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An efficacious degradation of pesticide by salt tolerant Streptomyces venezuelae ACT 1
Bioresource Technology 132 (2013) 378–382
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
An efficacious degradation of pesticide by salt tolerant Streptomyces venezuelae ACT 1 Balakrishnan Naveena, Gurusamy Annalakshmi, Nagarajan Partha ⇑ Department of Chemical Engineering, A.C. College of Technology, Anna University Chennai, Chennai 600 025, India
h i g h l i g h t s " Marine isolate, Streptomyces venezuelae ACT1 was used for the degradation. " Organophophorus hydrolase activity rate was evaluated using modified Gompertz model. " Substrate assimilation and inhibition were analyzed for the growth and the enzyme activity. " Influence of P0/X0 ratio on degradation as well as COD reduction rate was demonstrated.
a r t i c l e
i n f o
Article history: Received 30 July 2012 Received in revised form 2 November 2012 Accepted 2 November 2012 Available online 15 November 2012 Keywords: Organophosphorus pesticide P0/X0 ratio Substrate assimilation Substrate inhibition Streptomyces venezuelae ACT1
a b s t r a c t Degradation of the organophosphorus pesticide has been studied using the marine isolate, Streptomyces venezuelae ACT1. The organism exhibited a specific growth rate of 0.371 h1 and the organophosphorus hydrolase activity rate as 0.273 h1. Hence the organism was found to be very effective towards the pesticide degradation. Further the substrate assimilation and inhibition model of the organism were demonstrated using Monod and Haldane model equations which depicted that the inhibition model fits well for both the cell growth and enzyme activity. The maximum specific growth rate and the enzyme activity rate were found to be 0.571 h1 and 0.472 h1, respectively. Effect of P0/X0 ratio on degradation and COD reduction rate revealed that higher these ratios raise the degradation rate and the COD reduction rate. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Organophosphorus pesticides have been used extensively all over the world for agricultural purposes. Currently, their use is being phased out because of their toxicity, environmental persistence and accumulation in the food chain. Several degradation processes such as photocatalytic degradation, advanced oxidative processes, bioremediation, ozonation and photo- Fenton reactions have been proposed which are expensive and produce secondary pollution (Sassman et al., 2004). Therefore, biological processes have been explored and however, little information is available on the ability of organophosphorus pesticide biotransformation by Gram-positive microorganisms and particularly by actinomycete species. Pesticide degradation kinetics is essential for the better design of industrial level processes besides the improvement of the process control of pesticide wastewater treatment. It has been extrapolated that during batch fermentation, S0/X0 ratio influences the change in composition of microbial community ⇑ Corresponding author. Mobile: +91 9790445938. E-mail address: [email protected] (N. Partha). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.019
i.e., the larger the S0/X0 ratio, the greater the change in the structure of microbial community (Chudoba et al., 1992). Through this ratiocination, it has been suspected that as the biodegradation is associated with the enzyme production, the ratio of initial pesticide concentration (P0) to initial biomass concentration (X0) also influences the degradation rate. Hence, in this research, attempts were made to evaluate and the effect of P0/X0 ratio on degradation and Chemical oxygen demand (COD) reduction rate Streptomyces venezuelae ACT1. Furthermore, in order to minimize the pollution caused by the effluents generated by the pesticide manufacturing companies, the present study also aims to evaluate the relationship between the concentration of pesticide and specific growth rate, in order to effectively explain the biodegradation of pesticide. 2. Methods 2.1. Pesticide, microbial strain and medium for degradation The organophosphorus pesticide, Parathion ((O,O-diethyl-Op-nitrophenyl phosphorothioate) was purchased from the local market. Salt tolerant S. venezuelae ACT1 was isolated from marine
379
B. Naveena et al. / Bioresource Technology 132 (2013) 378–382
water sample (Naveena et al., 2012a). The gene sequence obtained through 16srRNA analysis was published in National centre for Biotechnology information (NCBI) (GenBank ID: JQ039359). Stock cultures were maintained in the medium containing (g/L) glucose, 4; yeast extract, 4; malt extract, 10; calcium carbonate, 2; agar, 12, pH 7. The starch casein broth (SCB) comprising (g/L), (component g/L) starch, 10; K2HPO4, 2; KNO3, 2; NaCl, 2; casein, 0.3; MgSO4 7H2O, 0.05; CaCO3, 0.02; FeSO47H2O, 0.01; pH 7, was used for the degradation studies. Experiments were done as triplicates in pH 7 and incubation temperature, 35 °C with rotary shaking at 150 rpm and their average were taken into consideration.
ln
X Xo
¼ ln
2
0
13
X max el exp 4 exp @ m ðk tÞ þ 1A5 Xo ln X max
ð1Þ
Xo
eE ðk tÞ þ 1 P ¼ A exp exp A
ð2Þ
where X0, Xmax, X, t, lm, k, P, A and E denote initial biomass concentration (mg/mL), maximum biomass concentration (mg/mL) and biomass concentration (mg/mL), incubation time (h), specific growth rate (h1), lag time (h), Enzyme activity (U/mL), Enzyme production potential (U/mL) and specific enzyme activity rate (h1), respectively.
2.2. Evaluation of growth and degradation rate 2.3. Evaluation of substrate utilization and inhibition kinetics 0.1 mL of the pure culture was inoculated in the sterile SCB supplemented with 100 mg/L of pesticide and incubated in the above mentioned experimental conditions. Samples were collected at every 3 h intervals. The biomass was calculated from the pellet weight and the specific growth rate was evaluated by modified Gompertz equation (1). The supernatant was used for organophosphorus hydrolase assay as proposed by Shimazu et al. (2001) at 410 nm using UV–Vis spectrophotometer. The degradation analysis was done at 590 nm and the rate was calculated according to Gopinath et al. (2011). Enzyme activity rate was calculated using the following modified Gompertz equation (2) (Naveena et al., 2012b).
The influence of initial pesticide concentration on cell growth and organophophorus hydrolase activity was analyzed by varying the pesticide concentration in the range of 100–500 mg/L using Monod model, Eqs. (3) and (4), respectively. Further the substrate inhibition was analyzed through the Haldane’s Eqs. (5) and (6).
l¼ E¼
lmax ½P K s þ ½P Emax ½P K s þ ½P
Fig. 1. Growth (A) and organophosphorus activity (B) kinetics of Streptomyces venezuelae ACT1 in the medium containing organophoshorus pesticide.
ð3Þ
ð4Þ
380
l¼
E¼
B. Naveena et al. / Bioresource Technology 132 (2013) 378–382
lmax ½P
ð5Þ
2
K s þ ½P þ ½P KI Emax ½P K s þ ½P þ
ð6Þ
½P2 KI
where [P] is the initial concentration of pesticide (mg/L), l is cific growth rate (h1), lmax is maximum specific growth (h1), E is the specific enzyme activity rate, Emax is maximum cific enzyme activity rate, Ks is half velocity constant (mg/L), substrate inhibition constant (mg/L).
sperate speKI is
C¼
C max ½P0 =X 0 K m=X þ ½P0 =X 0
ð8Þ
where [P0] is the initial concentration of pesticide (mg/L), [X0] is the initial concentration of biomass (mg/L), V is specific degradation rate (mg/L h), Vmax is maximum specific degradation rate (mg/L h), C is the COD reduction rate (% h1), Cmax is the maximum COD reduction rate (% h1), Km/X is substrate affinity constant (mg/mL). 3. Results and discussion 3.1. Effect of time on cell growth and enzyme activity
2.4. Effect of P0/X0 ratio on degradation and COD reduction kinetics The influence of various P0/X0 ratios on degradation of pesticide and COD reduction by S. venezuelae ACT1 was analyzed by varying P0 at constant X0 and by varying X0 at constant P0 using the following Eqs. (7) and (8), respectively. The initial pesticide concentration (P0) was varied from 100–500 mg/L and the initial biomass concentration (X0) was varied from 0.1 to 1 mg/mL. Aliquots were withdrawn at regular time intervals (every 3 h) and subjected to chemical oxygen demand (COD) analysis by open reflux method (APHA et al., 1995). The COD removal percentage was calculated from the data obtained.
V¼
V max ½P0 =X 0 K m=X þ ½P0 =X 0
ð7Þ
Cell growth of S. venezuelae was analyzed in the medium containing pesticide and the corresponding graph obtained is shown in Fig. 1A. Organophosphorus hydrolase activity was analyzed for every 3 h intervals during the biodegradation and the corresponding plot obtained is shown in Fig. 1B. The kinetic parameters such as specific growth rate, specific enzyme activity rate and lag time obtained for the analyses are 0.371 h1, 0.273 h1, 2.838 h (k) and 9.544 h (k), respectively. The organism showed better specific growth rate and the lag time was found to be low. This shows that the growth was highly exponential and hence suitable for biodegradation in short time. Likewise, the maximum enzyme activity was observed on 76 h, beyond that the enzyme activity was found to decrease. This is because of the increase in cell growth and hence it was inferred that the enzyme production was growth associated. The organophosphorus compound, p-nitrophenol was
Fig. 2. Substrate utilization and inhibition kinetics on cell growth (A) and enzyme activity (B).
381
B. Naveena et al. / Bioresource Technology 132 (2013) 378–382
The inhibition of cell growth by higher concentration of substrate was found to diminish the enzyme activity, simultaneously which could foster the reduction of degradation rate. Thus it was ascertained that a fall in specific growth rate due to substrate inhibition, lessens the enzyme activity rate as well as the degradation. It can also be resolved that at high concentration of the pesticide, the substrate molecules get accumulated and are competing for the active site of the enzyme organophosphorus hydrolase which results in decrease in specific growth rate and enzyme activity rate as described by Isik and Sponza (2005). Hence, these consequences may tend to decrease the pesticide degradation rate. The inhibition of an enzyme owing to substrate concentration conduce the nonmonotonicity of reaction rate expression with respect to the substrate concentration as described by Lee and Lim (1999).
the intermittent formed during parathion degradation metabolism which get further reduced to form nitrite as end product and this was confirmed by the assay reported by Siddaramappa et al. (1973). 3.2. Determination of substrate assimilation and inhibition Substrate assimilation and inhibition kinetics for the cell growth of S. venezuelae and organophophorus hydrolase activity were investigated and the plots obtained are shown in Fig. 2A and B, respectively. The correlation coefficients and the kinetic parameters obtained for both the models are given in Table 1. These investigations show that the specific growth rate and the enzyme activity rate increases with the initial concentration of pesticide until a maximum specific growth rate of 0.533 h1 was reached. As the Monod model is a non-inhibitory model, it fits well with lower concentration whereas at higher concentrations due to inhibition best fit was not observed. Hence Haldane’s model was tested which includes the inhibition effects and was found to offer best fit at higher concentration compared to lower concentrations.
3.3. Effect of P0/X0 ratio on COD and degradation rate The effect of P0/X0 ratio on degradation rate and COD reduction rate are exemplified in Figs. 3 and 4, respectively. The kinetic parameters obtained for these analyses are given in Table 1. The
Table 1 Kinetic parameters obtained for various kinetc analyses. S. no
Analyses
lmax
Emax
Ks
KI
Vmax
Ks/x
Cmax
Ks/x
R2
1 2 3 4 5 6
Monod analysis for cell growth Monod analysis for enzyme activity Haldane analysis for cell growth Haldane analysis for enzyme activity P0/X0 at constant X0 Constant P0
0.533 – 0.571 – – –
– 0.441 – 0.472 – –
62.54 91.01 181.5 192 – –
– – 766.6 685.6 – –
– – – – 19.34 6.965
– – – – 3346 407.8
– – – – 90.06 15.92
– – – – 3489 381.7
0.902 0.912 0.979 0.980 0.982 0.964
Fig. 3. Effect of P0/X0 ratio on specific degradation rate at constant X0 (A) and constant P0 (B).
382
B. Naveena et al. / Bioresource Technology 132 (2013) 378–382
Fig. 4. Effect of P0/X0 ratio on COD reduction rate at constant X0 (A) and constant P0 (B).
COD reduction profile was analogous to the trend of pesticide degradation. Figs. 3A and 4A extrapolates that increase in P0/X0 ratio enhances the degradation rate as well as the COD reduction rate, respectively, at constant X0. This can be explicated through the population-density-dependent growth (Wang et al., 2007). At a constant X0, a higher P0/X0 ratio signifies that biomass was rendered with a higher amount of substrate. Thenceforth, the initial level was higher, and consequently there was sufficient substrate for organophosphorus hydrolase activity during cell replication cycle. At an invariable initial pesticide concentration (P0), the degradation rate (Fig. 3B) and the COD reduction rate (Fig. 4B) were diminished with increase in initial biomass concentration, X0. Furthermore, Simkins and Alexander (1984) revealed that the initial cell density is greater than the number of new microorganisms; the microbial growth during the course of an experiment becomes insignificant on a proportional basis.
4. Conclusion This investigation concludes that S. venezuelae ACT1 as a potent source for parathion degradation. The specific growth rate and the enzyme activity rate were increased with the increase in initial concentration of pesticide up to a certain level, beyond that inhibition occurred. The degradation rate and the COD reduction rate were influenced by the P0/X0 ratio and conduces that increase in this ratio increases the degradation as well as COD reduction rate.
References APHA, AWWA, WEF, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. APHA, AWWA, WEF, Washington, DC. Chudoba, P., Capdeville, B., Chudoba, J., 1992. Explanation of biological meaning of the S0/X0 ratio in batch culture. Water Sci. Technol. 26, 743–751. Gopinath, K.P., Kathiravan, M.N., Srinivasan, R., Sankaranarayanan, S., 2011. Evaluation and elimination of inhibitory effects of salts and heavy metal ions on biodegradation of Congo red by Pseudomonas sp. mutant. Bioresour. Technol. 102, 3687–3693. Isik, M., Sponza, D.T., 2005. A batch study for assessing the inhibition effect of direct yellow 12 in a mixed methanogenic culture. Process Biochem. 40, 1053–1062. Lee, J.H., Lim, H.C., 1999. Multiplicity and stability analysis of continuous prefermentation of cheese culture. Process Biochem. 34, 467–475. Naveena, B., Sakthiselvan, P., Elaiyaraju, P., Partha, N., 2012a. Ultrasound induced production of thrombinase by marine actinomycetes: kinetic and optimization studies. Biochem. Eng. J. 61, 34–42. Naveena, B., Gopinath, K.P., Sakthiselvan, P., Partha, N., 2012b. Enhanced production of thrombinase by Streptomyces venezuelae: kinetic studies on growth and enzyme production of mutant strain. Bioresour. Technol. 111, 417–424. Sassman, S.A., Lee, L.S., Bischoff, M., Turco, R.F., 2004. Assessing N, N’-dibutylurea (DBU) formation in soils after application of n-butylisocyanate and benlate fungicides. J. Agric. Food Chem. 52, 747–754. Shimazu, M., Mulchandani, A., Chen, W., 2001. Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase. Biotechnol. Bioeng. 76, 318–324. Siddaramappa, R., Rajaram, K.P., Sethunathan, N., 1973. Degradation of Parathion by bacteria isolated from flooded soil. Appl. Microbiol. 26, 846–849. Simkins, S., Alexander, M., 1984. Models for mineralization kinetics with variables of substrate concentration and population density. Appl. Environ. Microbiol. 47, 1299–1306. Wang, J., Fang, F., Yu, H.Q., 2007. Substrate consumption and biomass growth of Ralstonia eutropha at various S0/X0 levels in batch cultures. Bioresour. Technol. 98, 2599–2604.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
An efficacious degradation of pesticide by salt tolerant Streptomyces venezuelae ACT 1 Balakrishnan Naveena, Gurusamy Annalakshmi, Nagarajan Partha ⇑ Department of Chemical Engineering, A.C. College of Technology, Anna University Chennai, Chennai 600 025, India
h i g h l i g h t s " Marine isolate, Streptomyces venezuelae ACT1 was used for the degradation. " Organophophorus hydrolase activity rate was evaluated using modified Gompertz model. " Substrate assimilation and inhibition were analyzed for the growth and the enzyme activity. " Influence of P0/X0 ratio on degradation as well as COD reduction rate was demonstrated.
a r t i c l e
i n f o
Article history: Received 30 July 2012 Received in revised form 2 November 2012 Accepted 2 November 2012 Available online 15 November 2012 Keywords: Organophosphorus pesticide P0/X0 ratio Substrate assimilation Substrate inhibition Streptomyces venezuelae ACT1
a b s t r a c t Degradation of the organophosphorus pesticide has been studied using the marine isolate, Streptomyces venezuelae ACT1. The organism exhibited a specific growth rate of 0.371 h1 and the organophosphorus hydrolase activity rate as 0.273 h1. Hence the organism was found to be very effective towards the pesticide degradation. Further the substrate assimilation and inhibition model of the organism were demonstrated using Monod and Haldane model equations which depicted that the inhibition model fits well for both the cell growth and enzyme activity. The maximum specific growth rate and the enzyme activity rate were found to be 0.571 h1 and 0.472 h1, respectively. Effect of P0/X0 ratio on degradation and COD reduction rate revealed that higher these ratios raise the degradation rate and the COD reduction rate. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Organophosphorus pesticides have been used extensively all over the world for agricultural purposes. Currently, their use is being phased out because of their toxicity, environmental persistence and accumulation in the food chain. Several degradation processes such as photocatalytic degradation, advanced oxidative processes, bioremediation, ozonation and photo- Fenton reactions have been proposed which are expensive and produce secondary pollution (Sassman et al., 2004). Therefore, biological processes have been explored and however, little information is available on the ability of organophosphorus pesticide biotransformation by Gram-positive microorganisms and particularly by actinomycete species. Pesticide degradation kinetics is essential for the better design of industrial level processes besides the improvement of the process control of pesticide wastewater treatment. It has been extrapolated that during batch fermentation, S0/X0 ratio influences the change in composition of microbial community ⇑ Corresponding author. Mobile: +91 9790445938. E-mail address: [email protected] (N. Partha). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.019
i.e., the larger the S0/X0 ratio, the greater the change in the structure of microbial community (Chudoba et al., 1992). Through this ratiocination, it has been suspected that as the biodegradation is associated with the enzyme production, the ratio of initial pesticide concentration (P0) to initial biomass concentration (X0) also influences the degradation rate. Hence, in this research, attempts were made to evaluate and the effect of P0/X0 ratio on degradation and Chemical oxygen demand (COD) reduction rate Streptomyces venezuelae ACT1. Furthermore, in order to minimize the pollution caused by the effluents generated by the pesticide manufacturing companies, the present study also aims to evaluate the relationship between the concentration of pesticide and specific growth rate, in order to effectively explain the biodegradation of pesticide. 2. Methods 2.1. Pesticide, microbial strain and medium for degradation The organophosphorus pesticide, Parathion ((O,O-diethyl-Op-nitrophenyl phosphorothioate) was purchased from the local market. Salt tolerant S. venezuelae ACT1 was isolated from marine
379
B. Naveena et al. / Bioresource Technology 132 (2013) 378–382
water sample (Naveena et al., 2012a). The gene sequence obtained through 16srRNA analysis was published in National centre for Biotechnology information (NCBI) (GenBank ID: JQ039359). Stock cultures were maintained in the medium containing (g/L) glucose, 4; yeast extract, 4; malt extract, 10; calcium carbonate, 2; agar, 12, pH 7. The starch casein broth (SCB) comprising (g/L), (component g/L) starch, 10; K2HPO4, 2; KNO3, 2; NaCl, 2; casein, 0.3; MgSO4 7H2O, 0.05; CaCO3, 0.02; FeSO47H2O, 0.01; pH 7, was used for the degradation studies. Experiments were done as triplicates in pH 7 and incubation temperature, 35 °C with rotary shaking at 150 rpm and their average were taken into consideration.
ln
X Xo
¼ ln
2
0
13
X max el exp 4 exp @ m ðk tÞ þ 1A5 Xo ln X max
ð1Þ
Xo
eE ðk tÞ þ 1 P ¼ A exp exp A
ð2Þ
where X0, Xmax, X, t, lm, k, P, A and E denote initial biomass concentration (mg/mL), maximum biomass concentration (mg/mL) and biomass concentration (mg/mL), incubation time (h), specific growth rate (h1), lag time (h), Enzyme activity (U/mL), Enzyme production potential (U/mL) and specific enzyme activity rate (h1), respectively.
2.2. Evaluation of growth and degradation rate 2.3. Evaluation of substrate utilization and inhibition kinetics 0.1 mL of the pure culture was inoculated in the sterile SCB supplemented with 100 mg/L of pesticide and incubated in the above mentioned experimental conditions. Samples were collected at every 3 h intervals. The biomass was calculated from the pellet weight and the specific growth rate was evaluated by modified Gompertz equation (1). The supernatant was used for organophosphorus hydrolase assay as proposed by Shimazu et al. (2001) at 410 nm using UV–Vis spectrophotometer. The degradation analysis was done at 590 nm and the rate was calculated according to Gopinath et al. (2011). Enzyme activity rate was calculated using the following modified Gompertz equation (2) (Naveena et al., 2012b).
The influence of initial pesticide concentration on cell growth and organophophorus hydrolase activity was analyzed by varying the pesticide concentration in the range of 100–500 mg/L using Monod model, Eqs. (3) and (4), respectively. Further the substrate inhibition was analyzed through the Haldane’s Eqs. (5) and (6).
l¼ E¼
lmax ½P K s þ ½P Emax ½P K s þ ½P
Fig. 1. Growth (A) and organophosphorus activity (B) kinetics of Streptomyces venezuelae ACT1 in the medium containing organophoshorus pesticide.
ð3Þ
ð4Þ
380
l¼
E¼
B. Naveena et al. / Bioresource Technology 132 (2013) 378–382
lmax ½P
ð5Þ
2
K s þ ½P þ ½P KI Emax ½P K s þ ½P þ
ð6Þ
½P2 KI
where [P] is the initial concentration of pesticide (mg/L), l is cific growth rate (h1), lmax is maximum specific growth (h1), E is the specific enzyme activity rate, Emax is maximum cific enzyme activity rate, Ks is half velocity constant (mg/L), substrate inhibition constant (mg/L).
sperate speKI is
C¼
C max ½P0 =X 0 K m=X þ ½P0 =X 0
ð8Þ
where [P0] is the initial concentration of pesticide (mg/L), [X0] is the initial concentration of biomass (mg/L), V is specific degradation rate (mg/L h), Vmax is maximum specific degradation rate (mg/L h), C is the COD reduction rate (% h1), Cmax is the maximum COD reduction rate (% h1), Km/X is substrate affinity constant (mg/mL). 3. Results and discussion 3.1. Effect of time on cell growth and enzyme activity
2.4. Effect of P0/X0 ratio on degradation and COD reduction kinetics The influence of various P0/X0 ratios on degradation of pesticide and COD reduction by S. venezuelae ACT1 was analyzed by varying P0 at constant X0 and by varying X0 at constant P0 using the following Eqs. (7) and (8), respectively. The initial pesticide concentration (P0) was varied from 100–500 mg/L and the initial biomass concentration (X0) was varied from 0.1 to 1 mg/mL. Aliquots were withdrawn at regular time intervals (every 3 h) and subjected to chemical oxygen demand (COD) analysis by open reflux method (APHA et al., 1995). The COD removal percentage was calculated from the data obtained.
V¼
V max ½P0 =X 0 K m=X þ ½P0 =X 0
ð7Þ
Cell growth of S. venezuelae was analyzed in the medium containing pesticide and the corresponding graph obtained is shown in Fig. 1A. Organophosphorus hydrolase activity was analyzed for every 3 h intervals during the biodegradation and the corresponding plot obtained is shown in Fig. 1B. The kinetic parameters such as specific growth rate, specific enzyme activity rate and lag time obtained for the analyses are 0.371 h1, 0.273 h1, 2.838 h (k) and 9.544 h (k), respectively. The organism showed better specific growth rate and the lag time was found to be low. This shows that the growth was highly exponential and hence suitable for biodegradation in short time. Likewise, the maximum enzyme activity was observed on 76 h, beyond that the enzyme activity was found to decrease. This is because of the increase in cell growth and hence it was inferred that the enzyme production was growth associated. The organophosphorus compound, p-nitrophenol was
Fig. 2. Substrate utilization and inhibition kinetics on cell growth (A) and enzyme activity (B).
381
B. Naveena et al. / Bioresource Technology 132 (2013) 378–382
The inhibition of cell growth by higher concentration of substrate was found to diminish the enzyme activity, simultaneously which could foster the reduction of degradation rate. Thus it was ascertained that a fall in specific growth rate due to substrate inhibition, lessens the enzyme activity rate as well as the degradation. It can also be resolved that at high concentration of the pesticide, the substrate molecules get accumulated and are competing for the active site of the enzyme organophosphorus hydrolase which results in decrease in specific growth rate and enzyme activity rate as described by Isik and Sponza (2005). Hence, these consequences may tend to decrease the pesticide degradation rate. The inhibition of an enzyme owing to substrate concentration conduce the nonmonotonicity of reaction rate expression with respect to the substrate concentration as described by Lee and Lim (1999).
the intermittent formed during parathion degradation metabolism which get further reduced to form nitrite as end product and this was confirmed by the assay reported by Siddaramappa et al. (1973). 3.2. Determination of substrate assimilation and inhibition Substrate assimilation and inhibition kinetics for the cell growth of S. venezuelae and organophophorus hydrolase activity were investigated and the plots obtained are shown in Fig. 2A and B, respectively. The correlation coefficients and the kinetic parameters obtained for both the models are given in Table 1. These investigations show that the specific growth rate and the enzyme activity rate increases with the initial concentration of pesticide until a maximum specific growth rate of 0.533 h1 was reached. As the Monod model is a non-inhibitory model, it fits well with lower concentration whereas at higher concentrations due to inhibition best fit was not observed. Hence Haldane’s model was tested which includes the inhibition effects and was found to offer best fit at higher concentration compared to lower concentrations.
3.3. Effect of P0/X0 ratio on COD and degradation rate The effect of P0/X0 ratio on degradation rate and COD reduction rate are exemplified in Figs. 3 and 4, respectively. The kinetic parameters obtained for these analyses are given in Table 1. The
Table 1 Kinetic parameters obtained for various kinetc analyses. S. no
Analyses
lmax
Emax
Ks
KI
Vmax
Ks/x
Cmax
Ks/x
R2
1 2 3 4 5 6
Monod analysis for cell growth Monod analysis for enzyme activity Haldane analysis for cell growth Haldane analysis for enzyme activity P0/X0 at constant X0 Constant P0
0.533 – 0.571 – – –
– 0.441 – 0.472 – –
62.54 91.01 181.5 192 – –
– – 766.6 685.6 – –
– – – – 19.34 6.965
– – – – 3346 407.8
– – – – 90.06 15.92
– – – – 3489 381.7
0.902 0.912 0.979 0.980 0.982 0.964
Fig. 3. Effect of P0/X0 ratio on specific degradation rate at constant X0 (A) and constant P0 (B).
382
B. Naveena et al. / Bioresource Technology 132 (2013) 378–382
Fig. 4. Effect of P0/X0 ratio on COD reduction rate at constant X0 (A) and constant P0 (B).
COD reduction profile was analogous to the trend of pesticide degradation. Figs. 3A and 4A extrapolates that increase in P0/X0 ratio enhances the degradation rate as well as the COD reduction rate, respectively, at constant X0. This can be explicated through the population-density-dependent growth (Wang et al., 2007). At a constant X0, a higher P0/X0 ratio signifies that biomass was rendered with a higher amount of substrate. Thenceforth, the initial level was higher, and consequently there was sufficient substrate for organophosphorus hydrolase activity during cell replication cycle. At an invariable initial pesticide concentration (P0), the degradation rate (Fig. 3B) and the COD reduction rate (Fig. 4B) were diminished with increase in initial biomass concentration, X0. Furthermore, Simkins and Alexander (1984) revealed that the initial cell density is greater than the number of new microorganisms; the microbial growth during the course of an experiment becomes insignificant on a proportional basis.
4. Conclusion This investigation concludes that S. venezuelae ACT1 as a potent source for parathion degradation. The specific growth rate and the enzyme activity rate were increased with the increase in initial concentration of pesticide up to a certain level, beyond that inhibition occurred. The degradation rate and the COD reduction rate were influenced by the P0/X0 ratio and conduces that increase in this ratio increases the degradation as well as COD reduction rate.
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