1、 Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 25902596ussedContents lists available at ScienceDirectJournal of the Taiwan Institutejou r nal h o mep age: w ww.els1. IntroductionPesticide persistence is one of the serious environmentalproblems. Over the decades, there are many rese
2、arch workswhich have explored the possibility of microorganisms inbiodegradation of pesticide. Oftentimes, scientists haveachieved partial degradation or complete mineralization of aparticular pesticide by biological means which has created aniche in accomplishing an environmentally and economically
3、efficient method when compared to other physical and chemicalmethods. There are works on degradation of organophosphorus,organochlorine, pyrethroids, etc. However, there are lacunae inknowledge regarding degradation of mixed pesticides so in thepresent investigation both organochlorine and organopho
4、spho-rus degradation was carried out.Organophosphorus pesticide generally act on acetylcholines-terase (AChE) thus inhibiting their function, and is responsible formore than two-thirds of deaths due to their high toxicity andwidespread use 1. They are strong inhibitors of cholinesteraseenzymes that
5、function as neurotransmitters, including acetylcho-linesterase, butylcholinesterase and pseudocholinesterase. Mostcommonly used organophosphorus pesticides for agriculture cropsin India are chlorpyrifos, monocrotophos, dimethoate, quinolphos,cyhalothrin and fenthion 2 among which chlorpyrifos andmon
6、ocrotophos were chosen as they are predominantly used inTamil Nadu, India. Chlorpyrifos O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate is an organophosphorus pesticide andits degradation metabolite 3,5,6-trichloro-2-pyridinol (TCP) ismore toxic than the parent compound by possessing anti
7、microbialproperty which inhibits the microbial growth 3. So it is crucialthat bacteria capable of degrading chlopyrifos to further degradeTCP and thereby achieve complete mineralization. Monocrotophosdimethyl-(E)-1-methyl-2 (methylcarbamoyl) vinyls phosphate isalso an organophosphorus, insecticide,
8、upon microbial degrada-tion formation of O-desmethylmonocrotophos monomethylReceived in revised form 14 June 2014Accepted 16 June 2014Available online 11 July 2014Keywords:OrganophosphorusOrganochlorineDegradationFermentorBacterial consortiummixed pesticides. Hence, in the present investigation biod
9、egradation of a mixture of bothorganophosphorus and organochlorine pesticide directed by microbial consortium was carried out ina fermentorbioreactor system. The pesticides chosen were chlorpyrifos, monocrotophos (organophos-phorus) and endosulfan (organochlorine). In order to accomplish an effectiv
10、e degradation, themicroorganisms were isolated from agricultural fields which were previously exposed to the pesticidesmentioned above. The bacteria were isolated by minimal meda with the individual pesticide as the onlycarbon source. The individual strains were identified by morphological, biochemi
11、cal and 16S rRNAcharacterization. The bacterial consortium comprised of 10 organisms which were as follows: Alcaligenessp. JAS1, Ochrobactrum sp. JAS2, Sphingobacterium sp. JAS3 which were screened and selected fromchlorpyrifos contaminated soil, Enterobacter ludwigii JAS17, Pseudomonas moraviensis
12、JAS18 and Serratiamarcescens JAS16 which were isolated from monocrotophos spiked soil and Klebsiella pneumoniae JAS8,Enterobacter cloacae JAS7, Halophilic bacteria JAS4, Enterobacter asburiae strain JAS5 were obtained fromendosulfan persistent soil sample. The fermentorbioreactor system containing 2
13、 L of sterile minimalmedium supplemented with 300 mg lC01of chlorpyrifos, 1000 mg lC01of monocrotophos, 1000 mg lC01ofendosulfan as the only source of carbon and inoculated with 20 ml of bacterial consortium(approximately 3 C2 106cells mlC01). The HPLC and GCMS studies were performed to record thede
14、gradation of mixed pesticides.C223 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.* Corresponding author. Tel.: +91 9843580709; fax: +91 4162243092.E-mail addresses: , (J. Abraham).http:/dx.doi.org/10.1016/j.jtice.2014.06.0141876-1070/C223 2014 Taiwan Ins
15、titute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.Simultaneous degradation of organophospesticides by bacterial consortiumJayanthi Abraham*, Sivagnanam Silambarasan, PeterMicrobial Biotechnology Laboratory, School of Biosciences and Technology, VIT University,A R T I C L E
16、 I N F OArticle history:Received 28 March 2014A B S T R A C TSeveral researchers have focviable option in remediationphorus and organochlorineLogeswariVellore 632014, Tamil Nadu, Indiaon degradation of pesticides by microorganisms, which is the mostof agricultural soil. However, very few have illust
17、rated the degradation ofof Chemical Engineersevier .co m/lo c ate/jtic e300 mg lC01of pesticide in a fermentor containing 2 L of sterileMSM with bacterial consortium of Alcaligenes sp. JAS1, Ochrobac-trum sp. JAS2 and Sphingobacterium sp. JAS3. Degradation ofmonocrotophos was carried out in 2 L MSM
18、containing1000 mg lC01monocrotophos with consortium of S. marcescensJAS16, E. ludwigii JAS17 and P. moraviensis JAS18. The endosulfan(1000 mg lC01) degradation experiment in liquid media wasconducted in a fermentor that contained 2 L of MSM inoculatedwith bacterial consortium of Halophilic bacteria
19、JAS4, E. asburiaeJAS5, E. cloacae JAS7 and K. pneumoniae JAS8. The degradationstudy of mixtures of pesticides was performed in fermentorbioreactor system containing 2 L of sterile MSM supplementedwith 300 mg lC01of chlorpyrifos, 1000 mg lC01of monocrotophos,1000 mg lC01of endosulfan as the only sour
20、ce of carbon andinoculated with 20 ml of bacterial consortium (approximately3 C2 106cells mlC01). Degradation of the mixture of pesticides wasconducted at 37 8C, 208 rpm agitation speed of two six-bladeimpellers, 0.7 vvm (volume of air per volume of mediumper minute) air flow rate. The samples were
21、periodically removedto determine the pesticides concentration and their mainJ. Abraham et al. / Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 25902596 2591phosphate, dimethyl phosphate, N-methylacetoacetamide andN-methylbutyramide has been reported 4. On the other hand,organochlori
22、ne pesticides are long persistent; they have atendency to bioaccumulate and exhibit potential toxicity towardsnontarget organisms for eg.: lindane, metalachlor, dichotodi-phenyltrichloroethane DDT, etc 5. Organochlorine compoundswork on insects by opening the sodium ion channel in theneurons or nerv
23、e cells of insects, causing them to firespontaneously. The insect will go into spasms and eventuallydie 6. Endosulfan is an extensively used cyclodiene organo-chlorine insecticide which is a combination of isomers alpha andbeta. Endosulfan is one of the controversial pesticides which hasbeen extensi
24、vely used in agriculture and is banned from 2011 inIndia due to the toxic effects incurred by it in Kasaragod districtof Kerala, India 7. Upon mineralization, the metabolites formedare endosulfan alpha, endosulfan beta, endosulfan diol andendosulfan sulfate. Endosulfan sulfate is found to be persist
25、entand more toxic than its parent compound.There is a long list of major pesticides which have attractedattention by the scientific community and effective microbialdegradation has been achieved, endosulfan by Bordetella sp. B9 8,Achromobacter xylosoxidans strain C8B 9, monocrotophos byAspergillus o
26、ryzae 10, Paracoccus sp. M-1 11, chlorpyrifos byKlebsiella sp. CPK 12, Spingobacterium sp. JAS3 13, actinobacter14. However, there is a dearth of scientific information regardingdegradation of mixed pesticides by microbes. So in the presentinvestigation, focus was given to simultaneous degradation o
27、fchlorpyrifos, monocrotophos (organophosphorus) and endosulfan(organochlorine) by a given microbial consortium. Hence, thepresent investigation was carried out with bacterial consortiumcomprising of 10 isolates against degradation of chlorpyrifos,monocrotophos and endosulfan.2. Materials and methods
28、2.1. ChemicalsCertified standard of analytical grade chlorpyrifos (99% chemi-cal purity), TCP (99% chemical purity), monocrotophos (99%purity), a-endosulfan (99% purity), b-endosulfan (99% purity),endosulfan sulfate (99% purity), were purchased from SigmaAldrich (St. Louis, MO, USA). The technical g
29、rade pesticides used inthis study were as chlorpyrifos of 20% emulsifiable concentratewas obtained from Isagro (Asia) Agrochemical Pvt. Ltd., Mumbai;endosulfan of 35% emulsified preparation was obtained fromHindustan insecticides limited, Kerala, India and monocrotophosof 36% emulsifiable concentrat
30、e was procured from UnitedPhosphorus Ltd., (Gujarat) India. All other reagents used in thisstudy were of high purity and analytical grade.2.2. MediaMinimal salt medium (MSM) containing (g LC01) (Na2HPO4, 5.8;KH2PO4, 3.0; NaCl, 0.5; NH4Cl, 1; MgSO4, 0.25 and pH 6.87.0) wasused in the process of pesti
31、cide degradation. The nutrient brothwas used for bacterial consortium preparation.2.3. Bacterial strains and consortium preparationThe pesticide degrading bacterial strains which were previouslyisolated from pesticide contaminated soil were used in this study.Chlorpyrifos degrading bacteria were Alc
32、aligenes sp. JAS1, Ochro-bactrum sp. JAS2 and Sphingobacterium sp. JAS3. Monocrotophosdegrading bacteria were Serratia marcescens JAS16, Enterobacterludwigii JAS17 and Pseudomonas moraviensis JAS18. Endosulfandegrading bacteria were Halophilic bacteria JAS4, Enterobacterasburiae strain JAS5, Enterob
33、acter cloacae JAS7 and Klebsiellapneumoniae JAS8.For the preparation of consortium, 100 ml of pure bacterialisolates were inoculated into 250 ml of Erlenmeyer flask contain-ing 50 ml of nutrient broth and incubated at 30 C6 2 8C for 120 rpmfor 2 d. This bacterial consortium (approximately 3 C2 106ce
34、lls mlC01)was used as inoculum for pesticide degradation.2.4. Laboratory scale fermentor systemA laboratory scale fermentorbioreactor system (Lark Innova-tive Fine Teknowledge, Hygene fermentor, India) was used in thisstudy (Fig. 1). The fermentorbioreactor system is equipped with acontrol processin
35、g unit capable of controlling the fermentationparameters, including temperature and agitation. The glass vesselhas a total volume of 3 L with maximum working volumes of 2.5 L.Foaming was controlled by addition of antifoam solution whenrequired. The Lark Innovative Fine Teknowledge, Hygene fermen-tor
36、 containing medium was sterilized in an autoclave at 121 8C for30 min2.5. Biodegradation of mixed pesticidesThe degradation experiments with individual pesticides andmixtures of pesticides were performed in a fermentorbioreactorsystem. Biodegradation of chlorpyrifos was carried out byFig. 1. Physica
37、l appearance of mixed pesticide degradation in the fermentor.metabolites. Thereafter the bacterial growth was regularlymonitored by a spectrophotometer at 600 nm2.6. Analytical methodsOn the day of extraction 510 ml of aqueous samples wererecovered and centrifuged at 7200 C2 g for 10 min to obtain a
38、 cell-free medium. Chlorpyrifos and TCP residues were extracted fromsupernatant using equal volume of dichloromethane (DCM) twice.Organic layer of DCM was aspirated, pooled and evaporated atroom temperature under nitrogen. The residues were dissolved inhigh performance liquid chromatography (HPLC) g
39、rade acetoni-of both chlorpyrifos and TCP were degraded by Paracoccus sp. TRPin 4 d of incubation was reported by Xu et al. 16. Sphingomonassp. DSP2, Stenotrophomonas sp. DSP4 degraded 100 mg lC01ofTable 1Bacterial isolates were obtained from pesticide treated soil.Pesticide Bacterial isolates Acces
40、sionnumberChlorpyrifos Alcaligenes sp. JAS1 JQ514558Ochrobactrum sp. JAS2 JQ514559Sphingobacterium sp. JAS3 JQ514560Monocrotophos Serratia marcescens JAS16 KF313550Enterobacter ludwigii AS17 KF528827Pseudomonas oraviensis AS18 KF528828Endosulfan Halophilic bacteria JAS4 KC509575Enterobacter asburiae
41、 strain JAS5 KC509576MSM + monocrotophos + BacterialconsortiumMSM + endosulfan + BacterialconsortiumCtC0 Co= C0261.6t C0 6.07 CtC0 Co= C0203.1t C0 126.03261.6 203.11.91 2.460.986 0.974J. Abraham et al. / Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 259025962592trile (1 ml) and the
42、n filtered through filter membrane (0.22 mmFH) to remove any particles. Endosulfan and its major metaboliteswere extracted by addition of equal volume of acetonitrile.Monocrotophos and its metabolites were extracted with equalvolume of ethyl acetate.The pesticide concentration was determined by HPLC
43、 (Waters1525 binary HPLC pump, Milford, USA) on a Symmetry C18column (Waters 5 mm, 4.6 mm C2 150 mm). Chlorpyrifos and itsmetabolite TCP was detected at 230 nm The isocratic mobilephase consisted of a mixture of methanol:water (85:15, V:V),which was pumped through the column at a flow rate of1 ml mi
44、nC01. The isocratic mobile phase for endosulfan and itsmetabolites comprised of a mixture of acetonitrile:water (65:35,V:V), which was pumped through the column at a flow rate of1 ml minC01, duration of cycle 20 min, and the breakdownmetabolites were detected at wavelength of 214 nm The isocraticmob
45、ile phase for monocrotophos composed of methanol:water(70:30, V:V), which was pumped through the column at a flowrate of 1 ml minC01, monocrotophos and its metabolite weredetected at 214 nmThe metabolites produced during the mineralization ofmixture of pesticide were analyzed by GCMS (Gas Chromatog-
46、raphy Mass Spectrometry). The sample was prepared by usingethyl acetate and it was injected into GCMS. Perkin Elmer Clarus680 gas chromatographic instrument equipped with a massspectrometer detector (Clarus 600 model) and an Elite-5MS(30.0 m, 0.25 mmID, 250 mm df) column was used. The carrier gasuse
47、d was helium at a flow rate of 1 ml minC01. The followingtemperature program was used: initially the oven temperaturewas held at 60 8C for 2 min and then ramped from 10 8C/min to300 8C with hold time for 4 min, total run time 30 min Thetemperature of the injector was maintained at 300 8C. The ion tr
48、apwas operated at 70 eV with a scan range of m/z from 50 to 600. Asample of 1 ml was injected in split mode (10:1). The intermediateand end product was identified based on the Wiley registry ofmass spectral data.Infrared spectra of pesticide degradations were recorded atroom temperature in the frequ
49、ency range of 4000400 cmC01with aFourier transform infrared (FTIR) spectrophotometer (8400Shimadzu, Japan, with Hyper IR-1.7 software for windows) witha helium neon laser lamp as a source of infrared radiation. PressedTable 2Kinetic parameters for the degradation of individual pesticides by bacterial consortium.Kinetic model Parameters TreatmentsMSM + chlorpyrifos + BacterialconsortiumZero order RegressionequationCtC0 Co= C0181.2t + 118.5 k (dC01) 181.2 DT500.82 R20.988 pellets were prepared by gr
