effet and mutual toxi effet between phenol and 2-CP. The results of the model demonstrate that the main interation between phenol and 2-CP is ompetitive inhibition as well as toxi inhibition. (2) The phenol has a ertain inhibitory effet on the degradation of 2-CP in the ometaboli substrates, and this effet grows with the inrease of the onentration of phenol. The 2-CP also has a ertain inhibitory effet on the degradation of phenol, and this effet grows with the inrease of the onentration of 2-CP. Whatever the onentration of phenol is, high or low, its prolonged presene will derease the removal of the 2-CP in the ometaboli system and will also ause a shift of the main arbon soure (that the miroorganisms in the dual-substrate system uses) from 2-CP to phenol. (3) The square of the orrelation oeffiient from the experiment was 0.97, indiating that 118 Xing-ping LIU Water Siene and Engineering, Sep. 2009, Vol. 2, No. 3,

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