Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor

Bioresource Technology xxx (2014) xxx–xxx

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Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor Maya Yadav a, Navnita Srivastva a, Ram Sharan Singh b, Siddh Nath Upadhyay b,1, Suresh Kumar Dubey a,⇑ a b

Department of Botany, Faculty of Science, Banaras Hindu University, Varanasi 221005, India Department of Chemical Engineering & Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi 221005, India

h i g h l i g h t s  Biodegradation of chlorpyrifos by bacteria are reported.  Continuous reactor give better performance than batch mode.  Continuous reactor shows plug-flow behaviour.  Accumulation of TCP is found to influence reactor performance.

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Bioreactor Chlorpyrifos TCP Biodegradation Removal efficiency

a b s t r a c t Biodegradation of chlorpyrifos (CP) by Pseudomonas (Iso 1) sp. was investigated in batch as well as continuous bioreactors packed with polyurethane foam pieces. The optimum process parameters for the maximum removal of CP, determined through batch experiments, were found to be: inoculum level, 300  106 Cfu mL1; CP concentration, 500 mg L1; pH 7.5; temperature, 37 °C and DO, 5.5 mg L1. The continuous packed bed bioreactor was operated at various flow rates (10–40 mL h1) under the optimum conditions. The steady state CP removal efficiency of more than 91% was observed up to the inlet load of 300 mg L1d1. The bioreactor was sensitive to flow fluctuations but was able to recover its performance quickly and exhibited the normal plug-flow behavior. Accumulation of TCP (3,5,6-trichloro-2-pyridinol) affected the reactor performance. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Chlorpyrifos (o,o-diethyl-o-3,5,6-trichloro-2-pyridin-yl phosphorothionate) (CP) is a broad spectrum insecticide widely used since 1960s, for the control of crop insects and house-hold pests (Bicker et al., 2005). Due to its long half life (60–120 days) and high residual concentrations (0.01–0.62 mg kg1), it has contaminated aquatic and terrestrial ecosystems and posed risk to public health (Ngan et al., 2005; Nawaz et al., 2011). Hence, removal of CP and other organophosphates have attracted global attention. It has been reported that various phylogenetically distinct bacterial communities degrade both CP and TCP through their co-metabolic activities and use them as the carbon source (Singh et al., 2011). Among the existing bioremediation approaches for decontamination of pesticide loaded soil or water, bioaugmentation technique has been successfully used for coumaphos (Mulbry et al., 1996), ⇑ Corresponding author. Tel.: +91 0542 2307147; fax: +91 0542 2368174. 1

E-mail addresses: [email protected], [email protected] (S.K. Dubey). DAE-Raja Ramanna Emeritus Fellow.

atrazine (Struthers et al., 1998), ethoprophos (Karpouzas et al., 2000), etc. The in situ treatment is slow, requires long duration for complete biodegradation, and is restrained by limiting factors such as low permeability and heterogeneity of the contaminated sites. The ex situ treatment permits effective control of various operating parameters, therefore, such studies in specially designed bioreactors of variety of configurations has attracted the attention of researchers (Maya et al., 2011). To the best of our knowledge, there is no reported work on the biodegradation of CP in aerobic continuous packed bed bioreactors. In the present study, the kinetics of biodegradation of CP has been first studied in a batch packed bed reactor to select the most effective strategy for bioremediation of CP. The performance of an aerated continuous packed bed bioreactor has been investigated under the optimized conditions obtained from the batch studies for the maximum removal of CP. Bacterial isolate Pseudomonas sp. (Iso 1) supported on polyurethane foam pieces has been used on the basis of its high capacity for CP degradation as demonstrated through our earlier study (Maya et al., 2011).

http://dx.doi.org/10.1016/j.biortech.2014.01.098 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Yadav, M., et al. Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.01.098

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M. Yadav et al. / Bioresource Technology xxx (2014) xxx–xxx

are CP concentrations at time t = 0 and t = t, respectively (Supplementary Fig. S1).

2. Methods 2.1. Selection of bacteria and inoculum preparation The bacterial species used in the present study was that isolated earlier from CP contaminated agricultural soil (Maya et al., 2011). The most efficient bacterial isolate (Pseudomonas sp. Iso 1) was used for the biodegradation experiment. The stock culture of the enriched bacterial isolate (Iso I) for all of the batch and continuous reactor experiments was prepared by growing bacteria in 50 mL of Luria Bertani (LB) media (Merck Company, Germany) overnight in a shake flasks. Cells were harvested by centrifugation at 5000g for 10 min at room temperature. 2.2. Packing material and bioreactors Polyurethane foam sheet of 1 cm thickness (Renuka Engineering Works, Pune, India) was cut into cubes of approximately 1 cm size using stainless steel scissors. The pieces were washed in distilled water, squeezed, dried in an air oven at 60 °C for 2 days and cooled in a dessicator. These pieces were used as packing media in the batch and continuous bioreactors. 2.2.1. Batch bioreactor The batch bioreactor consisted of a cylindrical borosilicate glass column (internal diameter = 5 cm; length = 30 cm) of 1000 mL working volume (total volume = 1200 mL). It was provided with inlet and outlet ports at suitable locations to facilitate feeding and withdrawal of reactants/products. The outlet of the reactor was located at a height of 20 cm from the bottom for venting out gases and withdrawal of treated liquid. All ports opening to atmosphere were closed with cotton plug to avoid contamination. The filtered air was supplied through silicon tubing sparger network placed 1 mm above the bottom of the reactor. The sparger network consisted of four radial arms at 90° apart and facilitated uniform distribution of air from the bottom of the reactor in the upward direction. The rising air bubbles kept the liquid in agitated state. Aeration rate was measured and regulated by a pre-calibrated rotameter. The polyurethane foam pieces soaked in aqueous solution of CP (99.9% pure, Accustandard Inc., USA) of known concentration and inoculum were filled in the reactor up to a height of 15 cm and the air flow was started. Biodegradation rate studies were conducted by monitoring the concentration of CP at regular intervals of time. Runs were made by varying the CP concentrations (100–700 mgL1), pH (6.0–9.0), temperature (25–45 °C), DO (3.2–7.8 mg L1, measured using a DO probe: YSI 5100, USA) and inoculum level (50–500  106 cells mL1). Experiments for optimizing all parameters were carried out at the CP concentration of 100 mg L1. All measurements were made in triplicate to obtain a representative value. The parameters were optimized by varying one and keeping all others constant. The optimum value thus obtained was used in the next experiment. A blank run without inoculum was used as the control and change in CP concentration with time due to adsorption/transfer to air stream was measured and was found to be insignificant. The residual CP concentration was measured using a high performance liquid chromatography (HPLC 600E, Waters Co. Milford, USA) (Maya et al., 2011) at 214 nm. For evaluating the kinetic parameters (Ks and Vmax) for biodegradation of chlorpyrifos under optimized conditions, the relation:

    1 S0 1 S0  S V max ¼ þ ln t Ks t S Ks

ð1Þ

obtained from Michaelis–Menten kinetics model, was used to fit the experimental data and evaluate the kinetic constants. Here S0 and S

2.2.2. Continuous bioreactor The continuous bioreactor packed with polyurethane foam cubes was fabricated from a borosilicate glass tube (internal diameter = 7 cm; length = 45 cm, working volume 1000 mL). The outlet of the reactor was located 26 cm from the bottom and was provided with a 0.45 lm bacterial filter to restrain the out flow of bacteria from the column. Effluent was collected in a 10 L plastic container. Provisions were made to add nutrient solutions and supply filtered air from the top of the reactor. Air was supplied by an aquarium pump and its flow rate was measured using a rotameter (Eureka, Pune) having range of 1–10 L min1. The chlorpyrifos solution (500 mg L1) was continuously supplied to the bed from a 10 L feeding tank by a peristaltic pump (Mc Lins PP10) at flow rates of 10–40 mL h1. The inoculum to the bioreactor was 5% of the holdup volume having 300  106 Cfu/mL. Previously optimized parameters obtained from the batch reactor studies were used for the operation of the bioreactor. The biodegradation was carried out for 42 days and its performance was evaluated in terms of:

% Removal efficiency ðREÞ ¼

Elimination capacity ðECÞ ¼

Sin  Sout  100 Sin

Q ðSin - Sout Þ V

ð2Þ

ð3Þ

at varying inlet loading rates

Inlet loading rate ðILÞ ¼

Sin Q V

ð4Þ

where, Sin and Sout are the inlet and outlet concentrations of CP in the bioreactor, Q is the volumetric flow rate and V is the hold-up volume of the reactor. 3. Results and discussion 3.1. Biodegradation in batch mode Batch experiments were carried out to study the effects of inoculum level, pH, temperature, DO and CP concentration on the percentage removal efficiency in aerated batch bioreactor. The degradation of CP increased rapidly as the inoculum level increased from 50  106 to 300  106 Cfu mL1 and thereafter attained a constant level of 89.8%. Thus, for further experiments the inoculum level of 300  106 Cfu mL1 was used. The % degradation of CP was low in the acidic condition (pH < 7) but increased to 91.2% at pH 7.5 and thereafter it decreased again in the alkaline range (pH > 7.5) due to the inhibitory effect on microorganisms (Singh et al., 2003). The maximum CP removal (93.9%) was obtained at 37 °C. However, with in the temperature range of 35–40 °C, slight variation in % removal was observed. The % CP removal increased rapidly to 94.2% up to the DO level of 5.5 mg L1 and then tended towards a constant value indicating O2 saturation (Venkata Mohan et al., 2004). The effect of initial concentration of CP on % removal was studied by varying it in the range of 100–700 mg L1. The % removal was found to be constant and more than 90% up to the CP concentration of 500 mg L1. Beyond this value, it decreased rapidly, indicating the possibility of substrate inhibition (Anwar et al., 2009). 3.2. Kinetics of biodegradation in batch reactor From the plot of

1 t


ln SS0 against

S0 S t




, the value of Ks, Vmax and

1

Vmax/Ks were found to be 270.3 mgL

, 50.78 mgL1d1 and 0.188

Please cite this article in press as: Yadav, M., et al. Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.01.098

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(d1) or 0.0078 h1, respectively. The value of Vmax/Ks obtained under optimized conditions is better than that obtained (0.11 d1) in our earlier work (Maya et al., 2011) when the other parameters were not optimized and the experiments were carried out at the CP concentration of 100 mg L1.

the RE is almost constant up to the loading rate of 300 mg L1d1 thereafter it is decreasing continuously. Similarly a near linear relationship is observed between the elimination capacity and the loading rate up to 300 mg L1d1. Beyond this, the elimination capacity increases slowly and reaches its maximum value of 289.8 mg L1d1 at the loading of 480 mg L1d1. TCP production has also increased sharply after the inlet loading rate of 300 mg L1d1. At low loading rates, the diffusional flux through the biofilm will also be low resulting in mass transfer limitations. Under this situation the innermost layer of the biofilm (close to the surface of packing media) will remain deficient in the substrate and the biodegradation capability of microbes will not be fully utilized. At higher loading rates, the diffusional flux is high and the process changes from mass transfer to bio-reaction controlling. From Fig. 2 it is seen that, at the loading rate of 300 mg L1 d1, the process is changing from mass-transfer to bio-reaction controlling. Increase in the loading rate from 0 to 300 mg L1 d1 results in more and more utilization of the biofilm leading to almost constant RE and rapid increase in EC. At higher concentrations of CP, substrate inhibition may result in the poor removal efficiency (Singh et al., 2010). For practical operations, the inlet loading rate at which mechanism in the bioreactor changes from mass transfer to bioreaction controlling and the point of intersection of removal efficiency and elimination capacity curves (Fig. 2) may be taken as an approximate estimate of the operating concentration range of the bioreactor. In the present study the optimum operating range has been found to be between 300 and 350 mg L1d1.

3.3. Biodegradation in continuous bioreactor The performance of continuous packed bed bioreactor was studied at varying flow rates using the optimum process parameters obtained through the batch experiments (Fig. 1). As the ambient temperature was nearly constant at around 34 ± 2 °C during the period of experiments no attempt was made to operate the reactor at a constant temperature. Initially the bioreactor was operated at a flow rate of 10 mL h1 to facilitate the proper microbial growth and to establish the steady-state condition. The steady-state was achieved on the 7th day of operation which is evident from the almost constant value of removal efficiency (91%) (Fig. 1), indicating that about 1 week is needed for the acclimation of supported Pseudomonas sp. for effective degradation of CP. On the 13th day, the flow rate was increased to 15 mL h1 and it was seen that after a sharp initial dip on the 14th day, the performance of bioreactor recovered quickly and became almost constant at 91% RE on the 16th day. On the 17th and 22nd days, the flow rates were again increased to 20 and 25 mL h1, respectively and a quick recovery in the performance was observed at both the flow rates. On the 28th day the flow rate was increased to 30 mL h1 and after a sharp dip the removal efficiency got stabilized at 78.4%. At flow rate of 40 mL h1, the steady-state RE value decreased sharply and stabilized at around 60%. Production of TCP during the degradation of CP was also monitored and the results are also shown in Fig. 1. It is seen that initially the production of TCP increased slowly with time and then stabilized at a constant value of around 30 mg L1 on 13th day of operation. Steady state values of TCP increased very slowly (30– 46.9 mg L1) as the flow rate of CP solution was increased up to 25 mL h1, but when it was increased further (first to 30 and then to 40 mL h1), a very sharp increase in TCP concentration was obtained. Fig. 2 shows the variation of the elimination capacity and removal efficiency with respect to the inlet loading rate of CP. The removal efficiency-inlet loading curve shows two distinct zones. With increase in the inlet loading rate (or flow rate) of CP,

3.4. Comparison of performance of the batch and continuous bioreactor The performance of the batch and continuous reactors was compared on the basis of amount of CP removed (mg d1). The amount of CP removed in 1 L batch reactor under the optimal conditions was 113.5 mg. In the continuous reactor of same volume under similar operating conditions, the removal increased rapidly from 109 mg d1 at the flow rate of 10 mL h1 to 270 mg d1 at 25 mL h1 and thereafter tended towards a nearly constant value (282 mg d1 at 30 mL h1 and 289 mg d1 at 40 mL h1). At high flow rate the removal was more than 2.6 times higher compared to that in the batch bio-reactor. At the low loading, all CP gets converted into TCP first and then both CP and TCP are consumed by bacteria. The amount

600

100 Flow 10 ml/h

15 ml/h

20 ml/h

25 ml/h

30 ml/h

40 ml/h

80

400

CP (mg/L)

60

TCP (mg/L) 300

% RE of CP 40

% RE of CP

Concentration (mg/L)

500

200 20

100

0

0 0

7

14

21

28

35

42

Time (days) Fig. 1. Bioreactor performance with change in inlet flow rate of CP solution.

Please cite this article in press as: Yadav, M., et al. Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.01.098

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M. Yadav et al. / Bioresource Technology xxx (2014) xxx–xxx

Fig. 2. Influence of inlet CP loading on the removal efficiency and elimination capacity of the CP and TCP accumulation.

of CP removed increases with flow rate but at high flow rates the substrate saturation takes place and TCP starts getting accumulated which inhibits the growth of the microorganism. The TCP formed keeps on accumulating and inhibiting the activity of microbes for CP degradation. In case of continuous bioreactor, under identical conditions the TCP formed flows out of the bioreactor resulting in higher degradation of CP.

the plug flow regime. The gradual accumulation of degradation product TCP affects the bioreactor performance.

3.5. Behaviour of continuous bioreactor

Acknowledgement

Assuming a first order rate expression at low substrate concentration, for a continuous reactor, one may write:

One of the authors (Maya Yadav) is thankful to ICMR, New Delhi, Government of India for the financial support in the form of JRF and SRF.

Se 1 ¼ ð1 þ khÞ Si

Conflict of interest All authors have no any actual or potential conflict of interest.

ð5Þ Appendix A. Supplementary data

for well mixed and

Se ¼ ekh SM

ð6Þ

for plug flow situation. Here Si is the inlet and, Se is the exit CP concentration, k is the first order rate constant, h is the hydraulic retention time = V/Q, V is the working volume of the reactor, and Q is the volumetric flow rate. The SSe vs h values were plotted for the entire i range of experimental data (Supplementary Fig. S2). The least squares analysis of the experimental data gave:

Se ¼ e0:0273h Si

ð7Þ

as the best fit relation (R2 = 0.96) indicating the plug flow behavior of reactor. 4. Conclusion Efficacy of Pseudomonas sp. supported on polyurethane foam for biodegradation of CP in batch and continuous aerated packed bed bioreactors have been demonstrated. Under optimum conditions the continuous packed bed bioreactor is capable of removing 91% of CP up to the inlet loading of 300 mg L1d1. The continuous bioreactor is sensitive to fluctuations in the concentration and flow rate, but is capable of regaining its steady state quickly. The continuous bioreactor gives a higher removal efficiency and operates in

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