Appl Biochem Biotechnol (2015) 176:636–644 DOI 10.1007/s12010-015-1601-7
Biodegradation of Acetochlor by a Newly Isolated Pseudomonas Strain Wei Luo 1 & Qiuya Gu 1 & Wenting Chen 1 & Xiangcheng Zhu 2 & Zhibing Duan 3 & Xiaobin Yu 1
Received: 16 July 2014 / Accepted: 27 March 2015 / Published online: 9 April 2015 # Springer Science+Business Media New York 2015
Abstract A novel microbial strain JD115 capable of degrading acetochlor was isolated from the sludge of acetochlor manufacture and was identified as Pseudomonas aeruginosa species. This strain was able to grow on acetochlor as the sole source of both carbon and nitrogen. The biodegradation of acetochlor by strain JD115 could be described either by the pseudo-firstorder or by the second-order kinetics models, while the latter gave a better performance. The strain optimally degraded acetochlor at a pH value of 7.0 and a temperature of 37 °C. Additional nutriments could greatly enhance the degradation rate of acetochlor up to 95.4 % in the presence of 50 mg acetochlor l−1. The metabolite analyses by GC-MS presumed that catechol was an intermediate product of acetochlor, which was finally degraded for 5 days of incubation. This study highlights the potential use of this strain for the bioremediation of an acetochlor-polluted environment. Keywords Acetochlor . Biodegradation . Isolation . Dynamic modeling . Bioremediation . Pseudomonas aeruginosa Mathematical Notations The substrate concentration at zero time S0 The substrate concentration at time t St
Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1601-7) contains supplementary material, which is available to authorized users.
* Wei Luo [email protected] 1
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, and School of Biotechnology, Jiangnan University, Wuxi 214122, People’s Republic of China
2
Xiangya International Academy of Translational Medicine, Central South University, Changsha 410013, People’s Republic of China
3
Department of Neuroscience & Cell Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA
Appl Biochem Biotechnol (2015) 176:636–644
DT50 k K2, K1, and K0 p h q
637
Half-life of acetochlor The rate constants The coefficients of the second-, first-, and zero-grade terms, respectively The maximum specific growth rate (day−1) The maximum concentration of substrate which can be used to form biomass The concentration of non-biodegradable substrate
Introduction Chloroacetamide herbicide residues are relatively common in watershed and groundwater aquifers in agricultural areas that have a history of herbicide applications. The prevalence of these compounds in the environment has stimulated investigations into the degradation of hazardous substances in water and contaminated soil. The biodegradation of acetochlor is very poor in the natural environment, since only 33 % of acetochlor were degraded after 1 month with a treatment of 10 mg acetochlor kg-1 of soil [1]. The biodegradation of acetochlor usually follows the first-order kinetics [2, 3]. A few microbial communities and pure culture responsible for the biodegradation of acetochlor have been isolated [4, 5]. In the present study, the culture capable of degrading acetochlor was isolated from acetochlor-contaminated soil. The objectives were to (1) identify the microbial strains able to decompose acetochlor, (2) characterize the biodegradation kinetics and determine the degradation rate constants, and (3) investigate the influence of environmental factors and nutriments on the degradation efficiency of acetochlor.
Materials and Methods Chemicals and Media Acetochlor (>99.5 %) was purchased from Sigma-Aldrich (Shanghai, China). Chemicals and solvents used in this study were of analytical grade or HPLC grade and were purchased from Sangon Biotech (Shanghai, China). The Luria-Bertani (LB) medium, pH 7.0, contained yeast extract 5 g l−1, tryptone 10 g l−1, and NaCl 10 g l−1. The mineral salts medium (MSM), pH 7.0, contained NaCl 1 g l−1, K2HPO4 1.5 g l−1, KH2PO4 0.5 g l−1, MgSO4 0.2 g l−1, and 1 ml trace elements solution (39.9 mg MnSO4·H2O, 42.8 mg ZnSO4·H2O, and 34.7 mg (NH4)6Mo7O4·4H2O per liter).
Growth and Degradation Experiments Enrichment and isolation of acetochlor-degrading bacteria was described in supporting information (Supplementary Data). The overnight LB-grown strain JD115 was harvested by centrifuging at 6,000 rpm for 10 min and washed twice with fresh MSM. Then, cells were re-suspended in 100 ml of MSM supplemented with 10 mg acetochlor l−1 in a 250-ml Erlenmeyer flask. The culture was incubated aerobically at 30 °C and 180 rpm in a rotary shaker. The samples were taken to determine the residual acetochlor and the growth of bacteria. Control experiments without strain inoculation were carried out under the same conditions.
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Biodegradation Kinetics Model The biodegradation experimental data were fitted to the pseudo-first-order and second-order equations using Origin 7.5 software (OriginLab, USA). Pseudo-first-order rate constants for the reactions were obtained using the equation as follows [6–8]: ln S t ¼ −kt þ ln S 0
ð1Þ
where S0 is the substrate concentration at zero time, and St is the substrate concentration at time t. The DT50 (days) values were calculated from the rate constants (k) as DT50 ¼
ln 2 k
ð2Þ
The second-order rate constants were obtained according to the Quiroga-Sales model [9]: −
dS ¼ k2S2 þ K1S þ K0 dt
ð3Þ
where S is the substrate concentration; and K2, K1, and K0 were the coefficients of the second-, first-, and zero-grade term, respectively. By integrating Eq. 3, the following equation was obtained: S¼
hðS 0 − qÞ − qðS 0 − hÞept ðS 0 − qÞ−ðS 0 − hÞept
p¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi K 21 −4K 0 K 2
ð4Þ
ð5Þ
q¼
ð−K 1 þ pÞ 2K 2
ð6Þ
h¼
−ðK 1 þ pÞ 2K 2
ð7Þ
where S0 is the substrate concentration at zero time; and p, q, and h are combinations of the coefficients of the second-degree polynomial which defines the substrate consumption rate. Here, p represents the maximum specific growth rate (day−1), h is the maximum concentration of substrate which can be used to form biomass (mg l−1), and q is the concentration of nonbiodegradable substrate (mg l−1). When S=S0/2, the half-life (DT50) can be expressed as ðS 0 −qÞðS 0 −2hÞ ln ðS 0 −hÞðS 0 −2qÞ ð8Þ DT50 ¼ p
Analytical Methods The cell growth was determined by measuring the optical density at 600 nm (OD600) using a US-Vis Spectrophotometer (Unico, Shanghai). The culture sample was centrifuged at
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12,000 rpm for 5 min, and the supernatant was filtered through a 0.22-μm membrane filter, then the concentration of acetochlor was analyzed by HPLC [10], equipped with a TC C18 column (250×4.6 mm, Agilent Technologies, USA). The eluent was methanol/water (80:20, v/v) with a flow rate at 1.0 ml min−1. The wavelength of absorbance detector and injection volume was set at 215 nm and 10 μl, respectively. For analyses of the biodegradation products of acetochlor, the samples were first extracted with equal volume of dichloromethane three times, and then the residual aqueous phase was acidified to pH 2.0 and extracted with an equal volume of ethyl acetate three times. The organic phase was passed through anhydrous Na2SO4 and evaporated to dryness on a rotary vacuum evaporator. The samples were dissolved in methanol for GC-MS analyses, which were performed in the electron ionization (EI) mode (70 eV) with a Varian gas chromatograph system equipped with a quadrupole MS/MS detector [11]. Gas chromatography was conducted using a DB-5 column (30 m×0.25 mm×0.25 μm), and helium was used as a carrier gas (1.6 ml−1 min−1). The MS was operated in the full-scan mode, which scanned from 33 to 450 m/z. The temperature of the program was dictated by the following protocol: hold at 80 °C for 2 min, raise at 10 °C min−1 to 280 °C, and finally hold at 280 °C for 8 min. The temperature of injection port and detector was 250 and 280 °C, respectively.
Statistical Analyses All the biodegradability tests were carried out independently in duplicate or triplicate, and the average values were calculated. The standard deviation for all the values was less than ±5 %.
Results and Discussion Isolation and Identification of Acetochlor-Degrading Strain After enrichment and three generations of growth, one strain designated as JD115 was selected for further study due to its quick growth and high degrading capacity (60.2 %) towards acetochlor. Colonies of JD115 grown on acetochlor-MSM agar were circular, convex, and pale yellow (Supplementary Fig. 1a). Gram staining indicated that it was a Gram-negative and rod-shaped bacterium (Supplementary Fig. 1b). Phylogenetic analysis of the 16S rDNA gene sequence (Supplementary data) revealed that strain JD115 grouped among Pseudomonas species and formed a subclade with Pseudomonas aeruginosa (similarity=100 %) [12]. Based on above data, the strain JD115 was preliminarily identified as P. aeruginosa.
Biodegradation Kinetics of Acetochlor by Strain JD115 As shown in Fig. 1, the residual acetochlor was decreased with the increment of the biomass of strain JD115, while no obvious degradation of acetochlor was observed in the control experiment. The biodegradation of acetochlor continued for 5 days until the cease of microbial growth. The kinetic data of acetochlor biodegradation were then fitted by the pseudo-firstorder and the second-order kinetics models (Table 1). The first-order rate constant and DT50 were comparable with those values reported by others [2]. Although previous studies indicated
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Fig. 1 Time courses of the growth of strain JD115 and biodegradation of acetochlor by JD115. Control represents the degradation of acetochlor in MSM without strain JD115. The culture conditions were shown in BMaterials and Methods^ Section
that the biodegradation of acetochlor conformed to the first-order kinetics [3, 13], the present results show that the acetochlor degradation can be better described by the second-order (Quiroga-Sales) kinetics rather than by the pseudo-first-order model, since the regression coefficients (r2) of the former (0.9824) was much high than that of the latter (0.9297) (Table 1). The obtained value for the parameter h was found to be very close to the initial concentration of acetochlor, which indicated that the kinetic model predicted the biodegradation well under the conditions being studied. The parameter q, representing the concentration of nonbiodegradable substrate, was in accord with the residual amounts of acetochlor in the broth that could not be decomposed by strain JD115. The predicted p value derived from Quiroga-Sales model (Table 1) was also close to the maximum specific growth calculated from the growth curve of JD115. Values K2 and K0 are always negative corresponding to the end of inhibition.
Table 1 Biodegradation kinetics constants of acetochlor by strain JD115
Parameters
Kinetics model Pseudo-first-order model
Quiroga-sales model
k (day−1)
0.1297
K0 (mg l−1 day−1)
/
K1 (day−1)
/
2.16
K2 (l mg−1 day−1)
/
−0.14
/ −6.60
p (day−1)
/
0.98
q (mg l−1)
/
4.20
h (mg l−1) r2
/ 0.9297
DT50 (days)
5.34
11.23 0.9824 6.35
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Moreover, the lower values of K2 in terms of K2S2 imply a correction that includes the lag phase of microorganisms until they adapt to the organic matter presented in the substrate to be degraded [14]. The experimental data of acetochlor degradation have also been fitted to another second-order kinetics model previously proposed for the biodegradation of azo dyes [15]. However, the low correlation coefficient value (r2) indicated that the model predicted poorly in the biodegradation of acetochlor (data not shown).
Effects of Environmental Factors and Additional Nutriments on the Degradation of Acetochlor Biodegradation of acetochlor by strain JD115 was pH- and temperature-dependent, with the optimal pH value of 7.0 and temperature of 37 °C, respectively (data not shown). Furthermore, biodegradation of acetochlor was also affected by the acetochlor concentration (Fig. 2). This strain could effectively degrade acetochlor with the concentration between 10 and 50 mg l−1, and could tolerate the concentration as high as 100 mg l−1 (Fig. 2). The low degradation rate of acetochlor at a high concentration (100 mg l−1) maybe ascribed to its toxicity towards microbial strain. Previous studies have suggested that acetochlor could inhibit the population of soil bacteria and the respiration of soil microbes to varying degrees and affect nutrient cycling processes [13]. On the other hand, deficient acetochlor in the broth also led to the lack of carbon and energy sources, limiting the biomass to degrade acetochlor. So the addition of glucose and yeast extract in the broth greatly improved the degradation rate of acetochlor (Fig. 2). Another report also indicated that additional supplementation of nutriments is a practical way to enhance the microbial degradation of herbicides [6, 16].
Fig. 2 Effects of acetochlor concentration on the degradation rate of acetochlor. Black box represents the MSM supplemented with 5 g glucose l−1 and 1 g yeast extract l−1 in addition to acetochlor; black triangle exhibits the MSM added with acetochlor as the sole source of carbon and nitrogen. Cells were grown in the media (pH 7.0) at 37 °C and 180 rpm for 5 days
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So far, the highest degradation rate (100 %) of acetochlor has been reported by the bacterial communities [4, 17], which may ascribe to the synergistic actions between interspecies. However, the culture and application of microbial consortium for xenobiotics transformation are complicated and difficult. A few pure strains isolated to degrade acetochlor have also been reported in the past decade. For instance, Pseudomonas oleovorans could degrade 98.0 % of acetochlor at a concentration of 7.6 mg l−1 with 7 days of treatment with the degradation efficiency of 1.07 mg l−1 day−1 [18]. Ensifer adhaerens was also able to utilize acetochlor as the sole nitrogen source, while the degradation rate was only 33.6 % in the presence of 10 mg acetochlor l−1 and 10 days’ incubation, with a degradation efficiency of just 0.33 mg l−1 day−1 [19]. They are much lower than the data (degradation rate 95.4 %, degradation efficiency 9.54 mg l−1 day−1, 50 mg acetochlor l−1) obtained in this study. To the best of our knowledge, this is the first investigation of P. aeruginosa on the biodegradation of acetochlor. It has been observed that this strain has the capacity to biodegrade polycyclic aromatic hydrocarbon, phenol, acetaminophen, synthetic polymers, and textile dye; thus, the isolated P. aeruginosa JD115 may be a potential strain to treat various environmental pollutions.
Identification of the Metabolites Produced from Acetochlor The degradation products of acetochlor in the culture medium extracts were identified by GCMS (Fig. 3) based on mass spectral data and fragmentation patterns. The main biodegradation
Fig. 3 GC-MS chromatogram of the extract obtained from the culture at 0 days (a), 3 days (b), and 5 days (c)
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metabolite identified in this experiment was presumed as catechol represented by the peak of 7.44 min in the 3-day sample (Fig. 3b). This peak subsequently disappeared in the 5-day culture (Fig. 3c), suggesting that it was finally degraded. Our results are much similar with previous report, in which catechol was indicated as the degradation products of butachlor [5].
Conclusion This study indicates that the isolated strain JD115 was able to degrade acetochlor whenever it was utilized as the sole source of carbon and nitrogen or co-growth substrate. The degradation kinetics complies with the second-order kinetics (Quiroga-Sales model) to a higher degree, rather than the pseudo-first-order model. This strain was able to degrade 95.4 % of 50 mg acetochlor l−1 with the supplementation of glucose and yeast extract. The metabolite produced from acetochlor was indicated as catechol, which was completely degraded after 5 days of incubation. Acknowledgments This work was supported by the Environmental Protection Project of Jiangsu Province, China, Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (Grant No. KLIB-KF201105), the 111 Project (No. 111-2-06), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
References 1. Jablonkai, I. (2000). Microbial and photolytic degradation of the herbicide acetochlor. International Journal of Environmental Analytical Chemistry, 78, 1–8. 2. Ye, C.-M., Wang, X.-J., & Zheng, H.-H. (2002). Biodegradation of acetanilide herbicides acetochlor and butachlor in soil. Journal of Environmental Sciences, 14, 524–529. 3. Dictor, M.-C., Baran, N., Gautier, A., & Mouvet, C. (2008). Acetochlor mineralization and fate of its two major metabolites in two soils under laboratory conditions. Chemosphere, 71, 663–670. 4. Xu, J., Yang, M., Dai, J., Cao, H., Pan, C., Qiu, X., & Xu, M. (2008). Degradation of acetochlor by four microbial communities. Bioresource Technology, 99, 7797–7802. 5. Zhang, J., Zheng, J.-W., Liang, B., Wang, C.-H., Cai, S., Ni, Y.-Y., He, J., & Li, S.-P. (2011). Biodegradation of chloroacetamide herbicides by Paracoccus sp. FLY-8 in vitro. Journal of Agricultural and Food Chemistry, 59, 4614–4621. 6. Luo, W., Zhao, Y., Ding, H., Lin, X., & Zheng, H. (2008). Co-metabolic degradation of bensulfuron-methyl in laboratory conditions. Journal of Hazardous Materials, 158, 208–214. 7. Teng, F., Liu, Z., Zheng, H., & Wang, P. (2014). Degradation kinetics of anthocyanins from Solanum nigrum L. Berries. Journal of Food Science and Biotechnology, 33, 548–559. 8. Bankar, S. B., Jadhav, S. B., & Singhal, R. S. (2014). Poly-ε-lysine amylase conjugates to increase the stability of enzyme. Food Bioscience, 5, 85–90. 9. Quiroga, J. M., & Sales, D. (1991). Degradation kinetics of surfactants. Tenside, Surfactants, Detergents, 28, 27–30. 10. Rai, A. K., & Appaiah, K. A. (2014). Application of native yeast from Garcinia (Garcinia xanthochumus) for the preparation of fermented beverage: changes in biochemical and antioxidant properties. Food Bioscience, 5, 101–107. 11. Aguirre, A., Borneo, R., & León, A. E. (2013). Antimicrobial, mechanical and barrier properties of triticale protein films incorporated with oregano essential oil. Food Bioscience, 1, 2–9. 12. Wu, Y., Ding, G., Xi, H., & Tian, Y. (2014). Strain with thermo-stable lysine aminopeptidase: isolation, identification and gene cloning. Journal of Food Science and Biotechnology, 33, 821–826. 13. Cai, X., Sheng, G., & Liu, W. (2007). Degradation and detoxification of acetochlor in soils treated by organic and thiosulfate amendments. Chemosphere, 66, 286–292. 14. Chen, X., Yang, X., Yang, L., Xiao, B., Wu, X., Wang, J., & Wan, H. (2010). An effective pathway for the removal of microcystin LR via anoxic biodegradation in lake sediments. Water Research, 44, 1884–1892.
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15. Barragán, B. E., Costa, C., & Carmen Marquez, M. (2007). Biodegradation of azo dyes by bacteria inoculated on solid media. Dyes and Pigments, 75, 73–81. 16. Luo, W., Zhu, X., Chen, W., Duan, Z., Wang, L., & Zhou, Y. (2014). Mechanisms and strategies of microbial cometabolism in the degradation of organic compounds—chlorinated ethylenes as the model. Water Science and Technology, 69, 1971–1983. 17. Li, Y., Chen, Q., Wang, C.-H., Cai, S., He, J., Huang, X., & Li, S.-P. (2013). Degradation of acetochlor by consortium of two bacterial strains and cloning of a novel amidase gene involved in acetochlor-degrading pathway. Bioresource Technology, 1548, 628–631. 18. Xu, J., Qiu, X., Dai, J., Cao, H., Yang, M., Zhang, J., & Xu, M. (2006). Isolation and characterization of a Pseudomonas oleovorans degrading the chloroacetamide herbicide acetochlor. Biodegradation, 17, 219– 225. 19. Dong, B., Wang, F., Lin, A., Yu, C., Guo, L., Xie, W., Feng, L., & Hu, Y. (2011). Isolation and degradation characteristics of acetochlor-degrading strain A-3. Environmental Sciences, 32, 542–547.
Biodegradation of Acetochlor by a Newly Isolated Pseudomonas Strain Wei Luo 1 & Qiuya Gu 1 & Wenting Chen 1 & Xiangcheng Zhu 2 & Zhibing Duan 3 & Xiaobin Yu 1
Received: 16 July 2014 / Accepted: 27 March 2015 / Published online: 9 April 2015 # Springer Science+Business Media New York 2015
Abstract A novel microbial strain JD115 capable of degrading acetochlor was isolated from the sludge of acetochlor manufacture and was identified as Pseudomonas aeruginosa species. This strain was able to grow on acetochlor as the sole source of both carbon and nitrogen. The biodegradation of acetochlor by strain JD115 could be described either by the pseudo-firstorder or by the second-order kinetics models, while the latter gave a better performance. The strain optimally degraded acetochlor at a pH value of 7.0 and a temperature of 37 °C. Additional nutriments could greatly enhance the degradation rate of acetochlor up to 95.4 % in the presence of 50 mg acetochlor l−1. The metabolite analyses by GC-MS presumed that catechol was an intermediate product of acetochlor, which was finally degraded for 5 days of incubation. This study highlights the potential use of this strain for the bioremediation of an acetochlor-polluted environment. Keywords Acetochlor . Biodegradation . Isolation . Dynamic modeling . Bioremediation . Pseudomonas aeruginosa Mathematical Notations The substrate concentration at zero time S0 The substrate concentration at time t St
Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1601-7) contains supplementary material, which is available to authorized users.
* Wei Luo [email protected] 1
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, and School of Biotechnology, Jiangnan University, Wuxi 214122, People’s Republic of China
2
Xiangya International Academy of Translational Medicine, Central South University, Changsha 410013, People’s Republic of China
3
Department of Neuroscience & Cell Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA
Appl Biochem Biotechnol (2015) 176:636–644
DT50 k K2, K1, and K0 p h q
637
Half-life of acetochlor The rate constants The coefficients of the second-, first-, and zero-grade terms, respectively The maximum specific growth rate (day−1) The maximum concentration of substrate which can be used to form biomass The concentration of non-biodegradable substrate
Introduction Chloroacetamide herbicide residues are relatively common in watershed and groundwater aquifers in agricultural areas that have a history of herbicide applications. The prevalence of these compounds in the environment has stimulated investigations into the degradation of hazardous substances in water and contaminated soil. The biodegradation of acetochlor is very poor in the natural environment, since only 33 % of acetochlor were degraded after 1 month with a treatment of 10 mg acetochlor kg-1 of soil [1]. The biodegradation of acetochlor usually follows the first-order kinetics [2, 3]. A few microbial communities and pure culture responsible for the biodegradation of acetochlor have been isolated [4, 5]. In the present study, the culture capable of degrading acetochlor was isolated from acetochlor-contaminated soil. The objectives were to (1) identify the microbial strains able to decompose acetochlor, (2) characterize the biodegradation kinetics and determine the degradation rate constants, and (3) investigate the influence of environmental factors and nutriments on the degradation efficiency of acetochlor.
Materials and Methods Chemicals and Media Acetochlor (>99.5 %) was purchased from Sigma-Aldrich (Shanghai, China). Chemicals and solvents used in this study were of analytical grade or HPLC grade and were purchased from Sangon Biotech (Shanghai, China). The Luria-Bertani (LB) medium, pH 7.0, contained yeast extract 5 g l−1, tryptone 10 g l−1, and NaCl 10 g l−1. The mineral salts medium (MSM), pH 7.0, contained NaCl 1 g l−1, K2HPO4 1.5 g l−1, KH2PO4 0.5 g l−1, MgSO4 0.2 g l−1, and 1 ml trace elements solution (39.9 mg MnSO4·H2O, 42.8 mg ZnSO4·H2O, and 34.7 mg (NH4)6Mo7O4·4H2O per liter).
Growth and Degradation Experiments Enrichment and isolation of acetochlor-degrading bacteria was described in supporting information (Supplementary Data). The overnight LB-grown strain JD115 was harvested by centrifuging at 6,000 rpm for 10 min and washed twice with fresh MSM. Then, cells were re-suspended in 100 ml of MSM supplemented with 10 mg acetochlor l−1 in a 250-ml Erlenmeyer flask. The culture was incubated aerobically at 30 °C and 180 rpm in a rotary shaker. The samples were taken to determine the residual acetochlor and the growth of bacteria. Control experiments without strain inoculation were carried out under the same conditions.
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Biodegradation Kinetics Model The biodegradation experimental data were fitted to the pseudo-first-order and second-order equations using Origin 7.5 software (OriginLab, USA). Pseudo-first-order rate constants for the reactions were obtained using the equation as follows [6–8]: ln S t ¼ −kt þ ln S 0
ð1Þ
where S0 is the substrate concentration at zero time, and St is the substrate concentration at time t. The DT50 (days) values were calculated from the rate constants (k) as DT50 ¼
ln 2 k
ð2Þ
The second-order rate constants were obtained according to the Quiroga-Sales model [9]: −
dS ¼ k2S2 þ K1S þ K0 dt
ð3Þ
where S is the substrate concentration; and K2, K1, and K0 were the coefficients of the second-, first-, and zero-grade term, respectively. By integrating Eq. 3, the following equation was obtained: S¼
hðS 0 − qÞ − qðS 0 − hÞept ðS 0 − qÞ−ðS 0 − hÞept
p¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi K 21 −4K 0 K 2
ð4Þ
ð5Þ
q¼
ð−K 1 þ pÞ 2K 2
ð6Þ
h¼
−ðK 1 þ pÞ 2K 2
ð7Þ
where S0 is the substrate concentration at zero time; and p, q, and h are combinations of the coefficients of the second-degree polynomial which defines the substrate consumption rate. Here, p represents the maximum specific growth rate (day−1), h is the maximum concentration of substrate which can be used to form biomass (mg l−1), and q is the concentration of nonbiodegradable substrate (mg l−1). When S=S0/2, the half-life (DT50) can be expressed as ðS 0 −qÞðS 0 −2hÞ ln ðS 0 −hÞðS 0 −2qÞ ð8Þ DT50 ¼ p
Analytical Methods The cell growth was determined by measuring the optical density at 600 nm (OD600) using a US-Vis Spectrophotometer (Unico, Shanghai). The culture sample was centrifuged at
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12,000 rpm for 5 min, and the supernatant was filtered through a 0.22-μm membrane filter, then the concentration of acetochlor was analyzed by HPLC [10], equipped with a TC C18 column (250×4.6 mm, Agilent Technologies, USA). The eluent was methanol/water (80:20, v/v) with a flow rate at 1.0 ml min−1. The wavelength of absorbance detector and injection volume was set at 215 nm and 10 μl, respectively. For analyses of the biodegradation products of acetochlor, the samples were first extracted with equal volume of dichloromethane three times, and then the residual aqueous phase was acidified to pH 2.0 and extracted with an equal volume of ethyl acetate three times. The organic phase was passed through anhydrous Na2SO4 and evaporated to dryness on a rotary vacuum evaporator. The samples were dissolved in methanol for GC-MS analyses, which were performed in the electron ionization (EI) mode (70 eV) with a Varian gas chromatograph system equipped with a quadrupole MS/MS detector [11]. Gas chromatography was conducted using a DB-5 column (30 m×0.25 mm×0.25 μm), and helium was used as a carrier gas (1.6 ml−1 min−1). The MS was operated in the full-scan mode, which scanned from 33 to 450 m/z. The temperature of the program was dictated by the following protocol: hold at 80 °C for 2 min, raise at 10 °C min−1 to 280 °C, and finally hold at 280 °C for 8 min. The temperature of injection port and detector was 250 and 280 °C, respectively.
Statistical Analyses All the biodegradability tests were carried out independently in duplicate or triplicate, and the average values were calculated. The standard deviation for all the values was less than ±5 %.
Results and Discussion Isolation and Identification of Acetochlor-Degrading Strain After enrichment and three generations of growth, one strain designated as JD115 was selected for further study due to its quick growth and high degrading capacity (60.2 %) towards acetochlor. Colonies of JD115 grown on acetochlor-MSM agar were circular, convex, and pale yellow (Supplementary Fig. 1a). Gram staining indicated that it was a Gram-negative and rod-shaped bacterium (Supplementary Fig. 1b). Phylogenetic analysis of the 16S rDNA gene sequence (Supplementary data) revealed that strain JD115 grouped among Pseudomonas species and formed a subclade with Pseudomonas aeruginosa (similarity=100 %) [12]. Based on above data, the strain JD115 was preliminarily identified as P. aeruginosa.
Biodegradation Kinetics of Acetochlor by Strain JD115 As shown in Fig. 1, the residual acetochlor was decreased with the increment of the biomass of strain JD115, while no obvious degradation of acetochlor was observed in the control experiment. The biodegradation of acetochlor continued for 5 days until the cease of microbial growth. The kinetic data of acetochlor biodegradation were then fitted by the pseudo-firstorder and the second-order kinetics models (Table 1). The first-order rate constant and DT50 were comparable with those values reported by others [2]. Although previous studies indicated
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Fig. 1 Time courses of the growth of strain JD115 and biodegradation of acetochlor by JD115. Control represents the degradation of acetochlor in MSM without strain JD115. The culture conditions were shown in BMaterials and Methods^ Section
that the biodegradation of acetochlor conformed to the first-order kinetics [3, 13], the present results show that the acetochlor degradation can be better described by the second-order (Quiroga-Sales) kinetics rather than by the pseudo-first-order model, since the regression coefficients (r2) of the former (0.9824) was much high than that of the latter (0.9297) (Table 1). The obtained value for the parameter h was found to be very close to the initial concentration of acetochlor, which indicated that the kinetic model predicted the biodegradation well under the conditions being studied. The parameter q, representing the concentration of nonbiodegradable substrate, was in accord with the residual amounts of acetochlor in the broth that could not be decomposed by strain JD115. The predicted p value derived from Quiroga-Sales model (Table 1) was also close to the maximum specific growth calculated from the growth curve of JD115. Values K2 and K0 are always negative corresponding to the end of inhibition.
Table 1 Biodegradation kinetics constants of acetochlor by strain JD115
Parameters
Kinetics model Pseudo-first-order model
Quiroga-sales model
k (day−1)
0.1297
K0 (mg l−1 day−1)
/
K1 (day−1)
/
2.16
K2 (l mg−1 day−1)
/
−0.14
/ −6.60
p (day−1)
/
0.98
q (mg l−1)
/
4.20
h (mg l−1) r2
/ 0.9297
DT50 (days)
5.34
11.23 0.9824 6.35
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Moreover, the lower values of K2 in terms of K2S2 imply a correction that includes the lag phase of microorganisms until they adapt to the organic matter presented in the substrate to be degraded [14]. The experimental data of acetochlor degradation have also been fitted to another second-order kinetics model previously proposed for the biodegradation of azo dyes [15]. However, the low correlation coefficient value (r2) indicated that the model predicted poorly in the biodegradation of acetochlor (data not shown).
Effects of Environmental Factors and Additional Nutriments on the Degradation of Acetochlor Biodegradation of acetochlor by strain JD115 was pH- and temperature-dependent, with the optimal pH value of 7.0 and temperature of 37 °C, respectively (data not shown). Furthermore, biodegradation of acetochlor was also affected by the acetochlor concentration (Fig. 2). This strain could effectively degrade acetochlor with the concentration between 10 and 50 mg l−1, and could tolerate the concentration as high as 100 mg l−1 (Fig. 2). The low degradation rate of acetochlor at a high concentration (100 mg l−1) maybe ascribed to its toxicity towards microbial strain. Previous studies have suggested that acetochlor could inhibit the population of soil bacteria and the respiration of soil microbes to varying degrees and affect nutrient cycling processes [13]. On the other hand, deficient acetochlor in the broth also led to the lack of carbon and energy sources, limiting the biomass to degrade acetochlor. So the addition of glucose and yeast extract in the broth greatly improved the degradation rate of acetochlor (Fig. 2). Another report also indicated that additional supplementation of nutriments is a practical way to enhance the microbial degradation of herbicides [6, 16].
Fig. 2 Effects of acetochlor concentration on the degradation rate of acetochlor. Black box represents the MSM supplemented with 5 g glucose l−1 and 1 g yeast extract l−1 in addition to acetochlor; black triangle exhibits the MSM added with acetochlor as the sole source of carbon and nitrogen. Cells were grown in the media (pH 7.0) at 37 °C and 180 rpm for 5 days
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So far, the highest degradation rate (100 %) of acetochlor has been reported by the bacterial communities [4, 17], which may ascribe to the synergistic actions between interspecies. However, the culture and application of microbial consortium for xenobiotics transformation are complicated and difficult. A few pure strains isolated to degrade acetochlor have also been reported in the past decade. For instance, Pseudomonas oleovorans could degrade 98.0 % of acetochlor at a concentration of 7.6 mg l−1 with 7 days of treatment with the degradation efficiency of 1.07 mg l−1 day−1 [18]. Ensifer adhaerens was also able to utilize acetochlor as the sole nitrogen source, while the degradation rate was only 33.6 % in the presence of 10 mg acetochlor l−1 and 10 days’ incubation, with a degradation efficiency of just 0.33 mg l−1 day−1 [19]. They are much lower than the data (degradation rate 95.4 %, degradation efficiency 9.54 mg l−1 day−1, 50 mg acetochlor l−1) obtained in this study. To the best of our knowledge, this is the first investigation of P. aeruginosa on the biodegradation of acetochlor. It has been observed that this strain has the capacity to biodegrade polycyclic aromatic hydrocarbon, phenol, acetaminophen, synthetic polymers, and textile dye; thus, the isolated P. aeruginosa JD115 may be a potential strain to treat various environmental pollutions.
Identification of the Metabolites Produced from Acetochlor The degradation products of acetochlor in the culture medium extracts were identified by GCMS (Fig. 3) based on mass spectral data and fragmentation patterns. The main biodegradation
Fig. 3 GC-MS chromatogram of the extract obtained from the culture at 0 days (a), 3 days (b), and 5 days (c)
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metabolite identified in this experiment was presumed as catechol represented by the peak of 7.44 min in the 3-day sample (Fig. 3b). This peak subsequently disappeared in the 5-day culture (Fig. 3c), suggesting that it was finally degraded. Our results are much similar with previous report, in which catechol was indicated as the degradation products of butachlor [5].
Conclusion This study indicates that the isolated strain JD115 was able to degrade acetochlor whenever it was utilized as the sole source of carbon and nitrogen or co-growth substrate. The degradation kinetics complies with the second-order kinetics (Quiroga-Sales model) to a higher degree, rather than the pseudo-first-order model. This strain was able to degrade 95.4 % of 50 mg acetochlor l−1 with the supplementation of glucose and yeast extract. The metabolite produced from acetochlor was indicated as catechol, which was completely degraded after 5 days of incubation. Acknowledgments This work was supported by the Environmental Protection Project of Jiangsu Province, China, Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (Grant No. KLIB-KF201105), the 111 Project (No. 111-2-06), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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