Bacterial assisted degradation of chlorpyrifos: The key role of environmental conditions, trace metals and organic solvents

Bacterial assisted degradation of chlorpyrifos: The key role of environmental conditions, trace metals and organic solvents

Journal of Environmental Management 168 (2016) 1e9 Contents lists available at ScienceDirect Journal of Environmental Management journal homepage: w...

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Journal of Environmental Management 168 (2016) 1e9

Contents lists available at ScienceDirect

Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman

Research article

Bacterial assisted degradation of chlorpyrifos: The key role of environmental conditions, trace metals and organic solvents Saira Khalid*, Imran Hashmi, Sher Jamal Khan Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering, National University of Sciences and Technology, Islamabad 44000, Pakistan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 September 2015 Received in revised form 10 November 2015 Accepted 13 November 2015 Available online xxx

Wastewater from pesticide industries, agricultural or surface runoff containing pesticides and their residues has adverse environmental impacts. Present study demonstrates effect of petrochemicals and trace metals on chlorpyrifos (CP) biotransformation often released in wastewater of agrochemical industry. Biodegradation was investigated using bacterial strain Pseudomonas kilonensis SRK1 isolated from wastewater spiked with CP. Optimal environmental conditions for CP removal were CFU (306  106), pH (8); initial CP concentration (150 mg/L) and glucose as additional carbon source. Among various organic solvents (petrochemicals) used in this study toluene has stimulatory effect on CP degradation process using SRK1, contrary to this benzene and phenol negatively inhibited degradation process. Application of metal ions (Cu (II), Fe (II) Zn (II) at low concentration (1 mg/L) took part in biochemical reaction and positively stimulated CP degradation process. Metal ions at high concentrations have inhibitory effect on degradation process. A first order growth model was shown to fit the data. It could be concluded that both type and concentration of metal ions and petrochemicals can affect CP degradation process. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Chlorpyrifos Trace metals Wastewater Petrochemicals Biodegradation

1. Introduction Chlorpyrifos (O,O-Diethyl O-3,5,6-trichloropyridine-2-yl phosphorothioate) an organophosphate (OPP) insecticide which is in use for more than sixty years for controlling crop pests. Use of OPPs has played an important role in a remarkable increase in agricultural productivity. CP in introduced into the environment through agricultural and industrial runoff. CP and its metabolites are detected in environmental samples globally (Chishti et al., 2013). CP poisoning can causes nausea, abnormal functioning of nervous system, sudden irregular movement of the body, paralysis and may cause death of insects, humans and other mammals (Yadav et al., 2014); hence its removal from the contaminated environment has attained immense attention. Pesticide industries in Pakistan release large volume of wastewater into nearby water bodies without sufficient treatment. This wastewater contains pesticides, their residues, organic solvents and many other harmful chemicals. In order to avoid environmental

* Corresponding author. Institute of Environmental Sciences and Engineering (IESE), School of Civil and Environmental Engineering (SCEE), National University of Sciences and Technology (NUST), H-12, Islamabad 44000, Pakistan. E-mail addresses: [email protected], [email protected] (S. Khalid). http://dx.doi.org/10.1016/j.jenvman.2015.11.030 0301-4797/© 2015 Elsevier Ltd. All rights reserved.

contamination, this wastewater should be treated. Microbial biotreatment is an important pathway for elimination of toxic organic compounds such as pesticides (Chishti et al., 2013). Bacterial species having organophosphorous hydrolase enzyme can use OPPs as a source of carbon and energy (Singh and Walker, 2006). Several studies have been carried out for removal of OPPs from wastewater using bacterial strains. Recently we reported biodegradation of organophosphate pesticide chlorpyrifos using Psychrobacter alimentarius (Khalid and Hashmi, 2015). Wastewater from pesticide industries contains variety of organic and inorganic compounds such as trace metals and petrochemicals (organic solvents). Research on pesticides mostly focuses on organic dimensions but there is a dearth in literature regarding inorganic dimensions. Inorganics such as metal ions are present in commercial formulations but unfortunately these are not reflected in theoretical structures and labels. Pesticides with trace metals in their chemical structures have been detected in groundwater samples globally (Kolpin et al., 2000a, b). Presence of trace metals may affect biodegradation reaction as they take part in biochemical reactions, effecting bioavailability (Shomar, 2006, Mahmood et al., 2015). Organic compounds such as benzene, toluene, phenols also called petrochemicals are used as solvents for making liquid pesticide formulations. Whether or not these organic solvents take

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part in metabolic reactions, needs investigation. However there are lacunae in literature on this aspect, hence experimental evidence is needed to demonstrate their influence on biodegradation. Environmental and nutritional factors play important role in substrate utilization, production and release of enzymes and formation of metabolites. Understanding role of these factors is important for designing a sustainable biotreatment facility (Chen et al., 2006). Current study was planned to isolate and identify bacterial specie capable of degrading CP. Attempts were made to find out effect of environmental parameters such as inoculum density, pH, initial CP concentration and nutrients availability on CP biodegradation and kinetic properties. Hydrolyzing metabolites 3, 5, 6-trichloro-2-pyridinol (TCP) and tricholoromethoxypyridine (TMP) were also detected. Concentration dependent effect of organics (petrochemicals) and inorganics (trace metals) on biodegradation of CP was also investigated. Data aggregation on this prospect could shape a premise for better plans and biological operation. 2. Materials and methods 2.1. Sample collection, isolation and taxonomic characterization Wastewater samples for isolation of bacteria were collected in sterilized containers from wastewater drain of NUST Islamabad Campus, Pakistan. GC/HPLC grade standards of CP, TCP and TMP were procured from Sigma Aldrich Corporation. Mineral salt medium (MSM) as described earlier in Khalid and Hashmi (2015) was used.10 ml wastewater was added to MSM supplemented with CP and put into incubation chamber at 37  C for 7days. Several fold dilutions were spread on MSM agar and single colonies were obtained. Several biochemical tests (oxidase, catalase, and growth on differential medium, analytical profile index kit) and morphological tests were performed for identification of chlorpyrifos degrading strains using standard procedures. For molecular identification SRK1 was grown on nutrient agar plates and biomass was washed. Biomass was used for DNA extraction through instance matrix (biorad USA). Amplification was performed using universal primers 27F and 1492 R. Montage PCR cleanup kit (Millipore) was used for purification of amplification products. Sequencing was carried out at Macrogen, South Korea for this Big dye terminator cycle sequencing kit (Applied Biosystems, USA) and Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, USA) was used. To get genus and specie names sequence comparison was made with already reported sequences at NCBI. The phylogenetic tree was constructed using software MEGA4. 2.2. Effect of environmental and nutritional factors on growth of SRK1 Effect of variable environmental conditions on growth of SRK1 was investigated. 100 ml of sterilized MSM amended with CP (100 mg/L) was added to 250 ml volumetric flask and inoculated with bacterial strains SRK1 differing in inoculum density (OD600 0.5,1, 1.5, 2). Under same environmental conditions growth was monitored under variable pH (3, 4, 5, 6, 7, 8, 9, 10) in MSM and nutrient broth. Effect of initial substrate concentration on bacterial growth was investigated in MSM with CP (50, 100, 150, 200, 250, 300 mg/L). Glucose, sucrose and nutrient broth (1 g/L) was added as additional carbon source in MSM supplemented with CP for investigating their effect on growth of SRK1. 1e5 mg/L of trace metals (Cu(II), Hg(II), Fe(II), Mn(II), and Zn(II) were added in MSM individually in order to check their effect on growth of SRK1. Effect of petrochemicals on bacterial growth was monitored by

supplementing MSM with 10 and 100 mg/L of benzene, toluene and phenol. Conditions for all experiments were inoculum density 1.5, pH 8, CP 100 mg/L and incubation time 48 h (unless mentioned). Optical density (growth) was measured using single beam spectrophotometer using cuvette of 1 cm path length. 2.3. Biodegradation of chlorpyrifos 2.3.1. Experimental setup and operation Bench scale bioreactors previously described in Khalid and Hashmi (2015) were used for biodegradation study. Briefly four bench scale bioreactor of 10 L, 40 cm and 24 cm working volume, length, internal diameter working volume were used in this study. Bioreactors were stirred magnetically for uniform mixing of biomass. Air pumps along with porous grit stone diffusors were used for air supply. For feeding medium inlet valves and for withdrawal of sample outlet valves were installed. For avoiding contamination cotton plugs were utilized. Batch system worked at room temperature. As seeding inoculum biomass developed from CP degrading strain SRK1 was used. Initial pH of medium was set to 8 (unless mentioned). Later the temperature and pH of system was not controlled, yet their values were checked. There were three replications of all experiments. Sample were collected, filtered, extracted and analyzed periodically for remaining CP concentration using GC-ECD (Shimadzu 2010). 2.3.2. Kinetics of CP biodegradation To determine zero order and first order rate constant following algorithms were used (Eqs. (1) and (2))

ln C ¼ a þ kt

(1)

C ¼ b þ k0 t

(2)

where ko and k are zero order and first order rate constant respectively. Substrate concentration and its degradation duration are represented by t and C (Yang et al. 2014, Khalid and Hashmi, 2015). Half life (t1/2) is the time required to reduce 50% of initial concentration. Half-life (t1/2) of CP in batch system was calculated using Eq. (3) (Yang et al., 2014)

t1=2 ¼

0:693 k1

(3)

In C was plotted against time t and straight line regression equation was obtained. Slope of regression equation gives value for rate constant “k”. R2, first order rate constant k (h1) and t1/2 (days) are presented in Table 1. 2.3.3. Effect of environmental factors on biodegradation The effect of various environmental factors on SRK1 degradation ability was investigated. Runs were made in bioreactor with MSM amended with CP (150 mg/L) inoculated with SRK1 at inoculum density at OD600 (0.5, 1, 1.5, 2), pH (3e10). Effect of additional carbon source on CP biodegradation was investigated using MSM with CP (150 mg/L) by supplementing it with 1 g/L of glucose, sucrose and nutrient broth separately. Effect of initial concentration on biodegradation was studied in MSM with initial CP (50e300 mg/L). For all experiments in coulum density 1.5, pH 8 and CP concentration was 150 mg/L (unless mentioned). Medium without inoculum served as control. Optimum values acquired for one parameter was utilized in further tests. Sample were collected, filtered, extracted and analyzed periodically for remaining CP concentration using GC-ECD (Shimadzu 2010).

S. Khalid et al. / Journal of Environmental Management 168 (2016) 1e9

2.3.4. Effect of petrochemicals on CP biodegradation Effect of presence of petrochemicals on biodegradation of CP was investigated. MSM (pH 8) containing CP (150 mg/L) was amended with different concentrations (10, 20, 100 mg/L) of benzene, toluene, phenol individually and inoculated with SRK1 (OD ¼ 1.5 at 600 nm). MSM without any petrochemical and/or SRK1 served as control. Sample were collected, filtered, extracted and analyzed periodically for remaining CP concentration using GC-ECD (Shimadzu 2010). 2.3.5. Effect of metal ions on CP biodegradation Effect of different concentrations of metal ions in medium on biodegradation of CP was investigated. MSM (pH 8) containing CP (150 mg/L) was amended with different concentration (1, 5, 10 mg/ L) of Cu (II), Mn(II), Fe(II), Hg(II), Zn(II) individually. MSM was inoculated with SRK1 (OD ¼ 1.5 at 600 nm). MSM without these metal ions served as control. Sample were collected periodically, filtered and extracted to analyze the remaining CP concentration. 2.3.6. CP biodegradation and metabolite production in relation to growth of SRK1 Biodegradation of CP and production of metabolites TCP and TMP in relation to growth of SRK1 was investigated. MSM (pH 8) was sterilized and added to bioreactor. CP (150 mg/L) was added and inoculated with SRK1 (OD ¼ 1.5 at 600 nm). Periodic sampling from bioreactors was practiced for determination of cell density at 600 nm, changes in pH, remaining concentration of CP, TCP and TMP after extraction with ethyl acetate and acetone respectively. In order to access volatilization and abiotic degradation of CP, bioreactors with MSM were operated without inoculum. Samples were collected, extracted using ethyl acetate and injected into GCECD for analysis of remaining CP, TCP and TMP concentration. 2.3.7. Cyclic operation Experiments have proved SRK1 as a potential candidate for biodegradation of CP in one cycle. However, agrochemical industries generate huge volume of water continuously thus in field conditions bioreactors have to run in cyclic mode for treating wastewater. Therefore biotreatment systems should be sustainable for longer period of time. For this reason bioreactor performance was evaluated for numerous cycles at HRT 48 h. At the end of every

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cycle concentration of CP was monitored. When the CP concentration was below 5e15 mg/L (at the end of cycle) CP was added following the method of Swissa et al. (2014). No other nutrient or bacteria was added in the medium. This experiment was conducted to investigate sustainability of the system. 2.4. Analytical methods To determine concentration of CP in medium, 1 ml samples was collected, mixed with ethyl acetate twice in volume and vortexed briefly. For TCP/TMP determination sample was mixed with acetone thrice in volume, vortexed and supernatant was collected. CP, TCP and TMP were analyzed using GC-ECD/FID (Shimadzu 2010). GC grade standard of CP, TCP, and TMP were dissolved in acetone and injected in GC, peak read and retention time was recorded. Samples were analyzed for remaining CP, TCP and TMP concentration at GC conditions described by Khalid and Hashmi (2015). Extracted sample (1 ml) was injected and peak area measurement was used to find out remaining concentration of CP and its metabolites. Dissolved Oxygen, pH and temperature were determined using (multimeter HACH). Cell growth was determined in terms of absorbance at 600 nm using single beam spectrophotometer (HACH). 3. Results and discussion 3.1. Isolation and identification Numerous bacterial strains capable of CP degradation were isolated from wastewater of NUST Islamabad Pakistan, using enrichment techniques. There were 5 bacterial strains SRK1, SRK3, SRK6, SRK7, SRK9 and SRK10. MSM amended with 150 mg/L of CP was used to select most efficient strain.10 ml of medium was added to a test tube and inoculated with SRK1, SRK3,SRK6,SRK7, SRK9,SRK10 (OD600nm ¼ 1) separately. Medium was incubated at 35  C for 96 h. After 96 h SRK3, SRK6, SRK7, SRK9and SRK10 has shown 27, 19, 23.9, 13.8 and 21.7% removal. SRK1 has shown >50% degradation of initial CP concentration. Bacterial strain SRK1 was found to be most proficient and was selected for further studies. Further the results of 16S rRNA sequence analysis confirmed SRK1 as Pseudomonas kilonensis (Fig. 1). Sequence of SRK1 was submitted

Table 1 Kinetics of SRK1 assisted biodegradation of chlorpyrifos. Experiment

Value

Regression equation

Inoculum density (600 nm)

0.5 1 1.5 2 3 4 5 6 7 8 9 10 50 100 150 200 250 300 CP CP þ G CP þ S CP þ NB

In In In In In In In In In In In In In In In In In In In In In In

pH

CP concentration (mg/L)

Carbon source (g/L)

C C C C C C C C C C C C C C C C C C C C C C

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

5.0580.0032t 5.03680.0073t 4.9660.0379t 4.8220.0375t 5.0220.0014t 5.0480.0029t 5.040.0048t 5.06480.0087t 5.1180.0365t 5.1680.0509t 5.1040.0136t 5.0250.0013t 4.0250.0815t 4.7340.0613t 5.050.0465t 5.3180.0232t 5.58260.0163t 5.780.0141t 5.1840.0469t 5.1580.055t 4.9540.028t 5.2060.0497t

Rate constant “k” (h1)

R2

t 1/2 (days)

0.0032 0.0073 0.0379 0.0375 0.0014 0.0029 0.0048 0.008 0.0365 0.0509 0.0136 0.0013 0.0815 0.0613 0.0465 0.0232 0.0163 0.0141 0.0469 0.055 0.028 0.049

0.8381 0.9572 0.9593 0.9568 0.9453 0.8584 0.922 0.8905 0.9813 0.9853 0.9689 0.9451 0.9897 0.9659 0.9933 0.9887 0.9894 0.9709 0.977 0.9759 0.9744 0.9679

6.01 3.6 0.78 0.56 20.62 9.95 6.01 3.6 0.78 0.56 2.12 22.21 0.35 0.47 0.62 1.24 1.77 2.04 0.615 0.525 1.03 0.58

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take longer time period for acclimatization. In contrast large population have short acclimatization period. Our findings indicated quicker acclimation at higher inoculum concentration. With further increase acclimation period was almost same. In MSM with CP maximum growth 1.142 and 1.149 (0D600nm) was observed at pH 7 and 8. At pH < 6 and > 9 a sharp decline in growth of SRK1 was observed (Fig. 2b). SRK1 has tolerance to wide pH range (6e9). However pH 7 and 8 was found optimal for growth of SRK1 in all tested media, that is, MSM with/without CP and nutrient broth with/without CP. It might be attributed to the fact that very alkaline or acidic pH has inhibitory influence on bacterial activities (Yadav et al. 2014). pH affected growth of SRK1 irrespective of the medium. Among all media used maximum growth was observed in nutrient broth supplemented with CP (Fig. 2b). Fig. 2c is showing growth of bacterial strains with different initial chlorpyrifos concentration. 1.02, 1.34, 2.37, 1.7, 1.9 and 1.3 OD at 600 nm was achieved at 50, 100, 150, 200, 250 and 300 mg/L. With increase in CP concentration an increase in growth was observed upto 150 mg/L. In contrast above 200 mg/L a decline in growth was observed (Fig. 2c). Possible reason for decline in growth at higher CP concentration can be substrate inhibition (Yadav et al., 2014). This might be inhibition of anionic transportation, cell acidification and undesirable substrate binding to cell parts (Olson et al., 2003). Presence of easily degradable carbon sources in the medium accelerated growth of SRK1. In MSM with CP and one of the carbon source (glucose, sucrose and nutrient broth) higher growth was observed as compared to medium with only one of them (Fig. 2d). From this we can conclude that SRK1 was able to use both carbon sources. CP degrading enzymes were expressed even in the

Fig. 1. Evolutionary relationships of Pseudomonas Kilonensis SRK1 to 9 taxa obtained from NCBI. Neighbor-Joining method was used for conclusion of evolutionary history. Phylogenetic analysis was executed in MEGA 4.

at NCBI GenBank and accession number was obtained. Accession number for P. kilonensis SRK1 is KT013088. 3.2. Effect of environmental conditions on growth of SRK1

1.5 1

0 hour 12 hour 24 hour 48 hour

a

Absorbance at 600nm

`

Absorbance at 600nm

With increase in inoculum density from 0.5 to 2, a sharp increase in growth was observed. Confluent growth (OD600nm ¼ 1.13 and 1.25) was achieved at initial inoculum density of 1.5 and 2 respectively (Fig. 2a). Acclimatization period is duration mandatory for bacterial culture to reproduce to a level enough to initiate biodegradation (Anwar et al., 2009). Smaller populations usually

0.5 0

2.5 2 1.5 1

0 3

Absorbance at 600nm

2 1.5

c

24 hour 48 hour 72 hour 96 hour

1 0.5 0

Absorbance at 600nm

50

3 2

3.5 3 2.5 2 1.5 1 0.5 0

e

1 mg/L 5 mg/L

1 0

2.5

G

10 mg/L

2

5 6 7 8 Initial medium pH

9

10

d

S NB CP+G CP+S CP+NB Cosubstrates

f

100 mg/L

1.5 1 0.5 0 B

Metals

4

0 hour 24 hour 48 hour

CP

100 150 200 250 300 Initial CP concentration (mg/L)

Absorbance at 6000nm

Absorbance at 600nm

3

b

0.5

0.5 1 1.5 2 Initial inoculum density (600nm)

2.5

MSM with CP MSM without CP NB with CP NB without CP

T

P CP B+CP T+CP P+CP Petrochemicals

Fig. 2. Growth of SRK1 in terms of absorbance at 600 nm at different environmental conditions (a) inoculum density; 0.5e2 (b) pH; 3e10(c) initial CP concentration; 50e300 mg/L (d) glucose, sucrose, nutrient broth; 1 mg/L (e) trace metals; 1e5 mg/L (f) Petrochemicals; 10e100 mg/L.

S. Khalid et al. / Journal of Environmental Management 168 (2016) 1e9

presence of easily degradable carbon sources. However we can't conclude which carbon source was preferred. Both low as well as high concentrations of metals (without CP in medium) have not shown any increase in bacterial growth. Reason for this could be absence of carbon source. Without addition of any metal OD600nm ¼ 2.04 ± 0.01 was observed (Fig. 2e). When CP was added in medium, 1 mg/L of Cu, Fe and Zn has shown 2.83, 2.57 and 2.49 (OD at 600 nm). On the other hand at 5 mg/L except Cu all other metals have negatively affected bacterial growth. Medium with only CP as carbon source has shown 2.06 ± 0.02 (OD ta 600 nm). With toluene as only carbon source 0.95 (OD at 600 nm) was observed (Fig. 2f). When 10 mg/L of toluene was added to MSM supplemented with CP, OD ¼ 2.23 at 600 nm was observed. No considerable growth of SRK1 was observed in MSM supplemented with 100 mg/L of benzene, toluene or phenol. Only toluene at low concentration has accelerated bacterial growth both in the presence or absence of CP. Benzene and phenol has suppressed bacterial growth in medium with and without CP. 3.3. Effect of environmental conditions on biodegradation of chlorpyrifos 3.3.1. Effect of inoculum density on chlorpyrifos degradation Impact of initial cell density (0.5e2 at 600 nm) on CP degradation was investigated. After 48 h of incubation 2.26, 23.3, 87.8, 88.2% removal was accomplished at OD 0.5, 1, 1.5 and 2 respectively (Fig. 3a). With the increment in inoculum density increment in CP degradation was noticed. After 96 h of incubation 26, 52, 96.4, 96.6% degradation was observed at OD 0.5, 1, 1.5 and 2 respectively. t-test was performed for the results mentioned in Figure 3a at different initial inoculum density. Results indicated biodegradation at inoculum density 0.5 and 1 was significantly less from 1.5 (p ¼ 0.05). Biodegradation at 1.5 was slightly higher than 2 but this difference was not statistically significant. So we can conclude that inoculum level has significant effect on biodegradation process. Results demonstrated quicker acclimation with increase in optical density. Time required by a population to reproduce sufficiently to begin effective biodegradation reaction is called acclimatization. InCP at different inoculum densities was plotted against time (Fig 3b) and straight line regression equation was obtained. Rate constant (h1) k0.5, k1, k1.5, k 2 were 0.0032, 0.0073, 0.0379, 0.0375 (Table 1) respectively (k0.5 means rate constant for initial OD of 0.5 at 600 nm, all other subscripts denotes initial OD in similar manner). With increase in initial inoculum density an increase in rate constant and decrease in half life was observed, this means fast degradation of CP with higher inoculum density. Cultures having large population size acclimatize faster. Degradation of CP was observed at all inoculum density but 1.5e2 was optimal. 3.3.2. Effect of pH on chlorpyrifos degradation At acidic pH 3, 4 and 5 CP removal efficiency was 9.3, 10, 11.3%. At basic pH 9 and 10 CP removal (%) was 37 and 3.86. About 23, 73.3 and 87% CP removal was observed with pH 6, 7 and 8 respectively at HRT 48 h (Fig. 3c). Negligible degradation in control (uninoculated) of each pH was observed. t-test was performed for the results mentioned in Figure (3c) at different initial pH. Results indicated significant increase in biodegradation when pH was increased from 3 to 8 (p ¼ 0.05). Biodegradation at pH 8 was slightly higher than 7 but this difference was not statistically significant. Above pH 8 a significant decrease in removal efficiency of CP was observed. So we can conclude that pH has significant effect on biodegradation process and pH 7e8 was optimal. Hydrogen concentration in medium affects bacterial growth, indirectly affecting degradation of xenobiotics (Reddy et al., 2014). Anwar et al. (2009) reported that maximum biodegradation of CP was observed at pH 8.5.

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InCP was plotted against time (Fig 3d) and straight line regression equation was obtained. A first order growth model was shown to fit data (R2 > 0.85) (Table 1). Highest degradation rate k8 (0.051) and shortest half-life (0.56 days) was observed when initial pH was 8. This indicates pH 8 is optimum for biodegradation of CP by SRK1. Kinetic analysis revealed first order rate k3, k4, k5, k6, k7, k9, k10 (h1) for CP removal was 0.0014, 0.0029, 0.0048, 0.008, 0.0365,0.0136 and 0.013 respectively (k3 means rate constant for initial pH of 3 and all other subscripts denotes pH in similar manner) (Table 1). Results indicated t1/2 value of 20.62, 9.95, 6.01, 3.6, 0.78, 2.12, 22.12 (days) for pH 3, 4, 5, 6, 7, 9and 10. Here we can conclude that pH 8 was optimum for enzyme activity therefore higher CP removal was observed at this pH. This support changes in kinetic properties k1, t1/2 and CP removal efficiency with change in pH. 3.3.3. Effect of initial substrate concentration on chlorpyrifos degradation Results of CP degradation with various CP concentrations are presented in Figure 3e. About 96% removal was achieved upto 100 mg/L of CP at HRT 48 h. Percentage removal efficiency for CP after 48 h was 100, 96, 86.6, 67, 52 and 38.3 with initial concentration 50, 100, 150,200, 250, 300 mg/L. Results indicated that initial concentration has significant effect on biodegradation. In uninoculated controls only 2% disappearance was observed. When HRT was increased to 96 h about 99.3, 90, 78.8, 75% removal efficiency for CP was achieved with initial concentration 150, 200, 250 and 300 mg/L t-test was performed for the results mentioned in Figure (3e) at different initial concentrations of CP. Results indicated biodegradation at CP from 50 upto 150 mg/L was significantly increased (p ¼ 0.05). When concentration was increased beyond 150 to 200,250 and 300 mg/L a significant decrease in CP removal efficiency was observed at p ¼ 0.05. So we can conclude that initial CP concentration has significant effect on biodegradation process. InCP was plotted against time (Fig. 3f) and straight line regression equation was obtained. With increase in substrate concentration decrease in rate constant, k50 ¼ 0.0815 > k100 ¼ 0.0613 > k150 ¼ 0.0465 > k200 ¼ 0.0232 > k250 ¼ 0.0163 > k300 ¼ 0.0141 h1 was observed (k50 means rate constant for initial CP concentration of 50 mg/L and all other subscripts denotes concentration in similar manner). Results indicated t1/2 value of 0.35, 0.47, 0.62, 1.24, 1.77 and 2.04 (days) for 50, 100, 150,200, 250 and 300 mg/L CP respectively (Table 1). At high concentration degradation rate constant decreased and half-life increased possibly because of substrate inhibition. Substrate inhibition results in decreased growth. Possible substrate inhibitory effects could be hindrance in ion passage and non-targeted binding to cell parts (Olson et al., 2003). 3.3.4. Effect of additional carbon source on chlorpyrifos degradation Presence of additional carbons source in degradation medium may decrease chances of CP to be preferred as a carbon source. For investigation of possible effect of easily biodegradable carbon sources on CP biodegradation 1 g/L of glucose, sucrose and nutrient broth were added to MSM separately. Degradation in abiotic control was not appreciable <2.3%. 84% removal was observed in the medium with CP as only carbon source. About 92% CP disappearance was observed in medium with glucose as co-substrate (Fig. 3g). When sucrose and nutrient broth (NB) were added to medium, SRK1 utilized 71 and 86% of CP respectively (Fig. 3g). t-test was performed for the results mentioned in Figure 2g. Results indicated biodegradation at CP was significantly increased (p ¼ 0.05) when glucose was added in the medium. With addition of nutrient broth the difference was not statistically significant. When sucrose was added as cosubstrate, a statistically significant decrease in CP removal efficiency was observed at p ¼ 0.05. So we

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S. Khalid et al. / Journal of Environmental Management 168 (2016) 1e9 0.5

1

1.5

a

2

6

180

0.5

140

1.5

b

2

4

120 100 80

3 2

60 40

1

20 0

0 0

24

48 HRT (h)

72

96

0

200

pH3

pH4

pH5

pH6

180

pH7

pH8

pH9

pH10

48 HRT (h)

72

96

pH3

pH4

pH5

pH6

pH7

pH8

pH9

pH10

d

5

160 140

4

120 100 80

3 2

60 1

40 20

0

0 0 350 300

24

48 HRT (h)

72

50 mg/L

100 mg/L

150 mg/L

200 mg/L

250 mg/L

300 mg/L

0

96

e

6

24

48 HRT (h)

72

50 mg/L

100 mg/L

150 mg/L

200 mg/L

250 mg/L

300 mg/L

96

f

5

250 4 200

In(CP)

Chlorpyrifos concentration (mg/L)

24

6

In(CP)

Chlorpyrifos concentration (mg/L)

c

150

3 2

100

1

50

0

0 0

180

24

48 HRT (h)

CP CP+Sucrose Control

160

1096

72

0

g CP+Glucose CP+Nutrient Broth

140

24

48 HRT (h)

CP CP+Nutrient Broth Control

6

72

CP+Glucose CP+Sucrose

96

h

5

120

4

100

In (CP)

Chlorpyrifos concentraion (mg/L)

1

5

160

In (CP)

Chlorpyrifos concentration (mg/L)

200

80 60

3 2

40 1

20 0

0 0

24

48 HRT (h)

72

96

0

24

48

HRT (h)

72

96

Fig. 3. (a) Biodegradation of CP and (b) relationship of In(CP) to time at t inoculum density; 0.5, 1, 1.5, 2, (c) biodegradation of CP and (d) relationship of In(CP) to time at initial pH; 3,4,5,6,7,8, 9,10, (e) biodegradation of CP and (f) relationship of In(CP) to time at initial CP concentration; 50, 100, 150, 200, 250,300 mg/L, (g) biodegradation of CP and (h) relationship of In(CP) to time with different cosubstrates. (pH; 8, CP; 150, OD; 1.5 unless mentioned).

3.4. Effect of organic solvents (petrochemicals) on biodegradation of chlorpyrifos Some petrochemicals are used as solvents for dissolving pesticides in commercial formulation, therefore these petrochemicals are often found in effluents of pesticide industry. The effect of presence of petrochemicals on biodegradation of chlorpyrifos was examined. Benzene, toluene and phenol were selected as model petrochemicals. At 0 mg/L of petrochemicals (control) 91.8% degradation was achieved. Treatment without addition of SRK1 served as abiotic control and didn't exceed 2%. At low concentration of petrochemicals (10 mg/L) 88, 96.6 and 88.6% degradation was achieved with addition of benzene, toluene and phenol respectively (Fig. 4a). Degradation rate was enhanced by 4.8% with toluene addition. In contrast 10 mg/L of benzene and phenol has slightly decreased degradation by 3.8 and 3.2% respectively. At 20 mg/L of benzene, toluene and phenol 87.3, 91.6, 84.6% CP was degraded respectively. It was interesting to observe that there was no effect on degradation rate when 20 mg/L toluene was added. Benzene and phenol inhibited degradation rate by 4.5 and 7.2%. About 57.3, 81.3, 46% degradation was achieved with 100 mg/L of three petrochemicals (Fig. 4a). Swissa et al. (2014) reported slight reduction in degradation rate of atrazine with the addition of toluene (100 mg/L) and phenol (400 mg/L). The reason for slow degradation and longer lag phase could be utilization of other carbon sources before using CP. CP was a preferred carbon sources for SRK1 even in the presence of toluene might because enzymes responsible for biodegradation such as organophosphate hydrolase (OPH) were expressed. At higher concentration sharp decrease in degradation rate was observed, possibly because these organic solvents may take part in metabolic reactions and effect extracellular enzyme production (Mahmood et al., 2015). With increase in benzene concentration rate constant decreased from 0.0457 at 10 mg/L to 0.0188 at 100 mg/L. For phenol k value decreased from 0.047 to 0.0136 (h1) by increasing concentration from 10 to 100 mg/L, this indicates an inverse relationship in

180

0 hour

160

12 hour

7

24 hour

48 hour

a

140 120 100 80 60 40 20 0 Control Benzene Toulene Phenol Benzene Toulene Phenol Benzene Toulene Phenol 0mg/l

0.08

10mg/l

20mg/l

Concentration of petrochemicals (mg/L)

Benzene

Toluene

100mg/l

Phenol

b

0.07

Rate constant "k"

can conclude that addition of cosubstrates has significantly affected CP biodegradation process. In our previous study, we observed an increase in degradation rate with addition of yeast extract as additional carbon source (Khalid and Hashmi, 2015). Anwar et al. (2009) reported enhanced degradation of CP with glucose addition in the medium. InCP with different carbon sources was plotted against time (Fig. 3h) and straight line regression equation was obtained. When CP was only carbon source rate constant has a value of 0.046 (h1) and t1/2 was 0.615 (days). When glucose, sucrose and nutrient broth were added, first order rate k1 (h1) for CP was 0.055, 0.028 and 0.049 respectively (Table 1). Results indicated t1/2 value of 0.525, 0.58 days for CP with glucose and NB respectively (Table 1). Half life was increased from 0.615 to 1.03 with the addition of sucrose; this indicates slow degradation in the presence of sucrose. Kinetic analysis revealed fastest degradation in the presence of glucose as additional carbon source. In contrast degradation rate was decreased in the presence of sucrose. SRK1 preferred use of CP even when multiple carbon sources were present and its degradation ability was positively affected by glucose. Possible reason for this could be expression of enzymes responsible for CP degradation even in the presence of additional carbon sources (Anwar et al., 2009). When growth of SRK1 was observed in the presence of only one carbon source i.e. CP, glucose, sucrose or nutrient broth considerable growth was observed. However when they were added as cosubstrate with CP growth was higher than any single carbon source (Section 3.2). This could be an indication thatSRK1 was able to use these carbon sources simultaneously.

Chlorpyrifos concentration mg/L

S. Khalid et al. / Journal of Environmental Management 168 (2016) 1e9

0.06 0.05

R² = 0.8193

0.04 0.03

R² = 0.9962

0.02 R² = 0.9931

0.01 0 0

50

Concentration of petrochemicals (mg/L

100

Fig. 4. Effect of the petrochemicals i.e. benzene (10e100 mg/L), toluene (10e100 mg/ L), phenol (10e100 mg/L on CP degradation by strain SRK1 in mineral salt medium(a), rate constant at different concentration (b). (Inoculum density; 1.5, pH; 8, CP concentration; 150 mg/L, HRT; 48 h).

degradation rate and phenol concentration. When 10 mg/L toluene was added rate constant was 0.0736, which is higher than control. However no effect was observed at 20 mg/L. Toluene addition has shown highest degradation rate at all given concentrations (Fig. 4b). It could be concluded that degradation of CP decreased with increase in concentration of all petrochemicals. 3.5. Effect of metal ions on biodegradation of chlorpyrifos Studies reported presence of metal ions in commercial formulations. Considerable amount of trace metals are added in pesticide formulation without any scientific reason. Pesticides may be considered as source of metals ions which may affect their biodegradation. Therefore effect of presence of metal ions on biodegradation of CP was examined. Type and concentration of metal ions strongly effected SRK1 efficiency to degrade CP. Cu(II) ion at concentration of 1 and 5 mg/L enhanced degradation by SRK1. About 98% CP was degraded in presence of 1 and 5 mg/L Cu(II), after 48 h HRT (Fig. 5a). When concentration of Cu(II) was increased to 10 mg/L CP degradation was inhibited. Addition of other metals such as Fe(II) and Zn(II) at 1 mg/L enhanced degradation rate i.e. 96.6 and 94.6% degradation was achieved after 48 h HRT (Fig. 5a). Lag phase was longer with 1 mg/L of Mn(II) and Hg(II) in media. In case of Mn(II) and Hg(II) contrary to strong effect at high concentration there was no effect on rate of degradation of CP by SRK1at low concentration (1 mg/L). Sarkouhi et al. (2012) reported that CP can be effectively degraded in the presence of Agþ. For Mn(II), Fe(II), Hg(II) and Zn(II) there was an inverse relationship among metal concentration and CP degradation. When concentration was increased to 5 and 10 mg/L CP degradation was strongly decreased. With addition of 1 mg/L of trace metals Cu(II), Mn(II), Fe(II), Hg(II), Zn(II) first order rate constant was increased to 0.083, 0.057, 0.073. 0.0575, 0.0623 (h1) as compared to 0.0542 (h1) with no metal addition. With increase in Cu (II) concentration to 5 mg/L rate constant increased to 0.091 h1. Concentrations of copper and degradation rate were observed to be directly proportional upto 5 mg/L after this no significant change in biodegradation rate was observed. Biodegradation rate at 10 mg/L was 0.023 > 0.02 > 0.011 > 0.009 > 0.005 with Mn(II), Hg(II), Fe(II), Cu(II) and Zn(II) respectively (Fig. 5b).

S. Khalid et al. / Journal of Environmental Management 168 (2016) 1e9

5 Concentration of metal ( mg/L)

10

Fe(II)

b

CP

TCP

TMP

OD

Cu(II)

Mn(II)

0.1

Hg(II)

Zn(II)

1

100

0.8

80

0.6

60 40

0.4

20

0.2 0 0

10

20

30

HRT (h)

40

50

In (CP)

R² = 0.7378

In(CP)

8 7

pH

6

InC = 5.1925+0.0534t k= 0.0534/h t = 0.54 days R² = 0.9864

5 4

0.04

60

b

7

R² = 0.9999

0.06

1.2

120

0

R² = 0.9835

0.08

1.4

a

140

6 5 4

3

0.02

R² = 0.7301

R² = 0.9303

2

3 0

0 0

5 Concentration of metal (mg/L)

It can be concluded that effect of metal addition was highly concentration dependent but no specific pattern was observed. When concentration of metal was increased to 10 mg/L a decrease in rate constant to 0.023, 0.011, 0.02, 0.005 h1 was observed with Mn(II), Fe(II), Hg(II) and Zn(II) (Fig. 5b). Degradation of chlorpyrifos in the presence of Agþ followed first order kinetics (Sarkouhi et al., 2012) High concentration of aluminum salt accelerated degradation of CP in the presence of zero-valent nano iron and an increase in rate constant from 0.24 to 0.60 was reported (Reddy et al., 2013). Biodegradation of CP is a biochemical reaction involving electrons, metals act as electron acceptor affecting reaction pathway. Cu(II) and Fe(II) may be necessary for bacterial growth and act as cofactor for CP degrading enzyme in low concentration, this increased CP degradation rate at low concentration (Pointing et al., 2000, Chu et al., 2006). In contrast the other metal ions have inhibitory effect, possibly because metal ions competed with cofactor of CP degrading enzyme on the active site. Another reason for inhibitory effect of metal could be that cations may be formation of complexes with released proteins and enzymes, this retards enzyme activity. All metal ions studied have concentration dependent usually inhibitory effect on CP degradation (Shomar, 2006, Mahmood et al., 2015).

3.5.1. CP biodegradation and formation of 3, 5, 6 trichloro-2pyridinol (TCP) and tricholoromethoxypyridine (TMP) Fig. 6 exhibits increase in bacterial growth with chlorpyrifos degradation which results in TCP generation. CP concentration (150 mg/L) was diminished to 66 mg/L after 24 h. Following 48 h, 12.3 mg/L concentration was observed, demonstrating a removal of 91.8% (Fig. 6a). CP degradation could result in production of TCP which is more toxic than parent compound. Therefore in addition to CP degradation, generation of metabolites (TCP, TMP) was assessed. At the beginning of batch experiment TCP was 0 mg/L. After 24 h an increase in TCP concentration to 67 mg/L was observed. After that a slow diminish in TCP concentration was noticed which indicate its decay. After 48 h HRT TCP in liquid medium was 38 mg/L (Fig. 6a). 19 mg/L TMP was detected at 24 h and after 48 h it was completely mineralized to water soluble products. The analysis of CP degradation, TCP and TMP formation

10

20

30

40

50

60

HRT (h)

10

Fig. 5. Effect of various metal ions (1e10 mg/L) on CP degradation by strain SRK1 in mineral salt medium at (a), changes in first order rate constant k with metal concentration (b). (Inoculum density; 1.5, pH; 8, CP concentration; 150 mg/L, incubation time; 0,12,24, 48 h).

Absorbance at 600nm

CP/TCP/TMP concentration (mg/L)

160

Zn (II)

Fe(II)

Hg (II)

Mn(II)

Cu(II)

Zn (II)

Hg (II)

Fe(II)

Cu(II)

a

48 hour

8

0.12 Rate constant "k"

Mn(II)

Zn (II)

Fe(II) 1

24 hour

pH

0

12 hour

Hg (II)

Cu(II)

Mn(II)

0 hour

180 160 140 120 100 80 60 40 20 0 -20

Control

Chlorpyrifos concentration (mg/L)

8

Fig. 6. CP degradation by strain SRK1 in mineral salt medium, formation of metabolites, optical density (a), relationship of In(CP) to time and pH fluctuation(b). (Inoculum density; 1.5, pH; 8, CP concentration; 150 mg/L, HRT; 48 h).

was accompanied by analysis of bacterial cell density. After 24 h of incubation 1.04 OD (600 nm) was obtained by SRK1 (Fig. 6a). After 48 h of incubation cell density of 1.15 at 600 nm was observed. Increase in bacterial cells density with decrease in CP concentration indicated efficient utilization of CP as source of carbon and energy. Biodegradation of CP results in production of common metabolite TCP keeping in view microbial specie capable of utilizing TCP produced during CP degradation was of one of the interests of current study. Results demonstrated that SRK1 is capable of TCP biodegradation. CP degradation by SRK1 could be attributed to expression of organophosphate hydrolase. At the start of biodegradation process when CP was added a drop in pH of batch system was noticed (Fig. 6b), however system was able to recover and attain a steady state. Enzymes activity is strongly affected by pH changes and this might stops outside a specific pH range (Kim et al., 2013). Change in temp and pH causes chemical or physical alteration in nontarget components leads to an increased affinity for inhibitory binding. 3.5.2. Kinetics of biodegradation Kinetics is useful tool to express progress of microbial degradation as it proceeds which is hyperbolic saturation function of substrate concentration (Yang et al. 2014) can be expressed as Eq. 4



Rm :C kþC

(4)

First order model for CP degradation was characterized by rate constant k ¼ 0.0534 h1 (Fig. 6b). The results obtained after calculation demonstrated that the first order model (Fig. 6b) gives a better fit for CP biodegradation using SRK1. Half-life (t1/2) of CP in batch system with SRK1 was 0.54 days for CP. First order model has shown R2 ¼ 0.9864 and therefore an excellent fit (Fig. 6b). Results demonstrated SRK1 as proficient candidate for CP biodegradation and 50% of initial concentration of CP was degraded in a short time of 0.54 days only. 3.5.3. Cyclic operation of bioreactor At the point when CP was 5e15 mg/L in the medium, its concentration was maintained to 150 mg/L through repeated addition and degradation ability of SRK1 was investigated. An insignificant

S. Khalid et al. / Journal of Environmental Management 168 (2016) 1e9

9

dx.doi.org/10.1016/j.jenvman.2015.11.030.

0.06 k

0.04

180

CP

CP concentraƟon (mg/L)

160

OD

1.6

R² = 0.9619

0.02

1.4

0 0

140

2

Cycle

4

6

120

1.2

References

1

100

0.8

80

0.6

60 40

0.4

20

0.2

0

0 0

50

100

150

Time (h)

200

250

300

350

Fig. 7. Biodegradation of CP by SRK1 in bioreactors for six cycles. First order ate constant for each cycle in inset.

diminish in CP removal capability was seen following every cycle (Fig. 7). 97.8% removal in 6th cycle demonstrates probability for more cycles. With decrease in CP concentration an increase in bacterial growth was observed with each cycle. In start of cycle when CP was added first a slow increase upto 5 h and then a sharp increase in growth was observed. Increase and decrease in bacterial growth was associated with changing CP concentration. R2 > 0.98 demonstrated appropriateness of first order growth model to express progress of biodegradation process. A decrease in rate constant was observed with every upcoming cycle (kC1 ¼ 0.052> kC2 ¼ 0.05 > kC3 ¼ 0.044 > kC4 ¼ 0.043 > kC5 ¼ 0.04 > kC6 ¼ 0.038) observed (kC1 means rate constant for cycle 1, all other subscripts denotes number of cycles in similar manner). An increment in halflife was observed with each cycle i.e. 0.554, 0.577, 0.65, 0.668, 0.72, 0.75 days for C1, C2, C3, C4, C5, C6 respectively. 4. Conclusion SRK1 has ability to mineralize CP to TCP and TMP under broad range of environmental conditions. Petrochemicals and metals ions present in effluents of pesticide industry could strongly affect treatment process however SRK1 could perform biodegradation activity at low concentrations effectively. Presence of petrochemicals such as toluene (10e20 mg/L) and metal ions (Cu(II), Fe(II), and Zn(II)) at low concentrations (1 mg/L) may be useful to enhance degradation of CP in wastewater of pesticide industry. Bioreactors could be successfully operated for removal of CP using SRK1 for repeated cycles with slight reduction in efficiency. Appendix A. Supplementary data Supplementary data related to this article can be found at http://

Anwar, S., Liaquat, F., Khan, Q.M., Khalid, Z.M., Iqbal, S., 2009. Biodegradation of chlorpyrifos and its hydrolysis product3, 5, 6-trichloro-2-pyridinol by Bacillus pumilus strain C2A1. J. Hazard. Mater. 168, 400e405. Chen, H.J., Guo, G.L., Tseng, D.H., Cheng, C.L., Huang, S.L., 2006. Growth factors, kinetics and biodegradation mechanism associated with Pseudomonas nitroreducens TX1 grown on octylphenol polyethoxylates. J. Environ. Manage 80, 279e286. Chishti, Z., Hussain, S., Khaliq, R., Arshad, K.R., Khalid, A., Arshad, M., 2013. Microbial degradation of chlorpyrifos in liquid media and soil. J. Environ. Manage. 114, 372e438. Chu, X.Y., Wu, N.F., Deng, M.J., Tian, J., Yao, B., Fan, Y.L., 2006. Expression of organophosphorus hydrolase OPHC2 in Pichia pastoris: purification and characterization. Protein Expr. Purif. 49, 9e14. Khalid, S., Hashmi, I., 2015. Biotreatment of chlorpyrifos in a bench scale bioreactor using Psychrobacter alimentarius T14. Environ. Technol. http://dx.doi.org/ 10.1080/09593330.2015.1069406. Kim, J., Chao, K.J., Han, G., Lee, C., Hwang, S., 2013. Effect of temperature and pH on the biokinetic properties of thiocyanate biodegradation under autotrophic conditions. Water Res. 47, 251e258. Kolpin, D.W., Barbash, J.E., Gilliom, R.J., 2000a. Pesticides in ground water of the United States, 1992e1996. Ground Water 38, 858e863. Kolpin, D.W., Thurman, E.M., Linhart, S.M., 2000b. Finding minimal herbicide concentrations in ground water? Try looking for their degradates. Sci. Total Environ. 248, 115e122. Mahmood, S., Khalid, A., Arshad, M., Ahmad, R., November 2015. Effect of trace metals and electron shuttle on simultaneous reduction of reactive black-5 azo dye and hexavalent chromium in liquid medium by Pseudomonas sp. Chemosphere 138, 895e900. Olson, G.J., Brierley, J.A., Brierley, C.L., 2003. Bioleaching review part B. Appl. Microbiol. Biotechnol. 63, 249e257. Pointing, S.B., Bucher, V.V.C., Vrijmoed, L.L.P., 2000. Dye decolorization by sub tropical basidiomycetous fungi and the effect of metals on decolorizing ability. World J. Microbiol. Biotechnol. 16, 199e205. Reddy, A.V.B., Madhavi, V., Reddy, K.G., Madhavi, G., 2013. Remediation of chlorpyrifos-contaminated soils by laboratory-synthesized zero-valent nano iron particles: effect of pH and aluminium salts. J. Chem. http://dx.doi.org/10. 1155/2013/521045. Reddy, G.V.S., Reddy, B.R., Tlou, M.G., 2014. Biodegradation of 2-hydroxyquinoxaline (2-HQ) by Bacillus sp. J. Hazard. Mater. 278, 100e107. Sarkouhi, M., Shamsipur, M., Hassan, J., 2012. Metal ion promoted degradation mechanism of chlorpyrifos and phoxim. Arab. J. Chem. http://dx.doi.org/10. 1016/j.arabjc.2012.04.026. Shomar, B.H., 2006. Trace elements in major solid-pesticides used in the Gaza strip. Chemosphere 65, 898e905. Singh, B.K., Walker, A., 2006. Microbial degradation of organophosphorus compounds. FEMS Microbiol. Rev. 30, 428e471. Swissa, N., Nitzan, Y., Langzam, Y., Cahan, R., 2014. Atrazine biodegradation by a monoculture of Raoultella planticola isolated from a herbicides wastewater treatment facility. Int. Biodeterior. Biodegrad. 92, 6e11. Yadav, M., Srivastva, N., Singh, S.R., Upadhyay, S.N., Dubey, S.K., 2014. Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor. Bioresour. Technol. 165, 265e269. Yang, S.F., Wang, C.C., Chems, C.H., 2014. Di-n-butyl phthalate removal by strain Deinococcus sp. R5 in batch reactors. Int. Biodeterior. Biodegrad. 95, 55e60.