This article was downloaded by: [New York University] On: 11 July 2015, At: 10:55 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter Alimentarius T14 a
Saira Khalid & Imran Hashmi
a
a
Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering, National University of Sciences and Technology, Islamabad 44000, Pakistan Accepted author version posted online: 06 Jul 2015.
Click for updates To cite this article: Saira Khalid & Imran Hashmi (2015): Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter Alimentarius T14, Environmental Technology, DOI: 10.1080/09593330.2015.1069406 To link to this article: http://dx.doi.org/10.1080/09593330.2015.1069406
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis Journal: Environmental Technology DOI: 10.1080/09593330.2015.1069406
Contribution to:
Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter
rip
t
Title:
Environmental Technology
Original article
Order of authors: Saira Khalid a,* , Imran Hashmi a a
Institute of Environmental Sciences and Engineering, School of Civil and
M an
Affiliations:
Environmental Engineering, National University of Sciences and Technology, Islamabad 44000, Pakistan. *
Corresponding author/First author: Saira Khalid
d
Institute of Environmental Sciences and Engineering, School of Civil and
pt e
Environmental Engineering, National University of Sciences and Technology, H-12, Islamabad 44000, Pakistan, Tel.: +923365384103
ce
(E-mail: [email protected] ; [email protected])
Acknowledgement: Authors acknowledge support by Higher Education Commission of Pakistan.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
Article type:
us c
Alimentarius T14
1
Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter Alimentarius T14
ce
pt e
d
M an
us c
Bacteria tolerant to high pesticide concentration could be used for designing an efficient treatment technology. Bacterial strains T14 was isolated from pesticide contaminated soil in mineral salt medium (MSM) and identified as Psychrobacter alimentarius T14 using 16S rRNA gene sequence analysis. Bench scale bioreactor was evaluated for biotreatment of high Chlorpyrifos (CP) concentration using P. alimentarius T14. Effect of various parameters on bioreactor performance was examined and optimum removal was observed at optical density (OD600nm) 0.8, pH; 7.2, CP concentration; 300 mg L-1 and hydraulic retention time (HRT) 48h. At optimum conditions 70.3/79 % of CP/COD removal was achieved in batch bioreactors. In addition P. alimentarius T14 achieved 95/91, 62.3/75, 69.8/64 % CP/COD removal efficiency with addition of CS (co-substrates), CS1 (yeast extract + synthetic wastewater), CS2 (glucose + synthetic wastewater) and CS3 (yeast extract) respectively. Addition of CS1 to bioreactor could accelerate CP removal rate upto many cycles with considerable efficiency. However accumulation of 3, 5, 6-trichloro-2-pyridinol(TCP) affects reactor performance in cyclic mode. First order rate constant k1 0.062 h-1 and t1/2 11.1 h demonstrates fast degradation. Change in concentration of total chlorine and nitrogen could be result of complete mineralization. Photodegradation of CP in commercial product was more than its pure form. Commercial formulation accelerated photodegradation process; however no effect on biodegradation process was observed. After bio-photodegradation negligible toxicity for seeds of Triticum aestivum was observed. Study suggests an efficient treatment of wastewater containing CP and its metabolites in batch bioreactors could be achieved using P. alimentarius. Keywords: Wastewater; Chlorpyrifos; Biodegradation; TCP; Organophosphate Pesticides
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Abstract
2
1.
Introduction
us c
M an
d
pt e
ce
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Dow chemical company in 1965 has introduced a non-systematic insecticide named as Chlorpyrifos (CP). [1] CP is used widely for controlling agricultural and household pests. [2] Inhibition of acetyl cholinesterase after CP exposure in human leads to accumulation of acetylcholine in synapses and neuromuscular junction; which results in confusion, convulsion paralysis and in severe cases death may occur. [3] In anaerobic environment CP is 6-7 times more persistent than aerobic environment. [4] One of the possible options for cleanup of CP from contaminated environment is biodegradation using microorganisms as it is cost effective and has less damage for other organisms. Removal of CP may occur due to adsorption instead of metabolism this leads to transfer of pollutant from one environment to another, this needs further investigation. For bioremediation ex situ and/or in situ approaches are used. The use of ex situ treatment has attained researcher’s attention, as it offers considerable benefits over in situ treatment in terms of effective control over various process parameters. [3] Main aim of degradation studies should be efficiency enhancement and reduced hydraulic retention time (HRT). However studies on CP removal using bioreactors have reported longer HRT. Possible reason for long HRT could be use of conventional sources of sludge for bioreactors operation. In contrast use of potential bacterial culture capable of biodegradation of specific compound is gaining importance as it reduces the dependence on conventional sources for biomass production and reduces startup time of biotreatment units. [5] Approaches for bioremediation through environmental modification like nutrient application (additional carbon sources), aeration, or addition of degrader microorganism(s) are gaining importance [6] as it increases potential for field application. Production of metabolites TCP (3, 5, 6-trichloro-2-pyridinol) and TMP (3, 5, 6trichloro-2-methoxypyridine) after CP degradation is viewed critically. These metabolites are more toxic, persistent and soluble in water this leads to widespread contamination. There is dearth of knowledge on complete biodegradation of CP along with its metabolites. • Therefore current study was planned to investigate the CP degradation by the indigenous aerobic bacterial culture Psychrobacter alimentarius T14 enriched from soil samples of agricultural field in specially designed bioreactors. To best of author’s knowledge no study has been reported yet on role of Psychrobacter genus for degradation of any pesticide. • Effect of variation in process parameters on bioreactor performance including optical density (OD600nm), pH, initial CP concentration and HRT was also investigated. • Field application fails because of death of microorganisms in substrate limited conditions; this needs presence of a co-substrate to support microbial community in substrate limited conditions. Therefore attempts were made to make system more efficient and to reduce HRT by supporting microbial growth using co-substrates. • Along with complete removal of parent compound, the formation and decay of metabolites TCP (3, 5, 6-trichloro-2-pyridinol) and TMP (3, 5, 6-trichloro-2methoxypyridine) was also investigated using Gas Chromatography with Electron Capture detector (GC-ECD). As measure of extent of mineralization (dechlorination and 3
•
t
•
ring breakage) total chlorine, nitrogen concentration and chemical oxygen demand (COD) analysis was performed. When studies are applied for biodegradation of commercial products, solvent, stabilizers and emulsifiers present in commercial products effect the biodegradation process. Therefore potential of T14 for degradation of commercial CP along with pure CP was investigated. For complete removal and toxicity reduction integrated approach of bio-photodegradation could be used. Photodegradation of pure CP and commercial CP was also investigated under variable light sources using analytical grade CP and its commercial product.
rip
2. Materials and Methods
pt e
d
M an
us c
Agricultural soil samples were collected in polythene bag (1kg) in December, 2013 from agricultural fields of district Chakwal, Punjab, Pakistan. The sampling site received CP treatment for decades. Collected soil was analyzed for pH, moisture content and texture using standard protocols. Analytical grade standards of Chlorpyrifos (CP), 3, 5, 6-trichloro-2-pyridinol (TCP), 3, 5, 6-trichloro-2-methoxypyridine (TMP) were obtained from Sigma Aldrich Corporation USA and their stock solution were prepared in acetone. MSM (gL-1) K2HPO4 (1.5); KH2PO4 (0.5); NaCl (0.5); MgSO4 (0.2); NH4NO3 (1.0) was used. For isolation 100 mg L−1 CP was added in MSM inoculated with 10 g of soil sample and incubated at 37 ˚C. After 7 days 10ml suspension was added to freshly prepared MSM. After three successive transfers culture suspension was spread on MSM agar plates containing CP using dilute plate technique and 15 well separated bacterial strains were purified. After screening finally selected pure strain T14 was subject to morphological, physiological, biochemical characterization and analytical profile index kit following Bergey’s Manual of Determinative Bacteriology. Molecular identification of T14 was carried out using 16S rRNA gene sequence analysis by previously described method. [7] The obtained sequence was compared to already known gene sequences at National Centre for Biotechnology Information (NCBI) using Basic Logic Alignment Search Tool (BLAST). Obtained sequence was submitted to NCBI Genbank under accession number KT013087.
ce
2.2 Bacterial growth optimization
To investigate effect of environmental and growth conditions batch experiments were conducted under sterile conditions. Growth was monitored at various process parameters including acidic to basic pH range (4.2 - 9.2), temperature (15-45 ˚C) and CP concentration (0400 mg L−1). Optical density (OD600nm)(growth) was monitored regularly by single beam spectrophotometer (HACH) at 600nm.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
2.1 Sample collection and isolation
2.3 Designing and fabrication of batch bioreactor Bench scale bioreactor used in this study consisted of an influent reservoir, aeration unit, settling tank, treated effluent reservoir (Figure 1). Dimensions for the tanks were length (40 cm), internal diameter (24 cm), total volume (14 L), and working volume (10 L). To maintain uniform level of biomass and CP throughout bioreactor a four plate magnetic stirrer (VELP Italy) was used. Bioreactors were fabricated and installed in laboratory (Figure 1). Air supplies were made 4
through air pump, pneumatic tubing network and porous grit stone diffusers (installed at bioreactor bottom). Inlet/outlet ports and effluent valves were part of the system to facilitate feeding and withdrawal. To avoid environmental contamination cotton plugs were used. Bioreactors were operated at ambient temperature. (Figure 1 near here) 2.3.1 Batch operation
us c
M an
d
pt e
2.3.2 Evaluation of CP volatilization/abiotic degradation and adsorption on bacterial cells
ce
Initially for the evaluation of volatilization and abiotic degradation batch system was operated with CP in liquid medium without inoculation (microbial culture was not added). Samples were collected and evaluated for CP concentration upto 48 h. From biodegradation experiments sample was collected to evaluate adsorption on bacterial cells. Sample was centrifuged and supernatant was removed. CP in cells was extracted using ethyl acetate. After addition of solvent, sample was vortex (10 sec) followed by shaking (10 min). [8] Obtained samples were analyzed for adsorbed CP.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
K2HPO4 and KCl 0.5 and 0.25 g L-1 respectively were added in sterilized tap water and CP was added at desired concentrations [5] to make synthetic wastewater. Biomass developed from isolated bacterial strain T14 was used as seeding inoculum .Runs were made to evaluate T14 potential for CP removal in synthetic wastewater (Table 1). In addition the medium was enriched with various co-substrates CS1 (yeast extract 2 g L-1 + synthetic wastewater); CS2 (glucose 2 g L-1 + synthetic wastewater); CS3 (yeast extract 2 g L-1 only) to support microbial community and accelerate CP/ COD removal. Effect of various process parameters on CP biodegradation was investigated by varying optical density (OD600nm) (0.2-1); pH (4.2-9.2); CP concentration (100-500 mg L-1); HRT (24-96 h).Optimum values obtained for one parameter were used in next experiment. For analytical work grab samples were collected periodically. Initial pH was adjusted to optimum value; afterwards the system worked without temperature and pH control, but their values were monitored. Solvents, stabilizers, emulsifier and other compound present in commercial formulations effect biodegradation of active ingredient as compared to pure compound. Therefore biodegradation potential of T14 was investigated for commercial product of CP under brand name “Chlorpyrifos®” (40% EC R.B. Avari Enterprises (Pvt.) Ltd®) was purchased from local market. Bioreactor was operated under same condition as for analytical grade CP using CS1 as co-substrate. Samples were collected periodically for analysis of CP using GC-ECD. (Table 1 near here).
2.3.3 Cyclic operation of bioreactors Bioreactor with CS1 as co-substrate was operated for repeated cycles. For this when CP concentration in reactor was decreased to 8-10mg L-1 CP was again added, and this was repeated for four cycles. 2.4 Photodegradation of pure and commercial CP
5
2.6 Kinetics of CP biodegradation
M an
us c
Wastewater samples from bioreactors were collected periodically and were analyzed for CP , TCP , TMP concentration, COD removal (closed reflux titrimetric method), pH changes (pH meter HACH) and OD600 ( single beam spectrophotometer HACH). Ethyl acetate was used as a solvent for extraction of CP at a sample to solvent ratio 1:2 (v/v), (TCP, TMP) were extracted in acetone at 1:3 (v/v). For CP analysis 1 μl samples after extraction was injected in GC-ECD with fused silica capillary column TRB-1 at conditions described by Zhang et al. [9] Injection port and detector temperature for TCP and TMP detection were 200 and 250 °C, respectively. Initial oven temperature was 70 °C (for 1 min) finally increased to 220 (10° C min1 ). [10] Standard solutions of various concentrations were prepared and observed peak area was plotted against known concentrations to obtain a standard curve. This curve was used for concentration determination of injected samples. Retention time of CP, TCP and TMP were 15.00, 18.85 and 19.12 minutes respectively.
ce
pt e
d
Following algorithms were used for rate constant determination = + (1) = + (2) is first order Where substrate concentration is represented by C, t is degradation period and rate constant and is zero order rate constant. [11] t1/2 values for batch systems were calculated using data obtained from kinetic calculations by following expression [11] . (3) / = Kinetics equation obtained by plotting In C against degradation t, R2, first order rate constant k1 (h-1), / (days) for all batch systems are presented in Table 2.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Experiments for photodegradation of CP were performed with analytical grade and commercial grade CP under ultraviolet UV, Visible and dark. Solution of analytical grade chlorpyrifos (CP) and commercial product of chlorpyrifos (Cm.P) were prepared in ultrapure water. Photo-reactor with three chambers i.e. UV, visible light (200-280 nm) source and one dark. Sample was stirred continuously for even mixing. Light sources were turned on before starting experiment to achieve maximum intensity. One set of treatment was exposed to UV lamp and other to visible light. One set of quartz immersion tube was placed in dark with no light availability. After 5 days sample was collected and remaining CP concentration was determined using GC-ECD. 2.5 Extraction and analysis
2.7 Data analyses
Removal efficiency (%) was calculated using following formula:
∗
(4)
Cinfluent and Ceffluent represents concentration of CP or COD in influent and effluent samples. All experiments were performed in triplicate. Arithmetic mean, standard deviation from three replications was calculated and results were presented in graphical form using Microsoft Excel 2010 with error bars showing standard deviation. 6
3. Results and Discussion 3.1 Isolation and identification of CP degrading bacteria
us c
3.2 Growth response of bacteria to varying environmental conditions
pt e
d
M an
Effect of initial CP concentration on growth of P. alimentarius T14 was studied. Maximum OD at 600 nm was 0.43, 0.56 and 0.73 with100, 200 and 300 mg L-1 CP. At CP 400 mg L-1 optical density (OD600nm) 0.21 was observed which indicates suppressed growth with further increase in concentration (data not shown). MSM without CP inoculated with T14 has shown negligible change in cell density. Growth response of P. alimentarius T14 towards pH change from 4.2 to 9.2 was investigated. P. alimentarius T14 was able to grow at all pH, but achieved maximum growth at pH 7.2. When growth was studied as function of temperature from 15 to 45˚C, increase in growth was observed with increasing temperature. Optimum growth was observed at 37˚C. Interestingly T14 has shown ability to grow at temperature as low as 15˚C and as high as 45˚C (data not shown). Reason for less growth at high substrate concentration could be substrate saturation and/or high TCP accumulation. [3]Substrate inhibition could occur as a result of cell acidification. Maximum growth at pH 7.2 is probably due to result of substrate bioavailability and/or optimum enzyme expression. T14 has shown ability to grow at 45˚C probably because membrane bound proteins have ability to resist denaturation at high temperature. [12]
ce
3.2.1 Volatilization/abiotic degradation of CP Bioreactor without bacterial inoculation was monitored for abiotic losses of CP. 100 mg L-1 CP was reduces to 97.8 mg L-1 in 48 h. However, when the bioreactors were operated with the bulk concentration of the CP, comparative loss was negligible. [3]
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Soil sample has pH was found to be 7.8± 0.1, moisture content 6.44% and texture was sandy loam. All 15 isolated strains demonstrate ability to grow with CP concentration (100 mg L1 ) and use it as carbon source. T14 (non-pigmented) was able to grow at wide pH and temperature range, found to be positive for oxidase, catalase , TDA , voges proskauer (acetoine production) , gelatin liquification , glucose utilization and negative for growth on pseudomonas citramide agar, simmon citrate agar , macconkey agar, motility , gram stain, arginine dihydrolase, lysine decarboxylase, citrate utilization, H2S production, urea hydrolysis, indole production and utilization of mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, lactose. After 16s rRNA gene sequence analysis chlorpyrifos (CP) degrading strain T14 was identified as Psychrobacter alimentarius T14. GenBank accession number for Psychrobacter alimentarius T14 is KT013087.
3.2.2
Effect of variable environmental conditions on CP removal
Effect of optical density (OD600nm) on CP removal was examined from 0.2 to 1 OD at 600 nm. Maximum CP removal was achieved at optical density (OD600nm) 0.8. Results indicated faster acclimation at optical density (OD600nm) 0.8. With the increase in optical density (OD600nm) increase in CP removal was observed, might because cultures with higher inoculum level acclimatize faster. In contrast small population observes longer lag phase. Acclimatization period reflects time required for small population to multiply to a level sufficient to start CP biodegradation. CP removal was observed as a function of pH from 4.2 to 9.2. CP removal 7
rip
t
increased rapidly with increase in pH upto 7.2. No considerable increase in CP removal was observed with further increase in pH (>7.2) (Figure 2b). Maximum growth at pH 7.2 could be result of substrate bioavailability and/or optimum enzyme expression. Another reason for this could be inhibitory effect of pH above or below 7.2 on microbial activity. [3] Effect of initial CP concentration from 100 to 500 mg L-1 was investigated. With increasing initial CP concentration, decrease in removal efficiency was observed. Although T14 was able to tolerate CP upto 500 mg L-1 but a sharp decrease in removal efficiency was observed beyond 300 mg L-1 (Figure 2c). Possible reason could be substrate inhibition because of cell acidification. [12] CP biodegradation was also examined as a function of HRT from 24 to 96 h and results are presented in Figure 2d. Maximum CP removal was observed at HRT 48 h. Doubling HRT has doubled CP removal upto 48h (Figure 2d). Beyond HRT (48 h) increase in CP removal was not significant.
us c
3.3.3
CP adsorption on bacterial cells
3.3.4
M an
The results obtained indicated an adsorption of 8, 6, 7.3 and 5.4% on bacterial cells after 24h of incubation without CS and with CS1, CS2 and CS3. Results inferred CP removal occurred because of metabolism not adsorption. Briceno et al. [8] reported 9-12% adsorption on cells. The use of co-substrates has negligible effect on cell adsorption mechanism. Biodegradation of Chlorpyrifos and TCP/TMP formation
ce
pt e
d
Chlorpyrifos degradation, TCP production and optical density (OD600nm) by T14 are presented in Figure 3 (a, b, c, d). Initial CP concentration (300 mg L-1) was reduced to178, 115, 168, 163 mg L-1 without CS and with CS1, CS2 and CS3 after 24 h respectively. CP concentration in batch system without co-substrate addition after 48 h was 89 mg L-1 showing disappearance of 70.3%. After 48 h 15, 113 and 90 mg L-1 concentration were detected showing a disappearance of 95, 62.3 and 69.8% (Table 2) with CS1, CS2 and CS3 respectively. CP degradation is a rate limiting step and once it is degraded to TCP further metabolism occurs rapidly [10]. Therefore formation of metabolites like TCP, TMP was also evaluated. TCP production was found to be 76.5, 114, 86, 66.8 mg L-1 at 24 h without CS, with CS1, CS2 and CS3 (Figure 3 a, b, c). After which a gradual decrease in TCP concentration was observed due to TCP degradation by T14. At HRT 48 h TCP concentration was 45, 56, 42 mg L-1 with CS1, CS2 and CS3 respectively. Formation and decay of TCP was faster in batch system with CS1 as compared to other systems. TMP was not found in any batch systems. After 24h of incubation 1.04, 0.96 and 0.055 OD (600 nm) was obtained by CS1, CS2 and CS3 respectively (Figure 3 b, c, d).However system without any co-substrate achieved 0.66 OD at 600nm.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
(Figure 2(a), 2(b), 2(c), 2(d) near here)
(Figure 3 (a), 3( b), 3(c), 3(d), 3(e), 3(f) near here)
Yadav et al. [3] reported 91% CP removal efficiency at inlet loading rate of 300 mg L-1dusing Pseudomonas sp. but TCP accumulation affected bioreactor performance. 82% of the initial concentration (2.78µM) of CP was degraded by mixed enriched from indigenous soil in aerobic aqueous environment. [10] 100mg L-1 of CP was reduced to 84% by Pseudomonas sp. in 10 days.[14]. However in current study Psychrobacter alimentarius T14 has shown high removal efficiency (95%) of initial CP concentration (400 mg L-1) along with metabolites TCP and/or 1
8
TMP using yeast extract (CS1) in very short duration (48 h). To our knowledge genus Psychrobacter is yet not reported for pesticide degradation. Organophosphate pesticide degradation involves expression of enzymes like OPH (organophosphorous hydrolase) and OPAA (organophosphorous acid anhydrolase) by microorganism.[13] Biodegradation of CP by P. alimentarius T14 could be attributed to expression of OPH and/or OPAA enzymes.
us c
3.3.5 Fluctuation in pH during bioreactor operation
3.3.6
pt e
d
M an
When CP was added in medium significant decrease in pH was observed. pH range for batch system without addition of any co-substrate was 7.2 -8.8. The range of pH (other than initial fall) with added co-substrate CS1, CS2 and CS3 was 9.08 - 7.2, 7.56 - 4.57 and 8.41-7.17 respectively (Figure 3f). Results imply that addition of co-substrates has effect on pH of the medium. System with glucose as co-substrate (CS2) has shown a sharp decline in pH (4.5), Kontro et al. [15] reported similar results. One possible reason for drop in pH could be the consequence of microbial glucose metabolism [6] which resulted in release of organic acids (excretion products) in extra cellular medium. Another reason for decrease in CP removal efficiency at low pH is fact that acidic pH has inhibitory effect on microbial activity. pH measurement revealed that this parameter is affected by addition of co-substrates. Kinetics of CP biodegradation
ce
Bacterial degradation reactions are usually hyperbolic saturation function of compound (to be degraded) concentration [11] can be expressed as equation 5 .
(5) = In case Ck zero order reaction respectively For first order reactions rate constant k1 is expressed as k1=Rm/k (6) For zero order reaction rate constant reaction ko can be expressed as (7) ko = Rm Where, C is substrate concentration and t is degradation duration. R and Rm is substrate degradation rate and maximum substrate degradation rate respectively. R2 for all systems was > 0.95 which confirms applicability of first order model (Figure 3e). First order rate constant kCS1, kCS2 and kCS3 was 0.0626, 0.023 and 0.026 h-1 respectively (Table 2). Batch system without CS addition rate constant k was 0.026 h-1.The time in which half CP
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Results indicated that yeast extract accelerated CP degradation and supported microbial community; in contrast glucose has inhibited CP removal. Order of CP removal efficiency was CS1>CS2>CS3. Negligible adsorption on the cells was observed this confirms metabolism is reason of CP removal not adsorption. Further TCP formation and then a gradual decrease in its concentration indicated P. alimentarius T14 has ability to utilize TCP, however a slight variation in removal efficiency with co-substrate addition was observed. TMP could not be detected possibly because it was unstable and/or completely mineralized to water soluble products. [10] Results indicated significant application potential of P. alimentarius T14 for management of pesticide contaminated wastewater. However further understanding enzyme mechanism may help complete application of study on large scale.
9
concentration was removed from batch system ( / ) was 11.1 , 30.1, 26.6 and 26.6 h for CS1, CS2 , CS3 and without co-substrate respectively (Table 2). Results obtained inferred that batch system with CS1 was most efficient in CP degradation. (Table 2 near here) 3.3.7 COD removal efficiency
us c
3.3.8. Mineralization
pt e
d
M an
CP is first degraded to TCP and TMP and these metabolites are used as carbon, phosphorus and nitrogen source, this result in mineralization. As chlorine atoms attached to CP ring (o,o-diethylo-3,5,6-trichloro-2-pyridin-yl phosphorothionate) remains attached in TCP and TMP, dechlorination is an indicator that TCP and/or TMP is further mineralized. To determine the extent of mineralization total chlorine and total nitrogen concentration was monitored during biodegradation. Significant decrease in total chlorine and nitrogen concentration was observed. Change in concentration of total chlorine and nitrogen was observed in batch systems which indicated subsequent degradation after TCP and/or TMP production. Change in concentration of total chlorine is an indicator of dechlorination step. Change in nitrogen concentration demonstrates possibility of ring breakage. [10] 3.4 Cyclic operation of bioreactors
ce
When CP concentration was reduced to 8-10 mg L-1 in batch system with CS1, CP was again added and its removal efficiency was monitored for 4 cycles. A slight reduction in CP degradation ability was observed after each cycle (Figure 4). 93% removal efficiency was observed in 4th cycle, this indicates possibility for more cycles. With increase in number of cycle rate constant decreased (kC1=0.07> kC2=0.066> kC3=0.061 >kC4=0.058). Half- life slightly increased with each cycle i.e. 9.9, 10.5, 11.3 and 11.9 h for C1, C2, C3 and C4 respectively. Repeated cycles could only be possible if microbial growth is supported even in substrate limited condition; addition of co-substrate has served this purpose. Raoultella planticola has been successfully used for biodegradation of atrazine for 10 cycles with slight decrease in efficiency.[17]
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Chemical oxygen demand is a measure of organic degradation. For overall estimation of organics in all batch systems variation in COD removal was calculated using equation.COD removal efficiency without co-substrate addition was 79%. Batch system with CS1, CS2 and CS3 achieved 91, 75 and 64% COD removal efficiency respectively. Comparison of effluent COD among batch systems indicates maximum COD removal using CS1 and role of using cosubstrate (wastewater + yeast extract) was significant. T-I4 is a filamentous bacteria and its growth in excess can increase the MLSS concentration. Media was continuously mixed using magnetic stirrer and continuous aeration which resulted in lack of biofilm formation. [16]
(Figure 4 near here) 3.5 Biodegradation of CP in commercial product CP has very less solubility in water; therefore its commercial formulation contains other solvents, stabilizers and emulsifiers etc. in addition to active ingredients. They can affect 10
properties of active ingredients like solubility and bioavailability. Therefore potential of T14 for biodegradation of CP in commercial product was also examined. P. alimentarius T14 has shown considerable removal efficiency (93.7%) for commercial product as compared to control -1 (Figure 5 a). Rate constant kcm.p and half -life (t1/2) were 0.0493(h ) and 14 h respectively (Table 2). R2 (0.95) indicated applicability of first order model (Figure 5b). Results indicated P. alimentarius T14 has potential for field application in the presence of other organic contaminants/solvents/emulsifiers with good removal efficiency in short period of time. (Figure 5(a) and 5(b) near here)
M an
(Figure 6 near here)
us c
Initial concentration was reduced to 43% for commercial product of CP (Cm.P) exposed to UV upto 5days (Figure 6). In contrast only 29% removal was observed in analytical grade CP. Possible reason for higher removal percentage with commercial product could be role of solvents, stabilizers and emulsifiers present in commercial grade pesticide as photocatalyst. For CP in commercial product when exposed to visible light 7% loss was observed. Negligible removal was observed for CP in commercial product in dark and for analytical grade CP in visible and dark.
ce
4. Conclusion
pt e
d
In another experiment sample from CS1 after 48 h was collected and exposed to UV in quartz immersion tube for 24h to decrease bacterial load. After 24h period 1ml sample was spread on agar and incubated at 37˚C. No bacterial colony appeared; this indicated complete removal of bacteria from sample. Lack of bacterial growth after UV exposure confirms germicidal properties of UV for Psychrobacter alimentarius T14. After UV exposure sample was used for toxicity test using seeds of Triticum aestivum (data not shown). Germination percentage, root and shoot length was monitored. Negligible toxicity was observed. Results indicated an integrated approach of biodegradation followed by photodegradation/photolysis could achieve complete removal. Effluent after combined bio- photo treatment could be used for irrigation purposes with reduced chemical or biological toxicity.
Efficacies of P. alimentarius T14 for biodegradation of parent compound CP along with its metabolites TCP and/or TMP to water soluble products (chlorine, nitrogen etc.) in batch bioreactors have been demonstrated for the first time. Under optimum conditions batch system with CS1 has achieved 95% CP and 91% COD removal efficiency in current study. P. alimentarius T14 can utilize TCP. However TCP accumulation has affected bioreactor performance. Bioreactors performance is strongly affected with varying pH, HRT, initial CP concentration and metabolites produced. Half of initial CP (300 mg L-1) was removed within 0.46 days under optimized conditions. Bioreactors could be operated in cyclic manner. Interestingly P. alimentarius T14 has successfully degraded CP in commercial product, with no considerable effect on biodegradation process. However comparison of photodegradation among CP in pure and commercial product revealed role of solvents and stabilizers as photocatalyst.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
3.6 Photodegradation of CP
References 11
[1] Worthing CR. The Pesticide Manual. 6th ed. The British Crop Protection Council: Croydon;1997. [2] Gabaldon JA, Maquieira A, Puchades R. Development of a simple extraction procedure of chlorpyrifos determination in food samples by immunoassay. Talanta. 2007;3:1001-1010. [3] Yadav M, Srivastva N, Singh SR, Upadhyay SN, Dubey SK. Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor. Bioresour. Technol. 2014;165:265269.
us c
[5] Padoley KV, Rajvaidya AS, Subbarao TV, Pandey RA. Biodegradation of pyridine in a completely mixed activated sludge process. Bioresour. Technol. 2006;97:1225–1236. [6] Cycon M, Wójcik M, Piotrowska-Seget Z. Biodegradation of organophosphorus insecticide diazinon by Serratia sp. and Pseudomonas sp. and their use in bioremediation of contaminated soil. Chemosphere. 2009;76:494-501.
M an
[7] Khalid A, Kausar F, Arshad M, Mahmood T, Ahmed I. Accelerated decolorization of reactive azo dyes under saline conditions by bacteria isolated from Arabian seawater sediment. Appl. Microbiol. Biotechnol. 2012;96:1599-606.
ce
pt e
d
[8] Briceno G, Fuentes MS, Palma G, Jorquera MA, Amoroso MJ, Diez MC. Chlorpyrifos biodegradation and 3, 5, 6-trichloro-2-pyridinol production by Actinobacteria isolated from soil. Int. Biodeterior. Biodegradation. 2012;73:1-7. [9] Zhang X, Shen Y, Yu X , Liu X-J. Dissipation of chlorpyrifos and residue analysis in rice, soil and water under paddy field conditions. Ecotoxicol. Environ. Saf. 2012;78:276–280. [10] Tiwari MK, Guha S. Kinetics of biotransformation of chlorpyrifos in aqueous and soil slurry environments.Water Res. 2014;51:73–83. [11] Yang SF, Wang CC, Chems CH. Di-n-butyl phthalate removal by strain Deinococcus sp. R5 in batch reactors. Int. Biodeterior. Biodegradation. 2014;95:55–60. [12] Olson GJ, Brierley JA, Brierley CL. Bioleaching review part B. Appl. Microbiol. Biotechnol. 2003;63:249-257. [13] Abraham J, Silambarasan S, Logeswari P. Simultaneous degradation of organophosphorus and organchlorine pesticides by bacterial consortium. J Taiwan Inst. Chem. Eng. 2014;45:2590– 2596.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
[4] Lu J, Wu L, Newman J, Faber B, Gan J. Degradation of pesticides in nursery recycling pond waters. J Agric Food Chem. 2006;54:2658–2663.
[14] Maya K, Singh RS, Upadhyay SN, Dubey SK . Kinetic analysis reveals bacterial efficacy for biodegradation of chlorpyrifos and its hydrolyzing metabolite TCP. Process Biochem. 2011;46:2130-2136. [15] Kontro M, Lignell U, Hirvonen MR, Nevalainen A. pH effect on 10 Streptomyces spp. growth and sporulation depend on nutrients. Lett Appl Microbiol. 2005;41:32-38. [16] Khan SJ, Ilyas S, Javid S, Javid, S,Visvanathan C, Jegatheesan V. Performance of suspended and attached growth MBR systems in treating high strength synthetic wastewater. Bioresour. Technol. 2011;102:5331–5336. 12
us c M an d pt e ce Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
[17] Swissa N, Nitzan Y, Langzam Y, Cahan R. Atrazine biodegradation by a monoculture of Raoultella planticola isolated from a herbicides wastewater treatment facility. Int. Biodeterior. Biodegradation. 2014; 92:6-11.
13
Values
Co-substrate
Wastewater only CS1 (wastewater+ yeast extract) CS2 (wastewater + glucose) CS3 (Yeast extract) 7. 2 Chlorpyrifos 300 mg L-1 Psychrobacter alimentarius 0.8 48 h
us c M an d pt e ce Ac
Downloaded by [New York University] at 10:55 11 July 2015
Initial pH Pesticide Pesticide concentration Bacterial species Optical density (OD600nm) Hydraulic retention time HRT
rip
Parameter
t
Table 1. Physiochemical/biological parameters of bench scale bioreactor system
14
Kinetic Equation
k1 (h-1)
R2
t1/2 (h)
Removal Efficiency (%)
Without CS CS1 CS2 CS3
In C= 5.7864-0.026t In C= 5.9814-0.0626t In C= 5.75-0.023t In C= 5.77-0.026t
0.026 0.0626 0.023 0.026
0.9792 0.9633 0.9702 0.983
26.6 11.1 30.1 26.6
70.3 95 62.3 69.8
Cycle 1 Cycle 2 Cycle 3 Cycle 4
In C=6.0273-0.07t In C=6.0041-0.066t In C=5.9893-0.061t In C=5.9629-0.0587t
0.07 0.066 0.061 0.058
0.9765 0.9529 0.9584 0.9615
9.9 10.5 11.3 11.9
96.6 96 94 93
Commercial product of CP
In C=6.2167-0.0493t
0.0493
rip
us c 0.9599
M an d pt e ce 15
t
Treatment
Ac
Downloaded by [New York University] at 10:55 11 July 2015
Table 2. Kinetic parameters calculated for CP degradation by Psychrobacter alimentarius T14
14.05
93.7
Figure Captions Figure1. Schematic diagram of bench scale bioreactor (1)Influent reservoir (2) Aeration unit (3) Settling tank(4) Treated effluent reservoir (5) Air pump with Pneumatic tubing network (6) DO meter (7) pH meter (8) Magnetic stirrer (9) Diffusors (10) Sampling port. Figure 2. (a) CP removal efficiency (%) with (a) optical density (OD600nm)(0.2-1) (b) pH (4.29.2) (c) initial CP concentrations (100-500mg L-1) (d) hydraulic Retention time (HRT 24-96 h). Data points are average of three replication and error bar represents standard deviation.
us c
.Figure 4. Removal of Chlorpyrifos and formation of TCP during four cycles with CS1; rate constant k for all cycles (inset).
M an
Figure 5. Relative Change in concentration of CP from commercial product (Cm.P) with CS1(a) Relation of In(CP) and t (b).
ce
pt e
d
Figure 6. Change in concentration of Chlorpyrifos removal efficiency (%) when exposed to ultraviolet light (UV); visible light; dark after 5 days.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Figure 3. Degradation dynamics of Chlorpyrifos CP (mg L-1), TCP formation (mg L-1), optical density (OD600nm) 0NM)0by strain T14 without co-substrates (a), CS1 (b) ,CS2 (c), CS3 (d), relationship of In(CP) and time (e), variation in medium pH (d) ( initial CP 300 mg L-1, initial pH 7.2, HRT 48 h).
16
ce
Ac d
pt e M an us c
Downloaded by [New York University] at 10:55 11 July 2015
Figure1
17
rip
t
60
40
100
1
4.2
6.2
7.2
8.2
9.2
Figure 2 (b)
100
(c)
90
5.2
pH
M an
0.4 0.6 0.8 Optical Density (OD600nm)
Figure 2 (a)
(d)
d
Removal efficiency (%)
90
80
pt e
70 60 50 40
ce
CS1 CS2 CS3 Without CS
100
80 70 60 50
CS1 CS2 CS3 Without CS
40 30
Ac
10
50
20
0.2
20
60
30
30
30
70
us c
50
80
t
70
(b)
CS1 CS2 CS3 Without CS
90 Removal efficiency (%)
80
40
Removal efficiency (%)
Downloaded by [New York University] at 10:55 11 July 2015
Removal efficiency (%)
90
100
(a)
CS1 CS2 CS3 Without CS
rip
100
200 300 400 500 Chlorpyrifos concentration (mg/L)
24
Figure 2 (c)
48
HRT (h)
Figure 2 (d)
18
72
96
0.8
200
0.6
150
0.4
100 0.2
50 0 Time (h)
40
100
0.4
50
0.2
0
20
1
CP TCP OD
(d)
350
200
0.8 0.6
150
0.4
100
0.2
50
300
0
0 0
20
40
60
Time (h)
Figure 3 (d)
CS1
10
CS2
9
CS3 Without CS
R² = 0.9702 R² = 0.983 R² = 0.9796
CS1 CS2 CS3 Without CS
(f)
8 7 6 5 4
R² = 0.9633
4
0.2
50
pH of medium
4.5
0.4
100
60
ce
Ac
In (CP)
5
0.6
150
Figure 3 (c)
(e)
0.8
200
d 40
pt e
20 Time (h)
1
CP TCP OD
250
0
0
5.5
1.2
M an
250
0
60
3 0
10
20 30 Time (h)
40
50
Optical density (OD600nm)
300
6
Time (h)
0
40
us c
1.2
Optical density (OD 600nm)
Concentration (mg/L)
0.6
Figure 3 (b)
(c)
350
0.8
150
0
Figure 3 (a)
1
CP TCP OD
200
60
Concentration (mg/L)
20
1.2
250
0 0
Downloaded by [New York University] at 10:55 11 July 2015
300
t
250
1.4
rip
CP TCP OD
Concentration (mg/L)
300
(b)
350
Optical density (OD600 nm)
1
(a)
Optical density (OD 600nm)
Concentration (mg/L)
350
60
0
20
40 Time (h)
Figure 3 (e)
Figure 3 (f) 19
60
ce
Ac d
pt e M an
CP /TCP concentration (mg/L)
Downloaded by [New York University] at 10:55 11 July 2015
150
100
rip
200
t
350
300
50
0 0 20 40 60 80
us c
0.08
CP 0.06
TCP
0.04
250 0.02
100 Time (h)
Figure 4
20 120
140
160
R² = 0.99
0
180
2
200
k
0 4 6
1 0.9
Cf/Ci
0.8 0.7
Cm.P with T-14
0.6
Cm.P without T-14
0.5 0.4
0.2 0.1 10
20
30 Time (h)
40
50
60
us c
0
M an
Figure 5 (a)
b
7
d
6
pt e
In(CP)
5 4 3
R² = 0.9599
2
ce
1 0
0
Ac
Downloaded by [New York University] at 10:55 11 July 2015
0
rip
t
0.3
10
20
30 Time (h)
Figure 5 (b)
21
40
50
60
ce
Ac d
pt e M an
Downloaded by [New York University] at 10:55 11 July 2015
10
5
rip
0 Analytical grade CP
Figure 6
us c
Removal efficiency (%) 15
t
50
45
40
35
30
25 UV
20 Visible
Dark
Commercial product (Cm.P)
22
Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter Alimentarius T14 Saira Khalid a,* , Imran Hashmi a a
Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering, National University of Sciences and Technology, H-12, Islamabad 44000, Pakistan,
rip
t
Corresponding author: * Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering, National University of Sciences and Technology, H-12, Islamabad 44000, Pakistan. Phone: +92-336-5384103 (E-mail: [email protected] ; [email protected])
us c
2.0
1.5
1.0
0.0
2.5
5.0
7.5
Figure S1 (a) uV(x 100, 000) C hrom at ogram
10. 0 9. 0 8. 0 7. 0 6. 0
M an
0.5
10.0
12.5
15.0
17.5
a
20.0
min
b
5. 0
d
4. 0 3. 0 2. 0
pt e
1. 0 2. 5
5. 0
Figure S1 (b) uV(x 1, 000, 000) C hrom at ogram
5. 0 4. 0 3. 0 2. 0
7. 5
10. 0
12. 5
15. 0
17. 5
20. 0
ce
6. 0
Ac
Downloaded by [New York University] at 10:55 11 July 2015
uV(x1,000,000) C hrom atogram
m in
c
1. 0 0. 0
2. 5
5.0
7. 5
10.0
12. 5
15.0
17. 5
20.0
m in
Figure S1 (c)
Figure S1. Typical GC chromatogram (a) Chlorpyrifos CP (retention time; 15.00min) (b) TCP (retention time; 18.85min) (c) TMP (retention time; 19.12 min).
23
t
120
rip
RE with CS2 RE with CS3
80
us c
RE without CS 60 40 20 0 5
10
M an
COD Removal Efficiency (%)
24
29
34
48
51
55
time (h)
ce
pt e
d
Figure S2. COD removal efficiency (%) in aqueous medium supplemented with (CP 300 mg L-1 , initial pH 7.2, hydraulic retention time (HRT 48 h) inoculated with T14. Data points are average of three replication and error bar represents standard deviation
Ac
Downloaded by [New York University] at 10:55 11 July 2015
RE with CS1 100
24
0.6 0.4 0.2
Chlorine
(b)
0.8 0.6 0.4
t
Relative change in concentration
0.8
1
0.2
Chlorine
Nitrogen
Nitrogen
0 40
0
60
20
us c
Time (h)
M an
20
(c)
Relative change in concentration
0.8 0.6 0.4 0.2 0
20
Time (h)
40
40
60
(d)
0.8 0.6 0.4 Chlorine
0.2
Nitrogen
0 60
ce
0
pt e
Nitrogen
d
Chlorine
Time (h)
1
Relative change in concentration
1
0
20
40 Time (h)
Figure S3. Relative Change in concentration of total chlorine and total nitrogen (a) Without cosubstrate CS, (b) CS1, (c) CS2, (d) CS3.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
0 0
rip
(a)
Relative change in concentration
1
25
60
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter Alimentarius T14 a
Saira Khalid & Imran Hashmi
a
a
Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering, National University of Sciences and Technology, Islamabad 44000, Pakistan Accepted author version posted online: 06 Jul 2015.
Click for updates To cite this article: Saira Khalid & Imran Hashmi (2015): Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter Alimentarius T14, Environmental Technology, DOI: 10.1080/09593330.2015.1069406 To link to this article: http://dx.doi.org/10.1080/09593330.2015.1069406
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis Journal: Environmental Technology DOI: 10.1080/09593330.2015.1069406
Contribution to:
Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter
rip
t
Title:
Environmental Technology
Original article
Order of authors: Saira Khalid a,* , Imran Hashmi a a
Institute of Environmental Sciences and Engineering, School of Civil and
M an
Affiliations:
Environmental Engineering, National University of Sciences and Technology, Islamabad 44000, Pakistan. *
Corresponding author/First author: Saira Khalid
d
Institute of Environmental Sciences and Engineering, School of Civil and
pt e
Environmental Engineering, National University of Sciences and Technology, H-12, Islamabad 44000, Pakistan, Tel.: +923365384103
ce
(E-mail: [email protected] ; [email protected])
Acknowledgement: Authors acknowledge support by Higher Education Commission of Pakistan.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
Article type:
us c
Alimentarius T14
1
Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter Alimentarius T14
ce
pt e
d
M an
us c
Bacteria tolerant to high pesticide concentration could be used for designing an efficient treatment technology. Bacterial strains T14 was isolated from pesticide contaminated soil in mineral salt medium (MSM) and identified as Psychrobacter alimentarius T14 using 16S rRNA gene sequence analysis. Bench scale bioreactor was evaluated for biotreatment of high Chlorpyrifos (CP) concentration using P. alimentarius T14. Effect of various parameters on bioreactor performance was examined and optimum removal was observed at optical density (OD600nm) 0.8, pH; 7.2, CP concentration; 300 mg L-1 and hydraulic retention time (HRT) 48h. At optimum conditions 70.3/79 % of CP/COD removal was achieved in batch bioreactors. In addition P. alimentarius T14 achieved 95/91, 62.3/75, 69.8/64 % CP/COD removal efficiency with addition of CS (co-substrates), CS1 (yeast extract + synthetic wastewater), CS2 (glucose + synthetic wastewater) and CS3 (yeast extract) respectively. Addition of CS1 to bioreactor could accelerate CP removal rate upto many cycles with considerable efficiency. However accumulation of 3, 5, 6-trichloro-2-pyridinol(TCP) affects reactor performance in cyclic mode. First order rate constant k1 0.062 h-1 and t1/2 11.1 h demonstrates fast degradation. Change in concentration of total chlorine and nitrogen could be result of complete mineralization. Photodegradation of CP in commercial product was more than its pure form. Commercial formulation accelerated photodegradation process; however no effect on biodegradation process was observed. After bio-photodegradation negligible toxicity for seeds of Triticum aestivum was observed. Study suggests an efficient treatment of wastewater containing CP and its metabolites in batch bioreactors could be achieved using P. alimentarius. Keywords: Wastewater; Chlorpyrifos; Biodegradation; TCP; Organophosphate Pesticides
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Abstract
2
1.
Introduction
us c
M an
d
pt e
ce
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Dow chemical company in 1965 has introduced a non-systematic insecticide named as Chlorpyrifos (CP). [1] CP is used widely for controlling agricultural and household pests. [2] Inhibition of acetyl cholinesterase after CP exposure in human leads to accumulation of acetylcholine in synapses and neuromuscular junction; which results in confusion, convulsion paralysis and in severe cases death may occur. [3] In anaerobic environment CP is 6-7 times more persistent than aerobic environment. [4] One of the possible options for cleanup of CP from contaminated environment is biodegradation using microorganisms as it is cost effective and has less damage for other organisms. Removal of CP may occur due to adsorption instead of metabolism this leads to transfer of pollutant from one environment to another, this needs further investigation. For bioremediation ex situ and/or in situ approaches are used. The use of ex situ treatment has attained researcher’s attention, as it offers considerable benefits over in situ treatment in terms of effective control over various process parameters. [3] Main aim of degradation studies should be efficiency enhancement and reduced hydraulic retention time (HRT). However studies on CP removal using bioreactors have reported longer HRT. Possible reason for long HRT could be use of conventional sources of sludge for bioreactors operation. In contrast use of potential bacterial culture capable of biodegradation of specific compound is gaining importance as it reduces the dependence on conventional sources for biomass production and reduces startup time of biotreatment units. [5] Approaches for bioremediation through environmental modification like nutrient application (additional carbon sources), aeration, or addition of degrader microorganism(s) are gaining importance [6] as it increases potential for field application. Production of metabolites TCP (3, 5, 6-trichloro-2-pyridinol) and TMP (3, 5, 6trichloro-2-methoxypyridine) after CP degradation is viewed critically. These metabolites are more toxic, persistent and soluble in water this leads to widespread contamination. There is dearth of knowledge on complete biodegradation of CP along with its metabolites. • Therefore current study was planned to investigate the CP degradation by the indigenous aerobic bacterial culture Psychrobacter alimentarius T14 enriched from soil samples of agricultural field in specially designed bioreactors. To best of author’s knowledge no study has been reported yet on role of Psychrobacter genus for degradation of any pesticide. • Effect of variation in process parameters on bioreactor performance including optical density (OD600nm), pH, initial CP concentration and HRT was also investigated. • Field application fails because of death of microorganisms in substrate limited conditions; this needs presence of a co-substrate to support microbial community in substrate limited conditions. Therefore attempts were made to make system more efficient and to reduce HRT by supporting microbial growth using co-substrates. • Along with complete removal of parent compound, the formation and decay of metabolites TCP (3, 5, 6-trichloro-2-pyridinol) and TMP (3, 5, 6-trichloro-2methoxypyridine) was also investigated using Gas Chromatography with Electron Capture detector (GC-ECD). As measure of extent of mineralization (dechlorination and 3
•
t
•
ring breakage) total chlorine, nitrogen concentration and chemical oxygen demand (COD) analysis was performed. When studies are applied for biodegradation of commercial products, solvent, stabilizers and emulsifiers present in commercial products effect the biodegradation process. Therefore potential of T14 for degradation of commercial CP along with pure CP was investigated. For complete removal and toxicity reduction integrated approach of bio-photodegradation could be used. Photodegradation of pure CP and commercial CP was also investigated under variable light sources using analytical grade CP and its commercial product.
rip
2. Materials and Methods
pt e
d
M an
us c
Agricultural soil samples were collected in polythene bag (1kg) in December, 2013 from agricultural fields of district Chakwal, Punjab, Pakistan. The sampling site received CP treatment for decades. Collected soil was analyzed for pH, moisture content and texture using standard protocols. Analytical grade standards of Chlorpyrifos (CP), 3, 5, 6-trichloro-2-pyridinol (TCP), 3, 5, 6-trichloro-2-methoxypyridine (TMP) were obtained from Sigma Aldrich Corporation USA and their stock solution were prepared in acetone. MSM (gL-1) K2HPO4 (1.5); KH2PO4 (0.5); NaCl (0.5); MgSO4 (0.2); NH4NO3 (1.0) was used. For isolation 100 mg L−1 CP was added in MSM inoculated with 10 g of soil sample and incubated at 37 ˚C. After 7 days 10ml suspension was added to freshly prepared MSM. After three successive transfers culture suspension was spread on MSM agar plates containing CP using dilute plate technique and 15 well separated bacterial strains were purified. After screening finally selected pure strain T14 was subject to morphological, physiological, biochemical characterization and analytical profile index kit following Bergey’s Manual of Determinative Bacteriology. Molecular identification of T14 was carried out using 16S rRNA gene sequence analysis by previously described method. [7] The obtained sequence was compared to already known gene sequences at National Centre for Biotechnology Information (NCBI) using Basic Logic Alignment Search Tool (BLAST). Obtained sequence was submitted to NCBI Genbank under accession number KT013087.
ce
2.2 Bacterial growth optimization
To investigate effect of environmental and growth conditions batch experiments were conducted under sterile conditions. Growth was monitored at various process parameters including acidic to basic pH range (4.2 - 9.2), temperature (15-45 ˚C) and CP concentration (0400 mg L−1). Optical density (OD600nm)(growth) was monitored regularly by single beam spectrophotometer (HACH) at 600nm.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
2.1 Sample collection and isolation
2.3 Designing and fabrication of batch bioreactor Bench scale bioreactor used in this study consisted of an influent reservoir, aeration unit, settling tank, treated effluent reservoir (Figure 1). Dimensions for the tanks were length (40 cm), internal diameter (24 cm), total volume (14 L), and working volume (10 L). To maintain uniform level of biomass and CP throughout bioreactor a four plate magnetic stirrer (VELP Italy) was used. Bioreactors were fabricated and installed in laboratory (Figure 1). Air supplies were made 4
through air pump, pneumatic tubing network and porous grit stone diffusers (installed at bioreactor bottom). Inlet/outlet ports and effluent valves were part of the system to facilitate feeding and withdrawal. To avoid environmental contamination cotton plugs were used. Bioreactors were operated at ambient temperature. (Figure 1 near here) 2.3.1 Batch operation
us c
M an
d
pt e
2.3.2 Evaluation of CP volatilization/abiotic degradation and adsorption on bacterial cells
ce
Initially for the evaluation of volatilization and abiotic degradation batch system was operated with CP in liquid medium without inoculation (microbial culture was not added). Samples were collected and evaluated for CP concentration upto 48 h. From biodegradation experiments sample was collected to evaluate adsorption on bacterial cells. Sample was centrifuged and supernatant was removed. CP in cells was extracted using ethyl acetate. After addition of solvent, sample was vortex (10 sec) followed by shaking (10 min). [8] Obtained samples were analyzed for adsorbed CP.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
K2HPO4 and KCl 0.5 and 0.25 g L-1 respectively were added in sterilized tap water and CP was added at desired concentrations [5] to make synthetic wastewater. Biomass developed from isolated bacterial strain T14 was used as seeding inoculum .Runs were made to evaluate T14 potential for CP removal in synthetic wastewater (Table 1). In addition the medium was enriched with various co-substrates CS1 (yeast extract 2 g L-1 + synthetic wastewater); CS2 (glucose 2 g L-1 + synthetic wastewater); CS3 (yeast extract 2 g L-1 only) to support microbial community and accelerate CP/ COD removal. Effect of various process parameters on CP biodegradation was investigated by varying optical density (OD600nm) (0.2-1); pH (4.2-9.2); CP concentration (100-500 mg L-1); HRT (24-96 h).Optimum values obtained for one parameter were used in next experiment. For analytical work grab samples were collected periodically. Initial pH was adjusted to optimum value; afterwards the system worked without temperature and pH control, but their values were monitored. Solvents, stabilizers, emulsifier and other compound present in commercial formulations effect biodegradation of active ingredient as compared to pure compound. Therefore biodegradation potential of T14 was investigated for commercial product of CP under brand name “Chlorpyrifos®” (40% EC R.B. Avari Enterprises (Pvt.) Ltd®) was purchased from local market. Bioreactor was operated under same condition as for analytical grade CP using CS1 as co-substrate. Samples were collected periodically for analysis of CP using GC-ECD. (Table 1 near here).
2.3.3 Cyclic operation of bioreactors Bioreactor with CS1 as co-substrate was operated for repeated cycles. For this when CP concentration in reactor was decreased to 8-10mg L-1 CP was again added, and this was repeated for four cycles. 2.4 Photodegradation of pure and commercial CP
5
2.6 Kinetics of CP biodegradation
M an
us c
Wastewater samples from bioreactors were collected periodically and were analyzed for CP , TCP , TMP concentration, COD removal (closed reflux titrimetric method), pH changes (pH meter HACH) and OD600 ( single beam spectrophotometer HACH). Ethyl acetate was used as a solvent for extraction of CP at a sample to solvent ratio 1:2 (v/v), (TCP, TMP) were extracted in acetone at 1:3 (v/v). For CP analysis 1 μl samples after extraction was injected in GC-ECD with fused silica capillary column TRB-1 at conditions described by Zhang et al. [9] Injection port and detector temperature for TCP and TMP detection were 200 and 250 °C, respectively. Initial oven temperature was 70 °C (for 1 min) finally increased to 220 (10° C min1 ). [10] Standard solutions of various concentrations were prepared and observed peak area was plotted against known concentrations to obtain a standard curve. This curve was used for concentration determination of injected samples. Retention time of CP, TCP and TMP were 15.00, 18.85 and 19.12 minutes respectively.
ce
pt e
d
Following algorithms were used for rate constant determination = + (1) = + (2) is first order Where substrate concentration is represented by C, t is degradation period and rate constant and is zero order rate constant. [11] t1/2 values for batch systems were calculated using data obtained from kinetic calculations by following expression [11] . (3) / = Kinetics equation obtained by plotting In C against degradation t, R2, first order rate constant k1 (h-1), / (days) for all batch systems are presented in Table 2.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Experiments for photodegradation of CP were performed with analytical grade and commercial grade CP under ultraviolet UV, Visible and dark. Solution of analytical grade chlorpyrifos (CP) and commercial product of chlorpyrifos (Cm.P) were prepared in ultrapure water. Photo-reactor with three chambers i.e. UV, visible light (200-280 nm) source and one dark. Sample was stirred continuously for even mixing. Light sources were turned on before starting experiment to achieve maximum intensity. One set of treatment was exposed to UV lamp and other to visible light. One set of quartz immersion tube was placed in dark with no light availability. After 5 days sample was collected and remaining CP concentration was determined using GC-ECD. 2.5 Extraction and analysis
2.7 Data analyses
Removal efficiency (%) was calculated using following formula:
∗
(4)
Cinfluent and Ceffluent represents concentration of CP or COD in influent and effluent samples. All experiments were performed in triplicate. Arithmetic mean, standard deviation from three replications was calculated and results were presented in graphical form using Microsoft Excel 2010 with error bars showing standard deviation. 6
3. Results and Discussion 3.1 Isolation and identification of CP degrading bacteria
us c
3.2 Growth response of bacteria to varying environmental conditions
pt e
d
M an
Effect of initial CP concentration on growth of P. alimentarius T14 was studied. Maximum OD at 600 nm was 0.43, 0.56 and 0.73 with100, 200 and 300 mg L-1 CP. At CP 400 mg L-1 optical density (OD600nm) 0.21 was observed which indicates suppressed growth with further increase in concentration (data not shown). MSM without CP inoculated with T14 has shown negligible change in cell density. Growth response of P. alimentarius T14 towards pH change from 4.2 to 9.2 was investigated. P. alimentarius T14 was able to grow at all pH, but achieved maximum growth at pH 7.2. When growth was studied as function of temperature from 15 to 45˚C, increase in growth was observed with increasing temperature. Optimum growth was observed at 37˚C. Interestingly T14 has shown ability to grow at temperature as low as 15˚C and as high as 45˚C (data not shown). Reason for less growth at high substrate concentration could be substrate saturation and/or high TCP accumulation. [3]Substrate inhibition could occur as a result of cell acidification. Maximum growth at pH 7.2 is probably due to result of substrate bioavailability and/or optimum enzyme expression. T14 has shown ability to grow at 45˚C probably because membrane bound proteins have ability to resist denaturation at high temperature. [12]
ce
3.2.1 Volatilization/abiotic degradation of CP Bioreactor without bacterial inoculation was monitored for abiotic losses of CP. 100 mg L-1 CP was reduces to 97.8 mg L-1 in 48 h. However, when the bioreactors were operated with the bulk concentration of the CP, comparative loss was negligible. [3]
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Soil sample has pH was found to be 7.8± 0.1, moisture content 6.44% and texture was sandy loam. All 15 isolated strains demonstrate ability to grow with CP concentration (100 mg L1 ) and use it as carbon source. T14 (non-pigmented) was able to grow at wide pH and temperature range, found to be positive for oxidase, catalase , TDA , voges proskauer (acetoine production) , gelatin liquification , glucose utilization and negative for growth on pseudomonas citramide agar, simmon citrate agar , macconkey agar, motility , gram stain, arginine dihydrolase, lysine decarboxylase, citrate utilization, H2S production, urea hydrolysis, indole production and utilization of mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, lactose. After 16s rRNA gene sequence analysis chlorpyrifos (CP) degrading strain T14 was identified as Psychrobacter alimentarius T14. GenBank accession number for Psychrobacter alimentarius T14 is KT013087.
3.2.2
Effect of variable environmental conditions on CP removal
Effect of optical density (OD600nm) on CP removal was examined from 0.2 to 1 OD at 600 nm. Maximum CP removal was achieved at optical density (OD600nm) 0.8. Results indicated faster acclimation at optical density (OD600nm) 0.8. With the increase in optical density (OD600nm) increase in CP removal was observed, might because cultures with higher inoculum level acclimatize faster. In contrast small population observes longer lag phase. Acclimatization period reflects time required for small population to multiply to a level sufficient to start CP biodegradation. CP removal was observed as a function of pH from 4.2 to 9.2. CP removal 7
rip
t
increased rapidly with increase in pH upto 7.2. No considerable increase in CP removal was observed with further increase in pH (>7.2) (Figure 2b). Maximum growth at pH 7.2 could be result of substrate bioavailability and/or optimum enzyme expression. Another reason for this could be inhibitory effect of pH above or below 7.2 on microbial activity. [3] Effect of initial CP concentration from 100 to 500 mg L-1 was investigated. With increasing initial CP concentration, decrease in removal efficiency was observed. Although T14 was able to tolerate CP upto 500 mg L-1 but a sharp decrease in removal efficiency was observed beyond 300 mg L-1 (Figure 2c). Possible reason could be substrate inhibition because of cell acidification. [12] CP biodegradation was also examined as a function of HRT from 24 to 96 h and results are presented in Figure 2d. Maximum CP removal was observed at HRT 48 h. Doubling HRT has doubled CP removal upto 48h (Figure 2d). Beyond HRT (48 h) increase in CP removal was not significant.
us c
3.3.3
CP adsorption on bacterial cells
3.3.4
M an
The results obtained indicated an adsorption of 8, 6, 7.3 and 5.4% on bacterial cells after 24h of incubation without CS and with CS1, CS2 and CS3. Results inferred CP removal occurred because of metabolism not adsorption. Briceno et al. [8] reported 9-12% adsorption on cells. The use of co-substrates has negligible effect on cell adsorption mechanism. Biodegradation of Chlorpyrifos and TCP/TMP formation
ce
pt e
d
Chlorpyrifos degradation, TCP production and optical density (OD600nm) by T14 are presented in Figure 3 (a, b, c, d). Initial CP concentration (300 mg L-1) was reduced to178, 115, 168, 163 mg L-1 without CS and with CS1, CS2 and CS3 after 24 h respectively. CP concentration in batch system without co-substrate addition after 48 h was 89 mg L-1 showing disappearance of 70.3%. After 48 h 15, 113 and 90 mg L-1 concentration were detected showing a disappearance of 95, 62.3 and 69.8% (Table 2) with CS1, CS2 and CS3 respectively. CP degradation is a rate limiting step and once it is degraded to TCP further metabolism occurs rapidly [10]. Therefore formation of metabolites like TCP, TMP was also evaluated. TCP production was found to be 76.5, 114, 86, 66.8 mg L-1 at 24 h without CS, with CS1, CS2 and CS3 (Figure 3 a, b, c). After which a gradual decrease in TCP concentration was observed due to TCP degradation by T14. At HRT 48 h TCP concentration was 45, 56, 42 mg L-1 with CS1, CS2 and CS3 respectively. Formation and decay of TCP was faster in batch system with CS1 as compared to other systems. TMP was not found in any batch systems. After 24h of incubation 1.04, 0.96 and 0.055 OD (600 nm) was obtained by CS1, CS2 and CS3 respectively (Figure 3 b, c, d).However system without any co-substrate achieved 0.66 OD at 600nm.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
(Figure 2(a), 2(b), 2(c), 2(d) near here)
(Figure 3 (a), 3( b), 3(c), 3(d), 3(e), 3(f) near here)
Yadav et al. [3] reported 91% CP removal efficiency at inlet loading rate of 300 mg L-1dusing Pseudomonas sp. but TCP accumulation affected bioreactor performance. 82% of the initial concentration (2.78µM) of CP was degraded by mixed enriched from indigenous soil in aerobic aqueous environment. [10] 100mg L-1 of CP was reduced to 84% by Pseudomonas sp. in 10 days.[14]. However in current study Psychrobacter alimentarius T14 has shown high removal efficiency (95%) of initial CP concentration (400 mg L-1) along with metabolites TCP and/or 1
8
TMP using yeast extract (CS1) in very short duration (48 h). To our knowledge genus Psychrobacter is yet not reported for pesticide degradation. Organophosphate pesticide degradation involves expression of enzymes like OPH (organophosphorous hydrolase) and OPAA (organophosphorous acid anhydrolase) by microorganism.[13] Biodegradation of CP by P. alimentarius T14 could be attributed to expression of OPH and/or OPAA enzymes.
us c
3.3.5 Fluctuation in pH during bioreactor operation
3.3.6
pt e
d
M an
When CP was added in medium significant decrease in pH was observed. pH range for batch system without addition of any co-substrate was 7.2 -8.8. The range of pH (other than initial fall) with added co-substrate CS1, CS2 and CS3 was 9.08 - 7.2, 7.56 - 4.57 and 8.41-7.17 respectively (Figure 3f). Results imply that addition of co-substrates has effect on pH of the medium. System with glucose as co-substrate (CS2) has shown a sharp decline in pH (4.5), Kontro et al. [15] reported similar results. One possible reason for drop in pH could be the consequence of microbial glucose metabolism [6] which resulted in release of organic acids (excretion products) in extra cellular medium. Another reason for decrease in CP removal efficiency at low pH is fact that acidic pH has inhibitory effect on microbial activity. pH measurement revealed that this parameter is affected by addition of co-substrates. Kinetics of CP biodegradation
ce
Bacterial degradation reactions are usually hyperbolic saturation function of compound (to be degraded) concentration [11] can be expressed as equation 5 .
(5) = In case Ck zero order reaction respectively For first order reactions rate constant k1 is expressed as k1=Rm/k (6) For zero order reaction rate constant reaction ko can be expressed as (7) ko = Rm Where, C is substrate concentration and t is degradation duration. R and Rm is substrate degradation rate and maximum substrate degradation rate respectively. R2 for all systems was > 0.95 which confirms applicability of first order model (Figure 3e). First order rate constant kCS1, kCS2 and kCS3 was 0.0626, 0.023 and 0.026 h-1 respectively (Table 2). Batch system without CS addition rate constant k was 0.026 h-1.The time in which half CP
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Results indicated that yeast extract accelerated CP degradation and supported microbial community; in contrast glucose has inhibited CP removal. Order of CP removal efficiency was CS1>CS2>CS3. Negligible adsorption on the cells was observed this confirms metabolism is reason of CP removal not adsorption. Further TCP formation and then a gradual decrease in its concentration indicated P. alimentarius T14 has ability to utilize TCP, however a slight variation in removal efficiency with co-substrate addition was observed. TMP could not be detected possibly because it was unstable and/or completely mineralized to water soluble products. [10] Results indicated significant application potential of P. alimentarius T14 for management of pesticide contaminated wastewater. However further understanding enzyme mechanism may help complete application of study on large scale.
9
concentration was removed from batch system ( / ) was 11.1 , 30.1, 26.6 and 26.6 h for CS1, CS2 , CS3 and without co-substrate respectively (Table 2). Results obtained inferred that batch system with CS1 was most efficient in CP degradation. (Table 2 near here) 3.3.7 COD removal efficiency
us c
3.3.8. Mineralization
pt e
d
M an
CP is first degraded to TCP and TMP and these metabolites are used as carbon, phosphorus and nitrogen source, this result in mineralization. As chlorine atoms attached to CP ring (o,o-diethylo-3,5,6-trichloro-2-pyridin-yl phosphorothionate) remains attached in TCP and TMP, dechlorination is an indicator that TCP and/or TMP is further mineralized. To determine the extent of mineralization total chlorine and total nitrogen concentration was monitored during biodegradation. Significant decrease in total chlorine and nitrogen concentration was observed. Change in concentration of total chlorine and nitrogen was observed in batch systems which indicated subsequent degradation after TCP and/or TMP production. Change in concentration of total chlorine is an indicator of dechlorination step. Change in nitrogen concentration demonstrates possibility of ring breakage. [10] 3.4 Cyclic operation of bioreactors
ce
When CP concentration was reduced to 8-10 mg L-1 in batch system with CS1, CP was again added and its removal efficiency was monitored for 4 cycles. A slight reduction in CP degradation ability was observed after each cycle (Figure 4). 93% removal efficiency was observed in 4th cycle, this indicates possibility for more cycles. With increase in number of cycle rate constant decreased (kC1=0.07> kC2=0.066> kC3=0.061 >kC4=0.058). Half- life slightly increased with each cycle i.e. 9.9, 10.5, 11.3 and 11.9 h for C1, C2, C3 and C4 respectively. Repeated cycles could only be possible if microbial growth is supported even in substrate limited condition; addition of co-substrate has served this purpose. Raoultella planticola has been successfully used for biodegradation of atrazine for 10 cycles with slight decrease in efficiency.[17]
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Chemical oxygen demand is a measure of organic degradation. For overall estimation of organics in all batch systems variation in COD removal was calculated using equation.COD removal efficiency without co-substrate addition was 79%. Batch system with CS1, CS2 and CS3 achieved 91, 75 and 64% COD removal efficiency respectively. Comparison of effluent COD among batch systems indicates maximum COD removal using CS1 and role of using cosubstrate (wastewater + yeast extract) was significant. T-I4 is a filamentous bacteria and its growth in excess can increase the MLSS concentration. Media was continuously mixed using magnetic stirrer and continuous aeration which resulted in lack of biofilm formation. [16]
(Figure 4 near here) 3.5 Biodegradation of CP in commercial product CP has very less solubility in water; therefore its commercial formulation contains other solvents, stabilizers and emulsifiers etc. in addition to active ingredients. They can affect 10
properties of active ingredients like solubility and bioavailability. Therefore potential of T14 for biodegradation of CP in commercial product was also examined. P. alimentarius T14 has shown considerable removal efficiency (93.7%) for commercial product as compared to control -1 (Figure 5 a). Rate constant kcm.p and half -life (t1/2) were 0.0493(h ) and 14 h respectively (Table 2). R2 (0.95) indicated applicability of first order model (Figure 5b). Results indicated P. alimentarius T14 has potential for field application in the presence of other organic contaminants/solvents/emulsifiers with good removal efficiency in short period of time. (Figure 5(a) and 5(b) near here)
M an
(Figure 6 near here)
us c
Initial concentration was reduced to 43% for commercial product of CP (Cm.P) exposed to UV upto 5days (Figure 6). In contrast only 29% removal was observed in analytical grade CP. Possible reason for higher removal percentage with commercial product could be role of solvents, stabilizers and emulsifiers present in commercial grade pesticide as photocatalyst. For CP in commercial product when exposed to visible light 7% loss was observed. Negligible removal was observed for CP in commercial product in dark and for analytical grade CP in visible and dark.
ce
4. Conclusion
pt e
d
In another experiment sample from CS1 after 48 h was collected and exposed to UV in quartz immersion tube for 24h to decrease bacterial load. After 24h period 1ml sample was spread on agar and incubated at 37˚C. No bacterial colony appeared; this indicated complete removal of bacteria from sample. Lack of bacterial growth after UV exposure confirms germicidal properties of UV for Psychrobacter alimentarius T14. After UV exposure sample was used for toxicity test using seeds of Triticum aestivum (data not shown). Germination percentage, root and shoot length was monitored. Negligible toxicity was observed. Results indicated an integrated approach of biodegradation followed by photodegradation/photolysis could achieve complete removal. Effluent after combined bio- photo treatment could be used for irrigation purposes with reduced chemical or biological toxicity.
Efficacies of P. alimentarius T14 for biodegradation of parent compound CP along with its metabolites TCP and/or TMP to water soluble products (chlorine, nitrogen etc.) in batch bioreactors have been demonstrated for the first time. Under optimum conditions batch system with CS1 has achieved 95% CP and 91% COD removal efficiency in current study. P. alimentarius T14 can utilize TCP. However TCP accumulation has affected bioreactor performance. Bioreactors performance is strongly affected with varying pH, HRT, initial CP concentration and metabolites produced. Half of initial CP (300 mg L-1) was removed within 0.46 days under optimized conditions. Bioreactors could be operated in cyclic manner. Interestingly P. alimentarius T14 has successfully degraded CP in commercial product, with no considerable effect on biodegradation process. However comparison of photodegradation among CP in pure and commercial product revealed role of solvents and stabilizers as photocatalyst.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
3.6 Photodegradation of CP
References 11
[1] Worthing CR. The Pesticide Manual. 6th ed. The British Crop Protection Council: Croydon;1997. [2] Gabaldon JA, Maquieira A, Puchades R. Development of a simple extraction procedure of chlorpyrifos determination in food samples by immunoassay. Talanta. 2007;3:1001-1010. [3] Yadav M, Srivastva N, Singh SR, Upadhyay SN, Dubey SK. Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor. Bioresour. Technol. 2014;165:265269.
us c
[5] Padoley KV, Rajvaidya AS, Subbarao TV, Pandey RA. Biodegradation of pyridine in a completely mixed activated sludge process. Bioresour. Technol. 2006;97:1225–1236. [6] Cycon M, Wójcik M, Piotrowska-Seget Z. Biodegradation of organophosphorus insecticide diazinon by Serratia sp. and Pseudomonas sp. and their use in bioremediation of contaminated soil. Chemosphere. 2009;76:494-501.
M an
[7] Khalid A, Kausar F, Arshad M, Mahmood T, Ahmed I. Accelerated decolorization of reactive azo dyes under saline conditions by bacteria isolated from Arabian seawater sediment. Appl. Microbiol. Biotechnol. 2012;96:1599-606.
ce
pt e
d
[8] Briceno G, Fuentes MS, Palma G, Jorquera MA, Amoroso MJ, Diez MC. Chlorpyrifos biodegradation and 3, 5, 6-trichloro-2-pyridinol production by Actinobacteria isolated from soil. Int. Biodeterior. Biodegradation. 2012;73:1-7. [9] Zhang X, Shen Y, Yu X , Liu X-J. Dissipation of chlorpyrifos and residue analysis in rice, soil and water under paddy field conditions. Ecotoxicol. Environ. Saf. 2012;78:276–280. [10] Tiwari MK, Guha S. Kinetics of biotransformation of chlorpyrifos in aqueous and soil slurry environments.Water Res. 2014;51:73–83. [11] Yang SF, Wang CC, Chems CH. Di-n-butyl phthalate removal by strain Deinococcus sp. R5 in batch reactors. Int. Biodeterior. Biodegradation. 2014;95:55–60. [12] Olson GJ, Brierley JA, Brierley CL. Bioleaching review part B. Appl. Microbiol. Biotechnol. 2003;63:249-257. [13] Abraham J, Silambarasan S, Logeswari P. Simultaneous degradation of organophosphorus and organchlorine pesticides by bacterial consortium. J Taiwan Inst. Chem. Eng. 2014;45:2590– 2596.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
[4] Lu J, Wu L, Newman J, Faber B, Gan J. Degradation of pesticides in nursery recycling pond waters. J Agric Food Chem. 2006;54:2658–2663.
[14] Maya K, Singh RS, Upadhyay SN, Dubey SK . Kinetic analysis reveals bacterial efficacy for biodegradation of chlorpyrifos and its hydrolyzing metabolite TCP. Process Biochem. 2011;46:2130-2136. [15] Kontro M, Lignell U, Hirvonen MR, Nevalainen A. pH effect on 10 Streptomyces spp. growth and sporulation depend on nutrients. Lett Appl Microbiol. 2005;41:32-38. [16] Khan SJ, Ilyas S, Javid S, Javid, S,Visvanathan C, Jegatheesan V. Performance of suspended and attached growth MBR systems in treating high strength synthetic wastewater. Bioresour. Technol. 2011;102:5331–5336. 12
us c M an d pt e ce Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
[17] Swissa N, Nitzan Y, Langzam Y, Cahan R. Atrazine biodegradation by a monoculture of Raoultella planticola isolated from a herbicides wastewater treatment facility. Int. Biodeterior. Biodegradation. 2014; 92:6-11.
13
Values
Co-substrate
Wastewater only CS1 (wastewater+ yeast extract) CS2 (wastewater + glucose) CS3 (Yeast extract) 7. 2 Chlorpyrifos 300 mg L-1 Psychrobacter alimentarius 0.8 48 h
us c M an d pt e ce Ac
Downloaded by [New York University] at 10:55 11 July 2015
Initial pH Pesticide Pesticide concentration Bacterial species Optical density (OD600nm) Hydraulic retention time HRT
rip
Parameter
t
Table 1. Physiochemical/biological parameters of bench scale bioreactor system
14
Kinetic Equation
k1 (h-1)
R2
t1/2 (h)
Removal Efficiency (%)
Without CS CS1 CS2 CS3
In C= 5.7864-0.026t In C= 5.9814-0.0626t In C= 5.75-0.023t In C= 5.77-0.026t
0.026 0.0626 0.023 0.026
0.9792 0.9633 0.9702 0.983
26.6 11.1 30.1 26.6
70.3 95 62.3 69.8
Cycle 1 Cycle 2 Cycle 3 Cycle 4
In C=6.0273-0.07t In C=6.0041-0.066t In C=5.9893-0.061t In C=5.9629-0.0587t
0.07 0.066 0.061 0.058
0.9765 0.9529 0.9584 0.9615
9.9 10.5 11.3 11.9
96.6 96 94 93
Commercial product of CP
In C=6.2167-0.0493t
0.0493
rip
us c 0.9599
M an d pt e ce 15
t
Treatment
Ac
Downloaded by [New York University] at 10:55 11 July 2015
Table 2. Kinetic parameters calculated for CP degradation by Psychrobacter alimentarius T14
14.05
93.7
Figure Captions Figure1. Schematic diagram of bench scale bioreactor (1)Influent reservoir (2) Aeration unit (3) Settling tank(4) Treated effluent reservoir (5) Air pump with Pneumatic tubing network (6) DO meter (7) pH meter (8) Magnetic stirrer (9) Diffusors (10) Sampling port. Figure 2. (a) CP removal efficiency (%) with (a) optical density (OD600nm)(0.2-1) (b) pH (4.29.2) (c) initial CP concentrations (100-500mg L-1) (d) hydraulic Retention time (HRT 24-96 h). Data points are average of three replication and error bar represents standard deviation.
us c
.Figure 4. Removal of Chlorpyrifos and formation of TCP during four cycles with CS1; rate constant k for all cycles (inset).
M an
Figure 5. Relative Change in concentration of CP from commercial product (Cm.P) with CS1(a) Relation of In(CP) and t (b).
ce
pt e
d
Figure 6. Change in concentration of Chlorpyrifos removal efficiency (%) when exposed to ultraviolet light (UV); visible light; dark after 5 days.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
rip
t
Figure 3. Degradation dynamics of Chlorpyrifos CP (mg L-1), TCP formation (mg L-1), optical density (OD600nm) 0NM)0by strain T14 without co-substrates (a), CS1 (b) ,CS2 (c), CS3 (d), relationship of In(CP) and time (e), variation in medium pH (d) ( initial CP 300 mg L-1, initial pH 7.2, HRT 48 h).
16
ce
Ac d
pt e M an us c
Downloaded by [New York University] at 10:55 11 July 2015
Figure1
17
rip
t
60
40
100
1
4.2
6.2
7.2
8.2
9.2
Figure 2 (b)
100
(c)
90
5.2
pH
M an
0.4 0.6 0.8 Optical Density (OD600nm)
Figure 2 (a)
(d)
d
Removal efficiency (%)
90
80
pt e
70 60 50 40
ce
CS1 CS2 CS3 Without CS
100
80 70 60 50
CS1 CS2 CS3 Without CS
40 30
Ac
10
50
20
0.2
20
60
30
30
30
70
us c
50
80
t
70
(b)
CS1 CS2 CS3 Without CS
90 Removal efficiency (%)
80
40
Removal efficiency (%)
Downloaded by [New York University] at 10:55 11 July 2015
Removal efficiency (%)
90
100
(a)
CS1 CS2 CS3 Without CS
rip
100
200 300 400 500 Chlorpyrifos concentration (mg/L)
24
Figure 2 (c)
48
HRT (h)
Figure 2 (d)
18
72
96
0.8
200
0.6
150
0.4
100 0.2
50 0 Time (h)
40
100
0.4
50
0.2
0
20
1
CP TCP OD
(d)
350
200
0.8 0.6
150
0.4
100
0.2
50
300
0
0 0
20
40
60
Time (h)
Figure 3 (d)
CS1
10
CS2
9
CS3 Without CS
R² = 0.9702 R² = 0.983 R² = 0.9796
CS1 CS2 CS3 Without CS
(f)
8 7 6 5 4
R² = 0.9633
4
0.2
50
pH of medium
4.5
0.4
100
60
ce
Ac
In (CP)
5
0.6
150
Figure 3 (c)
(e)
0.8
200
d 40
pt e
20 Time (h)
1
CP TCP OD
250
0
0
5.5
1.2
M an
250
0
60
3 0
10
20 30 Time (h)
40
50
Optical density (OD600nm)
300
6
Time (h)
0
40
us c
1.2
Optical density (OD 600nm)
Concentration (mg/L)
0.6
Figure 3 (b)
(c)
350
0.8
150
0
Figure 3 (a)
1
CP TCP OD
200
60
Concentration (mg/L)
20
1.2
250
0 0
Downloaded by [New York University] at 10:55 11 July 2015
300
t
250
1.4
rip
CP TCP OD
Concentration (mg/L)
300
(b)
350
Optical density (OD600 nm)
1
(a)
Optical density (OD 600nm)
Concentration (mg/L)
350
60
0
20
40 Time (h)
Figure 3 (e)
Figure 3 (f) 19
60
ce
Ac d
pt e M an
CP /TCP concentration (mg/L)
Downloaded by [New York University] at 10:55 11 July 2015
150
100
rip
200
t
350
300
50
0 0 20 40 60 80
us c
0.08
CP 0.06
TCP
0.04
250 0.02
100 Time (h)
Figure 4
20 120
140
160
R² = 0.99
0
180
2
200
k
0 4 6
1 0.9
Cf/Ci
0.8 0.7
Cm.P with T-14
0.6
Cm.P without T-14
0.5 0.4
0.2 0.1 10
20
30 Time (h)
40
50
60
us c
0
M an
Figure 5 (a)
b
7
d
6
pt e
In(CP)
5 4 3
R² = 0.9599
2
ce
1 0
0
Ac
Downloaded by [New York University] at 10:55 11 July 2015
0
rip
t
0.3
10
20
30 Time (h)
Figure 5 (b)
21
40
50
60
ce
Ac d
pt e M an
Downloaded by [New York University] at 10:55 11 July 2015
10
5
rip
0 Analytical grade CP
Figure 6
us c
Removal efficiency (%) 15
t
50
45
40
35
30
25 UV
20 Visible
Dark
Commercial product (Cm.P)
22
Biotreatment of Chlorpyrifos in a Bench Scale Bioreactor using Psychrobacter Alimentarius T14 Saira Khalid a,* , Imran Hashmi a a
Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering, National University of Sciences and Technology, H-12, Islamabad 44000, Pakistan,
rip
t
Corresponding author: * Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering, National University of Sciences and Technology, H-12, Islamabad 44000, Pakistan. Phone: +92-336-5384103 (E-mail: [email protected] ; [email protected])
us c
2.0
1.5
1.0
0.0
2.5
5.0
7.5
Figure S1 (a) uV(x 100, 000) C hrom at ogram
10. 0 9. 0 8. 0 7. 0 6. 0
M an
0.5
10.0
12.5
15.0
17.5
a
20.0
min
b
5. 0
d
4. 0 3. 0 2. 0
pt e
1. 0 2. 5
5. 0
Figure S1 (b) uV(x 1, 000, 000) C hrom at ogram
5. 0 4. 0 3. 0 2. 0
7. 5
10. 0
12. 5
15. 0
17. 5
20. 0
ce
6. 0
Ac
Downloaded by [New York University] at 10:55 11 July 2015
uV(x1,000,000) C hrom atogram
m in
c
1. 0 0. 0
2. 5
5.0
7. 5
10.0
12. 5
15.0
17. 5
20.0
m in
Figure S1 (c)
Figure S1. Typical GC chromatogram (a) Chlorpyrifos CP (retention time; 15.00min) (b) TCP (retention time; 18.85min) (c) TMP (retention time; 19.12 min).
23
t
120
rip
RE with CS2 RE with CS3
80
us c
RE without CS 60 40 20 0 5
10
M an
COD Removal Efficiency (%)
24
29
34
48
51
55
time (h)
ce
pt e
d
Figure S2. COD removal efficiency (%) in aqueous medium supplemented with (CP 300 mg L-1 , initial pH 7.2, hydraulic retention time (HRT 48 h) inoculated with T14. Data points are average of three replication and error bar represents standard deviation
Ac
Downloaded by [New York University] at 10:55 11 July 2015
RE with CS1 100
24
0.6 0.4 0.2
Chlorine
(b)
0.8 0.6 0.4
t
Relative change in concentration
0.8
1
0.2
Chlorine
Nitrogen
Nitrogen
0 40
0
60
20
us c
Time (h)
M an
20
(c)
Relative change in concentration
0.8 0.6 0.4 0.2 0
20
Time (h)
40
40
60
(d)
0.8 0.6 0.4 Chlorine
0.2
Nitrogen
0 60
ce
0
pt e
Nitrogen
d
Chlorine
Time (h)
1
Relative change in concentration
1
0
20
40 Time (h)
Figure S3. Relative Change in concentration of total chlorine and total nitrogen (a) Without cosubstrate CS, (b) CS1, (c) CS2, (d) CS3.
Ac
Downloaded by [New York University] at 10:55 11 July 2015
0 0
rip
(a)
Relative change in concentration
1
25
60
